Review Article

published: 25 April 2011

doi: 10.3389/fmicb.2011.00074

Virulence properties of the Legionella pneumophila

cell envelope
Olga Shevchuk†, Jens Jäger† and Michael Steinert*
Institut für Mikrobiologie, Technische Universität Braunschweig, Braunschweig, Germany

Edited by: The bacterial envelope plays a crucial role in the pathogenesis of infectious diseases. In this
Carmen Buchrieser, Pasteur Institute,
review, we summarize the current knowledge of the structure and molecular composition of the
France
Legionella pneumophila cell envelope. We describe lipopolysaccharides biosynthesis and the
Reviewed by:
Ivo Gomperts-Boneca, Institut Pasteur, biological activities of membrane and periplasmic proteins and discuss their decisive functions
France during the pathogen–host interaction. In addition to adherence, invasion, and intracellular survival
Joachim Reidl, Institute for Molecular of L. pneumophila, special emphasis is laid on iron acquisition, detoxification, key elicitors of the
Biosciences, Austria
immune response and the diverse functions of outer membrane vesicles. The critical analysis of
*Correspondence:
the literature reveals that the dynamics and phenotypic plasticity of the Legionella cell surface
Michael Steinert, Institut für
Mikrobiologie, Technische Universität during the different metabolic stages require more attention in the future.
Braunschweig, Spielmannstrasse 7, Keywords: Legionella pneumophila, bacterial envelope, phospholipids, membrane proteins, LPS, outer membrane vesicles
Braunschweig 38106, Germany.
e-mail: [email protected]
†
Olga Shevchuk and Jens Jäger have
contributed equally to this work.

Bacterial cell envelopes fulfill several basic functions: They protect Virulence properties of outer membrane components are par-
the bacterium from environmental hazards, they allow a selective ticularly important in regard to outer membrane vesicles (OMVs).
passage of nutrients into and a specific export of waste products Like most bacteria, L. pneumophila sheds these vesicles from its
and secretion system substrates out of the cell. Additionally, they outer membrane. OMVs are spherical lipid bilayers and contain
mediate the direct contact with other organisms. This holds par- outer membrane components and periplasmic proteins.
ticularly true for pathogenic bacteria, whose often highly specific The actual structure of the L. pneumophila cell envelope was
interactions with host organisms depend largely on their surface assessed in detail by electron microscopy shortly after the discovery
structures. Accordingly, the ability of the Gram-negative facul- of the bacterium (Rodgers and Davey, 1982). Both membranes and
tative intracellular bacterium Legionella pneumophila to cause the peptidoglycan layer were visualized by different methods, result-
Legionnaires’ disease hinges predominantly on the components ing in vivid images of the components that are, nowadays, analyzed
and characteristics of its cell envelope. mostly biochemically. The authors are also the first to show the
The cytoplasm of Gram-negative bacteria is bordered by the existence of OMVs of L. pneumophila, even though they are termed
inner membrane. It consists of a bilayer of two phospholipid leaflets “blebs” and explained as “condensed pili-related proteins or random
with integral and peripheral proteins and lipoproteins. It harbors structural proteins of the outer membranes.” An extensive study of
metabolic enzymes, components of the respiratory chain and parts L. pneumophila morphology including envelope architecture was
of the iron acquisition machinery (Figure 1). performed by Faulkner and Garduño (2002). They hypothesize the
The periplasm contains a relatively thin layer of peptidogly- existence of several morphological variants, each corresponding to
can and different proteins. Legionella peptidoglycan is strongly a certain growth phase or stage of the infection cycle. Interestingly,
crosslinked (Amano and Williams, 1983). The periplasm is the five different envelope structures are presented which vary in thick-
location of many detoxifying enzymes which degrade harmful ness, number of membrane layers, and electron density of individual
substances from the environment. Secretion machineries which components. As some of the morphological variants only occurred
cross two membranes also go through the periplasmic space. during intracellular growth, the authors propose that the develop-
The outer membrane is asymmetric with an inner leaflet of ment of these variants depends on host metabolites. This notion
mostly phospholipids and an outer leaflet of mostly lipopolysac- can explain the absence of these forms during extracellular growth
charides (LPS). It harbors proteins with diverse functions in viru- in liquid media. The impact of processing artifacts arising during
lence such as adhesion and uptake into host cells. Legionella LPS the preparation of the samples, however, remains to be clarified.
has a unique architecture, particularly concerning the hydrophobic Many secretion systems and outer membrane proteins with roles
O-antigen. in virulence have been excellently reviewed elsewhere and are not
Certain types of surface appendages such as pili and flagella, within the focus of this work. This includes T1SS and twin-arginine
which are required for bacterial motility and pathogenicity, are translocation (Tat) secretion (Lammertyn and Anne, 2004), T2SS
anchored in the inner membrane and protrude into the extracel- (Cianciotto, 2009), T4SS as well as their respective translocated
lular space (Liles et al., 1998; Stone and Abu Kwaik, 1998; Heuner effectors (Ninio and Roy, 2007). Finally, secreted phospholipases
and Steinert, 2003). connect Legionella virulence to host lipids (Banerji et al., 2008).

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Shevchuk et al. Legionella pneumophila envelope

Less attention was paid to other components of the Legionella cell The lipid composition of a crude inner membrane ­preparation
envelope which are not part of the aforementioned complexes. This of L. pneumophila was analyzed shortly after the discovery of the
review concentrates on these envelope components and how they bacteria (Hindahl and Iglewski, 1984). They described it to con-
mediate Legionella virulence properties. tain mainly phosphatidylethanolamine and phosphatidylcholine
with smaller amounts of cardiolipin and phosphatidylglycerol.
The inner membrane of L. pneumophila Contamination with outer membrane components cannot be
Starting from the inside and proceeding outward, the first layer is excluded due to methodical reasons. Thus, these data should be
the inner membrane, also termed cytoplasmic or plasma mem- interpreted carefully.
brane. It is a lipid bilayer with integrated components of various An important function of the inner membrane of L. ­pneumophila
systems, including the iron uptake machinery, the respiratory chain, is the regulation of iron acquisition, summarized elsewhere
and the detoxification system (Table 1). (Cianciotto, 2007). Iron uptake is a crucial process during all phases
of L. pneumophila growth. It is mainly carried out by the GTP-
dependent iron transporter FeoB, which mediates the uptake of
Fe(II) (Robey and Cianciotto, 2002; Petermann et al., 2010). The
protein is required for optimal growth under iron-limiting con-
ditions in liquid media as well as in iron-restricted amoeba and
macrophages. FeoB is required for efficient killing of macrophages
and full virulence in a mouse model of Legionnaires’ disease.
Another iron acquisition mechanism involves the proteins IraA
and IraB. IraA is described as a small-molecule methyltransferase.
It mediates iron uptake and is required for the infection of human
macrophages and guinea pigs. IraB is an integral protein of the inner
membrane with homology to bacterial peptide transporters. It is
suggested to mediate the uptake of iron ions into the cell, possibly
chelated by small peptides. The potential significance of the iraAB
locus for virulence is underlined by the fact that it is found almost
exclusively in pathogenic Legionella, but not in avirulent species
(Viswanathan et al., 2000).
The multi-copper oxidase MCO is tethered to the cytoplasmic
membrane of L. pneumophila. Recently, this enzyme was suggested
to oxidize ferrous iron, which could otherwise lead to the formation
of hydroxyl radicals under aerobic conditions (Huston et al., 2008).
An MCO-negative mutant displays normal intracellular replica-
tion within macrophages. Therefore the function of MCO seems
to be limited to extracellular growth, during which the authors
hypothesize it to be involved in the protection against iron-related
oxidative stress.
Figure 1 | Overview of the L. pneumophila cell envelope. CP, cytoplasm;
IM, inner membrane; PP, periplasm; OM, outer membrane; OMVs, outer
Inner membrane proteins often regulate cytoplasmic processes
membrane vesicles; LPS, lipopolysaccharides; PAL, peptidoglycan-associated such as gene expression and the synthesis of signal transduction
lipoprotein; FeoB, iron transporter; PlaB, phospholipase A/lysophospholipase A; molecules. One example of this is LadC, a putative adenylate cyclase.
MOMP, major outer membrane protein; Mip, macrophage infectivity potentiator. L. pneumophila expresses the corresponding gene exclusively during

Table 1 | Inner membrane proteins of L. pneumophila associated with virulence and survival.

Protein Molecular function Role in infection/required for Reference

FeoB GTP-dependent Fe(II) transporter Macrophage killing, full virulence in mouse Petermann et al. (2010), Robey
and Cianciotto (2002)
IraA/IraB Small-molecule methyl transferase/ Iron uptake, infection of human Viswanathan et al. (2000)
peptide transporter macrophages, and guinea pigs
Multi-copper Potential oxidation of ferrous iron Extracellular replication Huston et al. (2008)
oxidase
LadC Putative adenylate cyclase Adhesion to macrophages, intracellular replication, Newton et al. (2008)
putative modification of protein functions via cAMP
TatB T2S, additional function(s) Intracellular replication in human macrophages, Rossier and Cianciotto (2005)
growth under iron-limiting conditions, cytochrome
c-dependent respiration, export of PLC activity to supernatant

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Shevchuk et al. Legionella pneumophila envelope

intracellular infection. Its importance for virulence is underlined Peptidoglycan fragments of many bacteria are recognized by
by the finding that a LadC-negative mutant adheres to macro- members the NLR family of receptors (Nucleotide-binding domain,
phages less effectively and replicates less within mammalian cells Leucine-Rich repeat-containing proteins) in the host cytosol.
and amoeba. In contrast to most other bacterial adenylate cyclases, Their activation leads to inflammatory responses. Interestingly,
LadC does not alter transcriptional profiles, so it is assumed that L. pneumophila cell extracts and culture supernatants activate two
LadC produces cAMP which, in turn, modifies protein–protein members of this family, NOD1 and NOD2 (Nucleotide-binding,
interactions or regulates protein activities (Newton et al., 2008). Oligomerization Domain-containing proteins 1 and 2), only to a
The inner membrane is crossed by the Tat complex which very small extent (Hasegawa et al., 2006). Why L. pneumophila is
can be involved in type II secretion. One of the Tat components, only weakly detected by NLRs and the details of its recognition by
TatB, was found to have additional, unexpected functions (Rossier the host has to be the subject of future investigations.
and Cianciotto, 2005). Firstly, intracellular replication in human The periplasm harbors many enzymes which degrade harm-
macrophages is impaired in the absence of TatB independently of ful substances that enter the bacterial cell. One example is the L.
type II secretion. Secondly, TatB-negative mutants are defective in pneumophila copper–zinc–superoxide dismutase (Cu–Zn–SOD).
growth under iron-limiting conditions, both extracellularly and It was shown that this enzyme is essential for survival in the sta-
within amoeba. Moreover, TatB of L. pneumophila is also required tionary growth phase. As Legionella has to survive for long periods
for cytochrome c-dependent respiration and finally for the export when no host is available, the Cu–Zn–SOD may aid the bacte-
of a specific phospholipase C activity to the culture supernatant – ria to overcome oxidative stress encountered during this period.
possibly executed by PlcA. Interestingly, copper–zinc oxidases occur in many eukaryotes, but
The inner membrane is also the starting point of several secre- only in very few bacteria such as Haemophilus influenzae, Brucella
tion systems. The unique Dot/Icm system of Legionella has been abortus, and Escherichia coli. A general involvement of this enzyme
reviewed very well elsewhere as has the type II secretion system class in microbial virulence is discussed (Schnell and Steinman,
(Cianciotto, 2009; Hubber and Roy, 2010). 1995; St John and Steinman, 1996).
In summary, the inner membrane of L. pneumophila influences The hydrogen peroxide which is produced by the Cu–Zn–SOD
virulence functions rather indirectly via the mediation of iron can be converted to H2O and O2 by the periplasmic katalase KatA.
acquisition and other cellular processes such as protein secretion. KatA and its cytoplasmic counterpart KatB are both required for
The contribution of inner membrane components to virulence optimal infection cycles in primary macrophages and amoeba
may emerge from enhanced survival under hostile conditions – (Bandyopadhyay et al., 2003). The authors propose a model in
and infection processes may just be an example for this. From this which KatA and KatB maintain a low intracellular H2O2 level, which
perspective, findings which relate survival factors to virulence may is required for optimal function of the Dot/Icm apparatus and
simply be due to the fact that host cells and tissues are examples of many other processes.
hostile environments to most bacteria. One of the Dot/Icm machinery components, IcmX, was local-
ized to the L. pneumophila periplasm. This protein is required for
Periplasm the establishment of the Legionella-containing vacuole and pore
The periplasmic space is a gel-like layer composed of soluble pro- formation in macrophage cell membranes, yet these effects are
teins and strongly crosslinked peptidoglycan located between the independent of an intact Dot/Icm apparatus. A truncated form
outer and inner membranes. It is enriched in proteases and nucle- of IcmX is secreted into culture supernatants, but not into the
ases and other degradative enzymes. Thus, the periplasm has been cytoplasm of host cells (Matthews and Roy, 2000). Intriguingly,
called an “evolutionary precursor of the lysosomes of eukaryotic a sequence near the C terminus of the IcmX gene is annotated to
cells” (Silhavy et al., 2010). contain a DNA polymerase domain of the POLBc superfamily.
The presence of digestive enzymes was confirmed by recent If this holds true, the purpose of a periplasmic DNA polymerase
L. pneumophila membrane proteome data. It has been shown remains to be clarified.
that mainly enzymes were found in the periplasm, such as met- Many periplasmic components contribute to L. pneumophila
alloproteases, phosphatases, isomerases, the periplasmic compo- virulence and some may be involved in bacterial protection against
nents of the Dot/Icm machinery and other proteins involved in immune defense mechanisms (see Table 2).
L. ­pneumophila virulence that promote penetration of host cells
(Cirillo et al., 2000; Khemiri et al., 2008). The outer membrane
Legionella pneumophila peptidoglycan was shown to contain The OM is the distinguishing feature of all Gram-negative bacteria.
muramic acid, glucosamine, glutamic acid, alanine, and meso- It is a lipid bilayer composed of phospholipids, lipoproteins, LPS,
diaminopimelic acid (meso-DAP). Interestingly, extremely strong and proteins. Phospholipids are located mainly in the inner leaflet
crosslinking was observed, with approximately 85% of meso-DAP of the outer membrane, as are the lipoproteins that connect the
and 90% of alanine residues contributing to these crosslinks. outer membrane to peptidoglycan. The outer membrane is the
Peptidoglycan was found to be partially resistant to lysozyme treat- location of mature LPS molecules and the shedding of OMVs.
ment. The stable peptidoglycan layer is likely to promote survival in The phospholipids of L. pneumophila were analyzed shortly
hostile environments (Amano and Williams, 1983). The important after the discovery of the bacteria (Finnerty et al., 1979). They
role of peptidoglycan in virulence is underlined by the finding that are, in decreasing order of concentration, phosphatidylcholine,
DAP-auxotroph L. pneumophila mutants display impaired survival phosphatidylethanolamine, cardiolipin, monomethylphosphati-
within macrophages and amoeba (Amano and Williams, 1983). dylethanolamine, phosphatidylglycerol, and dimethylphosphati-

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Shevchuk et al. Legionella pneumophila envelope

dylethanolamine. It remains unclear whether the lipid composition A bioinformatic approach proposed around 250 proteins
of the outer membrane differs significantly from that of the inner in the L. pneumophila OM, however, most of their functions
membrane (Hindahl and Iglewski, 1984; Gabay and Horwitz, 1985). still need to be elucidated (Khemiri et al., 2008). With few
The discovery of phosphatidylcholine is striking as only about exceptions, the proteins of the OM can be divided into two
10% of all known bacteria contain this lipid in their membranes classes, lipoproteins and β-barrel proteins. Lipoproteins have
– mostly those bacteria that are closely associated with eukaryotes. lipid moieties attached to an amino-terminal cysteine residue
Examples include Pseudomonas aeruginosa, Agrobacterium tumefa- (Sankaran and Wu, 1994). β-barrel proteins are nearly all inte-
ciens, and B. abortus. Nevertheless the exact function of this phos- gral membrane proteins of the outer membrane. Most outer
pholipid in bacterial cell envelopes remains unknown (Sohlenkamp membrane proteins are involved in either attachment or inva-
et al., 2003). Intriguingly, the loss of phosphatidylcholine from the L. sion of host cells. Both classes of proteins are in direct contact
pneumophila envelope causes reduced cytotoxicity and lower yields of with the environment and host cells. They are therefore prefer-
bacteria within macrophages (Conover et al., 2008). Additionally the ential targets for vaccine development as well as for diagnosis
strains lacking this lipid bind to macrophages less effectively. Recently (Silhavy et al., 2010).
it was shown that Legionella bozemanae synthesizes phosphatidylcho- One such example is the 19-kDa peptidoglycan-associated
line from exogenous choline (Palusinska-Szysz et al., 2011). lipoprotein (PAL) which is a species-common immunodominant
In addition to phospholipid species, the fatty acid composition antigen for the diagnosis of Legionnaires’ disease (Kim et al., 2003;
of membranes also influences bacterial properties. In the station- Shim et al., 2009). This protein activates murine macrophages via
ary growth phase of L. pneumophila, the proportion of branched- toll-like receptor 2 (TLR2) and induces the secretion of proinflam-
chain fatty acids rises to over 60% and the average length of fatty matory cytokines such as IL-6 and TNF-α (Table 3).
acids in phospholipid molecules decreases compared to exponential Three of the T4SS components (DotD, DotC, IcmN) contain
growth. This change in fatty acid composition leads to an increased a lipobox motif at their N terminus and are predicted to be lipo-
tolerance to the antimicrobial peptide warnericin RK (Verdon et al., proteins. DotD and DotC are essential for bacterial intracellular
2011). The contribution of fatty acids to Legionella infection proc- survival (Yerushalmi et al., 2005; Nakano et al., 2010).
esses is still unknown. Future studies will shed more light on the For L. pneumophila, several outer membrane proteins are char-
influence of lipids on membrane protein structures, localization, acterized as important virulence factors. An example of an outer
and functions. In addition, the existence of distinct lipid domains membrane-associated and at least partially surface-exposed protein
in L. pneumophila membranes has not been described so far. with virulence functions is PlaB (major cell-associated phospholi-

Table 2 | Periplasmic proteins of L. pneumophila associated with virulence and survival.

Protein Molecular function Role in infection/required for Reference

Copper–zinc– Detoxification of Stationary growth survival St John and Steinman (1996)

superoxide dismutase superoxide radicals
KatA Degradation of H2O2 Optimal infection of macrophages and Bandyopadhyay et al. (2003)
amoeba (optimal function of the Dot/Icm apparatus)
IcmX Putative DNA polymerase Establishment of the Legionella-containing vacuole, Matthews and Roy (2000)
(POLBc superfamily) pore formation in macrophage cell membranes

Table 3 | Outer membrane proteins of L. pneumophila associated with virulence and survival.

Protein Molecular function Role in infection/required for Reference

PAL Activation of murine macrophages via Kim et al. (2003), Shim et al. (2009)
TLR2, induction of the secretion of
proinflammatory cytokines such as IL-6 and TNF-α
DotD, DotC, IcmN Intracellular survival Nakano et al. (2010),
Yerushalmi et al. (2005)
PlaB Phospholipase A/ Contact-dependent hemolytic activity and plays Schunder et al. (2010)
lysophospholipase A an important role in guinea pig infection
MOMP Porin attachment to host cells Bellinger-Kawahara and Horwitz
(1990), Krinos et al. (1999)
Hsp60 Attachment to and invasion of a HeLa cell Garduño et al. (1998), Hoffman
and Garduño (1999)
Mip Peptidyl–prolyl cis/ Efficient replication within host cells and transmigration Wagner et al. (2007), Debroy et al. (2006)
trans isomerase across an in vitro model of the lung epithelial barrier
Lcl Collagen-like protein Adherence to and invasion of host cells Vandersmissen et al. (2010)

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Shevchuk et al. Legionella pneumophila envelope

pase A/lysophospholipase A). It displays contact-dependent hemo-

lytic activity and plays an important role in guinea pig infection
(Schunder et al., 2010).
The L. pneumophila major outer membrane protein (MOMP)
is involved in the attachment to host cells (Gabay et al., 1985;
Bellinger-Kawahara and Horwitz, 1990; Mintz et al., 1995; Krinos
et al., 1999). The heat shock protein Hsp60 is also important for
attachment to and invasion of a HeLa cell model (Garduño et al.,
1998; Hoffman and Garduño, 1999).
Mip, the macrophage infectivity potentiator, is a membrane-
associated homodimeric protein that is mainly found on the bac-
terial surface (Riboldi-Tunnicliffe et al., 2001). The C-terminal
domain of Mip displays peptidyl–prolyl cis/trans isomerase
(PPIase) activity. It is related to the human FK506-binding pro-
tein and binds to collagen of types I, II, III, IV, V, and VI. The
protein is necessary for efficient replication within host cells.
Interestingly, it is also required for the transmigration of L. pneu-
mophila across an in vitro model of the lung epithelial barrier
(Wagner et al., 2007).
The substrate of Mip and its exact function in virulence have
not been identified yet. A step toward this goal was the finding that
Mip is required for the extracellular release of an phospholipase
C-like activity. Mip may mediate this by activating the secreted
enzyme – and potentially other proteins – directly after secretion
of one of the secretion machinery components by its PPIase activity
(Debroy et al., 2006).
The Legionella collagen-like protein Lcl contains an outer mem-
Figure 2 | Chemical structure of L. pneumophila LPS (modified from
brane motif and was shown to contribute to the adherence and Kooistra et al., 2002b). Structure indicates its various regions: O-specific chain,
invasion of host cells. Interestingly, the number of repeat units core region consisting of the outer core and inner core and lipid A. Leg, derivatives
present in the lcl gene has an influence on these adhesion charac- of legionaminic acid; 4e-Leg, derivatives of 4-epilegionaminic acid; Rha, rhamnose;
teristics (Vandersmissen et al., 2010). Man, mannose; QuiNAc, acetylquinovosamine; GlcNAc, acetylglucosamine; Kdo,
3-deoxy-d-manno-oct-2-ulosonic acid; P, phosphate; OAc, O-acetyl.
In summary, the outer membrane is the direct interface between
L. pneumophila and its host organisms. Some of its proteinaceous
components are directly involved in adhesion and invasion proc- core constitute the polysaccharide region of the LPS, whereas lipid
esses (Table 3). The influence of lipid composition on the functions A represents the part of the molecule which anchors the LPS in the
of OM virulence factors remains to be elucidated. outer membrane.
The O-chain of L. pneumophila LPS is a homopolymer of
L. pneumophila LPS the unusual sugar 5-acetamidino-7-acetamido-8-O-acetyl-
Lipopolysaccharides are located in the outer leaflet of the outer 3,5,7,9-tetradeoxy-l-glycero-d-galacto-nonulosonic acid, termed
membrane and they are a major immunodominant antigen of ­legionaminic acid (Palusinska-Szysz and Russa, 2009). This sugar
Legionella. Based on O-antigen architecture, the species L. pneu- molecule completely lacks free hydroxyl groups and is therefore
mophila can be divided into at least 15 serogroups (Helbig and very hydrophobic (Knirel et al., 1994; Helbig et al., 1995; Zähringer
Amemura-Maekawa, 2009). Within each serogroup, so-called et al., 1995; Kooistra et al., 2002a). The core region consists of the
monoclonal subgroups can be defined. For example, serogroup outer core and the inner core. The outer core of L. pneumophila
1 can be divided into 10 subgroups (Ciesielski et al., 1986). The is a oligosaccharide composed of rhamnose (Rha), mannose
species L. pneumophila accounts for about 90% of the cases of (Man), acetylquinovosamine (QuiNAc), and acetylglucosamine
legionellosis, and about 85% of these are caused by members of GlcNAc (Knirel et al., 1996, 1997). Like the O-chain it also exhib-
serogroup 1 (Helbig et al., 2002; Doleans et al., 2004; Gosselin et al., its hydrophobic properties, in contrast to the inner core, which is
2010; Napoli et al., 2010). For this reason, most researchers focus on hydrophilic. The inner core oligosaccharide of L. pneumophila LPS
serogroup 1, and this chapter, too, describes the chemical structure is characterized by a 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)
and functions of L. pneumophila serogroup 1 LPS. disaccharide [α-Kdo-(2α4)-α-Kdo-(2α6)] linked to lipid A which
In comparison to the LPS of other Gram-negative bacteria, the is conserved within many Gram-negative bacteria and is essential
L. pneumophila LPS has a unique structure. Due to high levels of for microbial growth (Moll et al., 1997).
long, branched fatty acids, and elevated levels of O- and N-acetyl Lipid A of L. pneumophila serogroup 1 contains unusual long-
groups, this LPS is highly hydrophobic (Figure 2). LPS molecules chain and branched fatty acids, which may be responsible for its low
consist of the O-specific chain, the core region, and the lipid A endotoxic potential (Moll et al., 1992; Neumeister et al., 1998). The
component, which is also called endotoxin. The O-chain and the structural function of lipid A is anchoring the LPS in the bacterial

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Shevchuk et al. Legionella pneumophila envelope

membrane. Unlike in other Gram-negative bacteria, L. pneumophila palmitoylation is believed to promote resistance to CAMPs by
lipid A does not function as a classical endotoxin. It was demon- decreasing membrane fluidity and preventing insertion of the
strated that L. pneumophila LPS is about 1000 times less potent in peptides (Guo et al., 1998; Bishop et al., 2000; Robey et al., 2001;
its ability to induce the secretion of proinflammatory cytokines Soderberg and Cianciotto, 2010). The structural modification of
from human monocytic cells than LPS of members of the family lipid A might help the bacteria to resist CAMPs released by the
Enterobacteriaceae (Neumeister et al., 1998). host immune system, or to evade recognition by TLR4, the innate
immune receptor. L. pneumophila rcp influences virulence and the
LPS biosynthesis and transport adaptation to Mg2+-limiting conditions (Wang and Quinn, 2010).
The biosynthesis of LPS is a complex process involving various After synthesis on the cytoplasmic face, both core-lipid A and
steps that occur at the inner membrane, following by assembly in O-antigen need to be transported to the periplasmic face of the
the periplasm and translocation of LPS molecules to the bacterial inner membrane. Little is known about the mechanisms of LPS
cell surface. polymerization and translocation in Legionella. After attach-
The genes involved in core oligosaccharide and O-chain bio- ment of the core, nascent core-lipid A is most probably flipped
synthesis are mainly localized on a 30-kb gene locus (Lüneberg to the periplasmic face of the inner membrane by the ABC trans-
et al., 2000). The excision of this region from the chromosome porter MsbA, where the O-antigen polymer is attached (Doerrler
leads to an alteration of the LPS epitope and a loss of virulence et al., 2004). Transport of the O-antigen may occur through
(Lüneberg et al., 1998, 2001). The L. pneumophila LPS gene locus an Wzt/Wzm ABC transporter. In all analyzed L. pneumophila
includes genes with products which are likely to be involved in LPS genomes, we have found Wzt and Wzm genes (Table 4). Wzm
core oligosaccharide biosynthesis (rmlA-D, glycosyltransferases, forms a channel in the inner membrane for the passage of the
acetyltransferase) as well as O-chain biosynthesis and transloca- lipid-linked O-antigen, and Wzt provides energy through its
tion (mnaA, neuB, neuA, wecA, wzt, wzm). The genes involved in ATPase activity (Lüneberg et al., 2000).
LPS biosynthesis and translocation and its distribution among It is not known how Legionella LPS is transported from the
five sequenced and annotated L. pneumophila genomes are sum- periplasm to the outer leaflet of the OM. Recently it was shown that
marized in Table 4. Interestingly, the gene cluster coding for the in E. coli the LptD/LptE complex performs this function (Ma et al.,
determinants of serogroup 1 LPS is present in diverse serogroups, 2008). The homolog of LptD/LptE was found in L. pneumophila,
suggesting that it is mobile and can be exchanged by horizontal therefore it can be speculated that the transport of LPS occurs by
gene transfer (Cazalet et al., 2008). The region encoding proteins a related mechanism.
involved in LPS biosynthesis can be subdivided in two blocks of
13 and 20 kb. Most of the genes in the 13-kb block are present in Functions of the Legionella LPS
all L. pneumophila strains, whereas the majority of genes in the Members of the TLR family in cells of the innate immune system
20-kb block are specific for serogroup 1. Three genes, coding for recognize specific conserved components of microbes, including
two O-antigen transporters (wzt and wzm) and one hypotheti- LPS. This initiates the cascade of the inflammatory response and
cal protein, might be used as markers for Legionella serogroup 1 activates adaptive immunity through the induction of cytokine
(Cazalet et al., 2008; Merault et al., 2010). production and synthesis of co-stimulatory molecules. LPS can
Lipid A can be modified, a process which alters the physical be recognized by TLR4, a receptor found on the surface of dif-
properties of the outer membrane (Albers et al., 2007). Some of ferent immune cells such as macrophages, neutrophils, and den-
these modifications are known to be under the control of the PmrA/ dritic cells (Mintz et al., 1992; Akira et al., 2001). The correlation
PmrB and/or the PhoP/PhoQ two-component systems in other between a TLR4 polymorphism and its influence on susceptibility
Gram-negative organisms (Miller et al., 1989; Guo et al., 1997, to Legionnaires’ disease was reported (Hawn et al., 2005). It is
1998; Gunn et al., 1998). Despite the detailed characterization of interesting to note that L. pneumophila requires TLR2 rather than
the PmrA/PmrB two-component system of L. pneumophila and its TLR4 to elicit the expression of CD14, which acts as a co-receptor
influence on gene expression of most of virulence determinants, for the detection of bacterial LPS. It is hypothesized that long-
the role of the this system in lipid A modification in Legionella has chain fatty acids and the high hydrophobicity of L. pneumophila
not yet been analyzed (Al-Khodor et al., 2009; Hovel-Miner et al., lipid A can abolish the interaction with the soluble LPS receptor
2009; Rasis and Segal, 2009). CD14 and the ability of LPS molecules to activate bone marrow
PhoP/PhoQ is a two-component system which regulates a cells (Neumeister et al., 1998; Girard et al., 2003). Remarkably, L.
number of lipid A-modifying enzymes in Salmonella enterica sero- pneumophila is known to up-regulate both, TLR2 and TLR4, and to
var Typhimurium. It was not detected in genomes of the genus activate CD40, CD86, and MHC class I/II molecules on dendritic
Legionella (Gibbons et al., 2005). Nevertheless, it is conceivable cells (Rogers et al., 2007).
that analogs with low protein homology or other two-component Recently it was demonstrated that LPS of L. pneumophila shed
systems regulate lipid A-modifying enzymes. One of the genes in liquid culture is able to arrest phagosome maturation in amoeba
which is transcriptionally activated by the PhoP/PhoQ system in and human macrophages. In particular, the presence of high-
Salmonella is pagP (Kawasaki et al., 2004). The inactivation of this molecular-weight LPS correlates with the inhibition of phagosome–
gene leads to a decreased resistance to cationic antimicrobial pep- lysosome fusion (Seeger et al., 2010). Another group has shown
tides (CAMPs). The Legionella homolog of pagP is called resist- that L. pneumophila LPS specifically interacts with pulmonary
ance to CAMPs (rcp) and functions as a palmitoyl transferase, collectins and surfactant proteins A and D, which play important
which transfers palmitate to lipid A molecules. The increased roles in innate immunity in the lung. The authors also propose that

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Shevchuk et al. Legionella pneumophila envelope

Table 4 | Paralogs of LPS biosynthesis and translocation proteins in L. pneumophila strains.

Enzyme Molecular function L. pneumophila strains

Corby Philadelphia-1 Lens Paris 2300/99 Alcoy

Lipid A biosynthesis
LpxA UDP–N-acetylglucosamine acyltransferase LPC_2835 Lpg0511 Lpl0549 Lpp0573 Lpa_00769
LPC_3254 Lpg2943 Lpl2874 Lpp3016 Lpa_04308
LpxC UDP–3-O-[3-hydroxymyristoyl] LPC_0533 Lpg2608 Lpl2531 Lpp2661 Lpa_03814
N-acetylglucosamine deacetylase
LpxD UDP–3-O-[3-hydroxymyristoyl] LPC_0119 Lpg0100 Lpl0100 Lpp0114 Lpa_00149
glucosamine N-acyltransferase LPC_2837 Lpg0508 Lpl0547 Lpp0571 Lpa_00766
LPC_3255 Lpg2944 Lpl2873 Lpp3015 Lpa_04309
LpxH UDP–2,3-diacylglucosamine hydrolase LPC_0973 Lpg1552 Lpl1474 Lpp1509 Lpa_02254
LpxB Lipid A disaccharide synthase LPC_0787 Lpg1371 Lpl1322 Lpp1325 Lpa_02021
LPC_3256 Lpg2945 Lpl2872 Lpp3014 Lpa_04311
LpxK Tetraacyldisaccharide 4′-kinase LPC_1262* Lpg1818* Lpl1782* Lpp1781* Lpa_02629*
Tetraacyldisaccharide-1-P-4′-kinase LPC_1374 Lpg1920 Lpl1884 Lpp1895 Lpa_02777
KdtA (WaaA) 3-Deoxy-d-manno-oct-2-ulosonic LPC_1808 Lpg2340 Lpl2261 Lpp2288 Lpa_03350
acid transferase
LpxL (WaaM) Lipid A acyltransferase LPC_2981 Lpg0363 Lpl0404 Lpp0428 Lpa_00577
LPC_3251# Lpg2940# Lpl2870# Lpp3012# Lpa_04304#
LPC_3252# Lpg2941# Lpl2871# Lpp3013# Lpa_04305#
Core region biosynthesis
WaaQ Heptosyl transferase LPC_0441 lpg2695 Lpl2622 Lpp2749 Lpa_03933
RmlA (RfbA) Glucose-1-phosphate LPC_2532 Lpg0760 Lpl0797 Lpp0826 Lpa_01168
thymidylyltransferase
RmlB (RfbB) dTDP–glucose 4,6-dehydratase RmlB LPC_2534 Lpg0758 Lpl0795 Lpp0824 Lpa_01166
RmlC dTDP–4-dehydrorhamnose 3,5-epimerase LPC_2536 Lpg0756 Lpl0793 Lpp0822 Lpa_01164
RmlD dTDP–6-deoxy-l-mannose dehydrogenase LPC_2535 Lpg0757 Lpl0793 Lpp0823 Lpa_01165
Glycosyltransferase LPC_2515 Lpg0779 Lpl0818 Lpp0843 Lpa_01190
Glycosyltransferase LPC_2516 Lpg0778 Lpl0817 Lpp0842 Lpa_01189
O-Chain biosynthesis
KdsA (NeuB) 3-Deoxy-d-manno-octulosonic LPC_0649 Lpg1182 Lpl1191 Lpp1185 Lpa_01838
acid (KDO) 8-phosphate synthase
HAD superfamily transporter hydrolase LPC_2456 Lpg0839 Lpl0870 Lpp0901 Lpa_01272
KdsB 3-Deoxy-manno-octulosonate LPC_1373 Lpg1919 Lpl1883 Lpp1894 Lpa_02777
GmhA Phosphoheptose isomerase LPC_3308 Lpg2993 Lpl2921 Lpp3064 Lpa_04384
HisB d,d-heptose 1,7-bisphosphate phosphatase LPC_1283 Lpg1838 Lpl1803 Lpp1802 Lpa_02656
WecE Aminotransferase, predicted pyridoxal LPC_0840 Lpg1424 Lpl1375 Lpp1379 Lpa_02088
phosphate-dependent enzyme
Lag-1 O-Acetyltransferase, acetylation LPC_2517 Lpg0777 Lpl0816 Lpp0841 Lpa_01188
of the O-polysaccharide
NeuC (NnaA) N-Acylglucosamine 2-epimerase LPC_2539 Lpg0753 Lpl0790 Lpp0819 Lpa_01161
NeuB N-Acetylneuraminic acid synthetase LPC_2540 Lpg0752 Lpl0789 Lpp0818 Lpa_01160
LPC_2524 Lpg0768 Lpg0809 Lpp0833 Lpa_01177
NeuA CMP–N-acetylneuraminic acid synthetase LPC_2541 Lpg0751 Lpl0788 Lpp0817 Lpa_01159
WecA O-Antigen initiating glycosyl transferase LPC_2530 Lpg0762 Lpl0799 Lpp0828 Lpa_01171
LPS translocation
MsbA Lipid A export ATP-binding/permease protein MsbA LPC_1263* Lpg1819* Lpl1783* Lpp1782* Lpa_02631*
Wzt** LPS O-antigen ABC transporter Wzt LPC_2519 Lpg0773 Lpl0814 Lpp0838 Lpa_01186
Wzm** LPS O-antigen ABC transporter Wzm LPC_2520 Lpg0772 Lpl0813 Lpp0837 Lpa_01184

The protein paralogs share a high level of homology. In general they have 96–100% of identity and 97–100% of positivity.
#
Indicates the proteins with lower homology (73–90%).
*The lpxK–msbA cluster exists in many Gram-negative bacteria. MsbA is known as a specific transporter, which exports core–lipid A from the cytoplasmic to the
periplasmic face of the inner membrane, while LpxK phosphorylates the 4′-position of lipid A.
**The genes wzm and wzt are specific for the Sg1 LPS gene cluster and can be used for rapid detection of L. pneumophila Sg1 in clinical and environmental isolates
(Cazalet et al., 2008).

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Shevchuk et al. Legionella pneumophila envelope

this interaction promotes the localization of L. pneumophila to an influenced by many environmental factors and is controlled by
acidic compartment, i.e., lysosomes, and intracellular growth of the a hierarchical cascade of regulators (Albert-Weissenberger et al.,
bacteria is subsequently inhibited (Sawada et al., 2010). 2010). The transition of the bacteria to the transmissive phase is
Interestingly, the LPS pattern of L. pneumophila grown in co-regulated with the expression of flagella. Regulators that control
broth has been found to be different from the pattern of bacteria flagellation also control important virulence traits such as lysosome
grown intracellulary in Acanthamoeba polyphaga (Barker et al., avoidance and cytotoxicity (Gabay et al., 1986; Byrne and Swanson,
1993). Moreover, during exponential growth, L. pneumophila LPS 1998; Molofsky et al., 2005). On the other hand cytosolic flagel-
is much more hydrophobic than in post-exponential cultures lin is described to trigger the macrophage response to a L. pneu-
(Seeger et al., 2010). mophila infection. This mechanism is mediated by Naip5/Birc1e,
In general, L. pneumophila LPS plays a crucial role in interaction a member of the NLR family. It activates the caspase-1-dependent
with host cells and modulation of intracellular trafficking, inde- cell-death pathway that restricts bacterial growth (Molofsky et al.,
pendently of the Dot/Icm secretion system. The unusual structure 2006; Ren et al., 2006). Information on the L. pneumophila flagel-
of lipid A might help the bacteria to avoid recognition by the innate lum is excellently reviewed in a recent publication (Heuner and
immune system. Albert-Weissenberger, 2008).

Flagella and pili Outer membrane vesicles

The first evidence of the presence of flagella and pili structures on Outer membrane vesicles are shed from the outer membrane by
the L. pneumophila surface was provided by Rodgers et al. (1980). L. pneumophila and most other Gram-negative bacteria. They are
The authors were also the first to observe that pili of L. pneumophila between 100 and 250 nm in diameter and consist of components
vary in length. Later the pili were divided into long (0.8–1.5 μm) from the outer membrane, including LPS, and the periplasm
and short (0.1–0.6 μm) forms (Stone and Abu Kwaik, 1998). The (Figure 3). L. pneumophila OMVs contain a disproportionately
PilE protein is the constituent of long type IV pili. It is involved high number of virulence-associated proteins and display lipolytic
in attachment and adherence to host cells as well as natural com- and proteolytic activities (Galka et al., 2008).
petence of L. pneumophila. At the same time a mutation in the In general, OMVs from other bacteria can mediate interbacte-
pilE gene does not affect the intracellular survival and replication rial contact and also the contact to eukaryotic cells. They can kill
of bacteria (Stone and Abu Kwaik, 1998; Stone and Kwaik, 1999). other bacteria by the delivery of harmful factors. Macromolecule-
Another protein responsible for type IV pili production is the degrading enzymes in association with OMVs can promote nutri-
prepilin peptidase PilD. Unlike PilE, PilD is important for suc- ent acquisition, i.e., by cleaving proteins into amino acids which are
cessful intracellular proliferation. This protein is also involved in then taken up by the bacterium. Modulations of biofilm formation
type II secretion activity (Aragon et al., 2000). Both mentioned and quorum sensing functions have also been assigned to OMVs.
pili proteins facilitate the formation of biofilms of L. pneumophila In the interaction with eukaryotic host cells, OMVs can deliver
(Lucas et al., 2006). Interestingly, despite microscopic evidence for toxins and other virulence factors and have been shown to adhere
the presence of the pili in liquid culture, some fimbrial synthesis to cell surfaces. In addition, the immune response – cytokine pro-
genes are induced only in host cells (Bruggemann et al., 2006). files, inflammation, innate immunity – is modified by contact to
Additionally to pili, L. pneumophila exhibits a single monopo- bacterial OMVs (Ellis and Kuehn, 2010).
lar flagellum, which is anchored within both membranes (OM, So far, OMVs of L. pneumophila have been studied to a lower
IM) and peptidoglycan by the basal body. This organelle plays an extent. A proteomic analysis revealed 74 different proteins. The
important role in cell motility, adhesion, and host invasion. It has export of 33 of these proteins occurs only via OMVs, but not indi-
also been described to be involved in biofilm formation (Heuner vidually via type II, III, or IV secretion systems. Of these OMV-
and Albert-Weissenberger, 2008). The expression of flagella is specific proteins, 18 are reported or predicted to contribute to

Figure 3 | Electron micrographs of L. pneumophila and outer membrane vesicles. L. pneumophila sheds OMVs (arrows) from its surface during growth in
liquid media (A) and within phagosomes of Dictyostelium discoideum (B).

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Shevchuk et al. Legionella pneumophila envelope

pathogenesis and virulence (Galka et al., 2008). This finding led Conclusion
to the conclusion that L. pneumophila employs OMVs as vehicles Legionella pneumophila inhabits fresh waters and biofilms.
for the transport of virulence factors toward the environment. Moreover, this pathogen parasitizes phylogenetically dis-
The exact mode of interaction of L. pneumophila OMVs and tant hosts such as protozoa and human cells, a process which
host cell surfaces remains to be elucidated. However, an associa- requires ­adhesion, invasion, and interactions within the phago-
tion of OMVs to the cytoplasmic membrane of human alveolar some. Under all these conditions the bacterial cell envelope is
epithelial cells has been shown. The contact between OMVs and the prime structure through which L. pneumophila interacts
the cells resulted in a change in cell morphology, leading to round with these fundamentally different environments. Although the
cells (Galka et al., 2008). potential properties of the cell envelope are ultimately deter-
Outer membrane vesicles can also elicit a specific cytokine mined by the information stored within the genome, it becomes
response from alveolar epithelial cells, resulting in the release of increasingly evident that molecular identities, spatial distribu-
interleukins-6, -7, -8, and -13 as well as G-CSF, IFN-γ, and MCP-1. tions, and biochemical activities of many envelope constituents
IL-7 and IL-8 are secreted only after stimulation with OMVs, but are highly dynamic and vary with L. pneumophila growth phases,
not after stimulation with individually secreted proteins. developmental differentiation processes as well as during the
Legionella pneumophila OMVs increase the growth of Acanthamoeba pathogen–host interaction. Therefore, the investigation of phe-
castellanii over the course of 72 h, rather than damaging the host cells notypic changes, which take place as the bacteria adapt to dif-
(Galka et al., 2008). As A. castellanii usually feeds on bacteria, ­membrane ferent conditions, holds great promise for the understanding of
vesicles are thought to serve as a source of nutrients, ­possibly to attract this pathogen. Proteins, carbohydrates, and lipids in the bacterial
amoeba toward bacteria, which then infect them. cell envelope serve both structural and signaling roles, but until
Latex beads which have been coated with L. pneumophila OMVs recently the main focus of biomedical research was on identifica-
can inhibit the fusion of Legionella-containing phagosomes to tion and analysis of proteins. Hereby we have learned that already
lysosomes, thereby preventing death of the bacteria (Fernandez- characterized proteins can have unexpected functions, suggesting
Moreira et al., 2006). This key feature of Legionella infections seems the need for more thorough investigations. Based on the cur-
to be mediated by OMVs, at least to a certain degree. The LPS on rent body of information there is also increased awareness that
the surface of OMVs is regulated similarly to LPS on the outer lipids, both of host and bacterial origin, choreograph pathogen
membrane. The phagolysosomal arrest is evoked more strongly by stability and host susceptibility to infection. The renewed inter-
soluble LPS shed into the bacterial surrounding. The arrest effi- est in these historically neglected effector molecules is currently
ciency seems to decrease over time (Seeger et al., 2010). fueled by the advances in lipidomics and glycomics technologies.
The inhibition of the fusion between phagosomes and lysosomes is Thus, identification of unique lipid entities and their biological
only one of the functions of L. pneumophila OMVs. They also display activities represent an enormously promising new frontier in the
proteolytic and lipolytic activities, though the fraction of individually infection biology of L. pneumophila.
secreted proteins features stronger degradative enzyme activities (Galka
et al., 2008). In this way, OMVs might contribute to the dissemination Acknowledgment
of the infection across tissue barriers such as the alveolar epithelium. This work was supported by the Deutsche Forschungsgemeinschaft
In conclusion, OMVs are believed to be a vehicle for the trans- (DFG) and the Federal Ministry of Education and Research
port of virulence factors to distant cells or host tissues. Their precise (BMBF). Jens Jäger is supported by the Foundation of German
contribution to L. pneumophila infections has not been determined Business (Stiftung der Deutschen Wirtschaft). The authors would
yet, but is under investigation. like to thank Simon Döhrmann for help with the images.

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