REVIEW

Uncovering the Hidden Credentials of Brucella Virulence

R. Martin Roop II,a Ian S. Barton,a Dariel Hopersberger,a Daniel W. Martina

Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, USA
a

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
VIRULENCE DETERMINANTS ACTING AT THE HOST-PATHOGEN INTERFACE . . . . . . . . . . . . . 4
Type IV Secretion System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Lipopolysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Omp25/Omp31 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Omp10, Omp16, and Omp19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Autotransporter Adhesins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Cyclic b -1,2-D-Glucan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Flagella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Phosphatidylcholine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Exopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
PHYSIOLOGIC ADAPTATION TO THE HOST ENVIRONMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Microaerobic Respiration and Denitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Carbon and Nitrogen Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

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Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Metal Acquisition and Detoxification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Urease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
PrpA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
BacA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
GadB, GadC, and GlsA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
REGULATION OF VIRULENCE GENE EXPRESSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
BvrRS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
N-Dodecanoyl Homoserine Lactone, VjbR, and BabR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
General Stress Response and LovhK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
CtrA and GcrA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
MucR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
GntR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
OtpR, CpbB, NodVW, TceSR, and PrlSR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
Stringent Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
c-di-GMP Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
sRNAs and Hfq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
AUTHOR BIOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44

SUMMARY Bacteria in the genus Brucella are important human and veterinary patho-
gens. The abortion and infertility they cause in food animals produce economic hard-
ships in areas where the disease has not been controlled, and human brucellosis is one
of the world’s most common zoonoses. Brucella strains have also been isolated from
wildlife, but we know much less about the pathobiology and epidemiology of these
infections than we do about brucellosis in domestic animals. The brucellae maintain pre- Citation Roop RM, II, Barton IS, Hopersberger
D, Martin DW. 2021. Uncovering the hidden
dominantly an intracellular lifestyle in their mammalian hosts, and their ability to subvert credentials of Brucella virulence. Microbiol Mol
the host immune response and survive and replicate in macrophages and placental Biol Rev 85:e00021-19. [Link]
trophoblasts underlies their success as pathogens. We are just beginning to understand MMBR.00021-19.
Copyright © 2021 American Society for
how these bacteria evolved from a progenitor alphaproteobacterium with an environ-
Microbiology. All Rights Reserved.
mental niche and diverged to become highly host-adapted and host-specific pathogens. Address correspondence to R. Martin Roop II,
Two important virulence determinants played critical roles in this evolution: (i) a type IV roopr@[Link].
secretion system that secretes effector molecules into the host cell cytoplasm that direct Published 10 February 2021
the intracellular trafficking of the brucellae and modulate host immune responses and

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Roop et al. Microbiology and Molecular Biology Reviews

(ii) a lipopolysaccharide moiety which poorly stimulates host inflammatory responses.

This review highlights what we presently know about how these and other virulence
determinants contribute to Brucella pathogenesis. Gaining a better understanding of
how the brucellae produce disease will provide us with information that can be used to
design better strategies for preventing brucellosis in animals and for preventing and
treating this disease in humans.

KEYWORDS Brucella, pathogenesis, virulence determinants

INTRODUCTION

M embers of the bacterial genus Brucella colonize a variety of mammals (1, 2). They
have also been found in reptiles (3, 4) and fish (5, 6). There are currently 12 recog-
nized Brucella species ([Link] and although these
bacteria are highly related at the genetic level, they display a remarkable degree of
host specificity (Fig. 1). Comparative genomic analyses have provided us with a fasci-
nating picture of how these bacteria evolved from an ancestral alphaproteobacterium
to become host-adapted pathogens, and several excellent reviews describe this evolu-
tionary path (7–11).
The best characterized Brucella species, B. melitensis, B. abortus, B. suis, B. canis, and
B. ovis, cause abortion and infertility in goats and sheep, cattle, pigs, dogs, and sheep,
respectively. These strains have long been recognized as economically important
pathogens by the agricultural and veterinary communities (1, 2). B. melitensis, B. abor-
tus, B. suis, and B. canis can also cause a chronic debilitating febrile illness in humans
exposed to infected animals or animal products (12), and human brucellosis is one of

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the world’s leading zoonotic diseases (13, 14). B. melitensis, B. suis, and B. abortus are
also of considerable concern from a biodefense perspective, because they have biolog-
ical characteristics that make them attractive for use as biowarfare or bioterrorism
agents (15).
The Brucella strains that infect domestic animals are highly host adapted and do
not survive for prolonged periods in the external environment (1, 2). The infections
caused by these strains also have distinctive characteristics. One is the highly infectious
nature of these bacteria for their natural hosts and their high degree of host specificity
(2, 16, 17). Another is the chronic nature of these infections, which in some cases can
be lifelong. The remarkable capacity of the brucellae to survive and replicate for pro-
longed periods in host macrophages underlies this persistence (18). Brucella infections
predominantly cause abortion and infertility in their natural hosts, where these bacte-
ria replicate prolifically in association with placental trophoblasts (Fig. 2). Exposure to
aborted fetuses and vaginal secretions serve as major routes of transmission, and vene-
real transmission is also important in some of these hosts. Offspring can also be infected
in utero or by consumption of contaminated milk. Although some Brucella strains are
highly infectious for humans, humans are an incidental host, and human-to-human
transmission is extremely rare (19).
Wildlife also serve as natural hosts for Brucella strains (Fig. 1), but we know much
less about the interactions of these strains with their hosts than we do about the
Brucella strains that infect domestic animals. Brucella ceti, B. microti, B. papionis, and the
unnamed Brucella species in the so-called BO2 clade appear to be overt pathogens (3,
4, 20–24), for instance, but whether B. neotomae, B. pinnipedialis, or B. vulpis cause dis-
ease in their natural hosts is presently unclear (25–27). B. ceti, B. neotomae, and BO2
strains including B. inopinata (28–35) have also been isolated from human infections,
which suggests that wildlife represent a potential zoonotic source of human brucellosis.
Over the past century, the importance of the Brucella spp. as veterinary and human
pathogens has prompted a considerable amount of research aimed at determining
how these bacteria produce disease. The implementation of molecular biology-based
approaches in the 1980s led to an acceleration of these efforts (36), and we now have
almost 800 complete genome sequences of Brucella strains from a wide range of hosts

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FIG 1 Natural hosts and zoonotic potential of Brucella strains. The Brucella species shown in blue are
recognized zoonotic pathogens, and the thickness of the solid arrows represents the relative frequency
with which these hosts serve as sources of human infection. The dashed arrows indicate that these
Brucella strains have been isolated from human disease but direct transmission from the
corresponding natural host to humans has not been documented. The question mark indicates that
the natural host for B. inopinata is unknown.

available for study. One thing that has become clear as we have examined the molecu-
lar basis of Brucella pathogenesis is that these bacteria have followed an evolutionary
pathway to virulence that is different from the pathways followed by other well-char-
acterized mammalian pathogens (8, 9, 37–39). Ignacio Moriyón and Edgardo Moreno
articulated this unique evolutionary pathway very well in a commentary entitled
“Brucella melitensis: a nasty bug with hidden credentials for virulence” (40) that accom-
panied the paper describing the completion of the B. melitensis 16M genome sequence

FIG 2 Natural disease cycle of brucellosis in domestic animals. The blue triangles denote the oral,
nasal, and venereal routes of infection.

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FIG 3 Contributions of the T4SS effectors, the LPS O-chain, Omp22, Omp25d, and cyclic b -1,2-D-
glucan (C b G) to the development of the replicative Brucella-containing vacuole in host macrophages.
The empty black and orange circles represent membrane vesicles trafficking from the endolysosomal
pathway, endoplasmic reticulum, and Golgi apparatus to the Brucella-containing vacuoles (BCVs). The
change in the colors of the BCV membranes represents their change in composition as they
transition from eBCVs to rBCVs. The outermost blue membrane of the aBCV represents engulfment of
the rBCV by the host cell autophagosomal pathway. eBCV, endosomal BCV; rBCV, replicative BCV;
aBCV, autophagosomal BCV.

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in 2002 (41). The intent of this review is to inform the reader of the progress that has
been made over the past 2 decades toward uncovering some of these “hidden creden-
tials” of Brucella virulence.

VIRULENCE DETERMINANTS ACTING AT THE HOST-PATHOGEN INTERFACE

Type IV Secretion System
The most extensively studied Brucella virulence determinant has been their type IV
secretion system (T4SS) (42). The Brucella T4SS consists of 11 proteins, 8 of which make
up the core of the transporter (VirB2, VirB3, and VirB5 through VirB10), 2 ATPases
(VirB4 and VirB11) that provide energy to drive effector secretion, and a lytic transgly-
cosylase (VirB1) that remodels the bacterial cell peptidoglycan layer during T4SS as-
sembly (43–52). The genes encoding the T4SS reside in an operon (42, 53) that is con-
served across Brucella strains, and B. abortus, B. melitensis, B. suis, B. canis, B. ovis, B.
microti, and B. neotomae virB mutants are highly attenuated in cultured mammalian
cells and experimental and natural hosts (42, 53–75). Brucella proteins that assist in the
assembly and function of the T4SS are also encoded by genes residing outside the virB
operon. The secretor activator gene A (SagA) protein, for instance, is a muramidase
that is thought to play an important role in remodeling the peptidoglycan layer during
assembly of the T4SS (76). The precise function of VirJ is unknown, but this periplasmic
protein interacts directly with T4SS substrates that have a periplasmic intermediate
during their export and is also required for proper assembly of the T4SS (77). B. abor-
tus sagA and virJ mutants both display significant attenuation in cultured mammalian
cells, but only the virJ mutant has been shown to be attenuated in the mouse model
(76, 77).
The brucellae maintain predominantly an intracellular residence in their mammalian
hosts (18), and one of the primary functions of the T4SS is to control the intracellular
trafficking of the Brucella-containing vacuoles in host macrophages so that these bac-
teria avoid killing and degradation in phagolysosomes (56–59). Upon entry into host
cells, the brucellae reside in acidified phagosomal compartments known as endosomal

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TABLE 1 Brucella T4SS effectors and their proposed functions

Effector ORFa Proposed function Reference(s)
VceA BAB1_1652 Unknown 81
VceC BAB1_1058 Modulation of host immune response 81, 95–97
RicA BAB1_1279 Intracellular trafficking of BCVs 82, 89
BPE005 BAB1_2005 Modulation of collagen deposition by hepatocytes 83, 106, 107
BPE043 BAB1_1043 Unknown 83
BPE123 BAB2_0123 Modulation of host cell enolase activity 83, 108
BPE275 BAB1_1275 Unknown 83
BtpA BAB1_0279 Modulation of host immune response, stabilization of host cell microtubules, 84, 98–100, 102–105
depletion of host cell NAD
BtpB BAB1_0756 Modulation of host immune response, stabilization of host cell microtubules, 84, 103, 105
depletion of host cell NAD
BspA BAB1_0678 Unknown 85
BspB BAB1_0712 Intracellular trafficking of BCVs 85, 87, 89
BspC BAB1_0847 Unknown 85
BspE BAB1_1675 Unknown 85
BspF BAB1_1948 Unknown 85
BspJ BAB2_0119 Inhibition of host cell apoptosis 85, 110
SepA BAB1_1492 Intracellular trafficking of BCVs 86
aOpen reading frame (ORF) designation in B. abortus 2308 genome sequence.

Brucella-containing vacuoles (eBCVs), which undergo limited interaction with the

lysosomal pathway (78, 79). The intracellular brucellae do not replicate in the eBCVs,
but the acidic pH of these compartments serves as a signal for the induction of the
genes encoding the T4SS (80). T4SS-mediated secretion of effectors into the host cell
then orchestrates a series of events that redirects the intracellular trafficking of the

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eBCVs, diminishing their interactions with the endolysosomal pathway and initiating
their extensive interaction with the host cell endoplasmic reticulum (ER) (Fig. 3). This
series of events leads to the formation of the so-called replicative BCVs (rBCVs), which
are slightly acidic/neutral pH compartments where the intracellular brucellae sustain
their chronic intracellular persistence in the host (78). These rBCVs eventually interact
with components of the host cell’s autophagy pathway, resulting in the formation of
autophagic BCVs (aBCVs), which are thought be important for bacterial egress and
cell-to-cell spread in the host (79).
The recent identification and characterization of several T4SS effectors have helped
us begin to understand how the T4SS controls the intracellular trafficking of Brucella
strains (81–86) (Table 1). BspB, for example, directly interacts with the conserved oligo-
meric Golgi (COG) complex in host cells (87). This multimeric protein complex controls
vesicle trafficking between the Golgi apparatus and the ER, and the interaction of BpsB
with the COG complex redirects Golgi apparatus-derived vesicles to the rBCV. The T4SS
effector RicA directly interacts with Rab2, a small GTPase that also modulates ER-Golgi
apparatus interactions in mammalian cells (82, 88). The exact mechanism by which
RicA contributes to rBCV development is unclear, but it has recently been shown that
the BCV traffic-altering function of BspB is totally dependent upon the activity of RicA
(89). Another T4SS effector, SepA, plays a role in exclusion of the lysosomal marker
LAMP-1 from eBCVs during their conversion to rBCVs (86), but the host cell target of
this effector has not been identified.
Another way that the Brucella T4SS contributes to virulence is by modulating the
host immune response (90–94) (Fig. 4 and 5). The T4SS effector VceC, for instance,
interacts with the host cell ER chaperone BiP (95). This interaction causes ER stress and
induces an unfolded protein response (UPR) in Brucella-infected cells, which stimulates
production of the inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor
alpha (TNF-a). VceC-induced production of these cytokines by macrophages induces
granuloma formation, which facilitates chronic infection. VceC-mediated inflammatory
cytokine production by placental trophoblasts also leads to host cell death and fetal
pathology in the pregnant mouse model, which has led investigators to postulate that
this T4SS effector may play an important role in transmission in natural hosts (96, 97).

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FIG 4 Brucella virulence determinants that influence the capacity of macrophages to modulate the
host immune response. fi, activation; a, inhibition. The dashed arrow indicates that the Brucella LPS
does not signal strongly through the TLR4 pathway and stimulates a diminished inflammatory response.
The red X indicates that the Brucella flagellin is not recognized by TLR5.

Unlike VceC, the T4SS effectors BtpA and BtpB inhibit inflammatory cytokine produc-

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tion by dendritic cells by interfering with the TLR-Myd88-MAL signaling pathway (84,
98–100). This ability of the T4SS effectors to both stimulate and inhibit host immune
responses has been proposed to allow the brucellae to stimulate enough of a response
to benefit their long-term intracellular persistence in the host and sufficient immuno-
pathology to facilitate their spread to other hosts but not elicit a strong enough
immune response in the host to result in sterilizing immunity and resolution of the
infection (101).
Evidence suggests that the Brucella T4SS effectors may also contribute to virulence
in other ways. BtpA and BtpB, for instance, stabilize microtubules, which likely has
impacts on host cell physiology beyond their ability to directly interfere with Myd88-

FIG 5 Brucella virulence determinants that impact the ability of dendritic cells to modulate the host
immune response. fi, activation; a, inhibition. The dashed arrow indicates that the Brucella LPS does
not signal strongly through the TLR4 pathway and stimulates a diminished inflammatory response.

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FIG 6 Genetic regulators and the corresponding stimuli that control expression of the genes encoding
the T4SS in Brucella. fi, activation; a, repression. The question mark indicates that the environmental
stimuli recognized by these regulators have not been determined. Both activation and repression of
virB expression have been reported for BabR.

MAL signaling (102, 103), and BtpA induces the unfolded protein response in infected
macrophages by an undefined mechanism (104). BtpA and BtpB have recently been
shown to have NAD1 hydrolase activities that allow the brucellae to reduce NAD1 lev-
els in host cells during infection (105). Because NAD1 plays such a broad-based role in
the physiology of eukaryotic cells, the NAD1 hydrolase activities of BtpA and BtpB may
help explain the multiple contributions that these T4SS effectors appear to be making

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to Brucella virulence. Studies with a human hepatic cell line and mice experimentally
infected with B. abortus 2308 have also established a link between the T4SS effector
BPE005 and collagen deposition and liver fibrosis (106, 107). In addition, the T4SS effec-
tor BPE123 directly interacts with the host cell glycolytic enzyme a-enolase during
HeLa cell infection and stimulates its activity (108). Normal levels of host cell a-enolase
are required for the wild-type replication of B. abortus 2308 in HeLa cells. This raises
the intriguing possibility that by modulating the activity of a host cell enzyme involved
in carbohydrate metabolism, the intracellular brucellae may be able to improve their
access to glucose. Experimental evidence suggests that glucose is the preferred carbon
source for these bacteria when they reside in alternatively activated macrophages dur-
ing the chronic stage of infection in mice (109). Finally, recent studies suggest that
BspJ may inhibit host cell apoptosis by directly interacting with NME/NM23 nucleoside
diphosphate kinase 2 and creatine kinase B in the nucleus of these cells (110).
The genes encoding the T4SS are tightly regulated (111, 112), and proper temporal
and stochiometric expression of these genes is essential for virulence (113, 114). The
same is presumably true for the T4SS effectors, although the expression patterns of the
individual genes encoding these effectors have not been as well characterized as those
of the virB genes. Strict temporal regulation of the genes encoding the T4SS and its
effectors makes sense, considering the orderly progression of the intracellular life cycle
of the brucellae in host cells (78, 79) and the observation that the T4SS effectors con-
tribute to the virulence of Brucella strains by means other than directing their intracel-
lular trafficking. Not surprisingly, multiple regulators control virB expression in Brucella
(115) (Fig. 6). The two-component regulator BvrRS, for instance, allows the brucellae to
recognize the acidic pH and nutrient deprivation they encounter in the eBCV and to
activate expression of the gene encoding the quorum-sensing (QS) regulator VjbR,
which in turn activates virB expression (112). The transcriptional regulator HutC and
stringent response regulator Rsh are also thought to allow Brucella strains to modulate
virB expression in response to their nutritional status (116, 117). Other regulators,
including MdrA (118), BabR (119–122), LovhK (123), MucR (124, 125), NolR (126), OtpR
(127), and small regulatory RNAs (sRNAs) (122, 128), have been implicated in regulating
virB expression in Brucella, but precisely how these regulators contribute to

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coordinating the proper expression of these genes and the environmental stimuli to
which they are responsive remains to be determined. There is also evidence that the
individual contributions of specific regulators to virB regulation may differ between the
Brucella species (129). B. ovis, for example, does not require acidic conditions to elicit
expression of its virB operon (70).

Lipopolysaccharide
Like most Gram-negative bacteria, Brucella strains produce a lipopolysaccharide
(LPS) molecule that plays an important role in maintaining the integrity of their cell en-
velope (130, 131). With two notable exceptions, they have a smooth LPS (S-LPS) con-
sisting of a polysaccharide O-chain, core, and lipid A. The exceptions are B. ovis and B.
canis, which naturally produce a rough LPS lacking the O-chain. The importance of the
O-chain for the virulence of naturally occurring smooth Brucella strains is well docu-
mented (132–145). One way that the LPS O-chain contributes to virulence is by pro-
tecting smooth Brucella strains from the bactericidal activities of complement (139,
143, 146) and the antimicrobial peptides they encounter during their interactions with
host phagocytes (147, 148). Another is by serving as an adhesin (Fig. 3). The interaction
of the O-chain with lipid rafts on the surface of mammalian cells allows smooth
Brucella strains to enter these cells by an endocytic pathway that avoids extensive
fusion of the BCVs with lysosomes (149). This route of entry is the first required step for
the formation of the replicative BCV by smooth strains that occurs prior to the partici-
pation of the T4SS effectors (150, 151).
O-chain-mediated uptake of smooth Brucella strains also plays an important role in
immune evasion, because this route of entry stimulates low levels of proinflammatory
cytokine production in macrophages and dendritic cells (152, 153) (Fig. 4 and 5).

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Smooth LPS shed by Brucella strains into BCVs is also resistant to degradation by mac-
rophages (154) and forms complexes with major histocompatibility complex class II
(MHC-II), inhibiting the capacity of these phagocytes to present antigens to T lympho-
cytes (155, 156) (Fig. 4). Still another way that O-chain-mediated entry of smooth
Brucella strains into macrophages contributes to virulence is by inhibiting caspase 2-
mediated apoptosis in these phagocytes (157–161). The precise mechanisms behind
this inhibition have not been determined, but the capacity of smooth strains to extend
the life span of macrophages likely enhances their ability to avoid immune clearance
and disseminate to different organs in their mammalian hosts.
Lipid A is often referred to as “endotoxin” because it is the component of LPS that
is recognized by the host pattern recognition receptor Toll-like receptor 4 (TLR4), and
the lipid A’s of many Gram-negative bacteria induce strong inflammatory responses
(162). It has long been known that Brucella strains produce an LPS with low endotoxin
activity (163–167). One reason that the lipid A of Brucella strains does not stimulate a
strong inflammatory response is that unlike its enteric counterparts, the Brucella lipid A
contains very-long-chain fatty acids (VLCFAs) (168, 169). These VLCFAs presumably pre-
vent the Brucella lipid A from forming the same strong interactions that other bacterial
lipid A molecules form with TLR4 (170) (Fig. 4 and 5). This poor recognition of the
Brucella lipid A by the innate immune response plays a critical role in the so-called
stealthy mode of virulence employed by these bacteria (167). Interestingly, the Brucella
lipid A also induces premature cell death in human neutrophils (171). Dead neutrophils
carrying intracellular brucellae are subsequently engulfed by macrophages and dendri-
tic cells via mechanisms that do not stimulate an inflammatory response. This has
been proposed to be yet another strategy that Brucella strains can employ to avoid
detection by the host immune system during the early stages of infection.
In many bacteria, the LPS core serves primarily as a structural link between the O-
chain and the lipid A. It also performs this function in Brucella (142), but in addition,
recent studies have shown that the LPS core plays an essential role in allowing these
bacteria to evade detection by the host immune response (172–175). Specifically, the
brucellae produce a core structure containing a lateral oligosaccharide side chain that
sterically shields the lipid A and inhibits its binding to TLR4 on host macrophages and

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dendritic cells (Fig. 4 and 5). This lateral side chain and the positive charge it imparts
on the LPS core also appear to be important for the resistance of both smooth and
rough Brucella strains to killing by complement and bactericidal peptides (172, 173,
175, 176).
The essential role that the LPS O-chain plays in the virulence of smooth Brucella
strains raises the intriguing question of why the naturally occurring rough strains are
such virulent pathogens in their natural hosts. Brucella ovis, for instance, is a major vet-
erinary pathogen in sheep-producing countries, where it causes epididymitis and infer-
tility in rams (177, 178). Brucella canis causes abortion and infertility in dogs, and B.
canis infections can have a severe impact on commercial breeding operations (179). B.
canis is also being increasingly recognized as a source of human infection (180). A con-
siderable amount of work has been done over the past 20 years to address how the
naturally occurring rough Brucella species compensate for the lack of an LPS O-chain.
One particularly informative finding is that despite their lack of an O-chain, B. ovis and
B. canis strains are almost as resistant to complement as smooth Brucella strains (181).
The pattern of expression of the Omp25/Omp31 proteins (which will be discussed
later) in the outer membrane of B. ovis and B. canis is different than that observed in
smooth strains (182), and experimental evidence suggests that it is this distinctive
Omp25/Omp31 composition that makes the rough strains resistant to complement
and other antimicrobial peptides (183). Unlike rough mutants derived from naturally
occurring smooth strains, B. ovis and B. canis strains are also fully competent for intra-
cellular replication in macrophages (70, 184, 185), and both establish and maintain
chronic spleen infections in experimentally infected mice (70, 183, 186–188). One ob-
servation that might explain this difference is that B. ovis and B. canis employ a lipid
raft-mediated route of entry into macrophages that follows a different endocytic path-

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way than smooth strains (189) (Fig. 3), and this unique route of entry appears to be im-
portant for allowing the BCVs containing B. canis and B. ovis strains to avoid fusion
with lysosomes (150, 190). This alternative pathway of entry into macrophages may
also explain why B. ovis and B. canis strains induce limited and ineffective inflammatory
responses in their natural hosts (71, 191–193).
The organization and composition of the LPS biosynthesis genes are highly con-
served across the Brucella species that infect domestic animals and many of the strains
that infect wildlife, and the absence of the O-chain from the B. ovis and B. canis LPS is
due to well-described genomic deletions (194–197). But a subset of the so-called early
diverging Brucella strains found in amphibians and isolated from human disease
employ an operon consisting of four genes, rmlABCD, to produce an LPS with a rham-
nose-based O-chain instead of the perosamine O-side chain that is found universally in
other smooth Brucella strains (198). Based on the documented role of the perosamine
O-chain in virulence, it has been postulated that acquisition of the genes encoding this
latter type of LPS O-chain played an important role in the evolution of Brucella strains
as mammalian pathogens (7, 199). Interestingly, serologic studies suggest that the LPS
core of some of the early diverging strains may also differ from that of the classical
Brucella strains (200).
A couple of gene products that play auxiliary roles in LPS production and are impor-
tant for Brucella virulence have also been identified. B. abortus ba14k (201) and romA
(202) mutants produce LPS with altered levels of O-chain, and both mutants display
significant attenuation in mice. RomA is a periplasmic protein thought to stabilize the
components of the LPS biosynthetic complex. BA14K is lectin-like protein, but the pre-
cise role that it plays in modulating LPS O-chain content has not been determined.

Omp25/Omp31
Brucella strains produce a family of highly conserved outer membrane proteins
(OMPs) known as Omp25, Omp25b, Omp25c, Omp25d, Omp31, Omb31b, and Omp22
that play important roles in maintaining the integrity of their cell envelope (203, 204).
These 25- to 30-kDa b -barrel OMPs work in conjunction with the LPS O-chain to pro-
tect these bacteria from complement and other antimicrobial peptides encountered in

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the host, and their contributions to virulence appear to be especially important for the
naturally occurring rough strains such as B. ovis (181, 183, 205–207). This distinction,
however, is not absolute, as B. abortus and B. melitensis omp25 mutants have been
shown to be attenuated in mice (205) and natural hosts (208, 209), and a B. melitensis
omp31 mutant is attenuated in cultured mammalian cells and mice (210, 211).
Beyond their structural roles in maintaining cell envelope integrity, the Brucella
Omp25/Omp31 proteins have also been shown to mediate direct interactions between
the brucellae and mammalian cells that are important for virulence. These latter func-
tions may help explain some of the disparate phenotypes reported for Brucella omp25
and omp31 mutants in virulence assays. Omp25d and Omp22, for instance, play impor-
tant roles in the entry of B. ovis into mammalian cells (207) (Fig. 3), but there is no evi-
dence that they perform this function in smooth strains (206), where the LPS O-chain
seems to be the predominant determinant in mammalian cell entry, as described in
the previous section. The Omp25 protein of B. abortus, a smooth strain, also directly
interacts with the SLAMF1 protein on the surface of dendritic cells and inhibits their
maturation and capacity to produce inflammatory cytokines (212) (Fig. 5). The capacity
of Omp25 to inhibit TNF-a production during Brucella infection was first reported by
Jubier-Maurin et al. (213), but only recently has the molecular basis for this activity
been clearly defined. There is also evidence suggesting that the Brucella Omp25 and
Omp31 proteins have the capacity to modulate other aspects of host cell function dur-
ing infection (214–219), but precisely how these other proposed Omp25/Omp31 func-
tions contribute to virulence remains to be determined.
One of the interesting features of the Omp25/Omp31 proteins is that they are
highly conserved across the Rhizobiaceae, and the Omp25/Omp31 orthologs are also
important for the successful interactions of other alphaproteobacteria with their re-

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spective eukaryotic hosts. The Omp25 ortholog AopB, for instance, is required for the
wild-type virulence of Agrobacterium tumefaciens in plants (220). The Bartonella heme-
binding proteins (Hbps) are also Omp25/Omp31 orthologs (221), and Bartonella hense-
lae hbp knockdown strains are attenuated in endothelial cell cultures (222). Notably,
the Brucella Omp31b has also been shown to bind hemin in vitro, and the gene encod-
ing this protein is iron regulated in B. suis (223). This raises the possibility that in addi-
tion to their other roles, the Omp25/Omp31 proteins may also contribute to virulence
by working in concert with the heme transporter BhuA (224) to allow the brucellae to
use heme as an iron source during residence in their mammalian hosts (225).
No single Brucella species has been shown to produce all seven of the Omp25/
Omp31 proteins (182). Some species, like B. abortus and B. ovis, lack genes encoding
specific Omp25/Omp31 proteins due to large genomic deletions (194, 226). Smaller
genetic disruptions appear to be responsible for the distinctive patterns of Omp25/
Omp31 production in other Brucella species (182). The global regulators BvrRS, VjbR,
and CtrA tightly coordinate the expression of the omp25 and omp31 genes in response
to both environmental stimuli and physiological cues (227–230), which is consistent
with the critical role that this family of OMPs plays in the basic physiology and viru-
lence of Brucella strains.

Omp10, Omp16, and Omp19

Brucella strains produce three outer membrane lipoproteins: Omp10, Omp16, and
Omp19 (231). Omp16 is a homolog of the peptidoglycan-associated lipoprotein (Pal),
which is highly conserved in Gram-negative bacteria (232). These proteins interact with
the components of the Tol complex and play critical roles in maintaining the structural
integrity and function of the outer membrane. The fact that omp16 is an essential gene
in Brucella is consistent with Omp16 playing the same role as other Pal homologs
(233–235). Omp19, on the other hand, is the most well characterized of the Brucella lip-
oproteins. Purified Omp19 has strong immunomodulatory activities affecting a wide
variety of host cells mediated by its interactions with TLR2, and these activities have
been proposed to be important not only for the capacity of the brucellae to evade
host immune responses but also their ability to induce immunopathology in selected

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tissues such as bone and the central nervous system (236–252) (Fig. 4 and 5). In addi-
tion, Omp19 shares significant amino acid homology with bacterial protease inhibitors
(253), and phenotypic analysis of a B. abortus omp19 mutant indicates that Omp19 pro-
tects the parental strain from proteases encountered in the intestinal tract after oral
infection and lysosomal proteases encountered during intracellular residence in host
macrophages (254). In addition, Omp19 also protects another immunomodulatory pro-
tein, Omp25, from degradation by proteases. Unlike Omp16 and Omp19, which show
homology to other bacterial proteins, Omp10 homologs only appear to be present in
Brucella and a limited number of other alphaproteobacteria (255) and the biological
function of this Omp is unknown. Interestingly, while B. abortus omp19 and omp10
mutants exhibit significant attenuation in mice (254, 256, 257), the corresponding B.
ovis mutants do not (233). But the fact that B. ovis omp10 omp19 double mutants can-
not be constructed suggests that these proteins may have a shared physiologic func-
tion (233).

Autotransporter Adhesins
Autotransporter (AT) adhesins play important roles in the attachment of many bac-
terial pathogens to mammalian cells (258). Five AT adhesins have been described in
Brucella. OmaA and BmaC are type I monomeric ATs (259, 260), BtaE and BtaF are type
II trimeric ATs (261–263), and BigA (264) shares structural domains with the Escherichia
coli adhesin intimin, which is considered an inverse AT adhesin (265). BmaC binds fi-
bronectin on the surface of host cells (260), and BtaE and BtaF bind hyaluronic acid
(261, 262), but the receptors for OmaA and BigA have not been described. Interestingly,
Brucella mutants lacking these AT adhesins exhibit reduced attachment to epithelial cells
but display wild-type intracellular replication in cultured macrophages. These mutants

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are also attenuated in mice when delivered by the intragastric or nasal routes but not
when administered intraperitoneally. These experimental findings suggest that the func-
tion of the AT adhesins is to facilitate attachment of the brucellae to the host at mucosal
surfaces during the early stages of infection. Notably, BigA appears to have a tropism for
eukaryotic cell junctions (264), and adhesion to and disruption of host cell junctions is a
strategy that other bacterial pathogens employ to cross mucosal barriers in the host
(266). A B. suis btaE btaF double mutant is also considerably more attenuated in mice
than a btaE or btaF mutant, indicating that BtaE and BtaF play complementary roles in
virulence (262). In addition to its role in attachment, there is also evidence that BtaF pro-
tects B. suis from the bactericidal activity of serum (262).
BmaC, BtaE, and BtaF are localized at one specific pole of the bacterial cell
(260–262), and the location of these AT adhesins at the new pole, coupled with the ob-
servation that Brucella cells in the G1 phase of the cell cycle are the predominant infec-
tious form (267), has led investigators to propose that these AT adhesins form an adhe-
sive pole on the Brucella cell (268, 269). Only a small proportion of the Brucella cells in
planktonic cultures produce these adhesins, suggesting that the corresponding genes
may be optimally expressed only upon exposure to a host-specific stimulus such as
contact with mammalian cells. This is consistent with the fact that several AT-encoding
genes appear to be regulated by the quorum-sensing regulator VjbR (119) and the
global regulator MucR (270, 271) and that a complex regulatory network has been
shown to control btaE expression in B. abortus (272, 273). There appears to be consid-
erable heterogeneity in the Brucella genes that encode AT-type adhesins (273), and as
noted previously, there is evidence indicating that these adhesins have overlapping
functions (262). Consequently, it will be important to study the contributions of the
Brucella AT adhesins in different species and strains and use mutants with multiple dis-
rupted genes to gain an accurate assessment of how these proteins contribute to
virulence.

Cyclic b-1,2-D-Glucan
Many Gram-negative bacteria produce polysaccharide polymers and secrete them
into their periplasm, where they perform a variety of physiologic functions (274).

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Brucella spp. and other alphaproteobacteria secrete a cyclic polymer consisting of 17

to 20 glucose residues known as cyclic b -1,2-glucan (C b G) into their periplasmic space
(275). In Sinorhizobium and Agrobacterium, the production of C b G is regulated by os-
motic conditions, suggesting a role for this molecule in osmoprotection (276). But C b G
production is not osmoregulated in Brucella (277, 278), and experimental evidence
suggests that this polysaccharide plays only a minor role as an osmoprotectant in
these bacteria (279). C b G does, however, play an essential role in Brucella virulence
(275, 280). Specifically, studies employing B. abortus C b G synthase mutants and puri-
fied C b G indicate that this molecule disrupts lipids rafts on the Brucella-containing
vacuoles, which is thought to prevent these vacuoles from sustaining their interactions
with lysosomes (281) (Fig. 3). C b G has also been shown to have a strong influence on
the capacity of macrophages and dendritic cells to produce both proinflammatory and
anti-inflammatory cytokines (279, 282–284) (Fig. 4 and 5). It is presently unclear how
C b B is released from its periplasmic location in Brucella cells to perform these biologi-
cal functions in vivo. Nevertheless, C b G appears to have dual functions in virulence. It
plays a critical role in the intracellular trafficking of the brucellae to their replicative
niche in host macrophages, and it fine-tunes the host immune response to allow their
chronic intracellular persistence. Given the proposed polar nature of the interaction of
Brucella cells with their mammalian hosts (268, 269), it is also notable that the C b G syn-
thase (Cgs) and transporter (Cgt) display polar localization on the bacterial cell (285).
Whether this polar localization of the C b G biosynthesis and transport machinery plays
any role in its contributions to virulence remains to be determined.

Flagella
Despite the fact that most Brucella strains are nonmotile, Halling reported the dis-

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covery of flagellar biosynthesis genes in B. abortus in 1998 (286). It was subsequently
determined that most, if not all, Brucella strains have the genetic capacity to produce
flagella (41, 287–289), but they lack chemotaxis genes and only a limited number of
strains in the BO2 clade appear to be able to use the flagella for motility (290).
Nevertheless, the isolation of B. melitensis fliF and flgF mutants in signature-tagged mu-
tagenesis screens for attenuation in mice (291) and pregnant goats (62) suggested that
these genes might play a role in virulence. The Letesson group demonstrated that B.
melitensis 16M does in fact produce a single sheathed polar flagellum that is covered
by an extension of the outer membrane (289, 292), and studies have confirmed that
flagellar biosynthesis genes are required for the wild-type virulence of B. melitensis and
B. abortus strains in the mouse model (289, 293). Interestingly, B. ovis mutants lacking
flagellar biosynthesis genes are fully virulent in mice, suggesting that the contributions
of flagella to pathogenesis may be strain and possibly host dependent (294).
One mechanism by which the flagella appear to contribute to virulence is by modu-
lating the host immune response (295). Unlike flagella from some other Gram-negative
pathogens, the Brucella flagellin is not recognized by TLR5. This contributes to the so-
called stealthy nature of Brucella infections. But experimental evidence indicates that
the Brucella flagellin ends up in the cytoplasm of infected host cells and stimulates an
inflammasome-mediated inflammatory response that is important for “limiting” the
extent of Brucella replication. Thus, the flagellum has been proposed to be yet another
virulence determinant that allows the brucellae to fine-tune the host immune response
in a manner that facilitates the establishment and maintenance of chronic infections.
The possibility also exists that these appendages have yet undiscovered roles in patho-
genesis, such as serving as adhesins and/or as sensors of surface attachment as they
do in other closely related alphaproteobacteria (296, 297). In addition, sheathed flag-
ella are relatively rare in bacteria, but studies have shown that they play a role in the
release of outer membrane vesicles (OMVs) (298), which is an interesting relationship
considering the roles that have been proposed for OMVs in host-pathogen interactions
during Brucella infections (299).
The flagellar biosynthesis genes are tightly regulated in Brucella, and while produc-
tion of the polar flagellum in B. melitensis 16M has been observed only in vitro in

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bacterial cells grown to early exponential phase in a rich medium, these genes are
readily expressed when this strain is replicating in mammalian cells (289). Subsets of
the Brucella flagellar biosynthesis genes are expressed in stages, but the regulatory
networks that coordinate the orderly temporal expression of these genes differ from
those found in other bacteria (300, 301). The quorum-sensing regulators VjbR and
BabR (also known as BlxR), the light-sensing regulator LovhK, the general stress
response regulator RpoE1, and cyclic di-GMP-mediated signaling also control flagellar
gene expression in Brucella (119, 120, 123, 302–305).
Bacteria that employ flagella for motility almost universally have chemotaxis genes
that allow them to control the direction of their flagellar rotation in response to gra-
dients of environmental stimuli (306). This allows them to swim toward nutrients and
away from toxic compounds. The fact that no chemotaxis genes have been detected
in Brucella strains is not too surprising given that most of these bacteria do not appear
to be using their flagella for locomotion. But it does raise some interesting questions
about how the motile BO2 strains direct their movements in their natural environments.

Phosphatidylcholine
The phospholipid phosphatidylcholine (PC) is a major component of eukaryotic cell
membranes but is not commonly found in prokaryotes (307). Almost 40 years ago, it
was reported that Brucella strains contains PC in their cell envelope (308), which led
investigators to suspect that the presence of this “eukaryotic” phospholipid in the OM
might contribute to virulence. Brucella strains produce PC by two different biochemical
pathways—the Pcs pathway, which directly produces PC from choline, and the Pmt
pathway, which produces PC by methylating the phospholipid phosphatidylethanol-

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amine (309–311). Independent studies performed in two different laboratories have
confirmed that PC plays an essential role in Brucella virulence. However, these studies
provided conflicting results regarding the relative contributions of the Pcs and Pmt
pathways to virulence, and further studies will be needed to get a clear picture of how
PC contributes to virulence. But, in general, the experimental evidence at hand sug-
gests that the PC/PE ratio in the outer membrane of Brucella strains impacts the ability
of these bacteria to resist killing by antimicrobial peptides and complement and that
PC may also play a role in modulating the host response to infection (309, 310, 312). It
is also notable that the incorporation of PC into the outer membrane is a distinctive
feature of the alphaproteobacteria in general and that this phospholipid also plays an
important role in the interactions of some of the other members of this group (e.g.,
Agrobacterium and the rhizobia) with their respective plant hosts (313).

Exopolysaccharides
Exopolysaccharides (EPSs) are polysaccharide polymers secreted by bacteria that
are either (i) attached tightly to the cell surface and form a capsule or (ii) loosely associ-
ated with bacterial cells forming an amorphous “slime layer” (314). EPSs play multiple
roles in bacterial pathogenesis. They can serve as adhesins and facilitate the attach-
ment of bacteria to eukaryotic cells (315). They can also protect bacteria from the bac-
tericidal activities of complement, neutrophils, and macrophages (316–318) and/or
allow them to evade recognition by host innate and acquired immune responses (316,
319). In addition, these polymers allow bacteria to form biofilms, which contributes to
their persistence in the environment and in mammalian hosts (320–322).
Brucella strains have the genetic capacity for EPS production (229, 270, 323), but ex-
perimental evidence suggests that the corresponding genes are tightly regulated. B.
melitensis 16M, for instance, does not produce a readily detectable EPS during routine
in vitro cultivation, but disruption of a putative quorum-sensing pathway causes this
strain to produce an apparent EPS detected by calcofluor staining and form “biofilm-
like” bacterial cell aggregates in liquid culture (229). EPS production and cellular aggre-
gation have also been described in a B. melitensis virB mutant (324) and a B. abortus
strain overexpressing the glycosyltransferase WbkA (325). Reports of “biofilm”

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FIG 7 Virulence determinants that allow the brucellae to resist the reactive oxygen and nitrogen
species they encounter during their intracellular residence in host phagocytes. Reactive oxygen and
nitrogen species are shown in red. OM, outer membrane; CM, cytoplasmic membrane.

formation by Brucella strains (326–328) and enhanced Congo red staining of a B. meli-
tensis mucR mutant (329) are also consistent with EPS production.
Because the specific genes required for EPS production have not been clearly defined,
we do not currently know if EPS plays an important role in Brucella virulence. But EPS pro-
duction is essential for the pathogenic and symbiotic interactions of the agrobacteria and
rhizobia with their respective plant hosts (330, 331). Considering the conserved strategies
that the alphaproteobacteria employ to sustain successful interactions with their eukaryo-

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tic hosts (9), it will be important to determine if EPS production also plays an important
role in Brucella virulence.

PHYSIOLOGIC ADAPTATION TO THE HOST ENVIRONMENT

Microaerobic Respiration and Denitrification
Brucella strains rely upon a strictly respiratory type of metabolism for growth (332).
Within the tissues of their mammalian hosts, these bacteria reside in microaerobic
environments, and bd- and cbb3-type cytochrome oxidases with very high affinities for
O2 play important roles in Brucella virulence (333–338). Interestingly, B. abortus 2308
requires the bd-type cytochrome oxidase for wild-type virulence in mice (334). In con-
trast, a B. suis 1330 mutant lacking the cbb3-type cytochrome oxidase displays marked
attenuation in mice, but a derivative of this strain lacking the bd-type cytochrome dis-
plays wild-type virulence (337). The basis for this differential requirement for the bd-
and cbb3-type cytochrome oxidases for the virulence of these two strains is not known.
But what is clear from both studies (334, 337) is that a respiratory chain with a high af-
finity for O2 is required for successful replication and persistence of the brucellae in
mammalian tissues.
Denitrification is a process that allows bacteria to reduce NO3 and use it instead of
O2 as a terminal electron acceptor for respiration (339). Most Brucella strains have the
four enzymatic pathways needed for the complete reduction of NO3 to N2 (340), and
experimental evidence indicates that the denitrification pathway contributes to viru-
lence (335, 340–342). One way it contributes is by allowing the brucellae to employ
NO3 as an alternative electron acceptor to sustain the respiration they need for chronic
intracellular persistence in the microaerobic environment of host tissues. Experimental
evidence also suggests that the denitrification enzymes provide Brucella strains with a
defense against the bactericidal activity of NO generated by host macrophages (340,
341, 343) (Fig. 7). In addition, the capacity of the intracellular brucellae to degrade NO
protects infected macrophages from apoptosis (343). Interestingly, the degree to which
denitrification contributes to virulence appears to differ between Brucella species that
have different natural hosts (344).
Expression of the genes encoding the bd- and cbb3-type cytochrome oxidases and

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denitrification enzymes is tightly coordinated by the two-component regulators PrrAB

(also known as RegAB) and NtrYX (345–348). Not surprisingly, PrrB and NtrY are redox
sensors, and in conjunction with their cognate regulators, they activate these genes in
response to low levels of ambient O2.

Carbon and Nitrogen Metabolism

Until recently, when genes associated with central metabolism were identified in
genetic screens for bacterial genes linked to virulence, these genes were often
assigned to the “housekeeping” category and not examined further. But it is now
widely appreciated that the capacity of bacterial pathogens to adapt their central me-
tabolism to take advantage of the carbon, nitrogen, and energy sources they encoun-
ter in the host plays an essential role in their virulence (349). It has also become evi-
dent that extensive metabolic reprogramming takes place in bacterial pathogens, the
host cells they inhabit, and the cells that modulate the host immune response during
the course of bacterial infections and that these metabolic adaptations in both the
pathogen and host often dictate the outcome of host-pathogen interactions (350–353).
Studies reported by Xavier et al. (109), Czy_z et al. (354), and Kerrinnes et al. (355) provide
striking examples of how metabolic reprogramming in host cells can influence the
course of Brucella infections.
Studies suggest that most Brucella strains rely on a balance of glucose catabolism
(55, 109, 356, 357) and the utilization of gluconeogenic substrates such as glutamate
and lactate (354, 358) to sustain their intracellular persistence in their mammalian
hosts. Selective use of the pentose phosphate pathway for glucose catabolism also
appears to have played an important role in the evolution of these bacteria as mam-
malian pathogens (359). Glucose catabolism appears to be especially important for

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these bacteria when they reside in alternatively activated macrophages during chronic
infection in mice, when these host phagocytes shift from a glycolytic catabolism to
one that relies on fatty acid oxidation, which makes glucose more readily available to
the intracellular brucellae (109). The enzymes pyruvate kinase (PykM) and pyruvate
phosphate dikinase (PpdK) play critical roles in glucose metabolism in Brucella (357,
358). PykM is critical for glucose catabolism, PpdK is required for gluconeogenesis, and
both are essential for the wild-type virulence of Brucella strains in mice. But ongoing
studies examining the role of glucose metabolism in Brucella virulence indicate that
we still have a lot to learn in this area. Experimental evidence, for instance, indicates
that the brucellae possess novel enzymes for converting fructose 1,6-bis-phosphate to
fructose-6-phosphate during gluconeogenesis in addition to the well-characterized
bacterial fructose bis-phosphatases Fbp and GlpX (358, 360). These bacteria also
appear to be capable of employing an atypical pathway for converting phosphoenol-
pyruvate (PEP) to pyruvate during the catabolism of hexoses in the absence of a func-
tional pyruvate kinase (357).
Erythritol is a four-carbon sugar alcohol that serves as a preferred carbon and
energy source for many Brucella strains (361–363). Substantial amounts of this carbohy-
drate are present in the placentas of ruminants during the latter stages of pregnancy,
and the capacity to catabolize erythritol has been proposed to be an important viru-
lence determinant for the Brucella spp., e.g., B. abortus and B. melitensis, that cause
abortion and infertility in cattle, sheep, and goats (364). Recent studies have better
defined the enzymes required for erythritol catabolism and shown that this carbohy-
drate is metabolized by a pathway that feeds into the pentose phosphate pathway
(365). At least one erythritol-responsive transcriptional regulator, EryD, has also been
identified (366), and this carbohydrate stimulates the expression of a variety of genes
in Brucella, including those involved in the production of the T4SS and flagella (367,
368). But gaining a clear picture of whether erythritol catabolism contributes to Brucella
virulence has remained elusive. Genetic studies, for instance, demonstrated a link
between production of the siderophore brucebactin, erythritol utilization, and the viru-
lence of B. abortus 2308 in pregnant cattle (369, 370), but a direct link between erythritol
catabolism and the virulence of this strain in cattle has not been confirmed

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experimentally. Erythritol catabolism genes have also been reported to be required for
the virulence of B. abortus and B. suis strains in mice and cultured murine and human
macrophages (234, 335, 371). But a recent comparative study of the virulence properties
of B. abortus mutants blocked at different steps in the erythritol catabolism pathway sug-
gests that the attenuation reported for B. abortus eryC, eryH, and eryI mutants and B. suis
eryB and eryC mutants was likely due to erythritol toxicity rather than their inability to
catabolize this four-carbon sugar alcohol (372). More importantly, this study showed that
a B. abortus eryA mutant which lacks the first enzyme in the erythritol catabolism path-
way and cannot catabolize this sugar alcohol displays only limited and early attenuation
in nonpregnant mice and wild-type virulence in bovine and human macrophages and
trophoblasts and pregnant mice. More extensive studies of the virulence properties of
eryA mutants in nonpregnant and pregnant natural hosts will be required before we
have a clear picture of the role that erythritol catabolism plays in Brucella virulence.
Evidence suggests that amino acids and NH41 provide the bulk of the nitrogen the
brucellae need to meet their physiologic requirements during residence in their mam-
malian hosts (373), although host-derived polyamines may serve as an important N
source for these bacteria in alternatively activated macrophages (355). Glutamate can
serve as both a sole C and N source for Brucella strains (374), and as observed in other
bacteria, the enzymes that interconvert a-ketoglutarate, glutamate, and glutamine and
the proteins that regulate these enzymes tightly coordinate NH41 assimilation in the
brucellae (373). The importance of this regulatory network for virulence is evidenced
by the fact that mutants lacking many of these enzymes (e.g., GlnA, GltB, GltD, and
GdhZ) and regulators (e.g., GlnD and GlnE) exhibit attenuation in cultured mammalian
cells and mice (54, 55, 234, 335, 342, 375). But one distinctive feature of N metabolism
in Brucella is that the two-component regulator NtrBC and alternative sigma factor

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RpoN, which play central roles in regulating nitrogen metabolism in many other bacte-
ria (376–379), do not appear to be required for wild-type virulence in Brucella
(380–382). One caveat here, however, is that the Brucella NtrBC and RpoN regulons
have not been extensively characterized, so it is not known if these regulators make
significant contributions to nitrogen metabolism or if the typical regulatory functions
of these proteins have been supplanted by other regulators.
Classical PEP-phosphotransferase (PTS) systems play both catalytic and regulatory
roles in bacterial carbohydrate transport and catabolism (383). The Brucella spp. do not
have a classical PTS, but they employ a related PTS known as the nitrogen-related PTS
(PTSNtr) (384) to coordinate their C and N metabolism (385), and Brucella mutants with
a disrupted PTSNtr are attenuated (234, 335, 375). But determining the precise role that
the Brucella PTSNtr plays in virulence is complicated by the fact that the hprK, ptsM, and
ptsO genes which encode principal components of the Brucella PTSNtr are cotranscribed
with the genes that encode the two-component regulator BvrRS. The observation that
Brucella mutants with a disrupted PTSNtr are defective in virB expression (385) suggests
that in addition to coordinating C and N metabolism in Brucella, PTSNtr may also be
influencing the regulatory activity of BvrR.

Antioxidants
Brucella strains utilize O2 as a terminal electron acceptor during their replication in
host tissues (334, 337). This puts these bacteria in a precarious position, because it pre-
disposes them to oxidative damage from both external and internal sources (Fig. 7).
During their interactions with neutrophils and macrophages, for instance, the brucellae
are exposed to the reactive oxygen species (ROS) generated by the respiratory burst of
these phagocytes (386, 387). In addition, they must also detoxify the superoxide (O22)
and hydrogen peroxide (H2O2) generated as by-products of their own aerobic metabo-
lism (388, 389).
Superoxide anion (O22) does not typically cross the cytoplasmic membrane due to
its negative charge (390), and two different superoxide dismutases (SODs) with differ-
ent cellular locations play distinct roles in detoxifying the exogenous O22 the brucellae
encounter during their interactions with host phagocytes and the endogenous O22

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they generate via their aerobic metabolism. The periplasmic Cu/Zn SOD SodC (391) is
responsible for detoxifying exogenous O22, and this enzyme plays an important role in
protecting the brucellae from the respiratory burst of host macrophages (392). The
cytoplasmic Mn SOD SodA, on the other hand, protects these bacteria from the O22
generated by their aerobic metabolism (389). Both enzymes are required for the wild-
type virulence of Brucella strains in mice (389, 392, 393).
In contrast to O22, H2O2 moves freely across cellular membranes (390), and the peri-
plasmic catalase KatE (394) and the cytoplasmic peroxiredoxin AhpC (388) work in con-
cert to ensure that the cellular levels of H2O2 generated by the aerobic metabolism of
Brucella strains are maintained below toxic levels. It should be noted, however, that
neither of these enzymes plays a major role in protecting the brucellae from the respi-
ratory burst of host macrophages (388). Brucella strains lacking KatE exhibit wild-type
virulence in mice (388, 395) and pregnant goats (396), and Brucella ahpC mutants are
also fully virulent in mice (388). On the other hand, B. abortus ahpC katE double
mutants are extremely attenuated in mice. These experimental findings coupled with
the attenuation observed with a B. abortus sodA mutant in mice indicate that the bru-
cellae must be able to protect themselves from ROS of both exogenous and endoge-
nous origin to produce infection in their mammalian hosts.
The transcriptional activator OxyR directly regulates katE expression in Brucella in
response to exposure to H2O2 (397, 398). But how ahpC, sodC, and sodA are regulated
is less clear. The redox-responsive two-component regulator RegAB and the stringent
response regulator Rsh have been reported to regulate sodC expression (348, 399), Hfq
(and presumably sRNAs) have been implicated in ahpC and sodC regulation (392, 400,
401), and VjbR has been linked to sodA regulation (119). Whether these regulatory links

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are direct or indirect, however, remains to be determined.

Metal Acquisition and Detoxification

Depriving pathogens of iron (Fe), manganese (Mn), and zinc (Zn) is a strategy
employed by the host to resist microbial infections (402, 403). The vast majority of the
Fe in mammalian tissues, for instance, is incorporated into cellular proteins or heme
and is not directly available for use by bacterial pathogens. Neutrophils also release
the metal binding proteins calprotectin and lactoferrin into the extracellular environ-
ment during the inflammatory response (404–406), and activated macrophages
employ the divalent cation transporter Nramp1 to pump Fe21, Mn21, and Zn21 out of
phagosomes containing intracellular pathogens (407). In addition, the liver produces
the hormone hepcidin during the inflammatory response, which inhibits the release of
Fe from the liver and spleen into the bloodstream (408).
Brucella strains rely on the siderophore brucebactin (409), the Fe21 transporter
FtrABCD (410), the heme transporter BhuA (224), the Mn transporter MntH (411), and
the Zn transporter ZnuA (412, 413) to overcome the Fe, Mn, and Zn deprivation they
encounter in their mammalian hosts (Fig. 8). Interestingly, the need for brucebactin
appears to be host dependent. This siderophore is not required for the virulence of B.
abortus 2308 in mice (409, 414, 415) but is essential for the virulence of this strain in
pregnant goats (416) and cattle (369). The basis for this differential requirement for
brucebactin in these hosts is unknown, but experimental evidence suggests that it
may be linked to erythritol catabolism (370).
Because Fe, Mn, or Zn can be toxic to bacterial cells when accumulated in excess,
the brucellae employ metal-responsive transcriptional regulators, storage proteins, and
exporters to prevent metal toxicity. The transcriptional regulators Irr, RirA, Mur, and
Zur ensure that the genes encoding high-affinity metal importers are selectively
expressed only when bacterial levels of specific metals fall below certain thresholds
(417–422). The Mn exporter EmfA (423) and Zn exporter ZntA also transport excess Mn
and Zn out of Brucella cells, and the Zn-specific transcriptional regulator ZntR tightly
regulates ZntA-mediated Zn export (422). The fact that Brucella irr (419), rirA (234, 424),
emfA (423), and zntR (422) mutants exhibit attenuation in mice strongly suggests that

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FIG 8 Fe, Mn, Zn, and Mg acquisition systems and defenses against metal toxicity that have been
shown to play a role in the virulence of Brucella. The solid arrows represent direct regulatory links
that have been documented experimentally, and the dashed arrows and lines denote suspected
regulatory links. OM, outer membrane; CM, cytoplasmic membrane.

in addition to overcoming the metal deprivation that they encounter with the host,
the brucellae must also avoid metal toxicity to be effective pathogens.
Nickel (Ni) and magnesium (Mg) transport also play important roles in Brucella viru-
lence. Ni is required for urease activity (see below), and Mg is the most abundant metal

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found in bacterial cells (425) and an essential micronutrient for Brucella (426). Two
high-affinity Ni transporters, NikABCDE and NikKLMQO, play functionally redundant
roles in supplying Brucella strains with the Ni required to support their urease activity
(427, 428). Although the Mg transporters in Brucella have not been well characterized,
a gene predicted to be involved in Mg transport (mgtB) was identified in a genetic
screen for B. melitensis mutants displaying attenuation in cultured mammalian cells
and mice (429). A B. suis mutant lacking a homolog of a Salmonella virulence determi-
nant involved in maintaining cellular Mg/ATP balance (MgtC) (430) is also attenuated
in cultured mammalian cells (431).

Urease
Most Brucella strains produce urease (432), and this enzyme is thought to protect
the brucellae from the extremely acidic conditions they encounter during passage
through the gastrointestinal tract following ingestion (63, 433, 434). Brucella ovis is an
exception, and the absence of urease activity in this bacterium has been proposed to
be one of the reasons for its limited host range and lack of transmission via the oral
route (196). There are two different genetic loci, ure1 and ure2, in Brucella strains con-
taining urease biosynthesis genes, but only one of these loci, ure1, encodes a func-
tional urease (433, 434). The ure2 locus encodes urea and Ni transporters that are
thought to be important for maintaining maximum urease activity at the low urea con-
centrations and acidic pH encountered in the mammalian gastrointestinal tract (428).

PrpA
Proline racemase protein A (PrpA) is a B cell mitogen secreted by Brucella into the
cytoplasm of infected macrophages, where it is palmitoylated by the host cell and
transported to the plasma membrane, where it interacts with the surface receptor
NMM-IIA (435–438) (Fig. 3). This interaction stimulates the production of the anti-
inflammatory cytokine IL-10 by these macrophages and represses their ability to pro-
duce proinflammatory cytokines. The B cell mitogen activity of PrpA also increases the
production of Brucella-specific IgG2a, which is thought to increase the opsonic uptake
of Brucella by host macrophages. The enzymatic activity of PrpA is essential for its
immune modulatory functions, but the mechanism behind this link is unknown. It is

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also unknown how PrpA is secreted into the host cell cytoplasm since this protein has
not been described as a T4SS substrate. The late-stage attenuation of a prpA mutant in
mice suggests that the immunomodulatory functions of PrpA may play a role in the
capacity of Brucella strains to sustain chronic infections in their mammalian hosts.

BacA
The plant symbiont Sinorhizobium meliloti is a close phylogenetic relative of
Brucella. During its symbiotic interactions with its plant host, S. meliloti imports small
cysteine-rich peptides known as NCRs (nodule-specific C-rich peptides) from the plant
into its cytoplasm via an ABC transporter known as BacA (439). NCR import serves two
beneficial functions for the rhizobia during symbiosis. It prevents the potentially bacte-
ricidal NCRs from damaging the bacterial cell envelope (440), and the NCRs serve as
signaling molecules that drive the intracellular rhizobia to differentiate into N2-fixing
bacteroids (441).
Brucella strains produce a BacA homolog, and a B. abortus bacA mutant is attenu-
ated in cultured murine and human macrophages and mice (39, 442, 443). The basis
for the attenuation of this mutant is presently unknown. The B. abortus bacA mutant
has reduced levels of very-long-chain fatty acids in its lipid A (169) and induces stron-
ger inflammatory responses in mice than its parent strain, which likely explains one as-
pect of its attenuation (443). But the plant NCRs are also considered to be homologs of
mammalian defensins (444), and the Brucella bacA gene can restore NCR import and
symbiotic capacity to an S. meliloti bacA mutant (445). This raises the possibility that
BacA may also be able to protect Brucella strains from the antimicrobial peptides they
encounter during their interactions with host phagocytes in a manner similar to that
by which this transporter protects S. meliloti from NCRs in plants.

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GadB, GadC, and GlsA
Many foodborne pathogens like E. coli and Salmonella rely on acid resistance (AR)
systems to help them overcome the low pH they encounter during passage through
the stomach (446). One of these is the AR2/AR2_Q system, which is comprised of a glu-
tamate carboxylase (GadB), a glutaminase (GlsA), and a g-aminobutyric acid (GABA)/
glutamate-glutamine antiporter (GadC). GlsA converts glutamine to glutamate, and
GadB converts glutamate into GABA. The latter reaction consumes a proton which
deacidifies the cytoplasm. GadC exports GABA to the exterior of the bacterial cell in
exchange for the import of glutamate or glutamine, which allows Gls and GadB to con-
tinue the deacidification cycle in the cytoplasm.
gadB, gadC, and glsA genes are present in all Brucella species, but they encode func-
tional AR2/AR2_Q systems only in B. microti, B. ceti, B. pinnipedialis, B. inopinata, and
the “atypical” Brucella strains isolated from amphibians (447–449). AR2/AR2_Q systems
allow these strains to withstand exposure to very acidic pH, and a B. microti gadBC mu-
tant is attenuated in mice infected by the oral route. It has been proposed that the
genes encoding a functional AR2/AR2_Q system have been retained only in the so-
called “ancestral” Brucella strains because these bacteria reside for prolonged periods
in the external environment, unlike the classical Brucella strains, which are highly host
adapted.

REGULATION OF VIRULENCE GENE EXPRESSION

BvrRS
The two-component regulator BvrRS (37) controls the expression of genes required
at multiple stages of the infectious process, and Brucella bvrR and bvrS mutants are
highly attenuated (37, 234, 335). BvrRS directly controls the expression of omp25,
omp22, and genes involved in LPS lipid A modification (174, 227). The altered cell enve-
lopes of Brucella bvrR and bvrS mutants make them extremely sensitive to killing by se-
rum (450), less invasive for mammalian cells than their wild-type counterparts (451),
and highly susceptible to the bactericidal activities of macrophages and dendritic cells
(452, 453). BvrS recognizes the acidic pH and nutrient deprivation encountered by the

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FIG 9 Overlapping regulation of genes encoding major virulence determinants in Brucella by BvrRS,
VjbR, RpoE, CtrA, and LovhK. BvrRS and LovhK are shown twice to reflect the fact that these regulators
have been shown to have both direct and indirect effects on genes encoding these virulence determinants.

brucellae in the endososomal BCVs and via BvrR directly activates the expression of the
genes encoding the T4SS (454). BvrRS also indirectly activates the expression of these
genes by activating the expression of the gene encoding the quorum-sensing regula-
tor VjbR (112) (Fig. 9). Experimental evidence suggests that many other Brucella genes
are regulated by BvrRS (228, 455–457), but deciphering the nature of these regulatory
links is complicated by the fact that BvrR lies upstream of VjbR in a regulatory network
that controls the expression of .200 Brucella genes (112, 119, 121). The bvrR and bvrS
genes are also transcribed in an operon with genes that encode a regulatory network
that coordinates C and N metabolism in these bacteria (385).
BvrRS is a member of a family of highly conserved two-component regulators that
play critical roles in the interactions of alphaproteobacteria with their respective eu-
karyotic hosts (458). The BvrRS homologs ChvIG and ChvI-ExoS, for example, are essen-

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tial for the pathogenic and symbiotic interactions of Agrobacterium tumefaciens and
Sinorhizobium meliloti with their plant hosts (459, 460), and BatRS is required for the
virulence of Bartonella henselae in cultured human endothelial cells (461) and
Bartonella tribocorum in rats (462). Although the sensor components of these regula-
tors appear to have evolved to recognize different host-relevant environmental stimuli,
the genes under the control of these regulators have highly conserved functions
directed at overcoming the innate immune responses of these hosts and establishing
long-term pathogenic or symbiotic relationships. Together with a periplasmic protein
known as ExoR, which modulates the regulatory function of the BvrRS homologs in
Sinorhizobium and Agrobacterium, these proteins comprise a regulatory circuit known
as the ExoR-ExoS-ChvI (RSI) invasion switch (458). This regulatory circuit is found only
in the alphaproteobacteria that infect eukaryotic hosts and is thought to have played
an important role in their evolution to their host-adapted lifestyles. Interestingly,
Brucella strains possess an exoR gene, but whether the corresponding gene product
plays a role in modulating the regulatory activity of BvrRS has not been reported.

N-Dodecanoyl Homoserine Lactone, VjbR, and BabR

Quorum sensing (QS) is a cell-to-cell signaling pathway that bacteria use to assess
their population density and trigger the density-dependent expression of genes affect-
ing a wide range of cellular behaviors (463, 464). A possible link between QS and
Brucella virulence was uncovered when the QS signaling molecule N-dodecanoyl
homoserine lactone (C12-HSL) was detected in the supernatant of B. melitensis cultures,
and it was found that the addition of exogenous C12-HSL to this strain repressed the
expression of its T4SS and flagellar biosynthesis genes (465). Subsequent studies iden-
tified the LuxR-type regulator VjbR as being responsible for this regulatory link (302),
and .150 Brucella genes are now known to be regulated by VjbR (81, 119, 121, 302,
466, 467). Brucella vjbR mutants are highly attenuated (65, 188, 234, 302, 468–470), and
one of the major reasons for this attenuation is that VjbR is required for the expression
of the virB operon which encodes the T4SS (Fig. 6 and 9). But VjbR also directly controls
the expression of genes encoding other important virulence determinants, including
Omp25, the autotransporter adhesin BtaE, the T4SS effector VceC, the cbb3-type

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FIG 10 Proposed role of C12-HSL signaling and VjbR in temporal regulation of virB expression during development of the Brucella-
containing vacuoles in host cells. It is thought that accumulation of C12-HSL in the spatial confines of the BCVs prevents virB
expression during the later stages of BCV development. The phagosome membrane is depicted in brown.

cytochrome oxidase, and FtcR, the master regulator of flagellar gene expression in
Brucella (81, 272).
Interestingly, the impact that C12-HSL binding has on the regulatory activity of VjbR
is the opposite of what is observed for canonical LuxR-type QS regulators in other bac-
teria. Specifically, the latter regulators generally turn on the expression of virulence
genes when they bind the QS signal (463, 464). In contrast, VjbR serves as an activator
of virB expression in Brucella, and binding of C12-HSL to VjbR causes it to disengage
from the virB promoter (466). Moreover, global studies of gene expression in wild-type
Brucella strains and isogenic vjbR mutants exposed to C12-HSL have also shown that
this QS signaling molecule has a negative impact on the regulatory activity of VjbR on
many other genes (119, 121, 234, 302). Based on these experimental findings, it has
been postulated that C12-HSL and VjbR do not comprise a classical QS pathway but

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rather a regulatory circuit that allows the intracellular brucellae to sense the spatial
confines of the Brucella-containing vacuole within host cells and turn off the expres-
sion of virB and other genes in a temporal fashion (119, 272, 302) (Fig. 10).
Another C12-HSL-responsive LuxR-type transcriptional regulator known as BabR
(also known as BlxR) also controls the expression of a subset of the VjbR-regulated
genes in Brucella, including the virB operon and flagellar biosythesis genes, and in
some cases these two regulators have opposite effects on these coregulated genes
(119–122, 471). BabR also regulates vjbR expression and vice versa. But in contrast to
vjbR mutants, Brucella babR mutants display little or no attenuation in experimental
models (119–121), leaving the importance of the regulatory overlap between VjbR and
BabR presently unclear.
It is clear that we still have a lot to learn about the roles that C12-HSL, VjbR, and
BabR play in the basic biology and virulence of Brucella. For instance, although a lacto-
nase that degrades C12-HSL and modulates its regulatory activity on VjbR has been
identified (472), the enzyme(s) responsible for the synthesis of the signaling molecule
has not. In addition, the regulatory activity of VjbR at some promoters is dependent on
the activities of other coregulators (272), but in only a few cases have these coregula-
tors been identified (111, 115, 116, 118, 273). There are also many genes involved in
basic physiologic processes such as metabolism and transport that are regulated by
VjbR and/or BabR (119–121), but the importance of these particular regulatory links to
virulence have not been determined. Finally, the fact that the vjbR and babR genes reg-
ulate each other (119–121) and are regulated by other global regulators (112, 123, 473)
often makes it difficult to pinpoint precisely where VjbR and BabR lie in the regulatory
pathways that control specific genes.

General Stress Response and LovhK

The general stress response (GSR) is a global change in gene expression that allows
the alphaproteobacteria to withstand a wide variety of environmental stresses (474).
Because some of these stresses, such as nutrient deprivation and exposure to reactive
oxygen species and acidic pH, are those that the brucellae encounter during their in-
tracellular life cycle in the host, the GSR has been proposed to play an important role

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FIG 11 Regulation of the general stress response (GSR) and other virulence determinants in Brucella
by LovhK. The yellow and red lightning bolts in panel B denote the stimuli thought to activate the
histidine kinase activity of Lovhk through its LOV and PAS domains. LovR has been proposed to
function as a phosphate sink and to modulate LovhK signaling. HK, histidine kinase domain; SL,
sigma factor-like domain; Rec, response regulator receiver domain.

in Brucella virulence (400). The alternative s factor RpoE1 controls the GSR in Brucella,
and the activity of RpoE1 is modulated by the anti-s factor NepR, the anti-anti-s factor
PhyR, and the HWE-type sensor histidine kinase (HK) LovhK (305, 475, 476) (Fig. 11).
Under nonstress conditions, NepR binds RpoE1 and prevents it from interacting with
RNA polymerase (RNAP). In response to undefined environmental or physiologic stim-
uli (see below), LovhK phosphorylates PhyR and PhyR-P titrates NepR away from
RpoE1. This allows RpoE1 to engage RNAP and regulate the .80 genes that comprise
the Brucella GSR regulon (303, 305, 381). Regulated proteolysis of PhyR and the activity
of another uncharacterized HWE-type sensor HK encoded by the gene immediately
downstream of rpoE1 also play important roles in fine-tuning the GSR in Brucella (304,
475).
Brucella rpoE1 and phyR mutants are more sensitive to oxidative stress and acidic
pH than wild-type strains (305, 474), and these mutants exhibit limited attenuation in
cultured mammalian cells and delayed clearance in the mouse model of chronic infec-
tion (305, 381, 475). These phenotypes are consistent with the proposed role of the
GSR in supporting the long-term intracellular persistence of the brucellae in host mac-
rophages. Specific genes in the Brucella GSR regulon that have individually been linked
to virulence include those encoding the flagella, urease, the bd-type cytochrome oxi-
dase, and the lectin-like protein BA14K (303, 305, 381) (Fig. 9).
Attempts to determine the environmental and/or physiologic stimuli that elicit the
GSR in Brucella have been complicated by the fact that LovhK has two sensory domains
that respond to different stimuli (475) (Fig. 11). One of these domains responds to blue
light, and the other is thought to be responsive to an undefined redox stimulus. The

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FIG 12 Unipolar cell division and coordination of cell division with the cell cycle in Brucella. The
polar locations of the AT adhesins BtaE, BtaF, and BmaC are shown, and the red boxes indicate that
Brucella cells in the G1 phase of the cell cycle are the most infectious for mammalian cells.

light-sensing domain is required for the wild-type intracellular replication of Brucella

strains in mammalian cells, but the redox-sensing domain is not (475, 477). But expo-
sure to light does not evoke the GSR in Brucella (476). The biological relevance of light

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as a stimulus for LovhK is also unclear considering the intracellular nature of Brucella
infections. One proposal that has been put forth is that recognition of light by LovhK
may occur during transmission between natural hosts, where exposure to aborted
fetuses in the external environment represents an important source of infection for na-
ive animals (476, 477) (Fig. 2).
LovhK also plays an important role in the regulation of virulence genes independ-
ent of its role in inducing the GSR (123, 475–477) (Fig. 11). The virB, vjbR, and babR
genes require LovhK for their wild-type expression, but these genes are not members
of the RpoE1 regulon, and Brucella lovhK mutants are considerably more attenuated in
mammalian cells and mice than rpoE1 and phyR mutants. The nature of the regulatory
links between LovhK and the virB, vjbR, and babR genes is currently unknown.

CtrA and GcrA

Like the other alphaproteobacteria, Brucella strains display a distinctive cell cycle
characterized by asymmetric cell division, cell polarity, and defined developmental
changes in gene expression (268, 269, 478, 479) (Fig. 12). Disruption of the cell cycle
has a negative impact on virulence (230, 480, 481), and as noted previously, Brucella
cells in the G1 phase of the cell cycle appear to be the most infectious for mammalian
cells (267, 482). The transcriptional regulator CtrA serves as the master regulator of
the cell cycle in Brucella (230, 481, 483), and the histidine kinase PdhS (484), CckA-ChpT
phosphorelay, and protease adaptor CpdR (481) modulate the regulatory activity of
CtrA is response to endogenous cell cycle signals (Fig. 13). Consistent with its role in
regulating the cell cycle, CtrA controls multiple genes involved in DNA replication,
chromosome segregation, and cell division in Brucella (230), and proper coordination
of the expression of these genes is undoubtedly important for sustaining the intracel-
lular persistence of these bacteria in mammalian cells. But CtrA also controls the
expression of omp25 and genes involved in LPS biosynthesis, so it is apparent that its
contributions to virulence in Brucella go beyond its role as a cell cycle regulator.
GcrA is another cell cycle regulator that allows s 70-bound RNA polymerase to rec-
ognize the promoters of genes that have been epigenetically “marked” by the DNA
methyltransferase CcrM (485). Recent studies have identified a GcrA ortholog in

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Roop et al. Microbiology and Molecular Biology Reviews

FIG 13 Regulation of CtrA and CpdR activity by PdhS and the CckA-ChpT phosphorelay in Brucella.
Arrows indicate the direction of phosphate transfer. The red X denotes CtrA degradation by ClpPX.
The multiple dashed arrows between PdhS and CckA indicate that the exact number and nature of
the regulatory steps between these two regulators have not been determined.

Brucella and shown that this gene is essential for wild-type virulence in cultured mam-
malian cells (486). But the specific contributions of individual GcrA-regulated genes to
virulence remains to be determined.

Downloaded from [Link] on 16 August 2025 by [Link].

MucR
The Zn finger protein MucR is a global regulator that plays an important role in the
virulence of Brucella strains (124, 270, 329, 375, 487). MucR serves predominantly as a
transcriptional repressor and has been shown to regulate the expression of a multitude
of Brucella genes required for virulence, including the virB operon, omp25, babR, and
genes involved in LPS, C b G, and flagellar biosynthesis, iron acquisition, and c-di-GMP
signaling. Experimental evidence suggests that one of the primary functions of MucR is
to work in concert with antagonistic transcriptional activators to ensure the proper
temporal expression of these genes during infection (271) (Fig. 14), and similar host-
specific regulatory functions have been proposed for MucR homologs in Agrobacterium
tumefaciens (488) and Sinorhizobium meliloti (489). MucR also works in concert with CtrA
to regulate the cell cycle in Caulobacter (490), and given the conservation of the cell
cycle genes across the alphaproteobacteria, it is likely that the Brucella MucR performs
the same function. Thus, it will also be important to determine how misregulation of the
cell cycle contributes to the attenuation of Brucella mucR mutants.

GntR
The GntR family is one of the most common types of transcriptional regulators
found in bacteria (491). Twenty-one GntR-type regulators have been described in
Brucella (126), and remarkably, 7 of these have been linked to virulence (126, 492). The
regulatory activities of GntR-type regulators are typically modulated by the allosteric
binding of metabolites, and correspondingly, most of the genes controlled by these
regulators are involved in metabolism (493). Notably, one of the GntR-type regulators
in Brucella is HutC, which regulates both histidine catabolism and expression of the
genes encoding the type IV secretion machinery (116). This relationship is intriguing,
because three other GntRs (GntR4, GntR12, and GntR17) have also been linked to
proper expression of the virB genes in Brucella (126, 473, 492, 494). GntR17 is also
required for wild-type expression of omp25 and the genes encoding the quorum-sens-
ing regulators VjbR and BabR in B. abortus (473, 494), and GntR12 is required for the
capacity of B. suis to elicit the unfolded protein response in cultured caprine alveolar
macrophages (492). Considering the clear role of the Brucella GntRs as virulence

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Uncovering Brucella Virulence Credentials Microbiology and Molecular Biology Reviews

FIG 14 Conserved roles that MucR/Ros-based transcriptional modules are proposed to play in
controlling host-specific gene expression in Agrobacterium, Sinorhizobium, and Brucella.

determinants, it will be important to better characterize the individual contributions of

these regulators to virulence, define the specific genes they regulate, and identify the
metabolites that control the regulatory activities of these GntRs.

OtpR, CpbB, NodVW, TceSR, and PrlSR

The histidine kinase CenK and response regulator CenR form a two-component reg-

Downloaded from [Link] on 16 August 2025 by [Link].

ulator that plays an essential role in regulating cell growth and division in Caulobacter
(495). The Brucella OtpR is a CenR homolog, and although otpR does not appear to be
an essential gene in Brucella, a B. melitensis otpR mutant displays growth and morpho-
logic defects and misregulation of peptidoglycan biosynthesis and cell division genes.
This suggests that the Caulobacter CenR and Brucella OtpR have similar physiologic
functions (496, 497). A B. melitensis otpR mutant is also extremely attenuated in both
cultured mammalian cells and mice. OtpR has been proposed to play an important
role in protecting Brucella from acidic pH, and indeed, over 500 genes display differen-
tial expression in B. melitensis 16M and an isogenic otpR mutant when these strains are
exposed to acidic pH (127, 128). But how many of these genes are directly regulated
by OtpR remains to be determined. It is also presently unclear if a CenK ortholog works
in concert with OtpR to comprise a two-component regulator in Brucella. Interestingly,
otpR resides upstream and in an operon with a gene encoding the putative cAMP-
binding protein CbpB, which also plays a role in the virulence of B. melitensis 16M.
Moreover, experimental evidence suggests that like OtpR, CbpB may also play an im-
portant role in regulating genes involved in peptidoglycan biosynthesis and cell divi-
sion (498).
NodVW and TceSR are two-component regulators that are highly conserved in
Brucella strains (499), but neither is found in their close phylogenetic relative,
Ochrobactrum anthropi, a soil bacterium that only infrequently causes disease in
humans. This observation led to the proposition that these regulators played an im-
portant role in the evolution of the brucellae as intracellular pathogens. This proposi-
tion is supported by the fact that Brucella nodVW and tceSR mutants are highly attenu-
ated in cultured mammalian cells and mice (500–502). Although the environmental
stimuli that the Brucella NodVW and TceSR respond to are unknown, it is interesting
that NodVW allows the closely related bacterium Bradyrhizobium japonicum to recog-
nize small molecules produced by its plant host and express genes required for symbi-
osis (503, 504).
PrlSR is a two-component regulator that allows B. melitensis 16M to form multicellu-
lar aggregates when exposed to environments of high ionic strength (505). Although
B. melitensis prlS and prlR mutants are not attenuated in cultured mammalian cells,

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Roop et al. Microbiology and Molecular Biology Reviews

these mutants cannot sustain chronic spleen infections in BALB/c mice. The genes that
are subject to PrlSR regulation have not been described, but the phenotypes reported
for the prlS and prlR mutants suggest that they are likely involved in exopolysaccharide
production or cell envelope modifications.

Stringent Response
When faced with intense nutrient deprivation, bacteria produce the alarmones gua-
nosine 39-diphosphate 59-triphosphate (pppGpp) and guanosine 39-diphosphate 59-
diphosphate (ppGpp), known collectively as (p)ppGpp. These signaling molecules bind
to RNA polymerase and cause programmatic changes in gene expression that allow
bacteria to undergo a physiologic transition known as the stringent response (506).
This transition shifts their metabolism from one directed toward cell growth and divi-
sion to a maintenance metabolism geared toward survival. Induction of the stringent
response also provides bacteria with resistance to a variety of different environmental
stresses other than nutrient deprivation. In addition, many bacterial pathogens rely on
the stringent response to induce the virulence determinants they need for successful
colonization of their mammalian hosts.
The (p)ppGpp synthetase/hydrolase Rsh controls the stringent response in Brucella
(117). Microarray analysis indicates that approximately 12% of the genes in the B. suis
1330 genome are regulated by the stringent response (399). Consistent with its expected
role in allowing the brucellae to adapt their physiology to nutrient deprivation, Rsh
represses the expression of rRNA genes and induces the expression of methionine bio-
synthesis genes in B. suis 1330 when this strain is exposed to nutrient-deprived condi-
tions. B. abortus, B. suis, and B. melitensis rsh mutants are also more sensitive to nutrient
deprivation than their parent strains (117, 507, 508), and depriving a CO2-dependent

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B. ovis strain of CO2 elicits the stringent response (509).
B. abortus, B. melitensis, and B. suis rsh mutants are highly attenuated in mice and
cultured mammalian cells (60, 117, 335, 507, 508), but evidence suggests that the con-
tributions of Rsh to virulence go beyond its capacity to allow the brucellae to with-
stand the nutrient deprivation they encounter in their mammalian hosts. The genes
encoding the type IV secretion system, cbb3-type high-affinity cytochrome oxidase, ni-
trate reductase, and Cu/Zn superoxide dismutase SodC, for instance, are regulated by
Rsh (117, 399). Constitutive production of (p)ppGpp also arrests Brucella cells in G1 of
the cell cycle (508), which as noted previously, is their most infectious form. It is
obvious that we still have a lot to learn about precisely how the stringent response
contributes to Brucella virulence.

c-di-GMP Signaling
The signaling molecule bis-(39–59)-cyclic diguanylic acid (c-di-GMP) regulates bac-
terial gene expression at multiple levels. c-di-GMP can bind transcriptional regulators
and modulate their regulatory activity, bind to riboswitches in mRNAs and control
the efficiency with which these transcripts are transcribed or translated, or bind to
proteins and regulate their function (510, 511). c-di-GMP-regulated genes and gene
products play critical roles in the virulence of many bacterial pathogens (512, 513),
and pioneering studies from the Splitter and Smith laboratories have provided con-
vincing evidence that the same holds true for Brucella (304, 327). Brucella mutants
lacking the diguanylate cyclase CgsB or the c-di-GMP-specific phosphodiesterases
BpdA or BpdB, for instance, display significant alterations in their virulence profiles in
cultured macrophages and mice, and microarray and proteomic analyses of B. meli-
tensis cgsB and bpdA mutants indicate that genes encoding important virulence
determinants such as the flagella and the type IV secretion system are regulated by
c-di-GMP. The mechanism(s) by which c-di-GMP regulates the expression of these
genes, however, has not yet been determined, as no c-di-GMP-responsive transcrip-
tional regulators, c-di-GMP-responsive riboswitches, or proteins with catalytic or
structural functions responsive to this signaling molecule have been identified in
Brucella. The phenotypic properties displayed by the B. melitensis bpdA mutant

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Uncovering Brucella Virulence Credentials Microbiology and Molecular Biology Reviews

suggest that c-di-GMP signaling controls many aspects of the basic biology of
Brucella strains, including their central metabolism and progression through the cell
cycle (327). This is consistent with the function of c-di-GMP as a global regulator in
bacteria in general (510, 511), and in the alphaproteobacteria in particular, where c-
di-GMP plays a major role in regulating cell cycle progression and cellular differentia-
tion in Caulobacter, Sinorhizobium, and Agrobacterium, as well as the symbiotic and
pathogenic interactions of the last two bacteria with their respective eukaryotic hosts
(514–517).
The innate immune sensor known as stimulator of interferon genes (STING) resides
in the ER of mammalian cells (518). This pattern recognition receptor recognizes c-di-
GMP produced by bacterial pathogens, and STING-induced inflammatory responses
can have both positive and negative impacts on bacterial infections (519). Therefore, it
is not surprising that recent studies indicate that one way that c-di-GMP production
contributes to Brucella virulence is by modulating the host immune response (520,
521). Specifically, c-di-GMP binding to STING stimulates the unfolded protein response
in the ER of infected cells, and the resulting inflammatory response facilitates granu-
loma formation, which is thought to be important for the persistence of chronic
infections.

sRNAs and Hfq

Small regulatory RNAs (sRNAs) play important roles in regulating prokaryotic gene
expression at the posttranscriptional level (522). sRNAs perform their regulatory func-
tions by binding to the mRNA transcripts of genes and blocking their translation or
causing folding changes in these transcripts that uncover ribosome binding sites and
enhance their translation. sRNAs binding to their cognate mRNA transcripts can also

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enhance the degradation of these mRNAs or enhance their stability, and this feature of
sRNAs also plays an important role in their regulatory function. Most sRNAs (i) range in
size from 50 to 300 nucleotides in length, (ii) are rich in secondary structure, (iii) regu-
late genes that reside remotely from the gene encoding the sRNA, and (iv) have limited
patches of nucleotides known as regulatory motifs that share imperfect complemen-
tarity with their mRNA targets. The limited complementarity of sRNAs with their target
transcripts allows them to regulate multiple genes, but it also means that most sRNAs
require the RNA chaperone Hfq to facilitate productive interactions with their target
mRNAs (523).
Hfq is required for the wild-type expression of many Brucella genes (122, 388, 392,
400, 524, 525), and B. abortus and B. melitensis hfq mutants exhibit pleiotropic pheno-
types and are highly attenuated in cultured mammalian cells and mice (400, 442, 443,
525–527). These experimental findings indirectly provide evidence for the importance
of sRNAs in the basic biology and virulence of Brucella strains. But the first sRNAs to be
identified and characterized in Brucella were the 110-nt sRNA AbcR1 and the 116-nt
sRNA AbcR2, and studies of the interactions of these sRNAs with their target transcripts
has provided us with important insight into how Brucella sRNAs function at the molec-
ular level (528, 529). AbcR1 and AbcR2, for example, play redundant roles in controlling
the expression of genes encoding ABC transporters involved in amino acid and poly-
amine transport, and a B. abortus mutant lacking both AbcR1 and AbcR2 is highly atte-
nuated. AbcR1 and AbcR2 orthologs are also found in other members of the Rhizobiales
order of the alphaproteobacteria along with a conserved LysR-type regulator known as
VtlR in Brucella, which coordinates the expression of these sRNAs. Importantly, these
sRNAs control multiple genes required for the successful interactions of these bacteria
with their respective hosts (530–532).
Since the discovery of AbcR1 and AbcR2, bioinformatics-based approaches and
RNA sequencing (RNA-seq) analysis have been used to identify other sRNA candidates
in Brucella genomes, and the existence of over 100 potentially authentic Brucella
sRNAs have been confirmed by reverse transcription PCR (RT-PCR) and/or Northern
blot analysis (128, 533–540). Some of these Brucella sRNAs are required for virulence in
cultured mammalian cells and/or mice, including BSR0602 (535), BSR0441 (537),

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Roop et al. Microbiology and Molecular Biology Reviews

BASI74 (538), Bmsr1 (539), and BSR1141 (540). Experimental evidence also suggests
that an sRNA designated BsrH plays a role in Brucella iron metabolism (534). More
extensive studies of these and other newly discovered sRNAs and the auxiliary proteins
such as Hfq and RNase E (541) that modulate their regulatory functions will help us
better understand how these genetic regulators contribute to the basic biology and
virulence of Brucella.

CONCLUDING REMARKS
Over the past 2 decades, we have uncovered many of the “hidden credentials” of
Brucella virulence. But there is obviously a lot more to learn about how these remark-
able bacteria produce disease in their natural hosts. We do not know, for instance,
how many of the T4SS effectors contribute to virulence. There are also unanswered
questions about how the brucellae coordinate the expression of genes encoding vir-
ulence determinants in response to external and endogenous signals received in the
host and how specific genetic regulators contribute to these patterns of gene expres-
sion. How the brucellae adjust their cellular metabolism to adapt to shifts in host cell
metabolism is also an area that needs more investigation (109, 354, 355). More exper-
imental work also needs to be done with natural hosts using routes of inoculation
that mimic those that occur naturally. Mice have been indispensable for gaining a
better understanding of the basic features of Brucella virulence (542), but studies
suggest that virulence determinants required in natural hosts may not always be
identified using the murine model (369). Specific virulence determinants required for
Brucella strains in their natural hosts may differ, and it is likely that the requirement
for individual virulence determinants may vary during different stages of the disease

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cycle in these hosts (e.g., chronic infection versus abortion and fetal pathology in
ruminants). From an evolutionary standpoint, it will be interesting to determine what
genetic changes allowed the brucellae to evolve from environmental bacteria with
phenotypic characteristics resembling those of their close present-day phylogenetic
relative Ochrobactrum to become the highly host-adapted and host-specific patho-
gens they are today (7). This review does not cover studies focused specifically on
host immune responses to Brucella infection, but this topic is covered by other
reviews (543–548). Moreover, it is obvious that a better understanding of these
responses is needed before we can truly gain a clear picture of the hidden credentials
of Brucella virulence.
From a translational perspective, gaining a better understanding of the hidden
credentials of Brucella virulence provides us with molecular targets that can be
exploited in our efforts to design better Brucella vaccines and novel antimicrobials
that can be used to prevent and treat brucellosis. Vaccines have been integral com-
ponents of programs that have been successfully used to control food animal brucel-
losis worldwide. But the currently available vaccines have their limitations and draw-
backs, and widespread efforts are under way to develop better Brucella vaccines for
use in both food animals and wildlife (549–552). There is also presently no safe and
effective vaccine that can be used to prevent human brucellosis, and this disease is
notoriously difficult to treat (553, 554). The development of new and better chemo-
therapeutic strategies for treating human brucellosis is an area that deserves greater
attention because it has the potential to have a tremendous impact on the status of
this disease as one of the world’s leading zoonoses and on the threat of Brucella strains
as agents of bioterrorism.

ACKNOWLEDGMENTS
The Brucella research community is a highly interactive group, and we express our
gratitude to the other members of this community for the free flow of information on a
real-time basis that continually stimulates progress and makes this an exciting field to
be a part of. We also thank Patrick Lane from ScEYEnce Studios for assisting us with the
figures used in this review.

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Uncovering Brucella Virulence Credentials Microbiology and Molecular Biology Reviews

Work in the laboratory of R.M.R. and D.W.M. is funded by grants (AI1414138 and
AI1418132) from the National Institute of Allergy and Infectious Diseases.

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R. Martin Roop II (Marty) obtained his Ian S. Barton is a postdoctoral fellow

Ph.D. in 1985 from Virginia Tech under in the Department of Microbiology and
the direction of Noel Krieg and was a Immunology at Brody School of Medicine at
postdoctoral fellow under the direction of East Carolina University. He received his B.A.
Hugo Veit and Gerhardt Schurig from 1985 in Biology from Hiram College and then
to 1988 at the Virginia-Maryland Regional obtained his Ph.D. from Indiana University in
College of Veterinary Medicine. In 1988, he 2019 while working with Clay Fuqua, where
joined the Department of Microbiology and he studied genome instability and plasmid
Immunology at the University of Arkansas conjugation in the alphaproteobacterial plant
for Medical Sciences and relocated to the pathogen Agrobacterium tumefaciens. Since
Department of Microbiology and Immunology joining the Roop laboratory, he has been
at Louisiana State University Health Sciences Center in 1992. In 2001, he investigating molecular mechanisms of virulence regulation in Brucella spp.
joined the faculty of the Department of Microbiology and Immunology at
Brody School of Medicine at East Carolina University. Dr. Roop has spent the
past 34 years studying the basic biology and virulence of Brucella.

March 2021 Volume 85 Issue 1 e00021-19 [Link] 44

Uncovering Brucella Virulence Credentials Microbiology and Molecular Biology Reviews

Dariel Hopersberger is currently a Ph.D. Daniel W. Martin is a Research Associate

candidate under the mentorship of Dr. R. Professor at Brody School of Medicine at East
Martin Roop II in the Department of Carolina University. He earned a B.S. and an
Microbiology and Immunology at Brody M.S. in Biology at The University of Texas at
School of Medicine at East Carolina University. Tyler. He obtained his Ph.D. in Microbiology
Her graduate project aims to characterize the in 1994 from the University of Texas Health
role of an uncharacterized exopolysaccharide Science Center at San Antonio while working
in Brucella virulence. She obtained her Bachelor with Dr. Vojo Deretic. His postdoctoral training
was performed at Stanford University with Drs.
of Science in Biochemistry from Old Dominion
Lucy Tompkins and Stanley Falkow from 1994
University in 2008.
to 1999. He has been studying Brucella biology
and pathogenesis for the past 16 years in collaboration with Marty Roop.

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March 2021 Volume 85 Issue 1 e00021-19 [Link] 45