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Theses & Dissertations Graduate Studies

Fall 12-16-2016

Staphylococcus Aureus Biofilms Interfere With Macrophage

Antimicrobial Responses Through Differential Gene Regulation,
Toxin Production, and Purine Metabolism
Tyler D. Scherr
University of Nebraska Medical Center

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 STAPHYLOCOCCUS AUREUS BIOFILMS INTERFERE WITH MACROPHAGE

ANTIMICROBIAL RESPONSES THROUGH DIFFERENTIAL GENE REGULATION,

TOXIN PRODUCTION, AND PURINE METABOLISM

By

Tyler Scherr

A DISSERTATION

Presented to the Faculty of

The Graduate College of the University of Nebraska Medical Center

In partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Department of Pathology and Microbiology

Under the supervision of Professor Tammy Kielian, Ph.D.

University of Nebraska Medical Center

Omaha, NE

September 2016
 1

Table of contents:

A. Acknowledgements 4

B. Abbreviations 6

C. List of Figures and Tables 10

D. Abstract 14

E. Chapter 1: Introduction 16

1) Staphylococcus aureus (S. aureus) biofilm infection 17

a) Methicillin-resistant S. aureus (MRSA) 17

b) S. aureus biofilms 18

c) S. aureus prosthetic joint infections (PJIs) 19

d) Mouse models of S. aureus prosthetic joint infection (PJI) 20

2) S. aureus virulence and host immune evasion 21

a) Accessory gene regulator (agr) 21

b) Microenvironment modulation 22

3) Innate immune response to S. aureus infection 23

a) Innate immune recognition of S. aureus 23

b) MΦ and PMN response to planktonic S. aureus 25

c) MΦ and PMN response to S. aureus biofilm 26

4) Overview of dissertation 27

F. Chapter 2: Materials and Methods 29

1) Bacterial strains and microbiological techniques 30

 2

2) Mouse strains 31

3) Cell culture techniques 33

4) Immune cell co-culture with S. aureus biofilms in vitro 35

5) Nebraska Transposon Mutant Library Screens 37

6) BMDMΦ treatments with biofilm-conditioned medium 39

7) Orthopedic implant S. aureus biofilm infection 40

8) Bacterial microarray analysis and qRT-PCR 43

9) Bacterial Proteomics 45

10) Bioflim extracellular DNA (eDNA) isolation 46

G. Chapter 3: Global transcriptome analysis of Staphylococcus aureus biofilms in

response to innate immune cells 49

Abstract 50

Introduction 51

Results 54

Discussion 84

H. Chapter 4: Staphylococcus aureus biofilms induce macrophage dysfunction through

leukocidin AB and α-toxin 88

Abstract 89

Introduction 90

Results 92

Discussion 118
 3

I. Chapter 5: purB affects eDNA release during Staphylococcus aureus biofilm

development to evade macrophage recognition 123

Abstract 124

Introduction 125

Results 127

Discussion 142

J. Chapter 6: Discussion and Future Directions 147

Key findings and conclusions 147

Future directions 152

L. References 156
 4

Acknowledgements

I would first like to thank my mentor, Dr. Tammy Kielian, for her constant support, guidance, and

encouragement. I came to UNMC hoping to have the opportunity to work on translational

research; to spend my time researching something that might eventually have a positive impact on

people. Everything Tammy does with her lab is always with the patient in mind, and I couldn’t

have asked for a better mentor. Her scientific curiosity and passion for translational research is

infectious and, through all of the ups and downs of scientific research, I’ve been proud to be a

small part of her lab and almost always looked forward to putting in a full-days’ work there.

I would also like to thank my supervisory committee, Dr. Kenneth Bayles, Dr. Paul Fey, Dr.

Curtis Hartman, Dr. Jessica Snowden, and Dr. Geoffrey Thiele for sharing their time and

expertise in providing invaluable guidance in shaping my educational experience and insightful

suggestions regarding my scientific projects.

Next, I need to thank all of the members of the Kielian lab past and present that I have had the

pleasure working alongside over the past 5 years. Mark Hanke, Cortney Heim, Debbie Vidlak,

Amy Aldrich, and Amanda Angle welcomed me into the lab and patiently showed me the ropes

and helped me considerably with completing my projects and maintaining my sanity. Richa

Hanamsagar, Jane Xiong, Nikolay Karpuk, Maria Burkovetskaya, Rachel Fallet, Jessica Odvody,

Megan Bosch, and Venkata Kakulavarapu have all provided valuable feedback on my research

and/or contributed directly to the success of my experiments. Finally, Casey Gries, Kelsey

Yamada, and Anna Staudacher have more recently been a pleasure to work with and near on the

Staph side of the lab, and I owe each of them a debt of gratitude for their assistance as well.
 5

I also owe a big thank you to several members of the Fey, Bayles, Chittezham Thomas, and

Snowden labs past and present for their assistance on various projects: Cortney Halsey, Carolyn

Schaeffer, Austin Nuxoll, Jill Lindgren, Marat Sadykov, Jennifer Endres, Derek Moormeier,

Vijaya Yajjala, Todd Widhelm, Matthew Beaver, Vinai Chittezham Thomas, and Sujata

Chaudhari. Several of our collaborators have also been instrumental to my project, including Dr.

Jeffrey Bose, Dr. Alex Horswill, Dr. Keer Sun, Dr. Victor Torres, and Dr. David James. I also

owe a special thanks to members of the UNMC Proteomics Facility (Dr. Pawel Ciborowski and

Jayme Horning) and the UNMC Flow Cytometry Facility (Dr. Charles Kuszynski, Dr. Phil

Hexley, Victoria Smith, and Sam Wall) for their patience and assistance with several

experiments.

Finally, I would like to thank my family and friends for their unceasing love and support. First

and foremost, to my beautiful wife Joelle, without whose constant support none of this would

have been nearly as successful. Next, my wild son Jax, who gives me a fresh perspective every

day and without whom none of this would be nearly as meaningful. To my parents, who shaped

me and without whom none of this would have been possible. To my family, who provided love

and support throughout my life and balance the past five years, especially my sister, Jennie,

brothers-in-law Ryan and Josh, my parents-in-law Tom and Kerry, and my Perry. And to all of

my UNMC friends who have made this journey in part or full with me (including the BRTP class

of 2011, Intramural Disc Golfers, 2015 GSA Officers, and UNMC Makers); I could have

definitely gotten my Ph.D. without you, but it most definitely would not have been as fun!
 6

Abbreviations:

Agr Accessory gene regulator

AIP Autoinducing peptide

ANOVA Analysis of variance

BHI Brain-heart infusion

BMDMΦ Bone marrow-derived macrophage

CA-MRSA Community-acquired Methicillin-resistant Staphylococcus aureus

CFU Colony forming units

DMEM Dulbecco’s Modified Eagle’s Medium

DNA Deoxyribonucleic acid

ECM Extracellular matrix

eDNA extracellular DNA

ELISA Enzyme-linked immunosorbent assay

Erm Erythromycin

EtOH Ethanol

FACS Fluorescence-activated cell sorting

FBS Fetal bovine serum

Fc Fragment, crystallizable

FITC Fluorescein isothocyanate

GFP Green fluorescent protein

GM-CSF Granulocyte-macrophage colony-stimulating factor

 7

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Hla α-hemolysin

HRP Horseradish peroxidase

IFN Interferon

Ig Immunoglobulin

IL Interleukin

iNOS Inducible nitric oxide synthase

i.p. Intraperitoneal

KO Knockout

K-wire Kirschner wire

LAC Los Angeles County

L-glut L-glutamine

LTA Lipoteichoic acid

LukAB Leukocidin AB

MΦ Macrophage

MACS Magnetic-activated cell sorting

M-CSF Macrophage colony-stimulating factor

MDSC Myeloid-derived suppressor cell

MRSA Methicillin-resistant Staphylococcus aureus

MSCRAMM Microbial surface components recognizing adhesive matrix molecules

MET Macrophage extracellular trap

 8

MyD88 Myeloid differentiation primary response gene 88

NADPH Nicotinamide adenine dinucleotide phosphate

NET Neutrophil extracellular trap

NOD2 Nucleotide-binding oligomerization domain-containing protein 2

NOS Nitric oxide synthase

OD Optical density

PAMP Pathogen associated molecular pattern

PBP2a Penicillin-binding protein 2a

PBS Phosphate-buffered saline

PCR Polymerase chain reaction

PE Phycoerythrin

PerCP Peridinin-chlorophyll-protein complex

PFA Paraformaldehyde

PGN Peptidoglycan

PIA Polysaccharide intercellular adhesion

PJI Prosthetic joint infection

PMN Polymorphonuclear cell

PRR Pattern recognition receptor

PVL Panton-Valentine Leukocidin

qRT-PCR Quantitative real time-polyemerase chain reaction

RNA Ribonucleic acid

 9

ROS Reactive oxygen species

RPMI Roswell Park Memorial Institute

S. aureus Staphylococcus aureus

s.c. Subcutaneous

SCC Staphylococcal cassette chromosome

S. epidermidis Staphylococcus epidermidis

TCR T cell receptor

TNF Tumor necrosis factor

TSA Tryptic soy agar

TSB Tryptic soy broth

WT Wild-type
 10

List of Figures and Tables

Figure S2.1. Fluorescent microspheres can be used as a surrogate for live S. aureus to assess MΦ

phagocytosis

Figure 3.1. S. aureus biofilm growth states

Figure 3.2. S. aureus biofilm-leukocyte co-culture paradigm

Figure 3.3. Differential responses of innate immune cells to S. aureus biofilms

Figure 3.4. Acute MΦ addition to S. aureus biofilms leads to the transcriptional repression of

numerous genes

Figure 3.5. Classification of genes significantly altered by leukocyte addition in S. aureus

biofilms

Figure 3.6. qRT-PCR validation of down-regulated genes in S. aureus biofilms identified by

microarray analysis

Figure 3.7. agr promotes S. aureus biofilm resistance to PMN challenge

Figure 3.8. Differential responses of MΦs and neutrophils to S. aureus biofilms are cell

autonomous

Supplemental Figure S3.1. PMNs phagocytose S. aureus biofilms throughout the co-culture

period

Table 3.2. Significantly down-regulated genes between 6 day-old S. aureus biofilm versus

biofilm-MΦ co-cultures for a 1 h period

Table 3.3. Differentially expressed genes between 6 day-old S. aureus biofilm versus biofilm-

MΦ co-cultures for a 24 h period

Table 3.4. Differentially expressed genes between 6 day-old S. aureus biofilm versus biofilm-

PMN co-cultures for a 1 h period

 11

Table 3.5. Differentially expressed genes between 6 day-old S. aureus biofilm versus biofilm-

PMN co-cultures for a 4 h period

Supplemental Table S3.1. Significantly increased genes between 6 day-old S. aureus biofilm

versus biofilm-MΦ co-cultures for a 1 h period

Supplemental Table S3.2. Significantly increased genes between 4 day-old S. aureus biofilm

versus biofilm-PMN co-cultures for a 1 h period

Figure 4.1. S. aureus biofilms secrete a proteinaceous factor(s) that inhibits MΦ phagocytosis

Figure 4.2. S. aureus biofilm-induced MΦ dysfunction is partially agr-dependent

Figure 4.3. SWATH-MS identifies potential biofilm factors responsible for MΦ dysfunction

Figure 4.4. LukA and Hla secretion is enhanced in S. aureus biofilms

Figure 4.5. LukAB and Hla play significant roles in biofilm-induced MΦ dysfunction

Figure 4.6. S. aureus Hla and LukAB act in concert to promote MΦ dysfunction

Figure 4.7. LukAB and Hla are important for S. aureus biofilm formation in vivo

Figure 4.8. Proposed model for S. aureus biofilm-induced MΦ dysfunction

Supplemental Figure S4.1. Bacterial counts and extracellular protein concentrations of S. aureus

WT planktonic, biofilm, and isogenic mutant biofilms are similar

Supplemental Figure S4.2. Validation of S. aureus Hla action on MΦ dysfunction

Supplemental Figure S4.3. Purified LukAB augments MΦ cytotoxicity

Supplemental Figure S4.4. Characterization of S. aureus biofilm growth in 1% casamino acids

Supplemental Table S4.1. Proteins identified to be in greater abundance in WT S. aureus

biofilm-conditioned medium compared to ∆agr biofilm-conditioned medium

 12

Supplemental Table S4.2. Proteins identified to be in greater abundance in WT S. aureus

biofilm-conditioned medium compared to WT planktonic-conditioned medium

Figure 5.1. Genes expressed during S. aureus biofilm growth that influence MΦ NF-κB

activation

Figure 5.2. purB is important for preventing MΦ invasion and phagocytosis of mature S. aureus

biofilms

Figure 5.3. eDNA is increased in S. aureus ΔpurB biofilms

Figure 5.4. S. aureus biofilm eDNA can be detected by MΦs and trigger activity

Figure 5.5. S. aureus purB is important for chronic biofilm establishment

Table 5.1. Genes expressed by S. aureus biofilms that influence MΦ NF-κB activation in vitro

Supplemental Figure S5.1. Predicted purine biosynthetic pathway in S. aureus

Supplemental Figure S5.2. purB activity effects Hla production in S. aureus biofilms

Supplemental Figure S5.3. Adenosine attenuates MΦ activation

Figure 6.1. Mechanisms by which S. aureus biofilms interfere with MΦ antimicrobial responses
 13

University of Nebraska Medical Center

Omaha, NE

September, 2016

STAPHYLOCOCCUS AUREUS BIOFILMS INTERFERE WITH MACROPHAGE

ANTIMICROBIAL RESPONSES THROUGH DIFFERENTIAL GENE REGULATION,

TOXIN PRODUCTION, AND PURINE METABOLISM

Tyler Scherr, Ph.D.

University of Nebraska Medical Center, 2015

Advisor: Tammy Kielian, Ph.D.

 14

Abstract

Staphylococcus aureus (S. aureus) is an opportunistic pathogen that is a leading cause of both

nosocomial and community-associated infections. Armed with a myriad of virulence factors and

the propensity to form a biofilm on native tissues and implanted medical devices alike, S. aureus

infections represent a very real public health threat, the treatment of which results in an excessive

economic burden. S. aureus biofilm infections are notoriously recalcitrant to antibiotic therapy

and adept at evading and neutralizing the host immune antimicrobial response. Previous studies

from our laboratory have shown that S. aureus biofilms are able to cause persistent infections, in

part, through the reprogramming of the macrophage (MΦ) immune response. While macrophages

are readily able to recognize and respond to S. aureus in a planktonic state, their ability to mount

a functional antimicrobial attack is thwarted upon encountering S. aureus biofilm. We have

observed that MΦs in close proximity to S. aureus biofilms are less phagocytic and skewed

towards an anti-inflammatory profile typified by arginase and IL-10 production. We have

demonstrated that the ability of S. aureus biofilms to cause chronic infections is due, in part, to

TLR2 or TLR9 evasion. However, we have shown that MyD88 signaling does provide some

benefit to the host in combating S. aureus biofilm infections, which may be attributed to IL-1

receptor signaling. To better understand how S. aureus biofilms subvert the MΦ antimicrobial

response, the work described in this dissertation assessed S. aureus transcriptional activity during

co-culture with MΦs, whether S. aureus biofilms inhibit MΦ activity through secreted molecules,

and performed a high-throughput screen of the Nebraska Transposon Mutant Library to identify

key genes involved in dampening the MΦ NF-κB-regulated proinflammatory response. We found

that S. aureus biofilms attenuate their transcriptional activity following MΦ exposure, augment α-

hemolysin (Hla) and leukocidin AB (LukAB) secretion to inhibit MΦ phagocytosis and induce

cell death, and rely on a functional purine biosynthetic pathway to prevent MΦ invasion and

phagocytosis, in part, through controlling the amount of eDNA available for MΦ recognition at
 15

the surface of the biofilm extracellular matrix (ECM). Collectively, these studies build upon our

previous observations by identifying key mechanisms whereby S. aureus biofilms are able to

thwart the MΦ antimicrobial response.

 16

Chapter 1: Introduction
 17

1) Staphylococcus aureus (S. aureus) biofilm infection

a) Methicillin-resistant S. aureus (MRSA)

Staphylococcus aureus (S. aureus) is a gram-positive, opportunistic pathogen that in the

past few decades has become a leading cause of both nosocomial and community-associated

infections (1). S. aureus is the most virulent of the staphylococcus genus, with the ability to cause

a wide range of diseases, from relatively minor skin and soft tissue infections (SSTIs) to more

severe pneumonia and sepsis potentially leading to toxic shock syndrome (2). S. aureus has

remained a successful pathogen throughout the antibiotic era due, in part, to its propensity to

rapidly acquire antibiotic resistance, and methicillin-resistant S. aureus (MRSA) is no exception.

Methicillin, a beta-lactamase insensitive beta-lactam that is bacteriocidal by inhibiting

peptidoglycan synthesis, began being used in the 1950s in response to the emergence of

penicillin-resistant S. aureus (PRSA). However, within ten years MRSA isolates began to emerge

in hospitals across the US, with one of the first known incidences occurring at Boston City

Hospital (3). Through the intervening decades, MRSA continued to slowly spread, but generally

only affected immune compromised patients, until the late 1990s when a dramatic surge in

MRSA rates began (4). It was around this time that community-associated MRSA (CA-MRSA)

began to occur outside of hospitals, inflicting otherwise healthy individuals (5) who, had

historically encountered primarily methicillin-sensitive S. aureus (MSSA) strains.

CA-MRSA infection is now considered a worldwide epidemic and SSTIs are common,

particularly among athletes, children, homeless persons, military personnel, and many other

groups (6). While exact numbers vary from study to study, some reports place the percentage of

CA-MRSA as responsible for over 50% of all CA-S. aureus infections (7). The emergence of

MRSA coincides with the ability of S. aureus to express a low-affinity penicillin binding protein

(PBP) known as PBP2a (3), which is encoded by the mecA gene and carried on a mobile genetic

element known as Staphylococcal Cassette Chromosome (SCC) mec (8), which also contains
 18

intact or truncated sets of the divergently transcribed regulatory genes, mecR1 and mecI (3). In

the presence of beta-lactams, such as methicillin, mecR1 cleaves mecI bound to the operator

region of the mecA promoter, thereby de-repressing PBP2a production (9). Since methicillin

cannot bind PBP2a, cell wall synthesis is able to proceed and S. aureus is able to replicate in its

presence (3). While both are MRSA, CA-MRSA usually differs from nosocomial MRSA in that it

harbors smaller SCCmec variants, Type IV and V, is less resistant to other types of antibiotics,

produces more toxins including Panton-Valentine Leukocidin (PVL), and most commonly causes

SSTIs (3).

b) S. aureus biofilms

Many bacteria can form biofilms, which are generally defined as an adherent community

of bacteria encased within a self-produced matrix typically composed of a combination of

exopolysaccharides, proteins, and extracellular DNA (eDNA) (10-12). The biofilm composition

is often dependent on environmental factors, such as nutrient availability and mechanical stress

(11). As biofilms represent a distinct lifestyle, biofilm-associated bacteria exhibit an altered

phenotype from planktonic cells with regard to metabolism, gene expression, and protein

production , undoubtedly in part due to nutrient, pH, and oxygen gradients present throughout the

characteristic tower-like biofilm structures (13). Biofilms represent a communal virulence

determinant to circumvent immune-mediated clearance and establish persistent infection (13-16).

Most medical device-associated biofilm infections are caused by S. epidermidis and S. aureus,

and both species can also establish biofilms on native host surfaces, such as heart and bone tissue

(17-19). The biofilm mode of growth has been recognized as a major mediator of infection,

contributing to an estimated 80% of all infections (20).

Biofilm formation is classically considered to occur in a stepwise fashion, consisting of

initial attachment, cell aggregation (21, 22) and proliferation, accumulation of an ECM and tower

formation, and detachment of cells and biofilm dispersal (10, 23, 24). Initial attachment is
 19

believed to be largely physico-chemical in nature, due to features such as hydrophobicity during

the process of passive adsorption (10, 24). S. aureus can form biofilms on both native tissue and

abiotic surfaces, and while the specific mechanisms for attachment likely differ between the two

types of surfaces, it is believed to be mainly governed by the interaction of bacteria with human

matrix proteins either present on native tissue or deposited on implanted medical devices shortly

after placement (24). Following initial attachment, S. aureus cells begin to accumulate and

aggregate into multiple layers in a process that is largely mediated by the expression of microbial

surface components recognizing adhesive matrix molecules (MSCRAMMs) (10). As the name

suggests, these are surface-attached proteins (e.g. fibronectin- and fibrinogen-binding

proteins)(25, 26) that help anchor the bacteria to the surface by linking with human matrix

proteins (24). Next, cells began to proliferate and form biofilm structures through the production

of exopolysaccharides (e.g. polysaccharide intercellular adhesion [PIA]) (27-29), proteins (e.g. S.

aureus surface proteins C and G [SasC, G]) (30, 31), and eDNA (10, 24, 32). The formation of

channels and tower-like structures are thought to facilitate nutrient and waste exchange within the

base of the biofilm (13, 23). Finally, upon full maturity biofilm-associated bacteria begin to

detach, facilitating biofilm dispersal in a process influenced by quorum-sensing (QS). QS is a

phenomenon whereby increased bacterial cell density triggers changes in gene expression and, in

S. aureus, is governed, in part, by the accessory gene regulator (agr) system (24).

c) S. aureus prosthetic joint infections (PJIs)

An increasing world population coupled with increased life expectancies has led to a

progressive rise in primary and revision arthroplasties (33). By 2030, the demand for primary

total hip and knee arthroplasties in the United States alone is estimated to increase by 174% and

673%, respectively, over the number of procedures performed in 2005 (17). Due to its commensal

nature, it has been estimated that 20% of healthy adults are S. aureus carriers, typically in the

nasal cavity, with another 60% experiencing transient carriage (34), placing a vast majority of the
 20

adult population at increased risk of infection (35). Infection is the major complication of

orthopedic implants, with incidences reportedly between 1-2% for both knee and hip

replacements (36-40), and S. aureus is a leading cause of orthopedic implant infections with

serious morbidity and mortality outcomes (17, 41) in large part due to its ability to form a

biofilm. Biofilm infections are recalcitrant to antibiotics (42, 43), which often necessitates

removal of the infected device or native tissue, and are associated with significant morbidity and

economic impact (17, 44, 45). Indeed, it has been estimated that approximately $1.8 billion is

spent annually in the US for the treatment and clinical management of orthopedic implant-related

infections (46, 47).

While the exact mechanisms of prosthetic infection are still unclear, studies have

indicated that the presence of a foreign body reduces the minimal inoculum of S. aureus required

to cause infection by a factor of greater than 105 (48, 49). Experimental evidence in animal

models, for instance, have shown that an infection can occur with fewer than 100 colony forming

units (CFU) of microorganisms, and despite the use of perioperative antimicrobial prophylaxis,

when a foreign body is present (50). These animal observations have seemingly held true in

human patients as well, with higher relapse of infection following bacteremia in patients with

orthopedic implants, 30-40% of which followed S. aureus bacteremia (51, 52).

d) Mouse models of S. aureus prosthetic joint infection (PJI)

There are currently two published murine models of S. aureus PJI, both of which explore

static biofilm growth and host-pathogen interactions during a persistent infection in an implant-

associated osteomyelitis setting, but which differ slightly in placement of the metal implant and

observed host response. In the mouse tibial implant model, C57BL/6 mice have been shown to

establish a chronic infection resulting in upregulation of IL-2, IL-12p70, TNF-α, IL-1β, IL-6, and

IL-17 (53). In this model, it has been proposed that early activation of the inflammatory response

may not only be ineffective at microbial clearance, but may also be detrimental to the host due to
 21

collateral tissue damage caused by neutrophil (PMN) activation and inflammatory mediator

influx. In contrast, the early inflammatory response gives way to a chronic anti-inflammatory

milieu during the femur implant model, typified by myeloid-derived suppressor cell (MDSC)

infiltration in part through IL-12p70-mediated recruitment, a dampening of proinflammatory

signaling (e.g. IL-1β and IL-6), an increase in anti-inflammatory signaling (e.g. IL-10) (54-56).

2) S. aureus virulence and host immune evasion

a) Accessory gene regulator (agr)

S. aureus regulates protein production via a number of two-component systems,

including a bacterial density-dependent mechanism of cell-to-cell communication known as QS.

QS operates through the secretion and detection of a signal molecule, in this case autoinducing

peptide (AIP), which triggers a cascade of cellular responses (57). In S. aureus, the QS system is

encoded by the agr locus, composed of four co-transcribed genes (agrA, agrC, agrD, and agrB)

encoding a two-component trans-membrane transduction complex, a pro-signaling peptide, and a

membrane transporter (58). In S. aureus, at least four agr allelic genes exist that are characterized

by a specific signaling peptide (numbered I to IV) and it has been observed that the agr activity

within a group inhibits that of the other groups (59). The effector molecule of the agr system is a

regulatory RNA, RNAIII, whose synthesis is dependent on agr activation and driven by the P3

promoter (10). Altogether, the agr system acts as a trigger, switching from the expression of

surface-associated proteins to secreted proteins and has been shown to regulate numerous

virulence factors (e.g. α-toxin, leukocidins, phenol-soluble modulins [PSMs], adhesins, proteases,

etc.) and even play a role in the biofilm maturation process (60, 61). For example, the DNA-

binding regulatory protein SarA is critical for biofilm formation and virulence factor expression

in S. aureus and SarA acts, in part, through the accessory gene regulator agr, which can thus be

thought of as an important regulatory switch between planktonic and biofilm lifestyles (62-65).

b) Microenvironment modulation
 22

Previous work has characterized numerous secreted virulence factors used by

staphylococci that target specific host cell populations. S. aureus, in particular, utilizes numerous

hemolysins (66), leukocidins (67, 68), and proteases to evade the host immune system (69).

While staphylococci certainly divert resources from secreted to structural proteins during early

biofilm growth, protein secretion is still maintained in biofilms. For example, several reports have

demonstrated that various bacterial biofilm-forming species secrete peptides and full-length

proteins (i.e. Esp) that interfere with biofilm development of competing organisms, presumably to

eliminate competition for a biofilm-friendly niche (70-72). Additionally, studies have

demonstrated evidence of host-biofilm cross-talk involving the QS molecules N-Acyl homoserine

lactones (AHL), presumably to facilitate biofilm formation and bacterial persistence (73, 74).

Therefore, it is likely that staphylococcal biofilms also secrete factors in vivo, perhaps under QS

system regulation, in an attempt to evade immune recognition and clearance. For example, we

have recently shown that S. aureus biofilms are uniquely enriched in Hla and LukAB in an agr-

dependent manner in vitro and that the ability of S. aureus biofilm-conditioned medium to inhibit

MΦ phagocytosis is partially dependent on an intact biofilm (75). However, the identity of such

molecules remains to be elucidated but could represent future attractive anti-biofilm agents (76).

Besides virulence factors, staphylococci also secrete molecules for nutrient procurement

and cell signaling. For example, siderophores are critical for iron acquisition (77), while several

signaling molecules are important for biofilm remodeling and dispersal, such as AIP (65),

nuclease (78), and PSMs (79). While the primary roles of these molecules are apparently

unrelated to immune interactions, recent evidence suggests the potential for alternative functions.

For example, nuclease, which mediates biofilm dispersal (64, 78), can also participate in

neutrophil extracellular trap (NET) degradation (80). Recently, nuclease in combination with

adenosine synthase has been implicated in the conversion of NETs to leukotoxic deoxyadenosine,

highlighting a clever means whereby S. aureus turns the immune response against itself (81). In
 23

addition, S. aureus PSM expression is induced following PMN phagocytosis, resulting in PMN

lysis and bacterial escape (79, 82, 83). This process is regulated by the stringent response

characterized by the synthesis of the intracellular signaling alarmone (p)ppGpp (83). Therefore,

the stringent response provides yet another means of staphylococcal adaptation in response to

select immune pressures, something that has previously been well-established in relation to

nutrient availability and metabolism (84-89). Indeed, recent evidence has implicated (p)ppGpp

and di-cyclic NTPs as critical signals in the switch from planktonic to biofilm growth (76, 90).

However, secreted factors are not the only means whereby staphylococcal biofilms could

regulate the host response in an exogenous manner. While bacteria have a plethora of

environmental sensory mechanisms at their disposal, host immune cells are also very sensitive to

their surrounding environment (91-93). Because biofilms represent a large biomass, the sum

metabolic activity of the bacteria themselves would be expected to have an impact on pH and

oxygen levels in the surrounding tissue microenvironment. Indeed, even subtle changes in pH

(92, 94) or oxygen (91) can significantly alter the nature of the immune response. Furthermore,

novel research with fungal and bacterial biofilms has identified a coordinated system of ROS

signaling for biofilm maturation (95). It will be interesting to see what, if any, impact this biofilm

ROS gradient has on the host immune response and whether host-generated ROS can also act as a

signaling molecule within the biofilm to influence target pathways, such as biofilm formation via

QS (76, 95).

3) Innate immune response to S. aureus infection

a) Innate immune recognition of S. aureus

The innate immune system is well equipped to recognize foreign invaders through germ-

line encoded pattern recognition receptors (PRRs) that identify broad, highly conserved microbial

patterns known as pathogen-associated molecular patterns (PAMPs) (96). One such class of PRRs

is the Toll-like receptors (TLRs), which are responsible for the recognition of several key PAMPs
 24

from both gram-positive and gram-negative bacterial pathogens (97, 98). Of particular

importance to S. aureus infections are TLR2 and TLR9, which recognize PGN and lipoproteins in

the bacterial cell wall and unmethylated CpG motifs characteristic of bacterial DNA, respectively

(99-102). TLR2 is expressed on the outer cell surface where it recognizes its ligands that are

naturally secreted and/or released from growing bacteria. TLR9, on the other hand, is an

intracellular receptor that could engage liberated bacterial DNA following phagocytosis and

degradation in the phagosome. In total, 12 TLRs have been identified in mice and 10 in humans,

each with their own unique ligand specificity (96, 103). While TLRs recognize different bacterial

PAMPs, most utilize a common signaling pathway through myeloid differentiation factor 88

(MyD88) and NF-κB that results in the transcriptional activation of several proinflammatory

cytokines and chemokines (104-107) that are critically important for coordinating an effective

antimicrobial immune response.

Although TLRs have been demonstrated to be critically important in mediating innate

immune recognition and clearance of S. aureus during planktonic growth (101, 108-111), several

recent studies have shown that S. aureus biofilms are able to circumvent TLR2 and TLR9

recognition (15, 56). These findings also agree with the observations that TLR2-deficient patients

show no increased risk of developing post-arthroplasty S. aureus infections (112). While the

mechanism responsible for TLR2/9 evasion by S. aureus biofilms are unknown, this could in part

be explained by ligand inaccessibility via the protective barrier provided by the complex biofilm

ECM (16). However, it is still possible that S. aureus biofilms may be recognized by alternative

PRRs, such as AIM2, DNA-dependent activator of IFN-regulator factors (DAI), and nucleotide-

binding oligomerization domain-containing protein 2 (NOD2) (113-116). Additionally, while

TLR2 and TLR9 do not appear to be important for S. aureus biofilm recognition and clearance,

IL-1β has been demonstrated to be critical for controlling early bacterial burdens in PJI (56).

Likewise, studies from our laboratory and others have shown that MyD88 signaling is critical for
 25

controlling bacterial burdens during S. aureus biofilm infections (15, 117, 118). Interestingly,

both the IL-1 receptor (IL-1R) and TLRs signal through the adaptor MyD88 for the eventual

induction of NF-kB-mediated transcription (16).

b) MΦ and neutrophil (PMN) response to planktonic S. aureus

The MΦ is a key innate immune responder that resides in nearly all tissues in the body

and arises upon maturation from extravasating monocytes from the peripheral circulation (119-

121). MΦs are highly plastic in that they can display a spectrum of functional states depending on

their environment. For example, in vitro experimentation has shown that monocytes treated with

granulocyte-macrophage colony-stimulating factor (GM-CSF) and inflammatory stimuli such as

PAMPs give rise to microbicidal M1 MΦs, whereas monocytes treated with macrophage colony-

stimulating factor (M-CSF) and IL-4 give rise to anti-inflammatory M2 MΦs that function in

tissue repair and fibrosis (122-124). In vivo, however, the situation is believed to be more

complex, with MΦ functionality existing on a dynamic spectrum of polarized states. Previous

work from our laboratory and others has shown that upon encountering S. aureus in a planktonic

state, MΦs become classically activated (M1) and exert their microbicidal effector functions, in

part, through the production of reactive oxygen and nitrogen species, in addition to

proinflammatory cytokines (125-128). MΦs also clear planktonic bacteria by phagocytosis, which

is tightly linked with proinflammatory cytokine and chemokine production to initiate adaptive

immune responses (129-131). Collectively, these innate leukocyte responses, coupled with

complement activation, usually ensure the successful clearance of planktonic staphylococcal

infections in an immune competent host.

The neutrophil (PMN) is typically the most prominent cellular component of the host

innate immune response to bacterial infections (132). PMNs are frequently the first responders to

microbial invaders and, as such, it has been observed that individuals with PMN defects (e.g.

chronic granulomatous disease) are highly susceptible to severe and life-threatening S. aureus
 26

infections (133). PMNs originate and mature in the bone marrow, maintain immune surveillance

by circulating in the peripheral vasculature, and are rapidly recruited to infected tissues through

chemotactic signals produced by host cells (e.g. IL-8, GRO-alpha, and MIP-2) (134-136) and/or

shed and secreted bacterial molecules (e.g. lipoteichoic acid or n-formyl peptides) (109, 137-141).

Upon encountering a foreign invader, PMNs will attempt to phagocytose the microbes, a process

which can be stimulated by PRRs but greatly enhanced when the pathogens are opsonized with

host serum molecules, including antibodies and cleavage products of the complement cascade

(142-146). Upon phagocytosis, PMNs use both oxygen-dependent (e.g. NADPH-dependent

oxidase) (147-150) and oxygen–independent (e.g. granules containing microbicidal agents such

as cathepsins) (151-154) processes to kill microbes. Recently, PMN and MΦ extracellular traps

(NETs and METs, respectively) have been identified as another means of antimicrobial action

(80, 155-157). This “beyond the grave” mechanism is typified by an extracellular net of DNA

released from dying phagocytes that contains localized islands of lytic enzymes that kill ensnared

extracellular bacteria. Collectively, these innate leukocyte responses, coupled with complement

activation, usually ensure the successful clearance of planktonic staphylococcal infections in an

immune competent host (76).

c) MΦ and PMN response to S. aureus biofilms

In contrast to planktonic S. aureus infections, a very different scenario has emerged

regarding biofilm infections. Recent studies have demonstrated that staphylococcal biofilms

actively skew host immunity toward an anti-inflammatory, pro-fibrotic response that favors

bacterial persistence (15, 158-160). This is typified by alternatively-activated (M2) MΦs and

arginase-1 (Arg1) activity, resulting in urea and ornithine production, which are involved in

collagen formation and tissue remodeling (127, 161, 162). Our laboratory has shown that MΦs

associated with S. aureus biofilms both in vitro and in vivo have decreased inducible nitric oxide

synthase (iNOS) concomitant with increased Arg1 expression, as well as attenuated cytokine and
 27

chemokine production (15, 159, 160). Similar findings have been reported in response to S.

epidermidis biofilms (158, 163-165) and biofilms from other bacterial species (166-169),

suggesting a conserved mechanism exists to thwart host immunity to ensure biofilm persistence

(76, 170).

4) Overview of dissertation

Initial experiments from our laboratory demonstrated that S. aureus biofilms were able to

cause persistent infections, in part, through the reprogramming of the MΦ immune response (15).

Specifically, we observed that MΦs in close proximity to S. aureus biofilms were less phagocytic

and skewed towards an anti-inflammatory M2 profile typified by Arg1 and IL-10 expression (15).

We hypothesized that this was due, in part, to the physical barrier presented by the biofilm ECM,

and that this matrix could be facilitating immune evasion by occluding otherwise recognizable

PAMPs as well as inducing “frustrated phagocytosis”. Additionally, we found that S. aureus

biofilms evade TLR2- and TLR9-mediated recognition in a murine model of biofilm infection

(15). However, we did demonstrate that MyD88 signaling does provide some benefit to the host

in combating S. aureus biofilm infections and that injecting exogenously activated M1 MΦs into

an established biofilm infection can promote clearance in vivo (159, 160).

During the course of my research, I have further identified multiple mechanisms that

contribute to S. aureus biofilm evasion of MΦ antimicrobial activity. First, I determined that S.

aureus biofilm-associated bacteria dampen their transcriptional activity upon encountering MΦs,

perhaps allowing the bacteria to limit the possibility of MΦ detection (171). Next, I observed that

S. aureus biofilm-conditioned medium is able to inhibit MΦ activation and induce MΦ cell death

due to heightened Hla and LukAB production (75). Finally, I performed a high-throughput screen

of the Nebraska Transposon Mutant Library and discovered the importance of purine biosynthesis

towards the ability of S. aureus biofilms to prevent MΦ phagocytosis. My research adds to the

growing literature describing the ability of S. aureus biofilms to circumvent the MΦ antimicrobial
 28

response and highlights some potential targets for novel treatments and prevention of chronic

biofilm related infections.

 29

Chapter 2: Materials and Methods

 30

1) Bacterial strains and microbiological techniques

Bacterial strains

S. aureus USA300 LAC is a community-associated methicillin-resistant (CA-MRSA) strain

isolated from a Los Angeles county (LAC) jail inmate with a SSTI and was also responsible for

the CA-MRSA outbreak of 2002 (172-175). We received the isolate from Dr. Frank DeLeo

(National Institute of Allergy and Infectious Diseases Rocky Mountain Laboratories, Hamilton,

MT) and cured it of its 27 kb LAC-p03 plasmid encoding erythromycin resistance (176) by

screening for spontaneous erythromycin sensitivity as previously described and was designated as

USA300 LAC 13C. For the purposes of this thesis, this wild type strain will be referred to as

USA300 LAC. The isogenic USA300 LAC agr mutant (Δagr) was provided by Dr. Alex

Horswill (University of Iowa Medical Center, Iowa City, IA) (11). The isogenic LAC ∆hla strain

was constructed by an insertion mutation using site-directed mutagenesis with the pE194

erythromycin resistance cassette (ermB) as previously described (177), with a hla complemented

strain (177) and a hla constitutively active strain (178) included to confirm the specificity of toxin

action. The USA300 JE2 Nebraska Transposon Mutant Library (NTML) (179) mutants, ∆lukA/H,

∆lukB/G, ∆lukD, ∆spl, and ∆purB were moved to the USA300 LAC 13C background by

transduction with Φ11 bacteriophage. Allelic replacement mutants of lukA and lukB and

complemented strains were generously provided by Dr. Victor Torres (New York University,

New York, NY). In vitro complementation of the USA300 ∆purB strain was performed by the

introduction of a functional purB gene on the pCM29 plasmid (180) under induction by the sarA

promoter, named pTS1. In vivo complementation of the USA300 ∆purB strain was performed by

the introduction of a functional purB gene with the native promoter on the pKK22 plasmid

generously provided by Dr. Jeffrey Bose (University of Kansas Medical Center, Kansas City,

KS). For confocal imaging, all strains were either transformed with pCM11 (erm10) or pCM29

(ca10) to express GFP driven by the sarA P1 promoter.

 31

Bacterial storage and preparation

Bacterial strains were stored as glycerol stocks at -80⁰C, struck out on fresh TSA plates

containing antibiotics for selection when necessary, and grown overnight at 37⁰C prior to

experimentation. A new streak plate was prepared for each experiment in an effort to avoid

mutation of bacteria by prolonged storage at 4⁰C. For experimentation, overnight cultures were

grown in the desired medium by selecting a single bacterial colony from the streak plate using a

sterile loop and incubating at 37⁰C overnight for 12-16 h with constant shaking at 250 rpm.

In vitro S. aureus biofilms

Sterile 12-well plates (Falcon, Corning, NY) or sterile 2-well glass chamber slides (Nunc,

Rochester, NY) were treated with 20% human plasma overnight at 4° C to facilitate bacterial

attachment. The following day, plasma coating buffer was removed and each chamber inoculated

with USA300 LAC at an OD600 of 0.05, whereupon bacteria were incubated at 37° C under static

aerobic conditions for a period of up to 6 days to generate mature biofilms. Our prior studies have

demonstrated that 6 day-old S. aureus biofilms propagated in RPMI-1640 are mature based on the

presence of tower structures and thickness (15, 160). Medium was carefully replenished every 24

h, and biofilms were visualized using a Zeiss laser scanning confocal microscope (LSM 510

META; Carl Zeiss, Oberkochen, Germany). Z-stacks were collected from beneath the glass slide

extending to above the point where bacteria could no longer be detected. Three-dimensional

images of biofilms and measurements to demonstrate biofilm thickness were performed using

Zen 2009 and 2012 software (Carl Zeiss).

2) Mouse strains

A breeding colony of C57BL/6 mice was established in Dr. Kielian’s laboratory after purchasing

animals from the National Cancer Institute (Frederick, MD) or Jackson Laboratories (Bar Harbor
 32

ME). These studies were performed in strict accordance with the recommendations provided in

the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH)

and were reviewed by the Institutional Animal Care and Use Committee of the University of

Nebraska Medical Center.

BALB/c NF-κB luciferase (NF-κB-luc) reporter mice (Caliper Life Sciences; Hopkinton, MA)

were generously provided by Caliper Life Sciences. These mice produce firefly luciferase under

the control of the mouse NF-κB promoter, allowing for the measurement of luminescence when

cells are provided with the substrate luciferin as an indicator of inflammatory status.

GFP transgenic (Tg) mice (C57BL/6-Tg[CAG-EGFP]) were purchased from The Jackson

Laboratory (Bar Harbor, ME). These mice produce GFP under the control of a chicken beta-actin

promoter and cytomegalovirus enhancer, which makes all of the tissues, with the exception of

erythrocytes and hair, appear green under excitation light.

TLR9 KO mice were obtained from The Jackson Laboratory (Bar Harbor, ME). These mice do

not express TLR9 and are therefore deficient in the ability of their immune cells to recognize

foreign, unmethylated CpG-DNA.

MyD88 KO mice (originally from Dr. S. Akira, Osaka University, Suita, Osaka, Japan) (26) were

purchased from the Centre de La Recherche Scientifique and have been previously backcrossed

with C57BL/6 mice for over 10 generations (29, 34). These mice do not express the adaptor

protein MyD88 which functions downstream of most TLR signaling pathways, connecting

bacterial PAMP recognition to NF-κB activity.

 33

3) Cell culture techniques

Primary mouse bone marrow-derived macrophage (BMDMΦ) culture

Adult BALB/c NF-κB-luc, C57BL/6 WT, CAG-EGFP, TLR9KO, and MyD88 KO mice were

euthanized with an overdose of inhaled isoflurane (Isothesia, VetUS, Dublin, OH) using a

euthanasia chamber and cervical dislocation as the secondary method of euthanasia. The

abdominal surface of each mouse was washed with an excess of 70% EtOH to minimize

contamination and a subcutaneous incision was made near the midline of the abdomen opening

up the peritoneum. Skin was separated until the hind limbs were exposed and excess muscle was

dissected away, allowing for the hind limbs to be removed at the hip joint and placed in 1X PBS

on ice. Hind limbs were then submerged in 70% EtOH, excess tissue and muscle was removed

with Kimwipes, and clean bones were placed in fresh 1X PBS on ice. The following steps were

performed under aseptic conditions in a biological safety cabinet with sterile autoclaved

instruments. Both ends of the bones were cut with scissors and bone marrow was flushed with

sterile, cold DMEM using a 26-gauge needle into a 50ml conical tube. Once all bones were

flushed, cells were pipetted to disrupt aggregates, filtered through a 70μm cell strainer, and

centrifuged at 1,200 rpm for 5 min at 4⁰C. The supernatant was aspirated and red blood cells

lysed by the addition of 900μl sterile water for 5 sec, followed by 100μl 10X PBS to restore

osmotic pressure. Finally, cells were washed with medium, centrifuged, and counted using trypan

blue (Lonza, Walkersville, Germany) on a hemacytometer. Cells were plated in 175mm2 tissue

culture dishes at a density of 107 cells/plate in 15ml of medium. BMDMΦ medium was composed

of Dulbecco’s modified eagle’s medium (DMEM, 4.5g/L glucose supplemented with 4mM L-

glutamine) containing heat-inactivated fetal bovine serum (10% v/v FBS, HyClone, Logan, UT;

inactivated at 55⁰C for 30 min, with mixing at 10 min intervals), 20% conditioned medium from

L929 cells (ATCC) as a source of MΦ colony stimulating factor (M-CSF) (181) or 40ng/ml M-

CSF (eBioscience Inc., San Diego, CA), 1% v/v HEPES, 1% v/v Glutamine (both HyClone,
 34

South Logan, UT), 0.1% v/v 50 mM Beta-mercaptoethanol (Fischer Scientific, Pittsburgh, PA)

and antibiotic/antimycotic solution (penicillin, streptomycin, and ampotericin B, final 1% v/v,

Mediatech Inc., Manassas, VA). Medium was changed on cultures at days 2, 4, and 6 after initial

plating and cells were harvested on day 7 for experimentation.

Neutrophil isolation from the mouse bone marrow

Adult C57BL/6 mice were euthanized and bone marrow was isolated as previously described

above and placed on a three-layer Percoll gradient (Amersham Pharmaca, Biotech, Uppsala,

Sweden). After filtration, cells were centrifuged at 400 x g for 10 min at 4⁰C, resuspended in 3 ml

of 78% Stock Isotonic Percoll (SIP, 100% SIP [9 parts Percoll to 1 part 10X PBS] in 1X PBS),

followed by layering 3 ml of 69% and 52% SIP on top. The three-layer gradient was then

centrifuged at 1500 x g for 30 min at 15⁰C with no brake. PMNs were carefully collected from

the 69%/78% interface and upper portion of the 78% layer, washed with PBS, centrifuged at 400

x g for 10 min at 4⁰C, resuspended in 1 ml 1X Lysing Buffer (BD Pharm Lyse, BD Biosciences,

Franklin Lakes, NJ) and incubated at room temperature for 2 min. Lysis was stopped by addition

of HBSS + 10% FBS and cells were centrifuged at 400 x g for 10 min at 4⁰C prior to

resuspsension in 2 ml buffer for magnetic-activated cell sorting (MACS, PBS without Ca, Mg, +

2% FBS), vortexed and counted with trypan blue on a hemacytometer. Magnetic labeling was

performed using a Miltenyi anti-Ly6G MicroBead Kit (Miltenyi Biotec, San Diego, CA)

according to manufacturer’s instructions. Magnetic separation was performed using an MS

column on a MACS Separator. Columns were prepared by rinsing with 500 μl buffer, whereupon

the cell suspension was added to the column. Unlabeled cells were flushed by washing the

column 3X with 500 μl buffer prior to flushing buffer through with a plunger to elute the fraction

of labeled cells into a collection tube. Cells were counted and at least 600,000 Ly6G+ cells were

removed to check purity by flow cytometry, while the remaining cells were immediately used for

experimentation.
 35

4) Immune cell co-culture with S. aureus biofilms in vitro

Co-cultures for microarray studies

BMDMΦs or PMNs (107 and 106, respectively) were co-cultured with S. aureus biofilms for

various periods to assess their impact on the biofilm transcriptome, which equated to a MOI of

10:1 (bacteria/MΦ) or 100:1 (bacteria/PMN). PMNs or BMDMΦs were incubated with biofilms

in RPMI-1640 supplemented with 10% FBS at 37° C under static aerobic conditions until they

were harvested by mechanical dissociation at two different time points (1 and 24 h for BMDMΦs;

1 and 4 h for PMNs) to collect RNA for microarray analysis.

For visualization of BMDMΦ- or PMN-biofilm interactions, leukocytes were labeled with 5 µM

CellTracker Orange or CellTracker Blue (both from Molecular Probes, San Diego, CA)

depending on the experimental setup. BMDMΦ and PMN cell death was assessed using

Propidium Iodide Staining Solution (eBioscience Inc., San Diego, CA). Leukocyte-biofilm

interactions were visualized using a Zeiss laser scanning confocal microscope (LSM 510 META

or LSM 710), where Z-stacks were collected from beneath the glass slide extending to above the

point where labeled BMDMΦs or PMNs could no longer be detected. Three-dimensional images

and measurements to demonstrate leukocyte invasion into the biofilm were performed using Zen

2009 software and Zeiss LSM Image Browser (both from Carl Zeiss).

Co-cultures for ELISA studies

To determine the effect of S. aureus biofilms on MΦ cytokine secretion profiles, 106 BMDMΦs

were incubated with mature USA300 LAC biofilms or planktonic bacteria for 6 h at 37°C under

static aerobic conditions as previously described above, whereupon supernatants were clarified by

centrifugation and filtration (0.2 µm) to quantitate TNF-α, IL-10, IL-1β (OptiEIA; BD

Biosciences, Franklin Lakes, NJ), and IL-1RA (DuoSet; R & D Systems, Minneapolis, MN)
 36

production by ELISA. To account for potential interference by S. aureus protein A (Spa) in

ELISA assays, two separate controls were utilized; first, biofilm-conditioned supernatants without

MΦs were tested, which resulted in minimal cytokine signals. Second, conditioned supernatants

from USA300 LAC Δspa biofilm-MΦ co-cultures were also examined and produced similar

results to what was observed for isogenic wild type biofilms (data not shown).

Co-cultures prior to FACS

To evaluate whether MΦs exposed to intact biofilms were refractory to further activation with

well-characterized microbial antigens, BMDMΦs from GFP Tg mice were co-cultured with

USA300 LAC static biofilms or an equivalent number of planktonic bacteria for 2 h as described

above and subsequently dissociated by trituration. Next, the suspension was incubated with the

vital dye 7-aminoactinomycin D (7-AAD; eBioscience, San Diego, CA) and viable BMDMΦs

(GFP+, 7-AAD-) were recovered by fluorescence-activated cell sorting (FACS). A total of 105

biofilm- or planktonic-exposed BMDMΦs were subsequently treated with 10 µg/ml

peptidoglycan (PGN), 0.1 µM CpG oligodeoxynucleotide (ODN), or 1 µg/ml of the synthetic

lipoprotein Pam3CSK4, or left untreated for an additional 24 h, whereupon supernatants were

evaluated for TNF-α and IL-10 production by ELISA (OptiEIA; BD Biosciences).

Co-cultures for eDNA/DNase treatments

For visualization of BMDMΦ-biofilm interactions, BMDMΦs were labeled with 5 μM

CellTracker blue (Molecular Probes, San Diego, CA) and added to biofilms in fresh medium for

4-6 h. Propidium iodide staining solution (eBioscience Inc., San Diego, CA) was used to assess

eDNA as well as MΦ and bacterial cell death. MΦ-biofilm interactions were visualized using a

Zeiss laser scanning confocal microscope (LSM 710 META), where z-stacks were collected from

beneath the glass slide extending to above the point where labeled MΦs could no longer be

detected. Three-dimensional imaging and measurements to demonstrate MΦ invasion and

phagocytosis of biofilm-associated bacteria were performed using Zen 2012 software and the
 37

Zeiss LSM Image Browser (both from Carl Zeiss). In some experiments, biofilms were pretreated

for 30 min with 100 U/ml DNase (Sigma, St. Louis, MO) or exogenous eDNA isolated from S.

aureus biofilm was added to the surface prior to co-culture. Biofilm height and live/dead

quantitation was performed using COMSTAT (182) via ImageJ.

5) Nebraska Transposon Mutant Library screens

The Nebraska Transposon Mutant Library (NTML) (179) is a collection of sequence-defined

mutants where 1,952 non-essential genes in the S. aureus USA300 LAC genome have been

disrupted by insertion of the mariner-based transposon, bursa aurealis (183). We screened the

NTML to identify genes expressed during biofilm growth that influenced MΦ phagocytosis,

inflammatory activity, and viability as described below. S. aureus mutants that displayed changes

in MΦ activity but still formed a biofilm were selected for further analysis.

BMDMΦ phagocytosis

The NTML was screened using a microtiter plate assay to identify biofilm-associated genes

regulating secreted products that inhibit BMDMΦ phagocytosis. Briefly, starter cultures of

NTML mutants were prepared from glycerol stocks and incubated for 16 h in RPMI-1640

supplemented with 10% FBS and 5 µg/ml erm at 37°C while shaking at 250 rpm under aerobic

conditions. 96-well microtiter plates were pre-coated with 20% human plasma diluted in sterile

carbonate-bicarbonate buffer (Sigma) at 4°C. The following day, plasma coating buffer was

removed and starter cultures were inoculated at a 1:200 dilution and incubated at 37°C under

static aerobic conditions for 4 days. Medium was carefully replenished every 24 h using an

epMotion 5075 LH robotics platform (Eppendorf, Hamburg, Germany) to minimize cross

contamination and prevent disruption of the biofilm structure.

 38

BMDMΦ s were seeded at 105 cells/well in tissue culture-treated 96-well plates (Becton

Dickinson, Franklin Lakes, NJ) and incubated overnight at 37°C, 5% CO2, whereupon BMDMΦs

were treated for 2 h at 37°C with biofilm-conditioned supernatants diluted 1:2 in fresh RPMI-

1640/10% FBS, followed by 1 h incubation with 106 yellow-green fluorescent microspheres (2.0

µm particle size; Molecular Probes, San Diego, CA) to assess phagocytic activity. After the 1 h

incubation period, BMDMΦs were washed extensively with sterile PBS to remove any residual

extracellular microspheres and phagocytic activity was assessed as a measure of total

fluorescence using a Victor3V 1420 plate reader (Perkin Elmer, Waltham, MA) or TECAN M200

PRO (Tecan Group Ltd., Mannedorf, Germany) running Magellan 7.0 software (Tecan Austria

GmbH, Grodig, Austria).

BMDMΦ NF-κB activation

The NTML was also screened to identify mutants that affected MΦ NF-κB activation during

biofilm growth. This assay utilized BMDMΦs from NF-κB-luciferase reporter mice (NF-κB-luc

BMDMΦs), where NF-κB promoter activity is detected with the substrate D-luciferin and

quantitated using a luminometer. Static biofilms were grown in luminescence-compatible 96-well

microtiter plates (Perkin Elmer, Waltham, MA) until mature (as described above), whereupon

NF-κB-luc BMDMΦs (105/well) were co-cultured with S. aureus biofilms for 4 h, after which

samples were lysed using ddH2O and 100 µl/well of D-luciferin (15mg/ml; Gold Biotechnology,

St. Louis, MO) was added to detect luciferase expression. Luminescence was quantified using a

Victor3V 1420 plate reader (Perkin Elmer). Positive and negative controls included NF-κB-luc

BMDMΦs treated with 10 µg/ml S. aureus PGN and untreated cells, respectively. Biofilms alone

produced minimal luminescence signal compared to background (data not shown).

6) BMDMΦ treatments with biofilm-conditioned medium

 39

Preparation of biofilm-conditioned medium

Static biofilms were generated in two-well glass chamber slides (Nunc, Rochester, NY) or 12-

well plates (Becton Dickinson, Franklin Lakes, NJ) with S. aureus grown in RPMI-1640 medium

supplemented with 1% casamino acids (CAA; Becton Dickinson) as previously described (15,

184, 185). Spent medium was replaced daily, whereupon conditioned medium for experiments

was collected from 6 day-old USA300 LAC biofilms 24 h following the last medium change and

filtered (0.2 µm). For comparisons, planktonic-conditioned medium was prepared by growing an

overnight culture of USA300 LAC to early (12 h) and late (18 h) stationary phase. As no

significant differences in phagocytosis or viability were observed when MΦs were treated with

conditioned medium from either early or late stationary phase cultures (data not shown), early

stationary phase was utilized throughout these studies. Conditioned medium was also collected

from 6 day-old USA300 LAC biofilms that were mechanically disrupted by trituration,

whereupon the suspension was incubated for another 24 h with fresh medium before collection as

described above. Where indicated, biofilm-conditioned medium was treated with 10μg/ml

proteinase K for 1h at 37⁰C to degrade proteins, or treated with either 10 μg/ml polyanethole

sodium sulfanate (PAS) or mechanically disrupted and incubated with 50 μg/ml lysostaphin (both

from Sigma, St. Louis, MO) 24 h prior to collection. In some experiments, fresh RPMI-1640

medium was spiked with 10 μg of purified S. aureus Hla (Sigma) or 25 μg of purified bioactive or

inactive LukAB (186) to examine effects on MΦ phagocytosis and viability. For some

experiments, biofilm-conditioned medium was incubated with rabbit anti-staphylococcal Hla

antiserum or control rabbit serum (both from Sigma) for 30 min prior to MΦ treatment.

BMDMΦ phagocytosis and cell viability assay

BMDMΦs were prepared and labeled with 5 µM CellTracker Blue (CTB; Molecular Probes, San

Diego, CA) as previously described (15, 187). CTB-labeled MΦs were added to sterile 2-well

glass chamber slides (5x106 cells/chamber) and allowed to adhere for 2 h. Next, MΦs were
 40

exposed to conditioned medium collected from S. aureus biofilms or planktonic cultures for 2 h at

37°C, whereupon red or green fluorescent microspheres (2.0 µm particle size; Molecular Probes)

were added at a concentration of 4.5x1010 microspheres/ml for 1 h to assess phagocytic activity.

Fluorescent microspheres were used instead of intact S. aureus, since pilot studies revealed that

similar results were obtained with both reagents (Supplemental Fig. S2.1). MΦs were treated with

undiluted biofilm- or planktonic-conditioned medium, since a stronger phenotype was observed

under these conditions (Supplemental Fig. S2.1). MΦ phagocytosis was assessed using a Zeiss

510 META laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) and

quantitated by the number of phagocytic events observed in at least 8, random fields of view

(63x) using ZEN 2009 software (Carl Zeiss). Data is expressed as either “percent phagocytosis”,

which indicates the percentage of phagocytic MΦs observed and manually counted within a given

experiment, or “total phagocytosed beads/63x field”, where the total number of phagocytic events

within a given experiment was calculated by an ImageJ plugin (ImageJ 1.47v, Wayne Rasband,

NIH, USA) based on an experimentally determined average pixel area and RGB color code

specific to the co-localization of phagocytosis. Pilot studies confirmed that identical results were

obtained with this calculation compared to manual counts (data not shown). While the number of

beads phagocytosed per MΦ was variable within any given incubation condition, we did not

observe any trends in this parameter between treatment groups and, therefore, this was not

quantified. MΦ viability was assessed by propidium iodide staining at the end of the 3 h

incubation period with the total number of viable MΦs quantitated in at least 8 random 63x fields

of view using ZEN 2009 software. Where reported, “total viable MΦs” were enumerated by an

ImageJ plugin based on an experimentally determined average pixel area and RGB color code

specific to viable MΦs and are reported per 63x microscopic field.

7) Mouse model of S. aureus orthopedic implant biofilm infection

 41

Preparation of inoculum

A single bacterial colony from a fresh streak plate was used to inoculate 25 ml of autoclaved

brain-heart infusion (BHI) broth in a baffled 250 ml flask (10:1 flask:volume ratio) with constant

shaking at 250 rpm at 37⁰C for 12-16 h. The overnight culture was then diluted 1:10 in fresh BHI

in order to estimate the total number of bacteria present by measuring OD600 (BioMate 3S

Spectrophotometer, Thermo Scientific, Waltham, MA). At the same time, 1 ml of the overnight

culture was centrifuged in a 1.5 ml Eppendorf microcentrifuge tube at 14,000 rpm for 5 min at

4⁰C to pellet the bacteria. The supernatant was removed the pellet was resuspended in 1 ml sterile

1X PBS and subsequently washed two more times by centrifuging as previously described. After

washing, the pellet was diluted in sterile PBS to a final estimated concentration of 5x105 cfu/ml

(1x103 cfu/2μl). The actual inoculum concentration was then determined by serial diluting the

washed culture in triplicate to 10-8 and plating 100 μl of the 10-7 and 10-8 dilutions on TSA.

Infection procedure

Age and sex-matched mice (8-10 weeks old) were weighed and anesthetized with

ketamine/xylazine (100 mg/kg and 5 mg/kg, respectively) by i.p. injection prior to the surgical

site being shaved and disinfected with povidone-iodine. A scalpel was then used to make an

incision along the midline of the knee exposing the patella. Next, a medial parapatellar

arthrotomy was performed with the scalpel to access the knee joint, allowing for a lateral

retraction of the patella to expose the entire distal femur. A 26-gauge needle was then used to

bore a hole through the trochlea into the intramedullary canal creating space for the insertion of a

precut and autoclaved 0.8-cm orthopedic-grade Kirschner wire (0.6 mm diameter, Nitinol [nickel-

titanium]; Custom Wire Technologies, Port Washington, WI) leaving approximately 1 mm

exposed as an inoculation site. A total of 103 cfu of the USA300 LAC strain was inoculated in 2

μl at the implant tip, and the patella was relocated prior to surgical wound closure with 6-0 metric

absorbable sutures and skin closure with 6-0 metric nylon sutures (both from Covidien,
 42

Mansfield, MA). Immediately following surgery and again at 24 h post-surgery, all animals

received Buprenex (0.1 mg/kg s.c.; Reckitt Benckiser, Hull, U.K.) for pain management, after

which all mice exhibited normal ambulation with no discernible pain.

Recovery of implant and surrounding tissues

Animals were sacrificed by an overdose of inhaled isoflurane followed by cervical dislocation.

The flank and left leg were then disinfected with 70% EtOH prior to the skin of the left leg being

carefully removed to expose the infected tissue. Excess underlying adipose tissue and muscle was

removed prior to collection of the tissue immediately proximal to the infection site, which was

then weighed and placed in 500 μl homogenization buffer (1X PBS + protease inhibitor cocktail

tablet, Roche, Indianapolis, IN) on ice. The tissue was then dissociated with the blunt end of a

plunger from a 30-cc syringe and filtered through a 35 μm filter (BD Falcon, Bedford, MA).

Next, a 150μl aliquot was removed for quantitation of bacterial burdens and potential assessment

of inflammatory mediator production (e.g. ELISA). The remaining filtrate was further processed

for flow cytometry as described below. Following tissue collection, the knee joint and femur were

collected, weighed, and placed in 500 μl homogenization buffer prior to being homogenized by a

combination of handheld Polytron homogenizer at the highest setting for 30 s and a Bullet

Blender (Next Advance, Averill Park, NY) with a combination of 0.9-2.0 mm diameter blend and

3.2 mm diameter stainless steel beads (Next Advance, Averill Park, NY). Implants were removed

from the femur and vortexed in 500 μl PBS for 5 min at 2000 rpm. Serial dilutions of effluents

from tissue, joint, femur, and implant were plated on TSA and grown overnight at 37⁰C to

determine bacterial colonization. For some experiments, the spleen, heart, and kidneys were

collected as described above to determine dissemination.

Flow cytometry

Characterization of immune infiltrates was performed via Fluorescence-activated cell sorting

(FACS). Animals were sacrificed with an overdose of inhaled isoflurane and tissues were excised
 43

and processed as previously described above. The tissue filtrate was washed with ice-cold 1X

PBS + 2% FBS and centrifuged at 1200 rpm for 5 min at 4⁰C, after which RBCs were lysed using

BD Pharm lyse (BD Biosciences, San Diego, CA) per the manufacturer instructions. After lysis,

cells were resuspended in 500 μl 1X PBS followed by Fc Block (2 μl/sample, eBioscience, San

Diego, CA) for 20 min at 4⁰C to minimize nonspecific antibody binding. 100 μl of each sample

was pooled and subsequently aliquoted into single color compensation and isotype control tubes

to identify gaiting thresholds and assess the degree of nonspecific staining, respectively. The

remaining 400 μl was divided between two tubes for subsequent staining with two panels (e.g.

innate and T cell) and q.s. to 500 μl with 1X PBS. Cells were then stained for 30 min at 4⁰C

protected from light with directly-conjugated antibodies for multi-color flow cytometry analysis,

prior to being washed with 1X PBS, centrifuged as previously described, and resuspended in 1X

PBS + 1% paraformaldehyde. The innate immune panel included: CD45-APC, Ly6G-PE, Ly6C-

PerCP-Cy5.5, and F4/80 PE-Cy7. The T cell panel included: CD3ε-APC, CD4-Pacific Blue,

CD8a-FITC, Ly6C-PerCP-Cy5.5, and TCR γδ-PE. All fluorochrome-conjugated antibodies were

purchased from either BD Biosciences or eBioscience. For the exclusion of dead cells, a

Live/Dead Fixable Stain Kit (Life Technologies, Eugene, OR) was also used, following

manufacturer’s instructions. Analysis was performed using BD FACSDiva software with cells

gated on the live CD45+ leukocyte population.

8) Bacterial microarray analysis and qRT-PCR

RNA isolation

At the appropriate intervals after biofilm-leukocyte co-culture, excess medium was removed from

biofilm chambers and 2X the remaining volume of RNAprotect (QIAGEN, Hilden, Germany)

was added. Biofilms were collected from the bottom of the chamber slide using a cell scraper.
 44

RNA isolated from biofilms alone (i.e. no leukocyte addition) was included as a control for

comparisons. The resulting suspension of biofilm cells was transferred to a tube and sonicated for

5 min to facilitate dispersal. After sonication, cells were pelleted by centrifugation for 5 min and

RNAprotect was decanted. The resulting pellet was resuspended in 700 µl of RLT Buffer

supplemented with β-mercaptoethanol and transferred to a 2 ml FastPrep lysing tube (MP

Biomedicals, Santa Ana, CA). Biofilm cells were lysed in a FastPrep high-speed homogenizer

(MP Biomedicals) for 20 sec on a speed setting of 6. The resulting lysate was incubated for 5 min

on ice and then centrifuged at 14,000 rpm at 4°C for 15 min. RNA was isolated from the clarified

supernatant using a RNeasy mini kit (QIAGEN) and contaminating DNA was removed by on-

column DNase digestion using the RNase-Free DNase Set (QIAGEN). RNA was isolated from

eight samples for each time point and co-culture condition with three independent experimental

replicates performed to assess the reproducibility of microarray results. RNA quality and quantity

were determined using an Agilent RNA6000 NANO kit and Agilent 2100 Bioanalyzer (Agilent

Technologies, Santa Clara, CA).

RNA labeling and DNA microarray analysis

Following the manufacturer’s recommendations, 75 ng of total RNA from each sample was

amplified using ExpressArt Bacterial mRNA amplification Nano kits (AmpTec GmbH,

Germany) and labeled using BioArray HighYield RNA Transcript Labeling kits (Enzo Life

Sciences, Inc., Farmingdale, NY). Three micrograms of resulting labeled RNA was hybridized to

a S. aureus GeneChip® following the manufacturer’s recommendations for antisense prokaryotic

arrays (Affymetrix, Santa Clara, CA) then washed, stained, and scanned as previously described

(22, 188). Commercially available GeneChips® were used in this study, representing > 3300 S.

aureus ORFs and > 4800 intergenic regions from strains N315, Mu50, NCTC 8325, and COL

(Affymetrix). GeneChip® signal intensity values for each biofilm sample at each replicate time

point (n ≥ 3) were normalized to the median signal intensity value for each GeneChip® and
 45

averaged using GENESPRING 7.2 software (Agilent Technologies, Redwood City, CA).

Transcripts that demonstrated 1) at least two-fold change in expression; 2) greater than

background signal intensity value and determined to be "Present" by Affymetrix algorithms; and

3) significant by Student's t-test (p value = 0.05) were considered differentially expressed.

Verification of differentially expressed genes by qRT-PCR

A subset of biofilm genes that were differentially regulated after leukocyte co-culture (i.e. sodA,

saeS, saeR, agrB, rsbU, atl, recA, and nuc) was verified by qRT-PCR using Sybr Green. Primers

were designed using Primer 3.0 software and melt-curve analysis was performed at the end of

each amplification run to verify signal specificity. Results are presented as the relative expression

compared to biofilms that were not co-cultured with leukocytes as a reference standard.

9) Bacterial proteomics

Sequential Windowed data-independent Acquisition of the Total High-resolution Mass

Spectra (SWATH-MS)

Conditioned medium from S. aureus WT and Δagr strains grown under biofilm or planktonic

conditions as described above were harvested, treated with a protease inhibitor cocktail (Roche,

Basel, Switzerland), and proteins precipitated with 20% TCA. Relative protein concentrations

were compared between groups using three independent replicates per sample by Sequential

Windowed data-independent Acquisition of the Total High-resolution Mass Spectra (SWATH-

MS) as previously described (189). A Z-transformation followed by a Z-test was performed on all

positively identified proteins (>98% confidence) between two sample sets at a time (i.e. WT

biofilm vs. Δagr biofilm or WT biofilm vs. WT planktonic) to assess significant differences in

relative protein abundance as previously described (189). Identified proteins were functionally

grouped by UniProt identifier utilizing the DAVID bioinformatics resource 6.7

 46

(http://david.abcc.ncifcrf.gov/) (190). Protein-protein interactions were predicted using STRING

9.05 (http://string-db.com) (191, 192).

Western blots

Conditioned medium from S. aureus WT and Δagr biofilm and planktonic growth conditions was

sterile-filtered and treated with a protease inhibitor cocktail (Roche) prior to storage at -80⁰C.

Upon thawing, samples were TCA precipitated overnight and suspended in 30 μl of Laemmli

buffer, whereupon 5 μl of each sample was loaded onto a gel, transferred to a PVDF membrane,

and probed for S. aureus LukA and Hla.

Enzyme-linked Immunosorbent Assay (ELISA)

Conditioned medium from WT S. aureus biofilm and planktonic cultures was sterile-filtered

(0.2µm) and analyzed for Hla concentrations by direct ELISA. Briefly, experimental samples or

serial dilutions of purified S. aureus Hla (Sigma), to generate a standard curve, were diluted in

carbonate-bicarbonate buffer and incubated in 96-well ELISA plates overnight at 4⁰C. The

following day, wells were washed extensively with 1X PBS/0.5% Tween and incubated with a

rabbit anti-Hla antibody followed by an anti-rabbit IgG-HRP antibody (both from Sigma) for

detection. Plates were developed using a TMB substrate (Becton Dickinson) with the reaction

halted using stop solution prior to reading at 450nm. Hla concentrations were normalized to total

protein as measured by a Pierce BCA Protein Assay.

10) Bioflim extracellular DNA (eDNA) isolation

Isolation of eDNA from static biofilms grown in 12-well plates was performed as described

previously (32). Briefly, day 6-old mature biofilms were chilled to 4⁰C followed by the addition

of 50 mM EDTA prior to removal of supernatant, resusupended and mechanically disrupted in

TES Buffer (Tris-HCL; pH 8.0/500mM NaCl). Next, a phenol:chloroform:isoamyl alcohol

 47

(25:24:1) extraction followed by a chloroform/isoamyl alcohol (24:1) extraction was performed

prior to storage overnight at -20⁰C in 100% ice-cold ethanol and 10% 3M NaAcetate. The next

day, the eDNA was pelleted and washed prior to a final resuspension in TE buffer. qRT-PCR was

performed on 1:10 dilutions of each sample with the LightCycler DNA Master SYBR Green I

(Roche) using gyrA.

 48

Supplemental Figure S2.1

Fluorescent microspheres can be used as a surrogate for live S. aureus to assess MΦ

phagocytosis. (A) Bone marrow-derived MΦ were incubated for 3 h with fresh medium or

conditioned medium collected from WT biofilms and left undiluted or diluted 1:5 with fresh

medium, whereupon phagocytosis was assessed by the uptake of planktonic S. aureus +

fluorescent microspheres (beads) or microspheres alone. Significant differences are denoted with

asterisks (***, p < 0.001; unpaired two-tailed student’s t-test). Results are representative of at

least two independent experiments.

 49

Chapter 3: Global transcriptome analysis of Staphylococcus aureus biofilms in response to

innate immune cells

Published in Infection and Immunity 81(12):4363-76, 2013

 50

Abstract

The potent phagocytic and microbicidal activities of PMNs and MΦs are among the first

lines of defense against bacterial infections. Yet Staphylococcus aureus is often resistant to innate

immune defense mechanisms, especially when organized as a biofilm. To investigate how S.

aureus biofilms respond to MΦs and PMNs, gene expression patterns were profiled using

Affymetrix microarrays. The addition of MΦs to S. aureus static biofilms led to a global

suppression of the biofilm transcriptome with a wide variety of genes downregulated. Notably,

genes involved in metabolism, cell wall synthesis/structure, and

transcription/translation/replication were among the most highly downregulated, which was most

dramatic at 1 h compared to 24 h following MΦ addition to biofilms. Unexpectedly, few genes

were enhanced in biofilms after MΦ challenge. Unlike co-culture with MΦs, co-culture of S.

aureus static biofilms with PMNs did not greatly influence the biofilm transcriptome.

Collectively, these experiments demonstrate that S. aureus biofilms differentially modify their

gene expression patterns depending on the leukocyte subset encountered.

 51

Introduction

Staphylococcus aureus produces numerous virulence factors that facilitate its ability to

invade, colonize, disseminate to distant sites, and impede host defenses to cause disease (84,

193). These characteristics can be amplified during biofilm formation, which represents a

complex multicellular community of organisms encased in a matrix composed primarily of

polysaccharides, extracellular DNA (eDNA), and proteins (10-12). S. aureus biofilm infections

are often difficult to treat due to their heterogeneity and altered metabolic and transcriptional

activity (194), which likely contributes to the chronic and recurrent nature of biofilm infections

(159, 195-197). Our recent studies have demonstrated that S. aureus biofilms interfere with

traditional microbial recognition and killing mechanisms by the innate immune system (159,

195). The subversion of these responses is another example of the remarkable success of S.

aureus as a pathogen and it is now clear that biofilm growth represents yet another immune

resistance determinant. However, our understanding of the cross-talk between S. aureus biofilms

and the immune response is limited.

PMNs are important antimicrobial effectors that possess an arsenal of bactericidal

compounds, including defensins, cathelicidins, and lysozyme (156, 198). In terms of their

microbicidal activity, PMNs are most notable for their ability to produce large amounts of

reactive oxygen intermediates catalyzed by NADPH oxidase. In addition, activated PMNs

degranulate and release PMN extracellular traps (NETs), a meshwork of DNA and enzymes that

facilitates the extracellular killing of S. aureus as well as other bacteria (199). However, the short

lifespan of PMNs requires their constant recruitment to sites of infection, and their transcriptional

capacity for inflammatory mediator production is more limited compared to other professional

phagocytes (i.e. MΦs and dendritic cells). MΦs reside in virtually all tissues and also serve as a

critical first line of defense against microbial invasion. In addition, MΦs are a major source of

proinflammatory mediators that are critical for amplifying leukocyte recruitment and activation
 52

cascades upon bacterial exposure, as well as providing potent phagocytic and antimicrobial

effects (200, 201). Like PMNs, MΦs can form MΦ extracellular traps (METs),which are believed

to exert similar antimicrobial activity (157). Both MΦs and PMNs are also equipped with an

arsenal of pattern recognition receptors that sense invariant motifs expressed across a broad range

of microbial species to trigger inflammatory mediator release (202, 203). Consequently, PMNs

and MΦs represent key antimicrobial effector populations and their interactions with S. aureus

biofilms is likely critical for dictating the outcome of infection. Our previous studies have

demonstrated that S. aureus biofilms impair MΦ phagocytosis and induce cell death (159, 195,

204); however the response of the biofilm itself to these leukocyte populations remains to be

defined.

While considerable progress has been made in defining S. aureus virulence factors and their

regulatory networks, less is known about the organism’s ability to cope with the host immune

response during biofilm growth (205-207). Genome-wide transcriptional profiling of planktonic

S. aureus following PMN exposure has previously been reported (208, 209); however the

transcriptional changes occurring in S. aureus biofilms in response to PMNs or MΦs has not yet

been investigated. We predicted that S. aureus biofilms modify their transcriptome in response to

these leukocyte subsets to subvert immune recognition and killing, thus favoring biofilm

persistence. This possibility was assessed by defining alterations in S. aureus biofilm gene

expression profiles after co-culture with MΦs or PMNs utilizing S. aureus Affymetrix GeneChip®

arrays. Here we report that S. aureus biofilms respond differently to these leukocyte populations,

with kinetic distinctions also observed. For example, MΦ addition induced a generalized

repression of the biofilm transcriptome within 1 h, whereas at a later interval this inhibition had

dissipated, which correlated with the biofilm’s ability to induce MΦ cell death. In contrast, the

biofilm transcriptome remained relatively stable following PMN addition, regardless of the

interval examined. These results indicate that S. aureus biofilms discriminate between leukocyte
 53

subsets and alter their transcriptional profile accordingly, presumably in favor of avoiding

detection by the host, leading to biofilm persistence.

 54

Results

Differences in neutrophil and macrophage interactions with S. aureus biofilms in vitro. To

evaluate the impact of leukocyte subsets on S. aureus biofilm transcriptional profiles, biofilms

that were propagated for either 4 or 6 d were selected for analysis based on their differences in

structural maturity (195). Specifically, 4 day-old biofilms were considered more immature in

terms of average thickness (32 µm) and irregular density (Fig. 3.1A and C), whereas 6 day-old

biofilms were classified as more mature based on a relatively uniform average thickness (47 µm)

and a more consistent density (Fig. 3.1B and C).

Previous studies from our group have demonstrated that S. aureus biofilms are capable of

circumventing MΦ phagocytosis and inducing MΦ death (159, 195, 204). Since the goal of this

study was to compare the impact of MΦs versus PMNs on the biofilm transcriptome, side-by-side

comparisons of how both populations interact with the biofilm were required. S. aureus USA300

LAC-GFP static biofilms were grown for 4 or 6 d, whereupon MΦs or PMNs were co-cultured

with biofilms for an additional 1, 4, or 24 h. Three-dimensional confocal microscopy images were

constructed to demonstrate the proximity of MΦs or PMNs from the biofilm surface and extent of

phagocytosis (Fig. 3.2A and 2B, respectively). MΦs incubated with S. aureus biofilms for either a

1 or 24 h period contained few internalized bacteria, a phenomenon that was independent of

biofilm age as we have previously described (Fig. 3.2A and 3.3) (195). In contrast, intracellular

bacteria were readily discernible in PMN biofilm co-cultures at 1, 4, and 24 h (Fig. 3.2B, 3.3, and

Supplemental Fig. 3.1). In addition, the majority of PMNs were found in close association with

the biofilm surface, whereas most MΦs remained distant from the biofilm (Fig. 3.2).
 55

Figure 3.1

S. aureus biofilm growth states. (A and B) USA300 LAC-GFP was inoculated into sterile 2-

well glass chamber slides and incubated at 37°C under static aerobic conditions for a period of 4

(A) or 6 (B) days in RPMI 1640 supplemented with 10% FBS with daily medium replacement.

Biofilms were visualized using confocal microscopy (magnification, X63; 1-μm slices), and

representative three-dimensional images were constructed. (C) Quantification of 4- and 6-day-old

biofilm thickness. Significant differences are denoted with asterisks (***, P < 0.001 using an

unpaired two-tailed Student t test; n = 30 biofilms/time point).

 56

Figure 3.2

S. aureus biofilm-leukocyte co-culture paradigm. USA300 LAC-GFP static biofilms (green)

were grown for 6 days, whereupon BMDMΦs (A) (orange) or bone marrow-isolated PMNs (B)

(blue) were incubated with biofilms for 24 h and 4 h, respectively. Biofilm co-cultures were

visualized using confocal microscopy (magnification, X63; 1-μm slices), and representative

three-dimensional images were constructed. Insets show a higher magnification to highlight the

absence of MΦ phagocytosis (A) and the presence of PMN phagocytosis (B) of staphylococcal

biofilms. Results are representative of two independent experiments examining three individual

biofilms each.
 57

Figure 3.3

Differential responses of innate immune cells to S. aureus biofilms. S. aureus USA300 LAC-

GFP static biofilms were grown for 6 days and visualized using confocal microscopy (63X

magnification, 1 µm slices). A total of 107 BMDMΦs or 5 x 106 bone marrow-isolated PMNs

were incubated with biofilms for 18-24 h, whereupon the percentage of leukocytes exhibiting

phagocytosis or death, were enumerated. Significant differences are denoted with asterisks (**, p

< 0.005 using an unpaired two-tailed Student’s t-test, n = 3).

 58

Macrophages and neutrophils induce differential gene expression profiles in S. aureus

biofilms. The disparity between the ability of MΦs and PMNs to phagocytose and invade S.

aureus biofilms suggested that these cell types may differentially influence biofilm transcriptional

activity. To investigate this possibility, Affymetrix S. aureus GeneChip® profiles were compared

between biofilms incubated in the presence or absence of either MΦs or PMNs. Analysis of the

number of differentially expressed genes revealed that MΦs caused the most substantive changes

in the biofilm, primarily at an early time point (i.e. 1 h; Fig. 3.4A, 3.5A, 3.5B, and Tables 3.2-3),

affecting genes involved in metabolism and transcription/translation/replication for both

immature and mature biofilms (Fig. 3.5). Unexpectedly, most biofilm genes were down-regulated

in response to acute MΦ exposure, with the exception of several hypothetical genes and a lone

gene involved in staphyloxanthin biosynthesis (Supplemental Table 3.1). However, the ability of

MΦs to induce global gene repression was transient, as the number of genes altered was

dramatically reduced after a 24 h co-culture period (Figs 3.4A, and Tables 3.2, 3.3, and

Supplemental Fig. 3.1). This is likely attributable to the fact that a large percentage (≥ 55%) of

MΦs are dead approximately 6 h after co-culture with S. aureus biofilms and presumably are no

longer able to produce factor(s) that influence the biofilm transcriptome (195). A subset of genes

identified as differentially expressed by biofilms upon MΦ addition in the microarray analysis

were verified by qRT-PCR (Fig. 3.6).

In comparison to MΦs, PMNs were more limited in their ability to affect S. aureus biofilm

gene transcription (Fig. 3.4B, 3.5C, 3.5D, and Tables 3.4, 3.5, and Supplemental 3.2). Of note,

many genes remained unaltered following MΦ or PMN addition to biofilms, suggesting that these

genes are important for biofilm maintenance in the face of an immune challenge. Nonetheless,

PMNs and MΦs differentially impact the S. aureus biofilm transcriptome as revealed by the

dichotomy in the observed gene expression patterns.

 59

Figure 3.4

Acute macrophage addition to S. aureus biofilms leads to the transcriptional repression of

numerous genes. The total number of genes significantly up- or down-regulated in response to

MΦ (A) or PMN (B) co-culture in immature (4 day-old) or mature (6 day-old) S. aureus biofilms

is shown, including those encoding for hypothetical proteins.

 60

Figure 3.5

Classification of genes significantly altered by leukocyte addition in S. aureus biofilms. S.

aureus USA300 LAC-GFP static biofilms were grown for 4 or 6 days, whereupon MΦs (A and

B) or PMNs (C and D) were incubated with biofilms for 1 h. The numbers of genes with defined

functions (grouped into cell wall/membrane, virulence/defense, regulation, metabolism,

transcription/translation/replication, and miscellaneous categories) significantly altered after MΦ

or PMN challenge are shown. Genes encoding for hypothetical proteins were not included.
 61

Table 3.2. Significantly down-regulated genes between 6 day-old S. aureus biofilm versus
biofilm-macrophage co-cultures for a 1 h period
Category
Virulence/defense
Fold
Change Common Locus Description
-30.01 nuc SA0860 thermonuclease precursor
-13.84 epiA SA1878 lantibiotic epidermin precursor
-12.43 SA2525 ABC transporter, ATP-binding protein
-9.54 SAS0388 exotoxin 3
-6.88 hlY SA1173 alpha-hemolysin precursor
BA000017
-6.40 hlb SA2003 phospholipase C
-5.59 chp SAR2036 chemotaxis-inhibiting protein
type I restriction-modification system, S subunit, EcoA
-4.99 SA0477 family, putative
-4.64 fmhA SA2409 fmhA protein
-3.16 SA2430 ABC transporter, ATP-binding/permease protein
-2.91 SA2445 fmtA-like protein
-2.78 SA1098 metallo-beta-lactamase family protein
-2.73 epiP SA1874 epidermin leader peptide processing serine protease
-2.39 entK SA0886 staphylococcal enterotoxin
-2.22 eprH SA1265 endopeptidase resistance gene
-2.04 set8 SA0384 exotoxin 8
-2.03 epiE SA1872 epidermin immunity protein F
Cell wall
Fold
Change Common Locus Description
-22.02 isdC SA1141 NPQTN cell wall surface anchor protein
-8.42 SA1994 ABC transporter, ATP-binding protein
-8.24 isdB SA1138 LPXTG cell wall surface anchor protein
-7.69 SA1779 transglycosylase domain protein
-7.66 spsB SA0969 signal peptidase IB
-6.44 isdD SA1142 hypothetical protein
-6.39 SA1932 transglycosylase domain protein
-6.23 SA0486 staphylococcus tandem lipoprotein
 62

-5.38 tagX SA0697 teichoic acid biosynthesis protein X

-4.62 abcA SA0700 ABC transporter, ATP-binding/permease protein
-4.40 SA1169 fibrinogen-binding protein precursor-related protein
-3.93 srtA SA2539 sortase
-3.46 opuBB SA0783 osmoprotectant ABC transporter, permease protein
-3.35 isaB SA2660 immunodominant antigen B
iron compound ABC transporter, iron compound-binding protein,
-3.34 SA0203 putative
-3.30 SA0611 glycosyl transferase, group 1 family protein
-3.27 SA0242 teichoic acid biosynthesis protein, putative
-3.19 dltA SA0935 D-alanine-activating enzyme/D-alanine-D-alanyl carrier protein ligase
-2.94 SA1806 cell wall surface anchor family protein
-2.93 SA1043 glycosyl transferase, group 1 family protein
-2.48 SA1951 Mur ligase family protein
-2.27 SA2431 ABC transporter, ATP-binding/permease protein
-2.27 cap1C SA2685 capsular polysaccharide biosynthesis protein Cap1C
-2.18 dltD SA0938 DltD protein
-2.13 SA2082 membrane protein, putative
-2.11 SA0119 cell wall surface anchor family protein
Transcription/translation/replication
Fold
Change Common Locus Description
-10.99 SA2089 single-stranded DNA-binding protein family
-9.90 SA1900 DNA repair exonuclease family protein
-7.17 gpxA SA1325 glutathione peroxidase
-6.75 rbf SA0725 transcriptional regulator, AraC family
-6.39 norR SA0746 transcriptional regulator, MarR family
-6.28 SA2058 PemK family protein
-6.18 rpsA SA1516 ribosomal protein S1
-6.13 SA2517 transcriptional regulator, MerR family
-5.85 gmk SA1221 guanylate kinase
-5.68 SA1919 transcriptional regulator, Fur family
-5.53 SA1060 transcriptional regulator, MarR family
-5.49 SA0550 S4 domain protein
-5.44 SA2203 ClpA-related protein
 63

-5.42 recF SA0004 recF protein

-5.40 SA1732 replication initiation and membrane attachment protein
-5.29 SA1939 phosphotyrosine protein phosphatase
-5.28 SA0305 ABC transporter, permease protein
-4.87 SA0980 transcriptional regulator, LysR family
-4.77 SA2407 lipoprotein, putative
-4.68 SA1803 pseudouridine synthase, family 1
-4.63 SA0028 IS431mec, transposase
-4.62 ctsR SA0567 transcriptional regulator
-4.51 licT SA1393 transcriptional antiterminator
-4.41 SA1997 transcriptional regulator, GntR family
-4.34 rpsU SA1632 ribosomal protein S21
-4.32 SA2297 monooxygenase family protein
-4.26 grpE SA1638 heat shock protein
-4.22 SA2290 transcriptional regulator, AraC family
-4.13 glyS SA1622 glycyl-tRNA synthetase
-3.97 SA0552 general stress protein 13
-3.88 tcaR SA2353 transcriptional regulator
-3.72 SA2713 rhodanese-like domain protein
-3.63 SA1897 protein export protein PrsA, putative
-3.62 SA1894 HIT family protein
-3.62 SA0526 DNA polymerase III, delta prime subunit, putative
-3.56 serS SA0009 seryl-tRNA synthetase
-3.52 ftsL SA1023 putative cell division protein
-3.44 SA2500 MutT/nudix family protein
-3.43 rpmG SA1369 ribosomal protein L33
-3.38 SA2325 transcriptional regulator, LysR family
-3.36 SA1541 transcriptional regulator, Fur family
-3.33 SA2317 phosphosugar-binding transcriptional regulator
-3.29 SA0535 primase-related protein
-3.24 SA1957 RNA methyltransferase, TrmA family
-3.24 rnr SA0846 exoribonuclease, VacB/RNase II family
-3.23 rpsP SA1254 ribosomal protein S16
-3.23 czrA SA2137 transcriptional regulator CzrA
 64

-3.23 gidB SA2736 glucose-inhibited division protein B

-3.20 rpmF SA1137 ribosomal protein L32
-3.20 nusB SA1569 N utilization substance protein B
-3.12 SA1794 thioredoxin, putative
-3.05 SA1285 N utilization substance protein A, putative
-3.04 SA1205 cell-division initiation protein, putative
-3.00 SA0563 transcriptional regulator, GntR family
-2.99 dinG SA1495 DNA polymerase III, epsilon subunit/ATP-dependent helicase
-2.96 SA0432 spoOJ protein
-2.95 SA1107 transcriptional regulator, Cro/CI family
-2.86 SA0540 endoribonuclease L-PSP, putative
-2.84 rexB SA0970 exonuclease RexB
-2.81 SA1839 transposase, IS200 family
-2.76 recQ SA1523 ATP-dependent DNA helicase RecQ
-2.67 gltC SA0513 transcriptional regulatory protein GltC
-2.61 SA1752 CBS domain protein
-2.61 SA2131 Dps family protein
-2.59 SA0314 conserved hypothetical protein
-2.58 miaA SA1323 tRNA delta(2)-isopentenylpyrophosphate transferase
-2.51 crcB SA1832 crcB protein
-2.51 SA1065 transcriptional regulator, putative
-2.48 SA1954 exonuclease
-2.45 nrdG SA2634 anaerobic ribonucleoside-triphosphate reductase activating protein
-2.45 dnaK SA1637 dnaK protein
-2.45 pcrA SA1966 ATP-dependent DNA helicase
-2.44 SA0024 5-nucleotidase family protein
-2.40 recG SA1241 ATP-dependent DNA helicase RecG
-2.39 SA0551 cell-division protein divIC, putative
-2.35 asnS SA1494 asparaginyl-tRNA synthetase
-2.27 dnaN SA0002 DNA polymerase III, beta subunit
-2.23 SA1771 OsmC/Ohr family protein
-2.21 htrA SA1028 serine protease
-2.20 hexB SA1316 DNA mismatch repair protein
-2.18 rnhC SA1150 ribonuclease HIII
 65

-2.10 parB SA2735 chromosome partioning protein, ParB family

-2.09 SA1153 DNA-dependent DNA polymerase family X
-2.07 pheT SA1149 phenylalanyl-tRNA synthetase, beta subunit
-2.06 SA1250 chromosome segregation SMC protein, putative
-2.05 map SA1946 methionine aminopeptidase, type I
-2.01 mscL SA1182 large-conductance mechanosensitive channel
Regulation
Fold
Change Common Locus Description
-8.53 agrD SA2024 accessory gene regulator protein D
-7.97 SA2359 sensor histidine kinase
-6.53 rsbW SA2055 anti-sigma B factor
-5.85 SA1354 sensor histidine kinase, putative
-5.73 sarZ SA2384 staphylococcal accessory protein Z
-5.61 rsbV SA2056 anti-anti-sigma factor RsbV
-5.41 icaR SA2688 intercellular adhesion regulator
-5.13 sarS SA0096 staphylococcal accessory regulator S
-4.95 vraR SA1942 DNA-binding response regulator
-4.91 rsbU SA2057 sigma factor B regulator protein
-4.82 graS SA0717 sensor histidine kinase
-4.50 arlS SA1450 sensor histidine kinase
-4.07 vraS SA1943 sensor histidine kinase
-3.82 sarY SA2289 staphylococcal accessory regulator Y
-3.64 sarA SA0672 staphylococcal accessory regulator A
-3.49 SA1906 sensor histidine kinase, putative
-3.37 agrB SA2023 accessory gene regulator protein B
-2.61 SA1355 DNA-binding response regulator, LuxR family
-2.53 arlR SA1451 DNA-binding response regulator
-2.39 agrC SA2025 accessory gene regulator protein C
Metabolism
Fold
Change Common Locus Description
-7.97 SA2167 iron compound ABC transporter, iron compound-binding protein
-7.58 isdE SA1143 iron compound ABC transporter, iron compound-binding protein, putative
-7.32 SA1111 spermidine/putrescine ABC transporter, spermidine/putrescine-binding prot
 66

-7.31 epiD SA1875 epidermin biosynthesis protein

-6.46 SA1920 D-isomer specific 2-hydroxyacid dehydrogenase family protein
-5.66 SA1592 rhodanese-like domain protein
-5.28 SA2363 L-lactate permease
-5.20 glmM SA2151 phosphoglucosamine mutase
-5.19 SA0024 5-nucleotidase family protein
-5.13 nrdF SA0793 ribonucleoside-diphosphate reductase 2, beta subunit
-5.11 cls1 SA1351 cardiolipin synthetase
-5.08 phoU SA1420 phosphate transport system protein
-5.06 SA0031 glycerophosphoryl diester phosphodiesterase, putative
-4.97 SA1787 chorismate mutase/phospho-2-dehydro-3-deoxyheptonate aldolase
-4.79 purB SA1969 adenylosuccinate lyase
-4.73 isdF SA1144 iron compound ABC transporter, permease protein, putative
-4.62 SA2459 carboxylesterase
-4.54 budA SA2617 alpha-acetolactate decarboxylase
-4.25 SA1753 universal stress protein family
-4.19 coaBC SA1223 phosphopantothenoylcysteine decarboxylase/phosphopantothenate--cystein
-4.10 SA2138 cation efflux family protein
-4.08 xpt SA0458 xanthine phosphoribosyltransferase
-4.06 proC SA1546 pyrroline-5-carboxylate reductase
-4.00 xseA SA1568 exodeoxyribonuclease VII, large subunit
-3.92 lacG SA2180 6-phospho-beta-galactosidase
-3.71 SA0876 arsenate reductase, putative
-3.62 sodA SA1610 superoxide dismutase
-3.58 zwf SA1549 glucose-6-phosphate 1-dehydrogenase
-3.58 SA1588 proline dipeptidase
-3.56 betA SA2627 choline dehydrogenase
-3.53 SA2375 transporter, CorA family
-3.47 SA1804 polysaccharide biosynthesis protein
-3.32 SA0666 iron compound ABC transporter, permease protein
-3.30 gcvH SA0877 glycine cleavage system H protein
-3.13 cdsA SA1280 phosphatidate cytidylyltransferase
-3.09 SA0024 5-nucleotidase family protein
-3.03 asd SA1429 aspartate-semialdehyde dehydrogenase
 67

-3.02 SA1818 3,4-dihydroxy-2-butanone-4-phosphate synthase/GTP cyclohydrolase II

-3.01 SA0155 cation efflux family protein
-2.98 SA2475 peptide ABC transporter, permease protein, putative
-2.97 SA0712 lipase/esterase
-2.97 SA0687 Na+/H+ antiporter, putative
-2.88 SA2278 acyl-CoA dehydrogenase-related protein
-2.84 rpe SA1235 ribulose-phosphate 3-epimerase
-2.78 SA0024 5-nucleotidase family protein
-2.76 SA1545 oxidoreductase, short-chain dehydrogenase/reductase family
-2.73 SA1553 glyoxalase family protein
-2.65 nagB SA0616 glucosamine-6-phosphate isomerase
-2.63 SA0024 5-nucleotidase family protein
-2.61 corA SA2342 magnesium and cobalt transport protein CorA, putative
-2.57 SA0409 conserved hypothetical protein
-2.57 bglA SA0251 6-phospho-beta-glucosidase
-2.57 SA1825 N-acetylmuramoyl-L-alanine amidase, family 4
-2.57 SA0200 phosphoglycerate transporter family protein
-2.54 tyrA SA1401 prephenate dehydrogenase
-2.52 ribD SA1820 riboflavin biosynthesis protein
-2.46 aroK SA1596 shikimate kinase
-2.42 arcC SA1182 carbamate kinase
-2.41 lysA SA1435 diaminopimelate decarboxylase
-2.40 thrB SA1364 homoserine kinase
-2.38 cidC SA2553 pyruvate oxidase
-2.31 SA1814 lysophospholipase, putative
-2.31 SA2303 inositol monophosphatase family protein
-2.28 yajC SA1693 preprotein translocase, YajC subunit
-2.27 SA0655 oxidoreductase, aldo/keto reductase family
-2.23 SA1109 spermidine/putrescine ABC transporter, permease protein
-2.15 nrdD SA2635 anaerobic ribonucleoside-triphosphate reductase
-2.13 menE SA1844 O-succinylbenzoic acid--CoA ligase, putative
-2.13 SA0308 carbohydrate kinase, PfkB family
-2.11 SA2195 M23/M37 peptidase domain protein
-2.08 SA0921 CBS domain protein
 68

-2.07 epiC SA1876 epidermin biosynthesis protein

-2.01 SA1309 pyruvate ferredoxin oxidoreductase, beta subunit
Miscellaneous
Fold
Change Common Locus Description
-15.57 SA2526 membrane protein, putative, authentic point mutation
-11.95 isdG SA1146 conserved hypothetical protein
-9.42 asp23 SA2173 alkaline shock protein 23
-9.15 SA0893 pathogenicity island protein
-8.72 SA0658 HD domain protein
-7.42 SA1439 acylphosphatase
-7.16 SA0821 HD domain protein
-6.87 SA0606 hydrolase, haloacid dehalogenase-like family
-6.33 SA2378 transcriptional regulator, AraC family
-6.30 SA0668 hydrolase, alpha/beta hydrolase fold family
integrase/recombinase, core domain family, authentic
-6.15 SA1573 frameshift
-5.80 SA0602 hydrolase, haloacid dehalogenase-like family
-5.75 SA1365 hydrolase, haloacid dehalogenase-like family
-5.51 SA0318 prophage L54a, integrase
-5.42 SA2666 N-acetylmuramoyl-L-alanine amidase domain protein
-5.20 SA2245 acetyltransferase, GNAT family
-4.77 SA2328 abortive infection protein family
-4.69 SA2159 drug transporter, putative
-4.65 SA0764 glycosyl transferase, group 2 family protein
-4.52 SA0892 pathogenicity island protein, authentic frameshift
-4.49 SA1806 probable ATP-dependent helicase
-4.49 SA0812 degV family protein
-4.46 SA2036 ABC transporter, ATP-binding protein
-4.46 dmpI SA1339 4-oxalocrotonate tautomerase
-4.31 SA1759 universal stress protein family
-4.26 SA2140 lytic regulatory protein, authentic frameshift
-4.24 SA1855 transposase, putative, degenerate
-4.23 SA0716 DNA-binding response regulator
-4.17 SA2142 SAP domain protein
 69

-4.15 scpA SA1538 segregation and condensation protein A

-4.02 SA1529 metallo-beta-lactamase superfamily protein
-4.02 SA0181 conserved domain protein
-3.89 SA1941 ribonuclease BN, putative
-3.89 SA2499 helicase, putative
-3.77 SA0630 amino acid permease
-3.76 SA2533 glyoxalase family protein
-3.76 SA0736 acetyltransferase, GNAT family
-3.63 cbf1 SA1898 cmp-binding-factor 1
-3.57 SA0747 cobalamin synthesis protein/P47K family protein
-3.44 SA1677 aminotransferase, class V
-3.41 SA0607 azoreductase
-3.38 SA1933 ThiJ/PfpI family protein
-3.34 SA1515 GTP-binding protein, Era/TrmE family
-3.33 SA1287 30S ribosomal protein L7 Ae
-3.30 SA1179 exotoxin 4, putative
-3.30 trxA SA1155 thioredoxin
-3.24 SA0803 lipoprotein, putative
-3.19 SA1047 conserved domain protein
-3.19 SA1180 exotoxin 3, putative
-3.17 SA0874 nitroreductase family protein
-3.09 SA1207 glyoxalase family protein
-3.03 SA1855 transposase, putative, degenerate
-3.00 bcr SA2437 bicyclomycin resistance protein
-2.97 SA0891 transcriptional regulator, putative
-2.95 SA2354 membrane protein, putative
-2.93 orfX SA0023 conserved hypothetical protein
-2.89 SAV1941 putative membrane protein
-2.87 SA0978 membrane protein, putative
-2.86 SA1412 hydrolase-related protein
-2.79 SA0553 MesJ/Ycf62 family protein
-2.79 SA1240 DAK2 domain protein
-2.67 SA1189 acetyltransferase (GNAT) family protein
-2.66 tcaA SA2352 tcaA protein
 70

-2.53 SA1708 type III leader peptidase family protein

-2.52 SA0875 thioredoxin, putative
-2.49 SA2339 DNA-3-methyladenine glycosylase
-2.49 SA2499 helicase, putative
-2.47 SA0262 Choloylglycine hydrolase family protein
-2.46 SA2522 DedA family protein
-2.41 SA1644 putative competence protein ComEC/Rec2
-2.40 SA0855 acetyltransferase, GNAT family
-2.39 SA1437 cold shock protein, CSD family
-2.38 SA2018 abortive infection protein family
-2.35 SAV1976 similar to phi PVL ORF 52 homolog
-2.34 SA1615 ATP-dependent RNA helicase, DEAD/DEAH box family
-2.30 SA1751 DHH subfamily 1 protein
-2.28 SA2014 phage terminase family protein
-2.28 int SA1810 integrase
-2.27 SA0595 peptidase, M20/M25/M40 family
-2.26 SA0201 DNA-binding response regulator, AraC family
-2.25 SA0250 PTS system, IIA component
-2.24 SA0744 ABC transporter, ATP-binding protein, MsbA family
-2.23 relA SA1010 GTP pyrophosphokinase
-2.19 SA1589 lipoprotein, putative
-2.18 SAV1976 similar to phi PVL ORF 52 homolog
-2.15 fmtC SA1396 fmtC protein
-2.02 SA2490 similar to N-hydroxyarylamine O-acetyltransferase
-2.02 SA1063 serine/threonine-protein kinase
 71

Table 3.3. Differentially expressed genes between 6 day-old S. aureus biofilm versus biofilm-
macrophage co-cultures for a 24 h period
Category
Upregulated
Metabolism
Fold
change Common Locus Description
11.68 SA0215 propionate CoA-transferase, putative
9.94 SA0214 long-chain-fatty-acid--CoA ligase, putative
5.14 SA0213 acyl-CoA dehydrogenase family protein
4.68 SA0211 acetyl-CoA acetyltransferase
2.88 gltS SA2340 sodium:glutamate symporter
Virulence/defense
Fold
change Common Locus Description
4.23 spa SA0095 immunoglobulin G binding protein A precursor
Cell wall
Fold
change Common Locus Description
2.55 pbp1 SA1194 penicillin-binding protein 1
2.46 dat SA1800 D-alanine aminotransferase
2.37 sdrD SA0520 Ser-Asp rich fibrinogen-binding, bone sialoprotein-binding protein
Transcription/translation/replication
Fold
change Common Locus Description
2.06 glnR SA1328 glutamine synthetase repressor
Miscellaneous
Fold
change Common Locus Description
2.73 SA1433 peptidase, M20/M25/M40 family
Category
Downregulated
Metabolism
Fold
Change Common Locus Description
 72

-3.85 SA1912 glucosamine-6-phosphate isomerase, putative

-3.04 SA2579 phytoene dehydrogenase
-2.42 SA0921 CBS domain protein
-2.30 SA2369 pyridine nucleotide-disulfide oxidoreductase
-2.27 SA2708 ABC transporter, ATP-binding protein
Transcription/translation/replication
Fold
Change Common Locus Description
-3.41 fabG SA2482 3-oxoacyl-(acyl carrier protein) reductase, authentic point mutation
-3.23 rpsA SA1516 ribosomal protein S1
-2.59 hutG SA2327 formiminoglutamase
-2.56 SA1753 universal stress protein family
-2.08 rpmB SA1238 ribosomal protein L28
Cell wall
Fold
Change Common Locus Description
-3.26 isdC SA1141 NPQTN cell wall surface anchor protein
-2.93 SA0764 glycosyl transferase, group 2 family protein
-2.20 cap5E SA0140 capsular polysaccharide biosynthesis protein
Virulence/defense
Fold
Change Common Locus Description
-2.91 SA1529 metallo-beta-lactamase superfamily protein
-2.76 nuc SA0860 thermonuclease precursor
-2.67 epiE SA1872 epidermin immunity protein F
-2.42 SA1797 metallo-beta-lactamase family protein
-2.37 SA2167 iron compound ABC transporter, iron compound-binding protein
Regulation
Fold
Change Common Locus Description
-2.26 arlR SA1451 DNA-binding response regulator
Miscellaneous
Fold
Change Common Locus Description
-3.21 asp23 SA2173 alkaline shock protein 23
-3.20 dmpI SA1339 4-oxalocrotonate tautomerase
 73

-3.05 SA1038 membrane protein

-2.82 SA2007 peptidase, M20/M25/M40 family, authentic frameshift
-2.68 SA0671 hydrolase, alpha/beta hydrolase fold family
-2.54 SA1576 traG protein, putative
-2.40 SA2434 membrane protein, putative
-2.12 SA1941 ribonuclease BN, putative
 74

Figure 3.6

qRT-PCR validation of down-regulated genes in S. aureus biofilms identified by microarray

analysis. A subset of genes identified by microarray analysis was confirmed by qRT-PCR

following either a 1 or 24 h co-culture period of MΦs with 4 day-old USA300 LAC static

biofilms. Results are presented as the relative gene expression after MΦ-biofilm co-culture

compared to biofilms that were not incubated with MΦs as a reference standard.
 75

Table 3.4. Differentially expressed genes between 6 day-old S. aureus biofilm versus biofilm-
neutrophil co-cultures for a 1 h period
Category
Upregulated
Metabolism
Fold
Change Common Locus Description
3.1 SACOL0200 phosphoglycerate transporter family protein
2.4 pyrE SACOL1217 orotate phosphoribosyltransferase
2.2 feoB SACOL2564 ferrous iron transport protein B
2.2 SACOL2322 peptidase, M20/M25/M40 family
2.0 carB SACOL1215 carbamoyl-phosphate synthase, large subunit
2.0 SACOL0217 ABC transporter, substrate-binding protein
Virulence/defense
Fold
Change Common Locus Description
2.0 SACOL2449 drug transporter, putative
Regulation
Fold
Change Common Locus Description
2.1 lytS SACOL0245 sensor histidine kinase LytS
2.0 SACOL2585 regulatory protein, putative
Miscellaneous
Fold
Change Common Locus Description
2.7 intergenic upstream of ORF sa_c4971s4276
2.5 intergenic upstream of ORF sa_c7173s10144
2.4 intergenic upstream of ORF sa_c8901s7819
Category
Downregulated
Metabolism
Fold
Change Common Locus Description
-3.7 SACOL2131 Dps family protein
-2.5 SACOL1952 ferritins family protein
 76

Regulation
Fold
Change Common Locus Description
-2.7 groES SACOL2017 chaperonin, 10 kDa
-2.4 grpE SACOL1638 heat shock protein
-2.3 groEL SACOL2016 chaperonin, 60 kDa
Miscellaneous
Fold
Change Common Locus Description
-2.6 intergenic upstream of ORF sa_c2703s2276
-2.6 intergenic upstream of ORF sa_c7269s10166
-2.2 reverse complement of intergenic upstream of ORF sa_c2703s2276
-2.1 reverse complement of intergenic upstream of ORF sa_c8455s7417
-2.1 intergenic upstream of ORF sa_c8544s7501

Table 3.5. Differentially expressed genes between 6 day-old S. aureus biofilm versus biofilm-
neutrophil co-cultures for a 4 h period
Category
Upregulated
Metabolism
Fold
Change Common Locus Description
3.4 purK SACOL1074 phosphoribosylaminoimidazole carboxylase, ATPase subunit
2.3 SACOL0240 4-diphosphocytidyl-2C-methyl-D-erythritol synthase, putative
2.1 gltB SACOL0514 glutamate synthase, large subunit
Cell wall
Fold
Change Common Locus Description
2.5 SACOL0191 M23/M37 peptidase domain protein
Miscellaneous
Fold
Change Common Locus Description
2.5 reverse complement of intergenic upstream of ORF sa_c10613s11068
2.2 SAS058 conserved hypothetical protein (all strains, N315)
2.1 intergenic downstream of ORF sa_c121s9459
 77

Increased agr transcription by S. aureus biofilms promotes resistance to neutrophil

challenge. agrA and agrB were both significantly enhanced in 4 day-old S. aureus biofilms

following a 1 h exposure to PMNs (Supplemental Table 3.2). To evaluate the importance of

increased agr transcriptional activity in the face of PMN challenge, we compared the extent of

biofilm phagocytosis and death of PMNs after co-culture with USA300 LAC or its isogenic agr

mutant (Δagr). While Δagr displayed significantly thicker biofilms as previously reported by

others (Fig. 3.7A) (64, 210), PMNs co-incubated with Δagr demonstrated significantly less cell

death and slightly enhanced phagocytosis of biofilm-associated bacteria, although the latter did

not reach statistical significance (Fig. 3.7B). These results suggest that while the physical nature

of the biofilm can limit the extent of phagocytosis, secreted factors also appear to play a role in

evasion of PMN effector functions, at least in vitro; a hypothesis further corroborated by the types

of genes known to be regulated by the agr operon (211, 212) as well as data previously reported

by others (180). This functional study further substantiates our microarray data by illustrating

how the enhanced expression of specific genes may facilitate biofilm evasion of innate immune

mechanisms.
 78

Figure 3.7

agr promotes S. aureus biofilm resistance to neutrophil challenge. S. aureus USA300 LAC-

GFP wild type (WT) and isogenic Δagr static biofilms were grown for 6 days and visualized

using confocal microscopy (63X magnification, 1 µm slices). (A) Quantification of 4 and 6 day-

old biofilm thickness. (B) PMNs (5 x 106) were incubated with biofilms for 20 h, whereupon the

percent of phagocytic and dead PMNs was enumerated. Significant differences are denoted with

asterisks, [(A) ***, p < 0.0001 using an unpaired two-tailed Student’s t-test, n = 60; (B) *, p <

0.05 using an unpaired two-tailed Student’s t-test, n = 3].

 79

Differential responses of macrophages and neutrophils to S. aureus biofilms are cell

autonomous. To determine whether the differences between MΦs and PMNs to phagocytose

biofilm-associated bacteria could be influenced by one another, both leukocyte populations were

co-cultured with WT USA300 LAC biofilms. The extent of MΦ and PMN invasion into the

biofilm was similar whether cells were added together (Fig. 3.8A) or separately (Fig. 3.8C).

Similarly, MΦs were incapable of phagocytosing biofilm-associated S. aureus whether co-

cultured with PMNs (Fig. 3.8B) or alone (Fig. 3.8D), whereas PMNs were equally phagocytic

under both conditions (Fig. 3.8B, D). This data demonstrates that the phagocytic ability of PMNs

over MΦs in regard to S. aureus biofilm is cell autonomous and is not influenced by the other

population in vitro.
 80

Figure 3.8

Differential responses of macrophages and neutrophils to S. aureus biofilms are cell

autonomous. S. aureus USA300 LAC-GFP static biofilms were grown for 6 days and visualized

using confocal microscopy (63X magnification, 1 µm slices). Quantification of invasion or

phagocytosis of MΦs and PMNs (5 x 106 each) cultured either together (A and B) or separately

(C and D) with 6 day-old biofilms at 4 and 24 h. Significant differences are denoted with asterisks

[(A and B) **, p < 0.01 using an unpaired two-tailed Student’s t-test, n = 4; ***, p < 0.0001 using

an unpaired two-tailed Student’s t-test, n = 4; (C and D) *, p < 0.05 using an unpaired two-tailed

Student’s t-test, n = 2; **, p < 0.01 using an unpaired two-tailed Student’s t-test, n =2].
 81

Supplemental Figure S3.1

Neutrophils phagocytose S. aureus biofilms throughout the co-culture period. USA300 LAC-

GFP static biofilms were grown for 6 days, whereupon bone marrow-isolated PMNs were

incubated with biofilms for 1, 4, or 24 h (A-C, respectively). Biofilm co-cultures were visualized

using confocal microscopy (63X magnification, 1 µm slices) and representative three-

dimensional images were constructed. Insets are provided at higher magnification to highlight the

presence of intracellular bacteria. Results are representative of at least two independent

experiments.
 82

Supplemental Table S3.1. Significantly increased genes between 6 day-old S. aureus biofilm
versus biofilm-macrophage co-cultures for a 1 h period
Category
Virulence/Defense
Fold
Change Common Locus Description
4.5 SA2291 staphyloxanthin biosynthesis protein
Miscellaneous
Fold
Change Common Locus Description
2.2 ssr161 Hypothetical
2.2 ssr162 Hypothetical
2.2 ssr169 Hypothetical
2.2 ssr166 Hypothetical
2.2 ssr167 Hypothetical
2.1 ssr168 Hypothetical
2.1 ssr159 Hypothetical
2.1 ssr160 Hypothetical

Supplemental Table S3.2. Significantly increased genes between 4 day-old S. aureus biofilm
versus biofilm-neutrophil co-cultures for a 1 h period
Category
Regulation
Fold
change Common Locus Description
5.6 agrB SA2381 accessory gene regulator protein B
4.7 agrA SACOL2026 accessory gene regulator protein A
Virulence/defense
Fold
change Common Locus Description
3.6 SACOL1187 antibacterial protein
Miscellaneous
Fold
change Common Locus Description
3.6 SA1773 hypothetical protein
 83

3.0 SACOL2565 conserved hypothetical protein

2.0 SACOL2557 conserved domain protein
2.0 SACOL2503 hypothetical protein
Transcription/translation/replication
Fold
change Common Locus Description
2.4 glnR SACOL1328 glutamine synthetase repressor
Cell Wall
Fold
change Common Locus Description
2.4 atl SACOL1062 bifunctional autolysin
2.1 SACOL0507 LysM domain protein
2.0 isaA SACOL2584 Immunodominant antigen A
Metabolism
Fold
change Common Locus Description
2.2 abcA SACOL0700 ABC transporter, ATP-binding/permease protein
2.1 SACOL1114 Mn2+/Fe2+ transporter, NRAMP family
 84

Discussion

One facet of S. aureus pathogenesis is the organism’s ability to maintain cellular

homeostasis while enduring immune-mediated stresses (174). S. aureus has been shown to

interfere with virtually every level of the host immune response, including increased resistance to

antimicrobial peptides, impairment of phagocyte recruitment, escape from NETs, resistance to

intracellular killing, and interference with complement function as well as antibody-mediated

opsonization (213). The objective of this study was to examine how relevant innate immune cell

populations, such as PMNs and MΦs, alter the S. aureus biofilm transcriptome. To our

knowledge, there have been no studies to date examining biofilm gene expression profiles after

incubation with innate immune cell populations with any species of bacterial biofilm. Based on

our preliminary data and the fact that PMNs are generally shorter-lived than MΦs, we chose early

(i.e. 1 and 4 h) and late (i.e. 1 and 24 h) time points for biofilm co-cultures, respectively.

Surprisingly, in subsequent experiments, we found that a significant number of PMNs remained

viable at 24 h after biofilm addition; therefore, PMN co-incubation periods were extended in

subsequent confocal experiments.

In the current study, the greatest transcriptional impact on S. aureus biofilms was achieved

during an early (i.e. 1 h) co-culture period with MΦs. This effect was independent of

phagocytosis, since we observed few internalized bacteria within MΦs incubated with S. aureus

biofilms (195, 204). Although numerous genes were repressed following biofilm exposure to

MΦs after 1 h of co-culture, surprisingly many genes remained unaltered. These unaffected genes

are likely important for biofilm maintenance, and may represent an essential core transcriptome

needed to maintain biofilm survival and/or evade host antimicrobial effector mechanisms in the

face of an immune challenge. In contrast, few genes were altered in S. aureus biofilms following

a 24 h exposure to MΦs, which may be explained by changes in MΦ viability following extended

biofilm co-culture periods. Specifically, our previous work demonstrated that most MΦs remain
 85

viable during a 1 h co-culture period with biofilms; however, by 6 h MΦ viability is significantly

reduced as measured by the live/dead stain 7-aminoactinomycin D (7-ADD)(195). Together,

these findings suggest that S. aureus biofilms rapidly transition into a transcriptionally dormant

mode after MΦ challenge, which is reversed once the pressure of viable MΦs has dissipated. We

cannot exclude the possibility that minor changes in biofilm gene expression could have occurred

following leukocyte addition that were not detected due to limitations in microarray sensitivity. In

addition, although extreme care was taken to ensure rapid processing of biofilms prior to RNA

isolation, it remains possible that some transcriptional changes may have occurred during this

interval due to the short half-life of many bacterial mRNAs (214). However, since we compared

the transcriptional profiles of biofilms alone concurrent with leukocyte co-culture conditions, any

changes in mRNA turnover would be expected to decay at the same rate.

Despite the extensive microbicidal mechanisms employed by PMNs, these cells did not

significantly alter the S. aureus biofilm transcriptome. This was unexpected, since we predicted

that PMN challenge would enhance bacterial virulence factor expression to interfere with

recognition/killing mechanisms. While recent studies have demonstrated the ability of S. aureus

to survive intracellularly within PMNs and, to a lesser extent, MΦs, one would expect this

adaptation to necessitate large scale transcriptional changes (215). One possibility to explain the

discrepancy between MΦs and PMNs in regulating biofilm transcriptional responses is that

PMNs were added to biofilms at a 10-fold reduced density than MΦs. However, our confocal

analysis indicated that PMNs invade the biofilm in greater numbers, remain viable for a longer

period than MΦs, and exhibit phagocytosis. Nonetheless, the latter appears to be futile, since

PMN phagocytosis of S. aureus biofilms does not significantly decrease bacterial numbers in

vitro (204). Additionally, PMNs have not proven to play a key microbicidal role in a S. aureus

catheter-associated biofilm model in vivo, showing minimal impact on bacterial burdens (197,

204).
 86

While MΦs and PMNs perform many overlapping functions in terms of bactericidal activity,

subtle specialization of labor could potentially account for the S. aureus biofilm transcriptional

differences reported in this study. For example, while PMNs are considered potent phagocytic

effectors against numerous extracellular bacteria, activated MΦs are recognized as a signaling

hub during bacterial infections through their secretion of numerous immune stimulatory and

bactericidal factors (200, 201, 216-218). Therefore, it is possible that S. aureus biofilms are more

responsive to this secreted milieu than physical disruption via phagocytosis, although this remains

speculative.

In addition to producing molecules to circumvent the host immune system, S. aureus biofilms

must adapt to nutrient-limiting conditions encountered during growth. Interestingly, MΦ addition

to S. aureus biofilms repressed numerous metabolism-associated genes, further suggesting that

biofilms transition into a dormant mode to evade immune killing pathways. Correspondingly,

MΦ exposure also lead to the repression of genes implicated in transcription, translation, and

replication as well as cell wall synthesis, which would be expected to conserve energy during a

decreased metabolic state. The repression of cell wall synthesis may also be a mechanism to

evade innate immune recognition.

The agr and sarA regulatory systems are among the many controlling the production of

staphylococcal virulence and defense factors. The agr locus encodes a QS system involved in

RNAIII production, which switches the synthesis of surface and adhesive molecules to toxin and

exoprotein expression (84). SarA is a DNA-binding regulator protein that influences the

expression of multiple genes, including those contributing to virulence and biofilm formation

(188, 219). In this study, sarA transcription was reduced in S. aureus biofilms following MΦ

exposure, which agrees with the generalized decrease in numerous genes regulated by SarA. In

contrast, agr transcription was significantly increased in S. aureus biofilms after PMN addition.

The enhanced expression of agr in this setting allowed us to examine its contribution to PMN
 87

reactivity using an isogenic agr mutant. The percentage of PMNs exhibiting phagocytic activity

was increased in Δagr biofilms, despite the significant increase in biofilm thickness. This

phenotype may be explained, in part, by alterations in phenol-soluble modulin (PSM) expression.

PSMs are surfactant peptides that have recently been implicated in bacterial biofilm formation

across a number of species, including S. aureus (79). PSM expression is intricately linked to

bacterial density and is agr regulated (79). Based on this evidence, it is reasonable to predict that

although Δagr biofilms are thicker than WT, they are likely less structurally complex and unable

to effectively circumvent PMN invasion. In addition, Δagr biofilms elicited less PMN death,

likely due to the reduced expression of lytic toxins, such as Hla and PSMs, which have been

shown to play key roles in S. aureus pathogenesis (82, 220). There were no significant effects on

MΦ phagocytosis or viability in response to Δagr biofilms (data not shown), implying that

enhanced agr transcription is selective for thwarting aspects of PMN function. This finding

further substantiates a potential selective role for impaired PSM action on PMNs, since its

cytotoxic effects primarily target this cell type(221, 222). Finally, as PSM expression is induced

by the stringent response resulting from the harsh conditions within the phagolysosome (83),

impaired PSM activity provides a potential explanation for the increased intracellular burden of

Δagr biofilms by PMNs. The impressive ability of S. aureus biofilms to adapt to PMN challenge

correlates with the modest transcriptome changes reported in this study.

In conclusion, the changes in S. aureus biofilm transcriptional profiles after leukocyte exposure

reported here provides a comprehensive view of the molecules that may impact S. aureus immune

evasion and survival during biofilm growth. Further investigations into the role of differentially

regulated genes will provide a better understanding of the ability of S. aureus to adapt to

environmental challenges and may provide novel strategies and therapeutic targets for

staphylococcal biofilm infections.

 88

Chapter 4: Staphylococcus aureus biofilms induce macrophage dysfunction through

leukocidin AB and α-toxin

Published in mBio 6(4), 2015

 89

Abstract

The MΦ response to planktonic S. aureus involves the induction of proinflammatory,

microbicidal activity. However, S. aureus biofilms can interfere with these responses, in part, by

polarizing MΦs toward an anti-inflammatory, pro-fibrotic phenotype. Here we demonstrate that

conditioned medium from mature S. aureus biofilms inhibited MΦ phagocytosis and induced

cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing identified the

active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry

identified Hla and LukAB as critical molecules secreted by S. aureus biofilms that inhibit murine

MΦ phagocytosis and promote cytotoxicity. A role for Hla and LukAB was confirmed using hla

and lukAB mutants and synergy between both toxins was demonstrated with a lukAB/hla double

mutant and verified by complementation. Independent confirmation of the effects of Hla and

LukAB on MΦ dysfunction was demonstrated using an isogenic strain where Hla was

constitutively expressed, a Hla antibody to block toxin activity, and purified LukAB peptide. The

importance of Hla and LukAB during S. aureus biofilm formation in vivo was assessed using a

murine orthopedic implant biofilm infection model, where the lukAB/hla strain displayed

significantly decreased bacterial burdens and increased MΦ infiltrates compared with each single

mutant. Collectively, these findings reveal a critical synergistic role for Hla and LukAB in

promoting MΦ dysfunction and facilitating S. aureus biofilm development in vivo.

 90

Introduction

Highly opportunistic pathogens possess attributes that facilitate persistent infections, in part,

by shielding themselves from immune-mediated attack (223-225). S. aureus is one such example,

and in addition to its well-known arsenal of virulence determinants, biofilm formation represents

another means to circumvent immune-mediated clearance in the host (15, 16). Biofilms are

heterogeneous bacterial communities encased in a complex matrix composed of extracellular

DNA (eDNA), proteins, and polysaccharides (11, 32, 226, 227). S. aureus has a propensity to

form biofilms on medical devices, such as prostheses and indwelling catheters, and the organism

remains a major cause of health care- and community-associated infections (194, 228, 229).

Many S. aureus virulence factors target innate immune pathways that are elicited during

acute planktonic infection, such as phagocytosis and proinflammatory transcription factor

activation (15, 160, 213, 230). Phagocytosis leads to the killing of extracellular pathogens as well

as proinflammatory cytokine and chemokine production, which collectively orchestrate local and

systemic inflammatory responses and initiate adaptive immunity (129-131). Recent studies have

demonstrated that biofilms formed by various bacterial species interfere with classical host anti-

bacterial effector mechanisms (170, 231-235). With regard to S. aureus, work from our laboratory

and others has shown that biofilms polarize MΦs towards an anti-inflammatory phenotype by

dampening proinflammatory responses and limiting MΦ invasion in vivo (15, 159, 160, 236,

237). This response is considered detrimental to biofilm clearance, since polarized MΦs possess

poor microbicidal activity and instead promote fibrosis (15). Similar findings of MΦ dysfunction

have been reported in response to S. epidermidis biofilms (158, 164, 165), suggesting the

existence of a conserved effort to thwart efficient biofilm recognition and clearance by the host.

However, the molecules responsible for the ability of S. aureus biofilms to attenuate MΦ

proinflammatory responses remain ill-defined.

 91

The objective of this study was to identify S. aureus biofilm-derived products that induce

MΦ dysfunction and facilitate biofilm persistence. Quantitative mass spectrometry identified Hla

and the bicomponent leukotoxin, LukAB, also known as LukGH, as potential candidates

responsible for inhibiting MΦ phagocytosis and promoting cytotoxicity, which was confirmed

using hla and lukAB mutants. A synergistic effect was demonstrated with a lukAB/hla double

mutant that also revealed decreased biofilm formation in vivo using a murine model of orthopedic

implant biofilm infection. The reduction in MΦ phagocytosis, concomitant with enhanced cell

death, likely facilitates the ability of S. aureus to avoid destructive host responses when organized

as a biofilm.
 92

Results

S. aureus biofilms secrete a proteinaceous factor(s) that inhibits macrophage phagocytosis.

Our previous studies demonstrated that MΦs are unable to phagocytose S. aureus biofilms (15,

160); however, the mechanism responsible for this phenomenon remained to be identified. While

it is known that the physical size of a biofilm is one factor that impedes phagocytosis (15), we

investigated the possibility that a secreted factor(s) was also involved. In order to assess the effect

of biofilm-conditioned medium on MΦ phagocytosis, we utilized fluorescent microspheres

instead of live bacteria, since live S. aureus actively secretes factors during planktonic growth

that would have been impossible to differentiate from biofilm-derived molecules. Using this

approach, we were able to readily distinguish differences in phagocytosis and viability of murine

MΦs exposed to fresh medium (Fig. 4.1A), S. aureus biofilm-conditioned medium (Fig. 4.1B),

and S. aureus planktonic-conditioned medium (Fig. 4.1C) using confocal microscopy. Of note,

similar effects of S. aureus biofilm-conditioned medium on MΦ phagocytosis were obtained with

fluorescent microspheres and intact S. aureus in pilot studies (Supplemental Fig. S2.1),

supporting the validity of this approach. MΦ phagocytic activity was significantly reduced after

treatment with conditioned medium from intact biofilms of the Methicillin-resistant S. aureus

(MRSA) clinical isolate USA300 LAC (172-175) (Fig. 4.1B and D), revealing a role for an

extracellular factor(s). To determine whether this effect relied on an intact biofilm structure, fresh

medium was added to mature biofilms that were disrupted by trituration, whereupon conditioned

medium was harvested 24 h later. Treatment of MΦs with supernatants collected from disrupted

biofilms had less impact on phagocytosis (Fig. 4.1D), suggesting that the putative extracellular

factor(s) is enriched in intact biofilms, perhaps via a QS system that is disturbed upon destruction

of the biofilm architecture. Similarly, conditioned medium from planktonic organisms was less

effective at blocking MΦ phagocytosis (Fig. 4.1C and D), even when cultures were grown to a

high cell density (i.e. late stationary phase; data not shown), demonstrating the enrichment of this
 93

secreted factor(s) in intact biofilms. Importantly, these differences did not result from alterations

in bacterial density or secreted protein levels, since titers and extracellular protein concentrations

of intact biofilms, disrupted biofilms, and planktonic cultures were similar (Supplemental Fig.

S4.1 and data not shown).

Whereas little information is currently available regarding the S. aureus biofilm secretome,

the importance of autolysis to biofilm formation has been well-established (20, 32, 78, 238-240).

To determine whether the putative biofilm extracellular factor(s) was actively secreted or a

byproduct of cell lysis, mature biofilms were treated for 24 h with polyanethole sodium sulfanate

(PAS) to inhibit lysis (241) or disrupted by trituration and treated with lysostaphin to artificially

induce lysis. Only biofilm-conditioned medium from PAS-treated supernatants maintained

inhibitory activity (Fig. 4.1F), suggesting that S. aureus biofilms actively secrete molecule(s) that

impede MΦ phagocytosis. To elucidate the chemical nature of secreted inhibitory factor(s),

conditioned medium from intact biofilms was treated with proteinase K prior to MΦ addition.

Proteinase K completely ablated the inhibitory effect of S. aureus biofilm-conditioned medium on

MΦ phagocytosis, implicating the action of a protein(s) in this phenomenon (Fig. 4.1H).

In addition to impaired phagocytosis, our prior report demonstrated that S. aureus biofilms

also induced MΦ cytotoxicity (15). The latter could result from frustrated phagocytosis based on

the inability of MΦs to physically engulf the bulky biofilm structure combined with the action of

secreted toxins, such as Hla or leukocidins with known cytotoxic activity (131, 213, 242).

Exposure of murine MΦs to conditioned medium from intact S. aureus biofilms induced

significant cell death, whereas minimal cytotoxicity was observed following treatment with

medium from either disrupted biofilms or planktonic S. aureus (Fig. 4.1A-C, and E). Similar to

the approach employed for phagocytosis, biofilms were treated with lysostaphin or PAS, where

only lysostaphin prevented the cytotoxic effects of biofilm-conditioned medium (Fig. 1G), again
 94

revealing the action of an actively secreted protein based on its proteinase K-sensitive nature (Fig.

4.1I).
 95

Figure 4.1

S. aureus biofilms secrete a proteinaceous factor(s) that inhibits macrophage phagocytosis.

(A-C) Representative 2D confocal images (63x) of BMDMΦ phagocytosis of fluorescent

microspheres (yellow-white) and cell death with propidium iodide stain (red-purple) after

exposure to (A) fresh medium, (B) S. aureus biofilm-conditioned medium, or (C) S. aureus

planktonic-conditioned medium. (D and E) BMDMΦs were exposed to fresh medium or

conditioned medium collected from an intact biofilm, a mature biofilm that was mechanically

disrupted, or a similar number of planktonic S. aureus. After a 3 h treatment period, (D) MΦ

phagocytosis of fluorescent microspheres and (E) cell viability was quantitated by confocal

microscopy. (F and G) Conditioned medium from biofilms treated with either PAS (10 μg/ml) or
 96

lysostaphin (50 μg/ml) were added to MΦs to assess the relative importance of active biofilm

secretion versus passive release of products via autolysis, respectively, on MΦ (F) phagocytosis

and (G) cell death. (H and I) Biofilm-conditioned supernatants were treated with proteinase K (10

μg/ml) prior to MΦ exposure to assess the chemical nature of the inhibitory molecule(s).

Significant differences are denoted with asterisks (***, p < 0.001; one-way ANOVA followed by

Bonferroni's multiple comparison test). Results are representative of at least two independent

experiments.
 97

S. aureus biofilm-induced macrophage dysfunction is partially agr-dependent. Our findings

that disrupted biofilms were not as effective at blocking MΦ phagocytosis or inducing

cytotoxicity suggested that QS systems enriched during biofilm formation may regulate the

putative inhibitory molecule(s). The expression of numerous virulence factors in S. aureus,

including secreted proteases, leukocidins, and Hla, is either directly or indirectly influenced by

two-component regulatory systems, such as the agr QS system (243). Agr modulates virulence

factor expression and is an important regulatory switch between planktonic and biofilm lifestyles

in S. aureus (64, 212, 244-246). Conditioned medium from a Δagr biofilm induced minimal MΦ

cell death (Fig. 4.2B), whereas the phagocytic block was significantly attenuated but only

partially influenced by agr (Fig. 4.2). Since the MΦ inhibitory phenotypes upon exposure to S.

aureus biofilms were partially agr-dependent, the Δagr strain was utilized for subsequent

proteomics comparisons with WT biofilms in an attempt to identify secreted proteins enriched

during biofilm growth that were capable of inducing MΦ dysfunction.

 98

Figure 4.2

S. aureus biofilm-induced macrophage dysfunction is partially agr-dependent. BMDMΦs

were exposed to fresh or conditioned medium from S. aureus WT or isogenic ∆agr biofilms for 2

h, whereupon (A) phagocytosis of fluorescent microspheres and (B) total viable MΦs were

quantitated by confocal microscopy. Significant differences are denoted with asterisks (***, p <

0.001; unpaired two-tailed student’s t-test). Results are representative of at least three

independent experiments.
 99

Sequential Windowed data-independent Acquisition of the Total High-resolution Mass

Spectra (SWATH-MS) as a tool to identify S. aureus biofilm factors that induce

macrophage dysfunction. We next employed a proteomics approach to identify candidate

molecules that might be responsible for biofilm-mediated MΦ dysfunction. Our proteomics

strategy utilized the ∆agr strain as a comparator with WT biofilm, since the MΦ inhibitory

phenotypes were partially agr-dependent (Fig. 4.2). A second comparison was made between

biofilm and planktonic conditions because the MΦ inhibitory factors were enriched during

biofilm growth (Fig. 4.1). To identify differentially expressed proteins between WT vs. ∆agr

biofilm- and planktonic-conditioned medium, TCA-precipitated proteins were analyzed by

quantitative SWATH-MS (189). As expected, conditioned medium from WT and ∆agr biofilms

displayed vastly different proteomic profiles, with 68 of 153 (44%) proteins significantly

enriched in WT biofilms, 23% of which were either secreted proteases or known virulence

factors, such as Hla (Fig. 4.3A and B; Table S4.1). In contrast, cell wall and structural proteins

were more abundant in ∆agr biofilm-conditioned medium compared to WT (Fig. 4.3A and B).

Similarly, proteomic comparisons between WT biofilm- vs. WT planktonic-conditioned medium

differed significantly, with 108 of 301 (36%) proteins enriched in WT biofilm, including several

secreted virulence factors such as toxins and proteases (Table S4.2). A functional proteomics

network constructed with overlapping hits from both comparisons (WT biofilm vs. ∆agr biofilm

and WT biofilm vs. WT planktonic) identified 17 proteins, including two serine proteases and

two leukocidin components, as candidates to account for the inhibitory effects of biofilm-

conditioned medium on MΦ function (Fig. 4.3C). Additionally, Hla was significantly enriched in

WT compared to ∆agr biofilms (Table S4.1), which represented another toxin of interest for its

potential role in regulating MΦ dysfunction. Importantly, both Hla and LukAB were significantly

enriched in biofilm- compared to planktonic-conditioned medium (Fig. 4.4), suggesting that these

two proteins may be responsible for biofilm-induced MΦ dysfunction. LukAB is a bi-component

leukotoxin also involved in S. aureus-mediated killing of host phagocytes (67, 247, 248).
 100

Therefore, Hla and LukAB were the focus of subsequent mechanistic studies as they likely play a

significant role in MΦ dysfunction and death in this setting.

 101

Figure 4.3

SWATH-MS identifies potential biofilm factors responsible for macrophage dysfunction.

Conditioned supernatants from either S. aureus WT or Δagr strains grown under planktonic or

biofilm conditions were harvested and TCA precipitated for protein isolation in triplicate.

Relative protein concentrations were compared between sample sets by Sequential Windowed

data independent Acquisition of the Total High-resolution Mass Spectra (SWATH-MS). (A) 68

of 153 (44%) identified proteins were significantly enriched in WT vs. Δagr biofilm, with the

largest percentage associated with metabolism and virulence. 85 of 153 (56%) identified proteins

were significantly enriched in Δagr biofilm vs. WT biofilm, with the largest percentage of

proteins falling into the functional category of cell wall proteins. (B) Direct comparison of

significantly expressed proteins in WT vs. Δagr biofilm by functional category, with more refined

groups of metabolism and virulence shown. (C) Functional protein association network of

SWATH-MS identified proteins significantly enriched in WT biofilm-conditioned supernatant vs.

planktonic and Δagr biofilm-conditioned supernatants. For all comparisons, statistical

significance was assessed using a Z-test (p < 0.05).

 102

Figure 4.4

LukA and Hla secretion is enhanced in S. aureus biofilms. (A) LukA and Hla levels in S.

aureus biofilm- vs. planktonic- conditioned medium were assessed by Western blots. (B)

Quantitation of Hla levels in conditioned medium from WT S. aureus biofilms versus planktonic

bacteria. Significant differences are denoted with asterisks (***, p < 0.001; unpaired two-tailed

student’s t-test). Results are representative of at least two independent experiments.

 103

LukAB and Hla play significant roles in biofilm-induced macrophage dysfunction. Biofilm-

conditioned medium from both ∆lukA and ∆lukB strains elicited minimal MΦ death compared to

WT biofilm (Fig. 4.5B). Likewise, the phagocytic block induced by the ∆lukA and ∆lukB strains

was less pronounced than WT biofilm-conditioned medium, although phagocytosis did not return

to baseline levels (Fig. 4.5A). Both ∆lukA and ∆lukB phenotypes could be complemented,

providing direct evidence for LukAB in modulating MΦ survival and phagocytosis (Fig. 4.5A

and B). In contrast, while serine proteases were also noted to be elevated by SWATH-MS,

biofilm-conditioned medium from serine protease mutants (∆spl or Δaur/spl/sspAB/scpA) as well

as another leukocidin mutant (∆lukD) behaved similarly to WT biofilms (data not shown).

Since LukAB did not account for the entire MΦ dysfunction phenotype and SWATH-MS

identified increased Hla levels in WT biofilm-conditioned medium (Table S4.1), we next

examined the contribution of Hla to MΦ dysfunction. Further justification for investigating Hla

stemmed from the vast literature on Hla regulation by the agr QS system (242, 249-251), the

finding that conditioned medium from ∆agr biofilms was less effective at inducing MΦ

dysfunction (Fig. 4.2), and that Hla secretion was significantly increased during biofilm growth

(Fig. 4.4). Hla inserts into host cell membranes and oligomerizes to form pores, leading to cell

death (242, 252). Indeed, MΦ survival was significantly improved following exposure to ∆hla

biofilm-conditioned medium compared to WT biofilm, which was complementable (Fig. 4.5D).

The effects of ∆hla on MΦ phagocytosis were less pronounced, but still reached statistical

significance (Fig. 4.5C). Furthermore, blockade of Hla activity in WT biofilm-conditioned

medium using a Hla neutralizing antibody phenocopied the findings with ∆hla (Fig. 4.5C and D).

Specificity of the Hla antibody was demonstrated by its ability to inhibit the effects of purified

Hla on MΦ survival and viability (Supplemental Fig. S4.3). Additional evidence to support Hla

action was provided by the ability of biofilm-conditioned medium from a S. aureus strain that
 104

constitutively expresses hla (hlaon) to induce significant MΦ death and inhibit phagocytosis (Fig.

4.5C and D).

Both Hla and LukAB expression were markedly increased in conditioned medium from WT

biofilms compared to planktonic bacteria (Fig. 4.4). Therefore, to assess whether LukAB and Hla

act cooperatively to effect MΦ activity, ∆lukA and ∆lukB biofilm-conditioned media were treated

with a Hla neutralizing antibody (Fig. 4.6). Interestingly, negating Hla action in ∆lukA and ∆lukB

biofilm-conditioned medium significantly improved MΦ phagocytosis compared to supernatants

where Hla was active (Fig. 4.6A). Similar findings were obtained with a ∆lukAB/∆hla mutant

(Fig. 4.6A). Hla blockade in ∆lukA and ∆lukB biofilm-conditioned medium had no additional

effect on MΦ survival, which was not unexpected since viability had nearly been restored with

each of the single mutants to levels observed with fresh medium (Fig. 4.6B). When MΦs were

treated with ∆lukAB biofilm-conditioned medium (that still produces Hla) in combination with

purified LukAB or a point mutant that lacks lytic activity (LukABE323A) (186), only bioactive

LukAB returned both phagocytic inhibition and cytotoxicity to levels observed with WT biofilm-

conditioned medium (Fig. S4.3). Collectively, these results demonstrate that LukAB acts in

concert with Hla to induce MΦ dysfunction.

 105

Figure 4.5

LukAB and Hla play significant roles in biofilm-induced macrophage dysfunction. (A and

B) BMDMΦs were exposed to fresh or conditioned medium from S. aureus WT, ∆lukA, ∆lukB,

or chromosomally complemented ∆lukAB (∆lukAB::lukAB) biofilms. After a 3 h treatment

period, (A) phagocytosis of fluorescent microspheres and (B) viable MΦs were quantitated by

confocal microscopy. (C and D) BMDMΦs were exposed to fresh or conditioned medium from S.

aureus WT biofilm + Hla antibody (α-Hla), ∆hla, plasmid complemented ∆hla [∆hla(pHla)], and

constitutively expressed hla (hlaon) biofilms. After a 3 h treatment period, (C) phagocytosis of

fluorescent microspheres and (D) viable MΦs were quantitated by confocal microscopy.

Significant differences are denoted with asterisks (*, p < 0.05; ***, p < 0.001; unpaired two-tailed

student’s t-test). Results are representative of at least three independent experiments.

 106

Figure 4.6

S. aureus Hla and LukAB act in concert to promote macrophage dysfunction. BMDMΦs

were exposed to fresh or conditioned medium from S. aureus WT or isogenic ∆lukA, ∆lukB and

∆lukAB/hla biofilms + Hla antibody (α-Hla). After a 3 h treatment period, (A) phagocytosis of

fluorescent microspheres and (B) viable MΦs were quantitated by confocal microscopy.

Significant differences are denoted with asterisks (***, p < 0.001; unpaired two-tailed student’s t-

test). Results are representative of at least three independent experiments.

 107

LukAB and Hla are important for S. aureus biofilm formation in vivo. Previous work from our

laboratory has demonstrated that augmenting MΦ proinflammatory activity is critical for biofilm

clearance in vivo (160). Therefore, to determine whether the functional role identified for LukAB

and Hla in mediating MΦ dysfunction in vitro would impact biofilm formation in vivo, we

utilized a murine model of S. aureus orthopedic implant biofilm infection (54, 55, 253). Similar

to our in vitro studies revealing cooperation between LukAB and Hla, the ΔlukAB/Δhla double

mutant displayed the greatest reduction in bacterial burdens in the knee joint, surrounding soft

tissue, and femur at days 3 and 7 post-infection compared to ΔlukAB or Δhla strains (Fig. 4.7A-

C). Furthermore, MΦ infiltrates were significantly increased in mice infected with ΔlukAB/Δhla

(Fig. 4.7D), although they represent a minor population in this model of orthopedic implant

biofilm infection, which is dominated by myeloid-derived suppressor cells (MDSCs) (54, 55).

Taken together, these results identify LukAB and Hla as important virulence factors for

modulating bacterial persistence and MΦ infiltrates during S. aureus biofilm formation in vivo.
 108

Figure 4.7

LukAB and Hla are important for S. aureus biofilm formation in vivo. Bacterial burdens

associated with the (A) soft tissue surrounding the knee, (B) knee joint, and (C) femur of mice

infected with WT S. aureus and isogenic ∆hla, ∆lukAB and ∆lukAB/hla strains at days 3 and 7

post-infection (n = 10 mice per strain for each time point). Results are expressed as the number of

CFU per gram of tissue to correct for differences in tissue sampling size. (B) Quantitation of

F4/80+ MΦs infiltrating the soft tissue of mice infected with WT S. aureus and isogenic ∆hla,

∆lukAB and ∆lukAB/hla strains. Significant differences are denoted with asterisks (*, p < 0.05;

**, p < 0.01; ***, p < 0.001; unpaired two-tailed Student’s t-test). Results are combined from two

independent experiments.
 109

Figure 4.8

Proposed model for S. aureus biofilm-induced macrophage dysfunction. S. aureus biofilms

produce an abundance of LukAB, which can associate with the cell surface as a toxin reservoir or

be actively secreted into the extracellular milieu. Biofilms also secrete Hla that acts

synergistically with LukAB to elicit MΦ dysfunction. Other secreted proteins, such as

lipoproteins, lantibiotics, or siderophores may also impact MΦ phagocytosis.

 110

Supplemental Figure S4.1

Bacterial counts and extracellular protein concentrations of S. aureus WT planktonic,

biofilm, and isogenic mutant biofilms are similar. (A) All S. aureus strains used in these

studies grew to comparable extents after 6 days of culture in RPMI-1640 supplemented with 1%

CAA as indicated by the number of viable bacteria determined by quantitative culture with results

expressed as colony forming units (CFU) per ml. (B) Quantitation of secreted proteins for each

strain. Data are representative of at least two independent experiments. Strains are abbreviated as

follows: ∆agr = accessory gene regulator mutant; ∆hla = α-hemolysin mutant; ∆hla (pHla) =

plasmid complemented α-hemolysin mutant; hlaon = WT strain constitutively expressing α-toxin;

∆lukA = leukocidin AB component single mutant; ∆lukB = leukocidin AB component single

mutant; ; ∆lukAB::lukAB = chromosomally complemented leukocidin AB double mutant;

∆lukAB/∆hla = leukocidin AB and α-hemolysin double mutant.

 111

Supplemental Figure S4.2

Validation of S. aureus Hla action on macrophage dysfunction. BMDMΦs were incubated for

2 h with fresh medium alone or fresh medium with purified S. aureus Hla + an isotype control or

Hla antibody (α-Hla). After a 2 h treatment period, (A) MΦ phagocytosis of fluorescent

microspheres and (B) total viable MΦs were quantitated by confocal microscopy. Significant

differences are denoted with asterisks (*, p < 0.05; ***, p < 0.001; unpaired two-tailed student’s

t-test). Results are representative of at least two independent experiments.

 112

Supplemental Figure S4.3

Purified LukAB augments macrophage cytotoxicity. BMDMΦs were exposed to fresh or

biofilm-conditioned medium from S. aureus WT or isogenic ΔlukAB + purified LukAB or

inactive LukABE323A. After a 3 h treatment period, the (A) percentage of MΦs phagocytosing

fluorescent microspheres and (B) percentage of viable MΦs were quantitated by confocal

microscopy. Significant differences are denoted with asterisks (***, p < 0.001; unpaired two-

tailed Student’s t-test). Results are representative of at least two independent experiments.
 113

Supplemental Figure S4.4

10 12
10 8

11
10

10 10 10 7

9
10

C F U /w e ll
C F U /w e ll

10 6
8
10

7
10
10 5
6
10

5
10
10 4
S u p e rn a ta n t
4
10
B io film
10 3
10 3
0 1 2 3 4 5 6

D a y s p o s t - in o c u la t io n

Characterization of S. aureus biofilm growth in 1% casamino acids. Total numbers of

dispersed (supernatant) vs. biofilm-associated bacteria in RPMI-1640 supplemented with 1%

casamino acids were assessed by serial dilution (CFU/well) throughout the 6 day biofilm

maturation process.
 114

Table S4.1 Proteins identified to be in greater abundance in WT S. aureus biofilm-

conditioned medium compared to ∆agr biofilm-conditioned medium

Magnitude Category Protein Identity

14.32 Translation RL5 Ribosomal protein
4.73 Translation Syn Asparagine-tRNA ligase
4.09 Translation RL13 Ribosomal protein
3.32 Translation RL332 Ribosomal protein
2.90 Translation EF-TU Elongation factor
2.72 Translation RL27 Ribosomal protein
2.56 Translation GatB Aspartyl/glutamyl-tRNA amidotransferase subunit B
2.42 Translation RS7 Ribosomal protein
2.1 Translation RL16 Ribosomal protein
2.01 Translation RL10 Ribosomal protein
25.93 Metabolism CysK Cysteine synthase
6.92 Metabolism CarB Carbamoyl phosphate synthetase subunit B
4.37 Metabolism OtcC Ornithine carbamoyltransferase
3.91 Metabolism KpyK Pyruvate kinase
3.63 Metabolism Pur8 Adenylosuccinate lyase
3.07 Metabolism HutG Formimidoylglutamase
2.87 Metabolism Pgk Phosphoglycerate kinase
2.50 Metabolism GlmS Glutamine--fructose-6-phosphate aminotransferase
2.42 Metabolism OdpB Pyruvate dehydrogenase E1 component subunit beta
2.29 Metabolism Ldh1 L-lactate dehydrogenase 1
42.01 Protease SplB Serine-like proteasea
33.61 Protease SplD Serine-like protease
24.78 Protease SspA Serine protease
24.78 Protease SspB Cysteine protease
14.23 Protease SspP Cysteine protease
9.02 Protease SplA Serine-like protease
6.48 Protease SplC Serine-like protease
2.17 Protease SplE Serine-like protease
11.97 Virulence LukDV/HlgB Leukocidin component
10.59 Virulence HlgA Gamma-hemolysin component A
9.71 Virulence Hld Delta-hemolysin
6.38 Virulence Hla Alpha-hemolysin
5.09 Virulence PsmA4 Phenol-soluble modulin alpha 4 peptide
3.90 Virulence LukB/H Leukocidin component
3.81 Virulence LukA/G Leukocidin component
3.09 Virulence ClfA Clumping factor A
9.98 Cell Stress Asp23 Alkaline shock protein
6.49 Cell Stress Cell Ch60 60 kDa chaperonin
5.92 Stress Cell ClpL ATP-dependent Clp protease ATP-binding subunit
5.08 Stress Cell OhrL Organic hydroperoxide resistance protein-like
2.29 Stress HchA Molecular chaperone Hsp31 and glyoxalase 3
23.69 Other Pnp Polyribonucleotide nucleotidyltransferase
23.39 Other Lip1 Lipase
6.92 Other Pbp Beta-lactam-inducible penicillin-binding protein
6.09 Other PpaC Probable manganese-dependent inorganic pyrophosphatase
 115

5.13 Other Lip2 Lipase

4.33 Other Ggi3 Antibacterial protein 3 homolog
4.25 Other AtpB ATP synthase subunit beta
3.98 Other AtpA ATP synthase subunit alpha
3.84 Other ButA Diacetyl reductase
3.72 Other Ispd2 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase 2
3.69 Other Y2518 Uncharacterized hydrolase
3.49 Other Y2365 Uncharacterized lipoprotein
3.44 Other Ptg3C PTS system glucose-specific EIICBA component
3.30 Other GcsT Aminomethyltransferase
3.17 Other FtsZ Cell division
2.94 Other PdxS Pyridoxal biosynthesis lyase
2.83 Other CatA Catalase
2.63 Other Y840 Uncharacterized protein
2.46 Other DldH Dihydrolipoyl dehydrogenase
2.45 Other ClpB Chaperone protein
2.44 Other PtgA Glucose-specific phosphotransferase enzyme IIA component
2.36 Other MtlD Mannitol-1-phosphate 5-dehydrogenase
2.21 Other Y370 Uncharacterized protein
2.20 Other Y941 NADH dehydrogenase-like protein
2.16 Other UP355 Uncharacterized protein
2.14 Other Dbh DNA-binding protein
2.10 Other Y1797 Uncharacterized protein
a
Proteins in red were selected for follow-up confirmation with mutant strains

Table S4.2 Proteins identified to be in greater abundance in WT S. aureus biofilm-

conditioned medium compared to WT planktonic-conditioned medium

Magnitude Category Protein Identity

36.16 Metabolism GlmS Glutamine--fructose-6-phosphate aminotransferase
19.56 Metabolism GlnA Glutamine synthetase
19.27 Metabolism PyrB Aspartate carbamoyltransferase
15.11 Metabolism Dha1 Alanine dehydrogenase 1
13.88 Metabolism Alf1 Fructose-bisphosphate aldolase class 1
10.66 Metabolism Odo1 2-oxoglutarate dehydrogenase E1 component
10.54 Metabolism Kad Adenylate kinase
10.16 Metabolism DaaA D-alanine aminotransferase
9.01 Metabolism TpiS Triosephosphate isomerase
8.12 Metabolism Pur1 Amidophosphoribosyltransferase
7.95 Metabolism Odp2 Dihydrolipoyllysine-residue acetyltransferase
7.59 Metabolism P5cr Pyrroline-5-carboxylate reductase
7.54 Metabolism PyrG CTP synthase
7.47 Metabolism HutG Formimidoylglutamase
6.98 Metabolism LdhD D-lactate dehydrogenase
6.74 Metabolism AckA Acetate kinase
6.71 Metabolism SdrD Serine-aspartate repeat-containing protein D
6.48 Metabolism Acp Acyl carrier protein
5.97 Metabolism RocA 1-pyrroline-5-carboxylate dehydrogenase
 116

4.97 Metabolism TdcB L-threonine dehydratase catabolic protein

4.86 Metabolism Pdp Pyrimidine-nucleoside phosphorylase
4.83 Metabolism Hps 3-hexulose-6-phosphate synthase
4.56 Metabolism K6pf 6-phosphofructokinase
4.00 Metabolism Mqo2 Probable malate:quinone oxidoreductase 2
3.94 Metabolism Pgk Phosphoglycerate kinase
3.62 Metabolism CarB Carbomoyl phosphate synthetase subunit B
3.56 Metabolism KpyK Pyruvate kinase
2.89 Metabolism OtcC Ornithine carbamoyltransferase, catabolic subunit
2.88 Metabolism PyrC Dihydroorotase
2.83 Metabolism F16pC Fructose-1,6-bisphosphatase class 3
2.66 Metabolism PurM Phosphoribosylformylglycinamidine cyclo-ligase
2.10 Metabolism OdpB Pyruvate dehydrogenase E1 component subunit beta
15.61 Translation RL29 Ribosomal protein
8.07 Translation RL3 Ribosomal protein
7.28 Translation RL25 Ribosomal protein
7.20 Translation RL14 Ribosomal protein
6.06 Translation RL24 Ribosomal protein
4.96 Translation RL7 Ribosomal protein
4.84 Translation GatB Aspartyl/glutamyl-tRNA amidotransferase subunit B
4.62 Translation RS2 Ribosomal protein
4.43 Translation RL16 Ribosomal protein
4.30 Translation RS15 Ribosomal protein
3.74 Translation RL5 Ribosomal protein
3.62 Translation RL11 Ribosomal protein
2.81 Translation RS7 Ribosomal protein
13.97 Virulence FnbA Fibronectin-binding protein A
9.19 Virulence GyrA DNA gyrase subunit A
8.05 Virulence LukDV/HlgB Leukocidin componenta
7.05 Virulence LukB/H Leukocidin component
6.59 Virulence BlaR Regulatory protein
4.03 Virulence HlgC Gamma-hemolysin component C
2.80 Virulence Emp Extracellular matrix protein-binding protein
2.48 Virulence Plc 1-phosphatidylinositol phosphodiesterase
66.31 Redox MtlD Mannitol-1-phosphate 5-dehydrogenase
13.95 Redox Dhe2 NAD-specific glutamate dehydrogenase
7.61 Redox Y2305 Putative 2-hydroxyacid dehydrogenase
6.05 Redox AhpF Alkyl hydroperoxide reductase subunit F
5.14 Redox Y542 Putative heme-dependent peroxidase
4.46 Redox Adh Alcohol dehydrogenase
4.19 Redox Azo1 FMN-dependent NADPH-azoreductase
7.29 Cell Stress SodM Superoxide dismutase
5.11 Cell Stress RecA Recombinase A
3.35 Cell Stress CspA Cold shock protein
2.75 Cell Stress GrpE Putative stress response protein
2.73 Cell Stress ClpB Chaperone protein
2.06 Cell Stress ClpC ATP-dependent Clp protease ATP-binding subunit
10.98 Structural Atl Bifunctional autolysin
2.90 Structural IsdC Iron-regulated surface determinant protein C
2.73 Structural Omp7 77 kDa membrane protein
2.57 Structural Rot Repressor of toxin, transcriptional regulator
 117

2.35 Structural IsdH Iron-regulated surface determinant protein H

2.00 Structural SarA Transcriptional regulator
8.33 Protease SplF Serine-like protease
5.74 Protease SplA Serine-like protease
5.46 Protease SplE Serine-like protease
3.91 Protease Aur Zinc metalloproteinase aureolysin
3.70 Protease PepVL Putative dipeptidase subunit
87.39 Other RsbW Serine-protein kinase
18.16 Other SrrA Transcriptional regulatory protein
15.64 Other Dpo3B DNA polymerase III subunit beta
14.54 Other PanB 3-methyl-2-oxobutanoate hydroxymethyltransferase
14.02 Other AtrF2 Putative acetyltransferase
13.22 Other RpoE Probable DNA-directed RNA polymerase subunit delta
11.32 Other Hem3 Porphobilinogen deaminase
11.29 Other RpoC DNA-directed RNA polymerase subunit beta'
8.30 Other AtpB ATP synthase subunit beta
7.27 Other GpsB Cell cycle protein
6.91 Other IlvE Probable branched-chain-amino-acid aminotransferase
6.71 Other HdoX2 Heme oxygenase (staphylobilin-producing) 2
5.82 Other Y680 Probable transcriptional regulatory protein\
5.75 Other CodY GTP-sensing transcriptional pleiotropic repressor
5.50 Other SceD Probable transglycosylase
5.46 Other Np30 30 kDa neutral phosphatase
4.60 Other ThlA Probable acetyl-CoA acyltransferase
4.49 Other Y1696 UPF0297 protein
4.33 Other RpoA DNA-directed RNA polymerase subunit alpha
4.27 Other Rnj1 Ribonuclease J 1
3.94 Other PdxT Glutamine amidotransferase subunit
3.93 Other FtsL Cell division protein
3.68 Other Oat2 Ornithine aminotransferase 2
3.56 Other Gsa2 Glutamate-1-semialdehyde 2,1-aminomutase 2
3.29 Other Y2518 Uncharacterized hydrolase
3.23 Other PtgA Glucose-specific phosphotransferase enzyme IIA component
3.07 Other Ptg3C PTS system glucose-specific EIICBA component
3.06 Other Y752 Uncharacterized protein
2.75 Other Y968 Uncharacterized protein
2.40 Other FabF 3-oxoacyl-[acyl-carrier-protein] synthase 2
2.37 Other Ldh2 L-lactate dehydrogenase 2
a
Proteins in red were selected for follow-up confirmation with mutant strains
 118

Discussion

S. aureus subverts the host immune response by numerous mechanisms, including increased

resistance to cationic antimicrobial peptides, impairment of phagocyte recruitment, interference

with antibody-mediated opsonization and complement activation, and resistance to intracellular

killing (213). In addition, biofilm formation further protects S. aureus from the host innate

immune response, representing a communal virulence determinant (15, 56, 159). We have

previously demonstrated that biofilm formation shields S. aureus from Toll-like receptor (TLR)

detection and interferes with MΦ activation in vivo (15, 160). Here we explored the genetic basis

of how biofilm growth prevents MΦ phagocytosis. Our earlier study showed that MΦs were

capable of phagocytosing bacteria from mechanically disrupted, but not intact biofilms,

suggesting that the size of the biofilm and/or density of its matrix represents a physical obstacle, a

phenomenon referred to as “frustrated phagocytosis” (15, 254, 255). Here we extend these

findings to demonstrate that S. aureus biofilms also secrete proteinaceous factors that actively

inhibit MΦ phagocytosis and induce cell death. Interestingly, this proteinaceous component was

mainly evident in intact biofilms, as conditioned medium from mechanically disrupted biofilms

or planktonic cultures grown to early or late stationary phase failed to prevent phagocytosis to the

same extent, although bacterial numbers and secreted protein concentrations were similar (Fig.

1D, Supplemental Fig. S4.1, and data not shown). The preferential ability of intact S. aureus

biofilms to inhibit MΦ phagocytosis suggested the involvement of QS mechanisms that are

enriched during biofilm growth and dissipate once the biofilm structure has been disrupted. This

was confirmed by the finding that biofilm-mediated MΦ dysfunction, in particular cell death, was

less pronounced following exposure to conditioned medium from a ∆agr biofilm. These

observations, combined with the fact that conditioned medium from PAS-treated biofilms

maintained inhibitory activity, whereas lysostaphin-treated biofilms did not, strongly implicated
 119

the importance of active protein secretion by the biofilm (based on proteinase K sensitivity) to

inhibit MΦ phagocytosis and induce cell death.

The identification of candidate proteins responsible for inducing MΦ death and inhibiting

phagocytosis was facilitated with a relatively new quantitative mass spectrometry technique,

namely SWATH-MS (189). After generating a protein library from our combined sample sets

(i.e. WT biofilm, Δagr biofilm, WT planktonic, and Δagr planktonic), comparisons were

performed to identify the most abundant proteins unique to WT biofilm-conditioned medium.

While this list included some proteins undoubtedly released as a result of cell lysis (i.e. metabolic

enzymes and ribosomal subunits), we focused on known secreted toxins and proteases that were

also detected. Of this list, LukB, as well as its partner component LukA, were shown to have a

significant impact on MΦ phagocytosis and viability. LukAB is unique among leukocidins for its

ability to either remain cell wall-associated or released into the extracellular milieu (67, 247,

248). A recent study has shown that human leukocytes are exquisitely sensitive to the cytolytic

actions of LukAB due to its specificity for CD11b (256) mediated by the binding of a specific

glutamic acid residue (323A) (186). While murine leukocytes are less sensitive to LukAB (256,

257), this toxin was still implicated in S. aureus pathogenesis in a murine renal abscess model

(67), which was confirmed in the current study using a murine S. aureus orthopedic implant

biofilm model. Therefore, although human cells display a greater sensitivity to LukAB, it is clear

from our report and work by others that this bi-component leukotoxin is also active towards

murine leukocytes.

In addition to LukAB, Hla also significantly contributed to biofilm-associated murine MΦ

death and phagocytosis. The toxic effects of Hla are well-known and, while a recent publication

has demonstrated the cytoprotective effects of S. aureus Hla within phagosomes (258), it is

important to note that this scenario is not applicable in our studies given that our phagocytosis

assay utilized microspheres and not viable bacteria. This strategy was employed to avoid
 120

confounds from toxins secreted by live planktonic S. aureus if they were used to measure MΦ

phagocytosis, which could not be discriminated from biofilm-secreted molecules. Interestingly,

the already potent cytolytic effects of S. aureus Hla were enhanced with the addition of purified

bioactive LukAB. Furthermore, treatment of ΔlukA and ΔlukB biofilm-conditioned medium with

a Hla neutralizing antibody significantly dampened the MΦ phagocytic block. These results

suggest a synergistic effect, whereby the presence of LukAB enhances or accelerates Hla-

mediated MΦ dysfunction, perhaps via enhanced binding, localization to the cell membrane, or

regulating intracellular signaling pathways.

Previous studies have demonstrated the importance of LukAB or Hla for S. aureus

pathogenesis in murine models of renal abscess (67), pneumonia (259, 260), skin infection (261,

262), bacteremia (263, 264), peritonitis (177, 262, 265), and other localized infection models

(242). However, it should be noted that a ΔlukAB single mutant displayed no attenuation of

virulence in murine models of skin infection and bacteremia (257). In support of our in vitro

findings, our study is the first to report that LukAB and Hla cooperate to regulate S. aureus

virulence in a murine orthopedic implant biofilm infection model. While ΔlukAB or Δhla single

mutants displayed decreased bacterial burdens in some tissues, ΔlukAB/Δhla showed the largest

reduction in bacterial numbers when compared to mice infected with the isogenic WT strain. In

further support of this synergistic effect, ΔlukAB/Δhla infected mice displayed the greatest

increase in MΦ infiltrates. While this in vivo data reveals an important synergistic role for LukAB

and Hla during S. aureus biofilm infection, it remains unclear whether these toxins are directly

altering MΦ survival (i.e. via cell lysis) or indirectly tailoring the immune response (i.e. eliciting

tissue damage resulting in altered cytokine signaling to promote MΦ phagocytosis and

proinflammatory activity). However, evidence against the former possibility was revealed by the

finding that biofilm-conditioned medium elicited similar cytotoxic effects towards

proinflammatory (classically activated) and pro-fibrotic (alternatively activated) MΦs (data not
 121

shown). We also investigated whether biofilm-conditioned medium augmented MΦ CD11b

expression, which binds LukAB, but found no evidence to support this possibility. Another

potential mechanism to link the synergistic effects of Hla and LukAB is the zinc-dependent

metalloproteinase ADAM10, since Hla is known to recognize ADAM10 on the host cell surface

(266). Once bound, Hla augments ADAM10 activity (267), which could result in increased

LukAB dissociation from the bacterial cell surface and, in turn, enhanced Hla activity. However,

it should be noted that this interaction may provide an explanation for our in vivo findings, but

fails to inform the apparent synergistic effect in our in vitro assay, since MΦs were treated with

biofilm-conditioned medium clarified of bacteria. The mechanism whereby LukAB and Hla

influence biofilm formation in vivo is an area of active investigation in our laboratory.

While the effect of S. aureus biofilm-conditioned medium on MΦ viability was largely

LukAB/Hla-dependent, it appears that part of the phagocytic block was not. SWATH-MS

identified other potential candidate proteins that could act in concert with Hla/LukAB to

maximally impair MΦ phagocytosis, including pyrimidine biosynthetic enzymes,

phosphotransferase proteins, pyruvate kinase, and histidine metabolic enzymes. Along these lines,

it is important to recognize that biofilms represent a diverse bacterial population influenced by a

myriad of complex gradients (e.g. nutrient, oxygen, pH,), metabolic activity, and virulence

potential (13, 84, 268, 269). For example, while our studies utilized conditioned medium

collected from static biofilms, a subpopulation of planktonic or “dispersed” cells is also present at

the air-liquid interface. While this cell population was 2-3 log lower than the biofilm

(Supplemental Fig. S4.4), it is probably naïve to disregard their impact; particularly in light of

recent studies demonstrating the secretory potential of biofilm-dispersed cells (270). Based on

this evidence, we posit that S. aureus biofilms prevent MΦ phagocytosis, in part, by inducing cell

death through LukAB and Hla production (Fig. 4.8). However, since the phagocytic block was

still evident even when MΦ viability was restored to 100% following LukAB/Hla inactivation,
 122

this suggests the action of additional proteins that act together with Hla/LukAB to maximally

inhibit MΦ phagocytic activity (Fig. 4.8).

Collectively, this study demonstrates that S. aureus biofilms have evolved mechanisms to

establish persistent infections, in part, by actively preventing MΦ phagocytosis and eliciting cell

death that is mediated by the synergistic actions of LukAB and Hla. These findings not only

identify a novel interaction for these secreted proteins, but also highlight the layers of redundancy

within the S. aureus virulence repertoire.

 123

Chapter 5: purB affects eDNA release during Staphylococcus aureus biofilm development to

evade macrophage recognition

Manuscript in preparation
 124

Abstract

The typical MΦ response to planktonic S. aureus involves the induction of

proinflammatory, microbicidal activity. However, S. aureus biofilms can interfere with anti-

bacterial mechanisms, in part, by polarizing MΦs toward an anti-inflammatory, pro-fibrotic

phenotype. Here we showed that MΦs exposed to S. aureus biofilms failed to induce the

proinflammatory transcription factor NF-κB, which translated into minimal inflammatory

cytokine production. We took advantage of this phenotype to screen the S. aureus Nebraska

Transposon Mutant Library to identify mutants that were no longer able to suppress MΦ NF-κB

activity. Among the top hits was purB, which encodes the enzyme adenylosuccinate lyase and

catalyzes two reactions in the purine metabolic pathway. In addition to no longer inhibiting MΦ

NF-κB activity, purB mutant biofilms were more susceptible to MΦ invasion and phagocytosis in

a MyD88-dependent manner. This was attributed, in part, to increased eDNA in the purB mutant

biofilm matrix that triggered MΦ invasion and phagocytosis of biofilm-associated bacteria via

Toll-like receptor 9 (TLR9). In vivo, the purB mutant displayed significantly decreased bacterial

burdens in a mouse orthopedic implant biofilm infection model concomitant with significantly

increased MΦ infiltrates that was dependent on TLR9 recognition during acute infection.

Collectively, these findings point towards a critical role for purB in facilitating biofilm evasion of

MΦ microbicidal effector functions.

 125

Introduction

S. aureus is an opportunistic pathogen recognized for its ability to cause both nosocomial

and community-associated infections (223). While S. aureus isolates encode a myriad of

virulence factors, including toxins, proteases, and nutrient acquisition systems that facilitate

colonization of numerous locations throughout the host, biofilm formation is another key

virulence determinant (15, 16). Biofilms are a heterogeneous population of bacteria surrounded

by a self-produced matrix composed of proteins, exopolysaccharides, and eDNA (32, 226, 227).

While the biofilm matrix likely provides some physical barrier from the host immune response,

heterogeneity within the bacterial population induced by signals such as nutrient and oxygen

gradients, likely provides another layer of protection via differential gene expression and protein

production throughout the biofilm proper (76). Indeed, our prior work demonstrated that S.

aureus biofilms alter their transcriptional profiles upon encountering MΦs (Chapter 3) and

biofilms are enriched in toxins that attenuate MΦ phagocytosis and induce cell death (Chapter 4)

(75, 171).

S. aureus has a number of mechanisms for sensing and responding to environmental

stimuli, including several characterized two-component regulatory systems. For example, the agr

QS system regulates the production of surface-associated versus secreted proteins according to

population density (84). In addition, the link between S. aureus metabolism and virulence is an

active area of investigation (86-89). While many connections are still unclear, previous studies

have demonstrated differential gene expression between S. aureus in the planktonic versus

biofilm lifestyles, including differences in metabolic gene expression (22). Interestingly, the

influence of bacterial metabolites on the host immune response is only beginning to be explored

and represents an area of investigation in our laboratory.

The innate immune system is well-equipped to recognize pathogens through a variety of

cell surface and PRRs. One example is the TLR family, which recognizes PAMPs such as
 126

lipoproteins and PGN in the bacterial cell wall (via TLR2) and unmethylated CpG motifs in

bacterial DNA (via TLR9). Once engaged by their microbial ligand, most TLRs (with the

exception of TLR3) initiate a proinflammatory signaling cascade through the adapter molecule

MyD88, culminating in NF-kB activation and the induction of a robust proinflammatory,

antimicrobial immune response. While PMNs are among the first innate immune responders to

planktonic bacterial infections such as those caused by S. aureus, there has been an increasing

appreciation for the role of resident tissue MΦs in this response. Although PMNs are highly

phagocytic and possess an arsenal of granules rich in antimicrobial peptides and enzymes (156),

MΦs are also professional phagocytes capable of producing high levels of reactive oxygen

species (ROS) and serve a critical role in leukocyte recruitment and activation through

chemokine/cytokine production as well as inducing adaptive immunity by antigen presentation

(125-127, 130).

Our previous work has established that S. aureus biofilms, in contrast to planktonic

organisms, prevent MΦ invasion and phagocytosis of biofilm-associated bacteria and drive MΦs

towards an anti-inflammatory state (15, 160). Recent studies have revealed a synergistic role for

the toxins Hla and LukAB in inhibiting MΦ phagocytosis and inducing cell death (75); however,

other factors are also involved. The purpose of this work was to identify key genes involved in

inhibiting MΦ proinflammatory activity by taking advantage of the NTML to screen for S. aureus

mutants that were still capable of biofilm formation but lost the ability to skew MΦs towards an

anti-inflammatory state, hence transforming them into proinflammatory, phagocytic cells. Our

results identified numerous hits in the purine regulatory pathway. In depth analysis of a purB

mutant (ΔpurB) revealed increased eDNA release at the outer surface of the biofilm that

triggered, in part, TLR9-mediated MΦ activation. In addition, ΔpurB was less virulent in a mouse

orthopedic implant biofilm model that was TLR9-dependent during the early stage of infection.
 127

Collectively, these findings suggest that S. aureus carefully regulate eDNA levels and

accessibility during biofilm formation to prevent MΦ recognition and proinflammatory activity.

 128

Results

Genes expressed during S. aureus biofilm growth that influence macrophage NF-κB

activation. Our previous work has shown that MΦs are unable to invade and phagocytose S.

aureus biofilm-associated bacteria and biofilm exposure drives MΦs towards an anti-

inflammatory phenotype (15, 160). Although our recent report demonstrated a synergistic role for

Hla and LukAB produced during S. aureus biofilm growth in preventing MΦ phagocytosis

(Chapter 4) (75), this study revealed that other proteins are also involved. To identify additional

molecules important for S. aureus biofilms to evade MΦ recognition, we utilized MΦs from NF-

κB-luciferase reporter mice to screen the NTML (158, 271, 272). NF-κB is a key transcription

factor that drives the expression of numerous cytokines/chemokines and is a widely used readout

of MΦ proinflammatory activity (125, 131, 230). To validate this screening approach, NF-κB

activation was minimal in MΦs exposed to intact S. aureus biofilms, whereas significant NF-κB

induction was observed in response to planktonic bacteria and S. aureus-derived PGN (Fig 5.1A).

The ability of biofilms to attenuate MΦ NF-κB activation is in agreement with significantly

impaired cytokine production (Fig 5.1B). Furthermore, after biofilm exposure, MΦs remain

refractory to subsequent stimulation by PAMPs, showing a reduction in both proinflammatory

and anti-inflammatory cytokines, similar to MΦs exposed to planktonic S. aureus (Fig 5.1C, D).

The NTML screen identified 22 mutants where MΦ NF-κB activity was significantly

increased compared to WT biofilms (Fig 5.1E, Table 5.1). S. aureus mutants were also evaluated

for their ability to form biofilms (Fig 5.1F), since impaired biofilm formation could lead to false

positive hits by the ability of planktonic bacteria to increase NF-κB activity (Fig 5.1A). Of note,

the numbers of mutants identified by our NF-κB screen were of relatively low abundance,

representing only 1.1% of the library, revealing the stringency of the assay, which was

corroborated by the identification of multiple hits within the purine pathway (i.e. purA, purB,

purF, purM, and purS) and other operons (Table 5.1 and data not shown). Of particular interest
 129

were purB and purA, since they represented the most robust inducers of MΦ NF-κB activity.

purB was selected for further analysis, since its phenotype was slightly larger, it participates in

two steps of the purine metabolic pathway (whereas purA catalyzes a single reaction;

Supplemental Fig. S5.1), and purB expression has been previously shown to be increased during

biofilm growth although functional assessments were not performed (22).

 130

Figure 5.1

Genes expressed during S. aureus biofilm growth that influence macrophage NF-κB

activation. (A) BMDMΦs from NF-κB-luciferase reporter mice were incubated with fresh

medium (Unstim.), PGN (10 µg/ml), or S. aureus planktonic or biofilm cultures for 4 h,

whereupon luciferase activity (counts per second) was measured as a function of NF-κB

activation and was normalized against PGN stimulated values (set to 100%). (B) MΦs were co-

cultured with biofilms or planktonic bacteria for 6 h, whereupon supernatants were collected to

quantitate TNF-α, IL-1β, IL-10, and IL-1RA by ELISA (N.D. = not detected). (C) MΦs were

incubated alone (non-exposed MΦ) or co-cultured with biofilms or planktonic bacteria for 2 h,

whereupon MΦs were separated from bacteria by FACS and treated with medium alone (no

treatment), the synthetic lipoprotein Pam3Cys (1 µg/ml), peptidoglycan (PGN; 10 µg/ml), or CpG

oligodeoxynucleotides (ODN; 0.1 µM) for an additional 24 h. At 24 h, supernatants were

collected for TNF-α and (D) IL-10 quantitation by ELISA [(-) = not detected]. (E) BMDMΦs

from NF-κB-luciferase reporter mice were incubated with WT or NTML biofilms for 4 h,

whereupon luciferase activity (counts per second) was measured as a function of NF-κB

activation. Luciferase activity elicited by each mutant was normalized against WT biofilm
 131

stimulated MΦs, which was set to 100% (solid red line). Red circles represent mutants where NF-

κB activity was increased (numbers refer to genes listed in Table 1). (F) Crystal violet staining

was utilized as a quantitative assessment of biofilm formation, with each mutant normalized

against WT biofilm, which was set to 100% (solid red line). Significant differences are denoted

by asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001; unpaired two-tailed student’s t-test).

Results are representative of at least three independent experiments.

 132

Table 5.1. Genes expressed by S. aureus biofilms that influence macrophage NF-κB

activation in vitro

% WT Locus Gene Identity Function Location

on Fig.
5.1E
418.05 SAUSA300_1889 purB adenylosuccinate lyase Purine 17
metabolism
417.83 SAUSA300_0017 purA adenylosuccinate synthetase Purine 1
metabolism
282.23 SAUSA300_1095 carA carbamoyl phosphate synthase small Pyrimidine 12
subunit metabolism
264.28 SAUSA300_0969 purS phosphoribosylformylglycinamidine Purine 11
synthase metabolism
213.39 SAUSA300_0957 Hypothetical protein hypothetical 10
207.64 SAUSA300_2060 atpA F0F1 ATP synthase subunit alpha metabolism 19
192.36 SAUSA300_0972 purF amidophosphoribosyltransferase Purine 9
metabolism
181.38 SAUSA300_1225 aspartate kinase Amino acid 14
metabolism
175.67 SAUSA300_1467 ipdA dihydrolipoamide dehydrogenase Amino acid 15
metabolism
157.96 SAUSA300_1105 priA primosomal protein N DNA 13
replication
156.74 SAUSA300_0973 purM phosphoribosylaminoimidazole Purine 8
synthetase metabolism
155.16 SAUSA300_0489 putative cell division protein FtsH Protease 4
154.37 SAUSA300_0787 aroD biosynthesis of aromatic amino acids Amino acid 7
metabolism
153.29 SAUSA300_0086 Hypothetical protein Hypothetical 2
151.66 SAUSA300_0618 ABC transporter substrate-binding Metal transport 6
protein
147.22 SAUSA300_0538 NAD-dependent Carbohydrate 5
epimerase/dehydratase family protein transport
146.53 SAUSA300_0213 oxidoreductase; Gfo/Idh/MocA Oxidoreductase 3
family
145.23 SAUSA300_1754 splE Serine-like protease Protease 16
145.22 SAUSA300_2631 Putative N-acetyltransferase Metabolites 20
142.94 SAUSA300_2030 Hypothetical protein Hypothetical 18
 133

purB is important for preventing macrophage invasion and phagocytosis of mature S.

aureus biofilms. To determine whether the ability of the purB mutant to augment MΦ NF-κB

activity translated into increased invasion and phagocytosis of biofilm-associated bacteria, MΦs

were co-cultured with WT, ΔpurB, and ΔpurB complemented USA300 LAC biofilms. Indeed,

MΦ invasion into mature ΔpurB biofilms was significantly increased compared to WT (Fig.

5.2A) concomitant with enhanced phagocytosis (Fig. 5.2B). To confirm that these changes were

not the result of increased planktonic growth of ΔpurB, biofilm formation was assessed. ΔpurB

was capable of biofilm formation and although biofilm height was significantly reduced (Fig.

5.2C), quantitation revealed a minor reduction in bacterial counts (i.e. < 2-fold; Fig. 5.2D). The

increases in MΦ invasion and phagocytosis of ΔpurB biofilms was complementable (Fig. 5.2A,

B), underscoring the importance of purB in inhibiting MΦ activity.

 134

Figure 5.2

purB is important for preventing macrophage invasion and phagocytosis of mature S.

aureus biofilms. BMDMΦs were co-cultured with mature WT, ΔpurB, or ΔpurB + pTS1 GFP

biofilms for 4-6 h, after which the percentage of MΦs (A) invading or (B) phagocytosing the

biofilms was enumerated using confocal microscopy. (C) Mature GFP biofilms alone were

observed by confocal microscopy to measure average biofilm height, (D) after which mature

biofilms were mechanically disrupted, serially diluted in PBS, and plated to quantitate viable

bacteria. Significant differences are denoted by asterisks (*, p < 0.05; **, p < 0.01; one-way

ANOVA, followed by Bonferroni’s multiple-comparison test). Results are representative of at

least three independent experiments.

 135

eDNA is increased in S. aureus ΔpurB biofilms. Since the ability of MΦs to invade and

phagocytose biofilms is likely dictated, in part, by the accessibility of immune stimulatory motifs

at the outer biofilm surface, we next focused on the biofilm ECM. As eDNA is required for S.

aureus biofilm development (32) and bacterial DNA is a known TLR9 ligand (101, 102),

USA300 LAC GFP biofilms were stained with PI to visualize eDNA (Fig. 5.3A). The ratio of PI

(eDNA/dead bacteria) to GFP (live) signal was quantitated in z-stacks acquired by confocal

microscopy, which revealed a significant increase in eDNA/dead bacteria in ΔpurB throughout

biofilm maturation (Fig. 5.3B). As PI is not specific for eDNA, but also stains dead cells present

in the biofilm, eDNA was isolated from mature biofilms and the housekeeping gene gyrA was

quantitated by qRT-PCR. Results confirmed that ΔpurB biofilms contained significantly

increased eDNA (Fig. 5.3C) and all ΔpurB phenotypes could be complemented (Fig. 5.3).

Another interesting finding was that more PI staining was observed at the outer surface of ΔpurB

biofilms, presumably making it more accessible to MΦ recognition, compared to the WT and

complemented strains where eDNA was mainly buried at the base of the biofilm (Fig. 5.3A). To

determine whether the failure of MΦs to invade and phagocytose WT biofilms resulted, in part,

from inaccessibility to immune stimulatory eDNA, purified biofilm-derived eDNA was added to

the surface of mature WT biofilms, which phenotypically transformed the MΦ response to what

was observed with ΔpurB, namely increased biofilm invasion and phagocytosis (Fig. 5.3D).
 136

Figure 5.3

eDNA is increased in S. aureus ΔpurB biofilms. (A) Propidium iodide was added to mature (6

day-old) WT, ΔpurB, or ΔpurB + pTS1 GFP biofilms, visualized by confocal microscopy (X63,

1-μm slices), and representative three-dimensional images were constructed. (B) GFP and PI

signals were quantitated by COMSTAT analysis to determine a PI/GFP signal ratio. (C) eDNA

was collected from mature biofilms and quantitated by performing qRT-PCR with gyrA. (D)

BMDMΦs were co-cultured for 4-6 h with mature WT GFP biofilms alone, biofilms pretreated

for 30 min with 100 U/ml DNase1, and biofilms with additional eDNA added to the surface after

which the percentage of MΦs (D) invading or (E) phagocytosing biofilms was enumerated using

confocal microscopy. Significant differences are denoted by asterisks (*, p < 0.05; **, p < 0.01;

one-way ANOVA, followed by Bonferroni’s multiple-comparison test). Results are

representative of at least three independent experiments.

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S. aureus ΔpurB biofilm eDNA can be detected by MΦs and trigger activation. MΦs are

equipped with a variety of PRRs. Among these are TLRs, including TLR9, which senses bacterial

DNA and signals via the adapter molecule MyD88 to trigger NF-κB activation and

proinflammatory properties (101, 102). Since ΔpurB augmented MΦ NF-κB activation, biofilm

invasion, and phagocytosis, we next investigated whether this was TLR9-dependent based on

increased eDNA in the ΔpurB biofilm matrix and its accessibility at the surface. Again, WT MΦs

displayed significantly increased invasion (Fig. 5.4A) and phagocytosis (Fig. 5.4B) of ΔpurB

biofilms; however, this was diminished upon co-culture of ΔpurB biofilms with TLR9 KO MΦs,

and completely abrogated in the presence of MyD88 KO MΦs (Fig. 5.4A, B). Collectively, these

results suggest that purB is important for regulating eDNA levels and localization in biofilms,

which is partially responsible for the ability of WT biofilms to evade MΦ detection by preventing

TLR9-dependent proinflammatory activity. However, additional MyD88-dependent signaling

receptors are also involved, the identity of which remains to be determined.

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Figure 5.4

S. aureus biofilm eDNA can be detected by MΦs and trigger activation. BMDMΦs from WT,

TLR9 KO, and MyD88 KO mice were co-cultured with mature WT, ΔpurB, or ΔpurB + pTS1

GFP biofilms for 4-6 h, after which the percentage of MΦs (A) invading or (B) phagocytosing the

biofilms was enumerated using confocal microscopy. Significant differences are denoted by

asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; one-way ANOVA, followed

by Bonferroni’s multiple-comparison test). Results are representative of at least three independent

experiments.
 139

S. aureus purB is important for chronic biofilm establishment. While the previous results

revealed a role for purB in the ability of S. aureus biofilms to evade MΦ recognition in vitro, we

next assessed the importance of purB in a model of orthopedic implant biofilm infection in WT

and TLR9 KO mice. Interestingly, ΔpurB displayed decreased bacterial burdens in WT mice

beginning at day 7, which became more pronounced over time, with some mice appearing to have

cleared the infection by day 28 (Fig. 5.5A). Decreased biofilm burdens in WT mice infected with

ΔpurB correlated with a significant reduction in anti-inflammatory MDSCs (Fig. 5.5B)

concomitant with increased MΦ infiltrates (Fig. 5.5B). While ΔpurB showed similarly decreased

biofilm burdens in TLR9 KO mice at later time points, concomitant with reduced MDSC and

increased MΦ infiltrates (Fig. 5.5C), importantly, there were no differences in titers between

ΔpurB and WT infected TLR9 KO mice at day 7 (black and grey squares). This suggests that

eDNA recognition in ΔpurB occurs via TLR9 but only during early biofilm development. By

extension, since WT biofilms have less eDNA that is not present at the outer biofilm surface and

accessible to invading MΦs, this may represent a mechanism whereby WT biofilms evade TLR9-

mediated recognition, in agreement with our earlier report (15). Importantly, the observed

decrease in bacterial burdens at day 14 was complementable (Fig. 5.5D), again underscoring the

specific importance of purB in this in vivo biofilm infection model. Complementation was not

assessed at other time points to limit the number of animals utilized.

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Figure 5.5

S. aureus purB is important for chronic biofilm establishment. (A) Bacterial burdens

associated with the soft tissue surrounding the knee of WT and TLR9 KO mice infected with WT

S. aureus and isogenic ΔpurB strains at days 7, 14, 21, and 28 post-infection. (B) Quantitation of

Ly6GhighLy6C+ MDSCs and F4/80+ MΦs infiltrating the soft tissue of C57BL/6 WT mice infected

WT S. aureus and isogenic ΔpurB strains at days 7, 14, 21, and 28 post-infection. (C)

Quantitation of Ly6GhighLy6C+ MDSCs and F4/80+ MΦs infiltrating the soft tissue of TLR9 KO

mice infected WT S. aureus and isogenic ΔpurB strains at days 7, 14, 21, and 28 post-infection.

(D) Bacterial burdens associated with the soft tissue surrounding the knee of WT and TLR9 KO

mice infected with WT S. aureus and isogenic ΔpurB, and complemented ΔpurB + pKK22:purB

strains at day 14 post-infection. Significant differences are denoted by asterisks (*, p < 0.05; **, p

< 0.01; ***, p < 0.001; one-way ANOVA, followed by Bonferroni’s multiple-comparison test).

Results are representative of at least two independent experiments.

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Supplemental Figure S5.1

Predicted purine biosynthetic pathway in S. aureus. 5-Phospho-alpha-D-ribose 1-diphosphate

(PRPP) is converted to 5-Phosphoribosylamine (PRA) by amidophosphoribosyltransferase

(purF), which is then converted to 5′-Phosphoribosylglycinamide (GAR) by

phosphoribosylamine-glycine ligase (purD), which is then converted to 5′-Phosphoribosyl-N-

ormylglycinamide (FGAR) by phosphoribosylglycinamide formyltransferase 1 (purN), which is

then converted to 2-(Formamido)-N1-(5′-phosphoribosyl)acetamidine (FGAM) by a combination

of phosphoribosylformylglycinamidine synthase (purS), phosphoribosylformylglycinamidine

synthase I (purQ), and phosphoribosylformylglycinamidine synthase II (purL), which is then

converted to Aminoimidazole ribotide (AIR) by phosphoribosylformylglycinamidine cyclo-

ligase (purM), which is then converted to (5-Phospho-D-ribosyl)-5-amino-4-

imidazolecarboxylate (CAIR) by 5-(carboxyamino)imidazole ribonucleotide synthase (purK) and

5-(carboxyamino)imidazole ribonucleotide mutase (purE), which is then converted to 1-(5′-

Phosphoribosyl)-5-amino-4-(N-succinocarboxamide)-imidazole (SAICAR) by

phosphoribosylaminoimidazole-succinocarboxamide synthase (purC), which is then converted to

1-(5'-Phosphoribosyl)-5-amino-4-imidazolecarboxamide (AICAR) by adenylosuccinate lyase

(purB), which is then converted to 1-(5'-Phosphoribosyl)-5-formamido-4-imidazolecarboxamide

(FAICAR) followed by Inosine monophosphate (IMP) both by

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phosphoribosylaminoimidazolecarboxamide formyltransferase / IMP cyclohydrolase (purH);

which is then converted to adenylosuccinate by adenylosuccinate synthase (purA), which is then

converted to Adenosine monophosphate (AMP) by adenylosuccinate lyase (purB), which can

then be eventually converted into Adenosine triphosphate (ATP) and used as energy, into adenine

or adenosine through adenosine synthase (adsA), or into RNA or DNA as needed.

 143

Discussion

S. aureus subverts the host immune response by numerous mechanisms, including

increased resistance to cationic antimicrobial peptides, impairment of phagocyte recruitment,

interference with antibody-mediated opsonization and complement activation, and resistance to

intracellular killing (213). In addition, biofilm formation further protects S. aureus from the host

innate immune response, representing a communal virulence determinant (15, 56, 159). The

objective of this study was to identify genes expressed by S. aureus biofilms that contribute to

MΦ dysfunction through the inhibition of NF-κB.

MΦs utilize phagocytic receptors to identify microbes and trigger immune defenses.

Infectious agents that cause persistent infections, such as S. aureus biofilms, must be able to

detect and subvert these defenses. We have previously demonstrated that biofilm formation

protects S. aureus from TLR detection and phagocytosis, reduces proinflammatory cytokine and

chemokine production, decreases inducible NO synthase (iNOS) expression, and interferes with

MΦ activation in vivo (15, 160). We were interested in the genetic basis of how biofilm growth

prevents MΦ phagocytic functions. Previously, we determined that MΦs were capable of

phagocytosing bacteria from mechanically disrupted, but not intact biofilms, suggesting that the

size of the biofilm and/or density of its matrix may form a mechanical obstacle that physically

prevents MΦ phagocytosis, a phenomenon leading to “frustrated phagocytosis” (15, 254, 255).

Subsequent studies revealed that toxins, including Hla and LukAB are also responsible for the

MΦ phagocytic block, but that additional factors are also involved, which is not unexpected given

the arsenal of S. aureus virulence determinants.

In response to planktonic bacteria, MΦs produce numerous proinflammatory mediators,

exhibit potent phagocytic and anti-microbial activity, and are critical for immune cell recruitment

and activation, serving as a first line of defense against microbial invasion (200, 201). However,

we have previously reported that MΦs are polarized towards an alternatively activated, anti-
 144

inflammatory, M2 phenotype upon contact with S. aureus biofilms (15, 160). Here we have

extended these findings to demonstrate that prior biofilm exposure makes MΦs refractory to a

subsequent microbial challenge, further highlighting the immune subversive properties of S.

aureus biofilms. Correspondingly, MΦ NF-κB activation was also significantly reduced after

contact with S. aureus biofilms, whereas a strong response was detected after exposure to

planktonic S. aureus or PGN. This indicates that in addition to impaired phagocytosis, another

mechanism utilized by S. aureus biofilms to subvert the host immune response results from the

ability to prevent MΦ NF-κB activation and resultant proinflammatory cytokine secretion.

Indeed, multiple pathogens employ similar strategies to interfere with MΦ microbicidal activity

either by hindering cytokine expression/secretion (273, 274) or by the production of virulence

factors that directly impede NF-κB activation (158, 275).

To identify genes expressed by S. aureus biofilms that inhibit MΦ NF-κB activation, a

screen of the NTML was performed. This approach utilized MΦs from transgenic mice where the

transcription factor NF-κB drives luciferase expression. Several S. aureus mutants in the purine

biosynthetic pathway (i.e. purA, purB, purF, purS, and purM) failed to block MΦ NF-kB

activation, implicating their involvement in thwarting proinflammatory functions. Previous work

demonstrated that S. aureus purine auxotrophs were less virulent in a murine abscess model of

infection (276). Likewise, purine biosynthesis also appears to have broad physiological effects in

S. aureus, including the modification of global patterns of virulence gene expression (276).

Accordingly, we found that ΔpurB biofilms secreted significantly decreased levels of Hla

compared to WT biofilms (Supplemental Fig. S5.2). Not surprisingly, ΔpurA and ΔpurB

displayed the most robust NF-κB activation in our experiments, as these genes represent more

distal steps in the purine biosynthetic pathway for converting inosine monophosphate (IMP) to

adenosine monophosphate (AMP). A major end product of the purine biosynthetic pathway,

AMP, can be converted to adenosine during staphylococcal infections (277). Adenosine is a

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potent extracellular messenger that is abundant at sites of hypoxia, trauma, and inflammation

(278). Moreover, adenosine has strong immunosuppressive effects, such as down-regulating

cytokine production and immune cell receptors, many of which are mediated by its ability to bind

A3 and A2A receptors on leukocytes (278-280). Accordingly, we found that adenosine attenuated

MΦ NF-κB activity in response to PGN stimulation, further indicating that products of the purine

biosynthetic pathway influence MΦ activation (Supplemental Fig. S5.3).

Interestingly, these studies indicate that purine biosynthesis is also important for S.

aureus regulation of eDNA in the outer biofilm matrix. Consistent with our previous studies,

disrupting the ability of MΦs to recognize bacterial eDNA via TLR9 does not alter invasion and

phagocytosis during co-culture with WT biofilms in vitro, nor does it result in a more severe

infection in vivo in association with WT S. aureus (15). However, disrupting purine biosynthesis

via ΔpurB results in increased eDNA, particularly at the biofilm surface, providing an accessible

PAMP for MΦ recognition and activation. While this phenomenon was complementable and

reproducible with the addition of eDNA to the WT S. aureus biofilm surface, we observed a

stronger phenotype in association with MyD88, implying an additive effect of other PAMP(s)

being recognized by other TLRs, the identity of which remains to be determined.

The results of the current study, concomitant with our previous findings, support the

conclusion that S. aureus biofilms have evolved mechanisms to establish persistent infections, in

part, by actively programming MΦs towards an anti-inflammatory phenotype and preventing

phagocytosis, presumably through the production of adenosine. Additionally, S. aureus biofilms

shield the eDNA component of the ECM from MΦ recognition at the biofilm surface, thereby

further evading MΦ detection.

 146

Supplemental Figure S5.2

purB activity effects Hla production in S. aureus biofilms. Quantitation of Hla levels in

conditioned medium from WT S. aureus biofilms versus isogenic ΔpurB, and complemented

ΔpurB + pTS1 strains. Significant differences are denoted with asterisks (****, p < 0.0001;

unpaired two-tailed student’s t-test). Results are representative of at least two independent

experiments.
 147

Supplemental Figure S5.3

Adenosine attenuates macrophage activation. Bone marrow-derived macrophages from NF-

κB-luciferase reporter mice were stimulated with 10 μg/ml peptidoglycan (PGN) for 2 h + 50 μM

adenosine, whereupon luciferase activity was measured as a function of NF-κB activation.

Significant differences are denoted with asterisks (*, p < 0.05; unpaired two-tailed student’s t-

test). Results are representative of at least three independent experiments.

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Chapter 6: Discussion and Future Directions

Key Findings and Conclusions:

The phagocytic and microbicidal activities of PMNs and MΦs are among the first lines of

protection from bacterial pathogens. However, S. aureus is often resistant to innate immunity,

especially when ensconced within the confines of the biofilm matrix. While previous studies in

our laboratory have shown that S. aureus biofilms are able to resist clearance by both PMNs and

MΦs, we wondered if this was accomplished by a conserved set of genes or through entirely

different means. In order to answer this question, we examined biofilm gene expression profiles

following co-culture with these two innate immune cell populations. We also compared the

relative ability of PMNs and MΦs to invade and phagocytose biofilm-associated S. aureus. To

our knowledge, this was the first study of its kind with any species of bacterial biofilm.

During co-culture with PMNs, we observed no significant alteration of the S. aureus

biofilm transcriptome at an early or late period (Chapter 3). This was unexpected, especially

taken together with our confocal data indicating that PMNs were readily able to invade the

biofilm matrix and phagocytose biofilm-associated bacteria. While it is known that S. aureus can

survive intracellularly in PMNs (215, 281, 282), PMN phagocytosis did not result in a significant

reduction in viable bacterial numbers in vitro, and one would expect this adaptation to necessitate

major transcriptional changes but this was not observed. While few transcriptional changes were

detected, interestingly agr transcription was significantly increased after PMN addition to

biofilms. agr regulates the production of numerous secreted virulence factors, including phenol-

soluble modulins (PSMs), which have been shown to play a role in biofilm maturation and are

known to elicit cytotoxic effects on PMNs (79, 283). In agreement with this finding, we noted

that Δagr biofilms were less able to inhibit PMN invasion and induce PMN cell death, despite

being significantly thicker on average.

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In contrast to PMNs, we observed a significant dampening of the biofilm transcriptome

during early co-culture with MΦs, including numerous metabolism-associated genes. This

transcriptional dormancy was negated at late co-culture periods, likely because a vast majority of

MΦs had already succumbed to cell lysis by biofilm-secreted toxins (Chapter 4). Also in contrast

to PMNs, we were able to document little/no MΦ invasion and phagocytosis of biofilm-

associated bacteria, perhaps in part due to successful “cloaking” of the biofilm. This curious

difference in the biofilm transcriptional response to PMNs and MΦs, despite invasion and

phagocytosis by the former and little to none by the latter, could be explained by the presence of a

MΦ secreted molecule being sensed by the biofilm.

As I had now confirmed previous experiments in our laboratory observing an inability of

MΦs to phagocytose biofilm-associated bacteria, the next question was whether molecules in the

biofilm-conditioned milieu contributed to this phagocytic block (Chapter 4). We first

demonstrated that S. aureus biofilm-conditioned medium could inhibit MΦ phagocytosis and

induce cell death, and that it was significantly more inhibitory and lytic than planktonic-

conditioned medium. Next, we established that the bioactive factors were actively secreted,

proteinaceous in nature, specific to intact biofilms, and at least partially agr-dependent. We then

performed proteomics analysis via SWATH-MS comparing conditioned medium from Δagr with

the WT strain, which identified Hla and LukAB as significantly enriched in WT biofilm-

conditioned medium. These results were further corroborated by Western Blot, ELISA, and a

series of MΦ treatments using combinations of neutralizing antibodies and Δhla ΔLukAB

mutants. Furthermore, hla and lukAB were found to act synergistically in vitro and in vivo in an

implant-associated biofilm infection model. Taken together, these studies identified a novel

interaction between these secreted proteins and highlighted another layer of redundancy within

the S. aureus virulence secretome.

 150

Having identified secreted proteins enriched in S. aureus biofilms that hinder macrophage

phagocytosis and induce cell death, we wanted to specifically ask how biofilms are able to skew

macrophages towards an anti-inflammatory, M2 profile. In order to answer this question, we

performed a high-throughput screen of the NTML in which we co-cultured BMDMΦs from NF-

κB reporter mice with S. aureus single-gene mutants to identify genes involved in inhibiting

proinflammatory, or M1, MΦ activation (Chapter 5). This screen identified over 20 genes, several

of which encode proteins involved in purine biosynthesis, including one of the top hits,

adenolysuccinate lyase (purB). In vitro, ΔpurB biofilms displayed a significantly decreased

ability to inhibit MΦ invasion and phagocytosis of biofilm-associated bacteria, which was

complementable. We next examined differences in biofilm eDNA content and found that ΔpurB

biofilms contained increased amounts of eDNA. Suspecting that increased eDNA at the biofilm

surface may be leading to increased MΦ activation and invasion, we next performed co-cultures

with BMDMΦs from TLR9 and MyD88 KO mice and found that both had a decreased ability to

invade ΔpurB biofilms. Interestingly, however, MyD88 KO MΦs showed the most significant

decrease, indicating a role for additional PAMPs in this co-culture paradigm in addition to the

CpG motifs present in biofilm eDNA that would be recognized by TLR9. Furthermore, we were

able to show that spiking additional eDNA onto WT S. aureus biofilms facilitated increased

invasion. Finally, we demonstrated the importance of purB in the establishment of an infection in

vivo in our murine orthopedic implant model, which we were also able to confirm by

complementation.

In total, my studies have helped identify some key factors that contribute to S. aureus

biofilm evasion of MΦ recognition and inhibition of MΦ antimicrobial activities (Fig. 6.1). These

experiments also serve to further highlight the immense challenge we face in attempting to more

effectively treat, and even prevent, S. aureus biofilm infections. The vast repertoire of virulence

factors and exquisite control over which S. aureus produces them is truly astounding. It is my
 151

hope that these studies may positively contribute to the ever growing corpus of S. aureus research

that should one day allow us to live in harmony with this commensal turned opportunistic

pathogen.
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Figure 6.1

Mechanisms by which S. aureus biofilms interfere with MΦ antimicrobial responses. 1) S.

aureus biofilms are able to uniquely detect and respond to MΦ over PMN insult by down-

regulating much of its transcriptional activity. 2) S. aureus biofilms are uniquely enriched over

planktonic cultures in Hla, LukAB, adenosine, and other factors that contribute to macrophage

antimicrobial inhibition and cell death. 3) S. aureus biofilms are able to regulate eDNA

expression in a purB-dependent manner thereby further preventing MΦ detection via TLR9.

 153

Future Directions:

Proteomics on innate immune cell and S. aureus biofilm co-cultures

While transcriptional profiling of the S. aureus biofilm after co-culture with either PMNs

or MΦs was novel and informative, a translational assessment of biofilm protein production could

help confirm some of the earlier data and pinpoint shifts in actual protein production in response

to immune insults. In addition, it would be exciting to simultaneously measure protein production

fluctuations from the PMN and MΦ populations after encountering a biofilm. This data collection

could also begin to shed light on potential players in host-pathogen crosstalk and could be

performed using SWATH-MS mass spectrometry. Significant S. aureus protein hits could be

isolated, purified, and used to treat both immune cell populations in vitro to assess the impact on

antimicrobial activity via phagocytic assay, qRT-PCR, and/or Western Blot. Similarly, individual

PMN or MΦ protein hits could be used to treat biofilms to assess the impact on metabolic and

transcriptional activity as measured by qRT-PCR and confocal microscopy via reporter constructs

of key genes. Finally, it may also be interesting to perform immune cell co-cultures with

planktonic S. aureus to gain a fuller understanding of how biofilms alter the innate immune

response. To my knowledge, this would be a completely novel study and should be feasible by

careful identification of bacterial and eukaryotic proteins via consultation with separately

acquired reference libraries.

Identify biofilm proteins that directly inhibit macrophage phagocytosis

Although the effect of S. aureus biofilm-conditioned medium on MΦ viability was

largely Hla/LukAB-dependent, part of the phagocytic block was not. While SWATH-MS

identified other potential candidate proteins including pyrimidine biosynthetic enzymes,

phosphotransferases, pyruvate kinase, histidine metabolic enzymes, and serine-like proteases,

 154

these were all identified in a comparison between WT and Δagr mutant biofilms and part of the

phagocytic block was not agr-dependent. However, we know this phenotype is protein-dependent

as treatment of the biofilm-conditioned medium with Proteinase K fully reversed the phagocytic

block. With this in mind, other two-component regulators could be tested, in conjunction with

neutralizing Hla and LukAB antibody, to identify a regulator of the protein of interest. Upon

identification, SWATH-MS could be performed as before with the new comparator to identify the

protein(s) involved in directly inhibiting MΦ phagocytosis. Alternatively, a phagocytic screen of

the NTML could be performed assessing the inhibitory effect of conditioned medium from every

mutant biofilm after treatment with the neutralizing antibodies. This study could lead to the

identification of a novel drug target, as we have previously shown that, once activated and

phagocytic, MΦs are capable of decreasing bacterial burdens in an established biofilm infection

in vivo.

Assess agr activity across various medium formulations

Though my original co-culture experiments were performed with RPMI-1640

supplemented with 10% FBS, the MS proteomics studies required supplementation with 1%

casamino acids, since the proteins in FBS would have masked the detection of most bacterial

proteins. Importantly, pilot studies demonstrated similar biofilm formation between FBS and

casamino acids and no adverse effects on MΦ function or viability. However, I have observed

apparently increased levels of sarAP1 promoter activity during biofilm growth in casamino acids

as determined by fluorescence intensity of the sarAP1-GFP plasmid used to visualize the biofilm,

even when normalized for any differences in culturable bacteria. In addition, I have measured

significantly increased levels of Hla production in biofilms grown in casamino acids by ELISA,

even when normalized for any differences in total protein production. As agr regulates numerous

virulence factors including Hla, it would be interesting to note any significant differences in its
 155

activity between the two media formulations. I could also include more traditional

microbiological media formulations (e.g. TSB), with the expectation that it may promote even

greater agr activity than casamino acids, since it is more nutrient-rich. These studies could be

very interesting and help inform medium selection for in vitro experimentation, particularly when

attempting to draw correlations between growth in media and growth during infection in a

patient. Finally, it would be very interesting to identify any molecules present in the FBS that

inhibit agr activity, as these could potentially be used therapeutically. What about examining

changes when grown in dilute blood to mimic colonization/growth during wound formation?

Characterize the ΔpurB blood agar phenotype

During my experiments with ΔpurB, I noticed that the mutant had a significant growth

defect on TSA + 10% sheep blood in that it was unable to form single, isolated colonies, although

growth was observed in concentrated areas of bacterial lawns. Curiously, this phenotype

disappears after a few days of static biofilm growth in vitro or after 7 days of infection in the in

vivo model of orthopedic implant biofilm infection. In order to determine the responsible genes, I

could perform transcriptomics comparing ΔpurB with the WT strain pre- and post- in vitro or in

vivo biofilm growth. Alternatively, I could perform DNA sequencing to look for any mutations

that may be occurring during these growth conditions that suddenly confer growth on blood agar.

S. aureus infections are challenging to treat as it seems to be uniquely adapted to

circumvent the host antimicrobial response, with the ability to colonize multiple sites on the

human body with ease. Furthermore, S. aureus seems equally adept at benignly living on the

human body as a commensal or chronically persisting inside the body as a pathogen. While many

scientists are attempting to prevent S. aureus infections through the development of vaccines

targeting key virulence factors or through manipulating the bacterial virulence factor regulatory

pathways themselves, the diversity of states in which S. aureus can survive (e.g. planktonic vs
 156

biofilm, proliferative vs persister) has made the identification of a common denominator to target

unclear. What is becoming clearer, however, is that a multifactorial approach, combining some

form of antibiotics with an immunostimulatory boost, will likely be required. Therefore, there is a

continued pressing need to better understand all of the ways in which S. aureus biofilms thwart

innate immunity.
 157

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