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Institut Pasteur, Unité des Interactions Bactéries Cellules, Paris F-75015, France; INSERM, U604, Paris F-75015, France; and INRA, USC2020, Paris F-75015, France
SUMMARY INTRODUCTION THE L. MONOCYTOGENES CELLULAR ENVELOPE Membrane Peptidoglycan Cell Wall Secondary Polymers: Teichoic Acids and Lipoteichoic Acids CLASSIFICATION OF LISTERIAL SURFACE PROTEINS ACCORDING TO THEIR ANCHORING MECHANISMS Proteins Covalently Linked to the Peptidoglycan: Sortase Substrates LPXTG sorting signal. NXXTX sorting signal. Proteins with Noncovalent Association to the Cell Wall GW modules. WxL domain. LysM domain. Peptidase peptidoglycan binding domain. Membrane-Bound Proteins Proteins with hydrophobic tails. Lipoproteins. Nonconventional Secreted Surface Proteins FUNCTIONS OF SURFACE PROTEINS AND THEIR RELEVANCE IN PATHOGENESIS Cell Wall Metabolism Autolysins. (i) N-Acetylglucosaminidases and N-acetylmuramidases. (ii) Amidases. (iii) gamma-D-Glutamyl-(L)-meso-diaminopimelate peptidases. (iv) Phage endolysins. PBPs: transglycosylases, transpeptidases, ß-lactamases, and D-alanyl-D-alanine carboxypeptidases. Deacetylase. Enzymes involved in assembly and modification of TAs and LTAs. Protein Processing, Folding, and Anchoring Adhesion to Host Cells Collagen binding domain. Fibronectin binding domain. Proteoglycan binding domain. RGD motif. Mucin binding domains. Ig-like fold domains. (i) CnaB-like repeat domain. (ii) LRR-adjacent Ig-like domain. (iii) PKD repeat domain. (iv) Big 3 domain. Invasion Motility Flagellar movement. Intracellular movement. Other Functions Noninvasin internalin-like proteins. Transporters. CONCLUSIONS AND FUTURE DIRECTIONS Pathogenic Potential Spatio-Temporal Expression Localization Strain and Species Variability ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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To annotate protein domains, we have combined sequence information from the database dedicated to the analysis of the genomes of L. monocytogenes (strain EGDe) and of its nonpathogenic relative Listeria innocua (strain CLIP 11262) (http://genolist.pasteur.fr/ListiList [67]) with that from the Pfam database (http://www.sanger.ac.uk/Software/Pfam [11]). We also used information from the NCBI Conserved Domain database (194) and the Interpro database (98). Sequence comparisons were performed using a PSI-BLAST analysis.
| THE L. MONOCYTOGENES CELLULAR ENVELOPE |
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90 Å thick and is composed of 55 to 60% protein, 30 to 35% lipid, and 1.3 to 2.3% carbohydrate (glucose, galactose, ribose, and arabinose) (65). Lipids include 80 to 85% phospholipids, such as phosphatidylglycerol, diphosphatidylglycerol, and phosphoglycolipid (185). The L. monocytogenes membrane fatty acid composition is dominated to an unusual extent by branched-chain fatty acids (>90% of the total fatty acid content) (6) and is modulated upon temperature variation. For instance, upon cold shock the anteiso-C15:0 content rises to maintain optimal membrane fluidity (6, 51, 132). However, nothing is yet known about the variation of the plasma membrane composition during Listeria cellular infection and how it affects anchoring of membrane proteins. 
-D-glutamyl-meso-diaminopimelyl-D-alanine-D-alanine, are bound to the MurNAc residue and are connected by a direct link between the D-Ala residue of one lateral peptide and the meso-diaminopimelyl residue of the other stem peptide (44, 57, 89, 162). Partial deacetylation of GlcNAc residues by PgdA enzyme is another characteristic of Listeria peptidoglycan (21, 89) (see below). TAs are electronegative polymers of ribitol-phosphates substituted with D-Ala residues and diverse sugars, which vary depending on the serotype (57-59, 145, 155). For instance, in serovar 1/2 (e.g., serotypes 1/2a and 1/2b), GlcNAc and rhamnose are present as substituents on the ribitol, whereas in serovar 4, GlcNAc is integral to the TA chains. Interestingly, serotype 4b strains are unique in bearing both galactose and glucose substituents on the GlcNAc of TA. This particularity is related to the presence in these strains of specific genes, such as gtcA, gltA, and gltB (106, 144).
The distinct glycosylations of TAs are probably of importance for their specific recognition by listerial proteins or phage endolysins (113) (see below). LTAs are polymers of glycerophosphate substituted with Ala, galactose, and lipid residues. The glycerophosphate chain of LTAs is uncovalently anchored to the outer leaflet of the plasma membrane through a lipid anchor, composed of galactose bound to glycerol and substituted with fatty acids (69, 79, 145). The LTAs are both membrane associated and secreted in the growth medium (58). Their X-ray structure suggests that they adopt a compact, organized micellar form with a length of 10 to 20 nm (101).
| CLASSIFICATION OF LISTERIAL SURFACE PROTEINS ACCORDING TO THEIR ANCHORING MECHANISMS |
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Through extensive in silico analysis of the genome of L. monocytogenes EGDe, Trost et al. (179) were able to predict a total of 525 sequences coding for proteins carrying a signal peptide, which corresponds to 18.4% of the total number of protein-coding genes. Among them, the "surface proteins" discussed in this review are those carrying motifs enabling strong or weak interactions with at least one component of the bacterial envelope (Fig. 1).
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NXXTX sorting signal. A second sortase type, sortase B (SrtB), was originally identified in S. aureus and specifically recognizes and cleaves a sorting signal different from LPXTG (121, 124). Staphylococcal SrtB anchors its only known substrate, IsdC, by cleaving a C-terminal NPQTN motif, between threonine and asparagine, and linking threonine to the pentaglycine cross bridges. The genes encoding SrtB and IsdC are both part of the isd locus, which is involved in bacterial heme iron uptake (121, 124). L. monocytogenes has also an alternative sortase B, whose coding sequence maps to an operon containing two genes encoding putative substrates, Lmo2185 (formerly SvpA) and Lmo2186 (16). Both proteins share homology to S. aureus IsdC and bear as putative sorting motifs NAKTN (Lmo2185) or NKVTN or NPKSS (Lmo2186), respectively. Inactivation of srtB impairs sorting of Lmo2185 (16). Lmo2185 and Lmo2186 are the only two SrtB substrates present in the L. monocytogenes cell wall proteome, consistent with the fact that there is no other protein with an NXXTX sorting signal encoded in the L. monocytogenes genome (146).
Work with S. aureus and analysis of other complete gram-positive genomes suggest that SrtB enzymes may have evolved to specifically target some proteins involved in iron uptake (49, 121). IsdC and Lmo2186 each contain one NEAT (near transporter) domain, and Lmo2185 contains three NEAT domains that are expected to play a role in iron transport (Fig. 2) (5). Interestingly, the L. monocytogenes operon containing the lmo2185, lmo2186, and srtB genes is regulated by the iron-responsive transcriptional repressor Fur and is induced under iron-deficient conditions (131). However, neither Lmo2185 nor Lmo2186 allows hemin, hemoglobin, or ferrichrome utilization (131). The exact functions of these two surface proteins, which are conserved in all Listeria species, remain to be characterized more precisely.
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80 amino acids, which are termed GW modules as they begin with the dipeptide Gly-Trp (25). GW modules are necessary and sufficient to anchor InlB to the bacterial surface by binding LTAs (86). These modules also interact with eukaryotic molecules, namely, glycosaminoglycans (GAGs) and gC1q-R (87, 119). Eight additional proteins carrying a variable number of GW modules are encoded in the L. monocytogenes EGDe genome; seven are putative autolysins containing cell wall hydrolase domains, while Lmo2713 is of unknown function (Table 1; Fig. 3A). Among the autolysins are Ami and Auto, which also are implicated in interactions with eukaryotic cells (32, 127). Auto is the only autolysin of this subfamily that is absent in L. innocua. Interestingly, the aut gene, encoding Auto, is in a locus comprising several genes presumably involved in TA synthesis, some of which are absent in L. innocua. Whether these genes products are required for association of Auto with the cell wall is at present unknown.
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GW modules like those in Listeria promote surface localization of several staphylococcal surface autolysins, such as AtlC from Staphylococcus caprae (2), AtlE from Staphylococcus epidermidis (77), and Aas from Staphylococcus saprophyticus (78). Domains containing 20-amino-acid repeat units beginning with GW but weakly related to Listeria GW modules are present in S. pneumoniae surface proteins, such as LytA (55, 56, 183) and PspA (197), or in clostridial proteins, such as CspA of Clostridium acetobutylicum (158) or the toxin ToxB of Clostridium difficile (56). These modules are responsible for binding of the protein to choline residues that decorate the TAs and LTAs in these species (29, 63).
WxL domain. A novel type of cell wall association domain of 160 to 190 amino acids, termed the WxL domain since it contains two conserved sequence motifs with the Trp-x-Leu signature, was recently identified in surface proteins of Lactobacillus plantarum and Enterococcus faecalis (28, 168). WxL proteins are present in many low-GC gram-positive bacteria and belong to the so-called Csc family of surface proteins. Csc gene clusters typically encode CscA, a protein with a conserved DUF916 domain of unknown function and a C-terminal transmembrane anchor; CscB and CscC, which display a C-terminal WxL domain; and CscD, a small LPXTG protein (Fig. 4). Csc proteins are proposed to form a multicomponent complex at the bacterial surface, although there is still no evidence for such a hypothesis (168). The EGDe L. monocytogenes genome contains two Csc-like gene clusters, among which four genes encode WxL domain-containing proteins (Lmo0549, Lmo0551, Lmo0585, and lmo0587). Lmo0549 contains an N-terminal LRR region and hence is a member of the internalin family. Two E. faecalis WxL proteins, EF2686 and EF2250, are also internalin-like proteins. The WxL domain of EF2686 promotes the association of the protein at the bacterial surface by interacting with peptidoglycan (28). It is tempting to speculate that listerial WxL proteins are attached at the bacterial surface by a similar mechanism, although this hypothesis remains to be experimentally addressed.
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40 amino acids called the lysine motif (LysM, Pfam PF01476) domain (Table 1). The LysM domain is found in a variety of enzymes involved in bacterial cell wall degradation and is also present in many other bacterial proteins with various enzymatic or binding activities (10). Several LysM-containing proteins, such as staphylococcal immunoglobulin G (IgG) binding proteins and Escherichia coli intimin, are involved in bacterial pathogenesis. LysM domains are also present in some eukaryotic proteins, possibly as a result of horizontal gene transfer from bacteria. The LysM domain is thought to be a general peptidoglycan binding module. Indeed, the C-terminal domain of the Enterococcus faecalis N-acetylglucosaminidase AtlA, which contains six LysM modules, bind to highly purified peptidoglycan (50). The LysM domain can adopt a beta-alpha-alpha-beta conformation, with the beta strands forming an antiparallel beta sheet and the two alpha helices packing on one side of this sheet (10). These structures show no similarity to other bacterial cell surface domains. The peptidoglycan binding activity of LysM domains has not been demonstrated for any listerial proteins. However, proteomic analyses of L. monocytogenes extracts suggest that LysM domain-containing proteins are surface exposed. The autolysins P60 and MurA, which contain two and four LysM domains, respectively, are present in purified cell wall fractions (34). Lmo1303 and Lmo2522, which contain one and two LysM domains, respectively, are detected in supernatant fractions, indicating that these proteins can translocate across the plasma membrane (179). One LysM domain is also found in Lmo1941, a protein with a transmembrane hydrophobic domain that is detected in the L. monocytogenes membrane fraction (193). Finally, a LysM domain is present in Lmo0880, a cell wall LPXTG protein (Fig. 2) (146). In this case, the LysM domain could play a role in the topological distribution within the cell wall of a protein attached by a sortase A-dependent mechanism.
Peptidase peptidoglycan binding domain.
One protein, Lmo1851, is predicted to possess an N-terminal signal peptide (179), a transmembrane domain, and a C-terminal domain that is also found at the N or C termini of a variety of peptidases (Table 1) (Fig. 5). This domain of 70 amino acids is composed of three alpha helices and may have a general peptidoglycan binding function (see INTERPRO entry IPR002477 and Pfam PF01471). Many of the proteins having this domain are uncharacterized. Lmo1851 is detected in the L. monocytogenes membrane fraction (193), suggesting that it may be associated with the membrane via its N-terminal hydrophobic region and stabilized inside the cell wall via its C-terminal peptidoglycan binding domain. However, this hypothesis requires experimental validation.
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Lipoproteins.
Lipoproteins are anchored to the membrane by covalent N-terminal lipidation. This process is directed by a specific signal peptide sequence characterized by a lipobox with a conserved cysteine residue (174). Two steps are necessary and sufficient for maturation of lipoproteins. Following signal peptide-directed export of the prolipoprotein, the prolipoprotein diacylglycerol transferase (Lgt) catalyzes the transfer of a diacylglycerol moiety from phosphatidylglycerol present in the membrane to the thiol of the conserved cysteine in the prolipoprotein. Subsequently, the signal peptide is removed by a specific lipoprotein signal peptidase (SPase) II (Lsp) enzyme, which cleaves within the lipobox to release the mature lipoprotein. Based on identification of a lipobox, it was possible to predict 68 lipoproteins in the L. monocytogenes EGDe genome sequence, the largest family of surface proteins (67). Recently, Baumgartner and colleagues were able to experimentally verify the genome predictions for 26 lipoproteins, which were specifically released in supernatants of a 
lgt strain (13). Listerial lipoproteins include 28 putative substrate binding proteins (SBPs) of ABC transporter systems, 19 proteins predicted to be involved in different enzymatic activities or other function, and 21 proteins of unknown function, the genes for six of which (Lmo0255, Lmo0460, Lmo0617, Lmo1340, Lmo2594, and Lmo2595) are absent from the L. innocua genome (Table 3).
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The autolysin-like protein Lmo0129 (Fig. 3) does not contain any obvious signal peptide, although it is detected in Listeria supernatant fractions (179). Interestingly, the gene encoding Lmo0129 is adjacent to a gene encoding a putative holin of the TcdE family, Lmo0128 (43), raising the possibility that Lmo0129 could be exported by a holin-like pathway (reviewed in references 70 and 191).
Another possible mechanism is deduced from electron microscopy studies performed 40 years ago that revealed the presence of small vesicles attached to the listerial membrane. These vesicles could be derived from small membrane extrusions (65). This observation is reminiscent of a well-described phenomenon in gram-negative bacteria. Many of them release vesicles from their outer membranes as secretory vehicles for proteins, lipids, and immunomodulatory compounds (189; for a review, see reference 99). These vesicles mediate bacterial binding and invasion, cause cytotoxicity, and modulate the host immune response. It is tempting to speculate that such phenomenon occurs in gram-positive bacteria. L. monocytogenes would be a nice model with which to test this hypothesis.
Finally, some proteins may also reach the cell surface through cosecretion and interaction with other surface proteins. Together, these results highlight the possibility of Sec-independent mechanisms of secretion and surface association that clearly deserve future attention.
| FUNCTIONS OF SURFACE PROTEINS AND THEIR RELEVANCE IN PATHOGENESIS |
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Knowledge of the enzymatic activities involved in cell wall assembly and turnover is important, as peptidoglycan synthetases are targets for many antibiotics and cell wall components are potent determinants of inflammation. L. monocytogenes cell wall derivatives are recognized by membrane-bound immune sensors, such as the macrophage scavenger receptor class A and Toll-like receptors (TLRs), or by intracytosolic sensors, such as Nod proteins, thereby stimulating host immunity. Macrophage scavenger receptor class A- and TLR2-deficient mice are highly susceptible to L. monocytogenes infection (85, 178). Nod2-deficient mice are susceptible to oral bacterial infections (92). Evidence for sensing of L. monocytogenes by Nod1 in vitro has also been recently reported (134). Together, these observations suggest that enzymes promoting the release of bacterial components are likely to be important modulators of the host innate (20) as well as adaptative (122) immune responses.
Autolysins. By digesting the cell wall peptidoglycan, autolysins are involved in numerous cellular processes, including cell growth and division, cell wall turnover, motility, chemotaxis, protein secretion, differentiation, and pathogenicity (140, 169). These hydrolytic enzymes are also responsible for the active release of immunogenic cell wall components. There are several types of autolysins, categorized by the nature of the bonds hydrolyzed in the peptidoglycan (Fig. 3B). Based on domain homology search data and a few studies using zymogram assays, we can expect the existence of several autolysins in L. monocytogenes. However, a direct demonstration of cleavage activities of these enzymes at specific bonds will require further biochemical studies.
(i) N-Acetylglucosaminidases and N-acetylmuramidases. Two types of enzymes hydrolyze the sugar backbone of peptidoglycan between the alternating MurNAc and GlcNAc residues of the glycan chains. N-Acetylmuramidases, which are related to lysozyme, cleave the ß-1,4 glycosidic bond between MurNAc and GlcNAc (Fig. 3B), whereas N-acetylglucosaminidases hydrolyze the bond between GlcNAc and MurNAc by liberating free reducing groups of GlcNAc. Three listerial proteins, Lmo0717, Lmo1104, and Lmo2522, possess a putative N-terminal N-acetylmuramidase domain. Six proteins, among which are the GW protein Auto and the LysM protein MurA (Fig. 3A), possess a domain with similarities to the catalytic domain of N-acetylglucosaminidases. Both Auto and MurA display cell wall hydrolase properties (31, 37). However, an aut deletion mutant has a morphology similar to that of the wild-type strain and has no defect in septation and cell division, while a murA mutant grows as long chains during exponential growth. Interestingly, Auto is required for entry of L. monocytogenes into different nonphagocytic eukaryotic cell lines, and an aut mutant displays a reduced virulence (31). How Auto specifically contributes to pathogenicity is unknown, though it may be linked to the maintenance of the cell surface architecture and/or to the release of immunologically active cell wall components.
(ii) Amidases. The Listeria genome encodes four proteins with a putative amidase domain known to hydrolyze the bond between the glycan chain and the peptide side chain of peptidoglycan. Two are GW proteins, Ami and Lmo1521, and two are proteins without any obvious modules for targeting at the bacterial surface, Lmo0129 and Lmo0849. Ami is the only amidase for which autolysin activity was reported (25). Like in InlB, the GW modules appear to promote Ami adhesion to host cells (127). Since the InlB GW modules bind cellular matrix proteoglycans (87), it is possible that Ami GW modules exert a similar function. Thus, Ami may act as a complementary adhesin during infection.
(iii) -D-Glutamyl-(L)-meso-diaminopimelate peptidases.
Another important group of L. monocytogenes autolysins is the P60 subfamily. P60, encoded by the iap (invasion-associated protein) gene, is a major 60-kDa autolysin first described as being involved in the L. monocytogenes invasion process (100). P60-defective mutants display an abnormal morphology, characterized by the presence of filamented cells containing fully formed septa (72, 196). The four members of the P60 family, Spl (P45), Lmo0394, Lmo1104, and P60 itself, share in their C-terminal region an NlpC/P60 domain (Pfam PF00877) related to the CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain, which is present in many proteins predicted to function in peptidoglycan hydrolysis (12, 151). For instance, this domain is present in Bacillus subtilis LytE and LytF and in S. aureus LytA and LytN autolysins (4). All CHAP superfamily enzymes characterized to date hydrolyze diverse substrates that contain a -D-glutamyl moiety. Although not yet biochemically validated, it is proposed that the P60 family fulfils the same function as Bacillus sphaericus endopeptidase II, which hydrolyzes the -D-glutamyl-meso-diaminopimelate linkage in the cell wall peptides (Fig. 3B) (169).
Besides P60, P45 (Spl) is the only protein of this family in L. monocytogenes known to exhibit peptidoglycan-lytic activity (163). Interestingly, P45 combines the C-terminal CHAP domain with an N-terminal domain related to the SMC (structural maintenance of chromosomes) family of proteins. The SMC proteins are essential for successful chromosome transmission during replication and segregation of the genome in all organisms (80). They form heterodimers and are core components of large multiprotein complexes. Lmo2504 is another putative surface protein with a C-terminal peptidase domain and an SMC-related N-terminal domain (Fig. 3). It is possible that P45 and Lmo2504 somehow couple the peptidoglycan remodeling to the segregation of the nucleoid, although further studies are required to test this hypothesis. Lmo1104 also combines the C-terminal CHAP domain with another N-terminal module. In this case, the N-terminal region shows similarity to a lytic transglycolsylase domain (Pfam PF01464). From this, Lmo1104 may thus have a dual function in hydrolyzing the cell wall. Lmo1104 is the only putative autolysin of the group that is absent in L. innocua.
A link between this class of surface proteins and Listeria pathogenicity has been established in the case of P60. Rough mutants expressing lower levels of P60 enter less efficiently into certain eukaryotic cells, suggesting a role for P60 in invasion (100). A P60-deficient mutant is attenuated in virulence after intravenous infection of mice and is impaired in its intracellular motility due to mislocalization of the actin-polymerizing factor ActA (107, 138). In addition, P60 plays an important role in the immune response against L. monocytogenes. P60-specific antibodies act as opsonins and might play a role in preventing systemic infections in immunocompetent individuals (95). P60 is also a major protective antigen that induces both T-CD8 and Th1 protective immune responses (64, 75). Finally, it was recently reported that P60 contributes to subversion of NK cell activation and production of gamma interferon (83).
(iv) Phage endolysins.
The genome of L. monocytogenes EGDe contains one prophage inserted in the comK locus (67, 111). This cryptic phage is highly homologous to phage A118 (111, 112, 114), a temperate bacteriophage attacking L. monocytogenes serovar 1/2. Like A118, the 
EGDe element possesses a gene encoding a putative endolysin (Lmo2278, homologous to Ply118 [115]) followed by a gene encoding a holin (homologous to Hol118 [115]) thought to trigger secretion of the endolysin (188). The EGDe endolysin is not characterized but could function like Ply118 endolysin, an L-alanyl-D-glutamate peptidase that cuts the amide bonds between L-Ala and D-Gln within the peptidoglycan peptide bridges (Fig. 3A) (115). Interestingly, endolysin Ply500 from phage A500 has the same enzymatic activity as Ply118, although it targets the cell wall of Listeria serovars 4, 5, and 6. The specificities of the endolysins are conferred by their different cell wall binding domains, which are thought to recognize Listeria-specific surface carbohydrates (113). Supporting this idea, phage PSA endolysin has a cell wall binding domain closely related to that of Ply500 and targets the same serovars, while having different enzymatic activity than Ply500 (96).
PBPs: transglycosylases, transpeptidases, ß-lactamases, and D-alanyl-D-alanine carboxypeptidases. Peptidoglycan assembly is a multistep process carried out by different enzymatic activities. Transglycosylase enzymes catalyze the insertion of peptidoglycan precursors into the growing end of the bacterial cell wall. Subsequently, transpeptidase enzymes join the peptide of a precursor with that of the preexisting peptidoglycan in order to cross-link the sugar chains (Fig. 3B). Penicillins and cephalosporins block the formation of the peptide cross-links and induce osmotic lysis of the bacterium by binding to the transpeptidase enzyme. Bacteria may resist the action of these ß-lactam antibiotics by mutating transpeptidases or by expressing ß-lactamase, an enzyme hydrolyzing ß-lactam rings. All these enzymes belong to the family of PBPs.
A first study reported that L. monocytogenes had five PBPs, as detected by their ability to bind benzylpenicillin (186). By searching the genome, Guinane and colleagues recently found seven surface proteins with similarities to PBPs (71). Our own in silico research identified 10 PBP-like proteins, which we classified into four families as represented in Fig. 3A. The first family, including PBPA1 and PBPA2 (formerly PBP1 and PBP4 [186]), corresponds to class A PBPs, which are bifunctional proteins with an N-terminal transglycosylase domain and a C-terminal transpeptidase domain. The second family, including PBPB1, PBPB2, (formerly PBP2 [186]), and PBPB3, corresponds to class B PBPs, which have a C-terminal transpeptidase domain and an N-terminal PBP dimerization domain (Pfam PF03717) involved in interaction with other proteins during cell morphogenesis. The third family encompasses two putative PBPs, PBPC1 and PBPC2, which contain a ß-lactamase class C domain (Pfam PF00144). PBPC1 is expected to be at the bacterial surface, while PBPC2 lacks any known cell surface association domain. Finally, the fourth family includes three proteins, PBPD1, PBPD2, and PBPD3, that contain a D-alanyl-D-alanine carboxypeptidase domain. This domain may catalyze the removal of the C-terminal D-alanine residue from peptidoglycan pentapeptides (Fig. 3B). PBPD1 (formerly PBP5 [186]) and PBPD2 are related to the peptidase S11 family (Pfam PF00768), represented by E. coli PBP5, while PBPD3 is related to the VanY DD-carboxypeptidase family (Pfam PF02557).
L. monocytogenes clinical isolates are resistant to cephalosporins but are, fortunately, susceptible to penicillin and ampicillin, which constitute the major treatment for listeriosis in combination with the aminoglycoside gentamicin. However, several recent findings provide evidence of the emergence of multidrug-resistant L. monocytogenes strains, including strains with resistance to penicillin and ampicillin (38, 143, 157, 170). Dissemination of these resistant traits in clinical strains of L. monocytogenes could have important medical consequences. It is therefore import to characterize listerial PBPs.
Recent studies have confirmed the enzymatic activity of PBPD1 (Lmo2754) (97) and of PBPA2 (Lmo2229) (198). Moreover, analysis of mutant strains suggests that PBPB3 (Lmo0441) and, to a lesser extent, PBPA2 (Lmo2229) are central to ß-lactam resistance in Listeria (71). Of interest is that PBPB3 shares similarities with MecA, the key determinant of ß-lactam resistance in methicillin-resistant S. aureus strains (109). Furthermore, disruption of some pbp genes attenuates L. monocytogenes virulence, probably due to alteration of the cell wall metabolism (71).
Deacetylase. A last important peptidoglycan-modifying enzyme is PgdA (Lmo0415), an enzyme recently characterized in L. monocytogenes as a GlcNAc deacetylase (21). pgdA mutants of L. monocytogenes, like those of S. pneumoniae (187), are hypersensitive to lysozyme, one of the first-line defense mechanisms in the human host against invading bacteria. Inactivation of pgdA also dramatically attenuates virulence and induces a massive beta interferon response in a TLR2- and Nod1-dependent manner. Peptidoglycan deacetylation is therefore essential for resistance against the innate immune response (21).
Enzymes involved in assembly and modification of TAs and LTAs. In addition to the assembly of cross-linked peptidoglycan, several enzymes are required for the synthesis of polyanionic TAs and LTAs, which make up an important polyanionic network with ion-exchange properties at the bacterial surface. However, polymerization of TAs and LTAs occurs intracellularly (14). Therefore, enzymes involved in cell wall polymer synthesis are expected to be cytosolic and not surface exposed, although a signal peptide is proposed to be present in two of them in L. monocytogenes, TagO (Lmo2519) and TagB (Lmo1088) (179). Enzymes involved in D-alanine esterification, a common modification found in TAs and LTAs of many gram-positive bacteria, are cytosolic or membrane-bound proteins (130). The synthesis of D-alanyl-LTA requires four proteins that are encoded by the dlt operon (1): dltA (lmo0974) encodes a cytoplasmic D-alanine-D-alanyl carrier protein ligase (designated DltA or Dcl); dltB (lmo0972) encodes the D-alanyl carrier protein Dcp, which is secreted; and dltC (lmo0973) and dltD (lmo0971) encode membrane-associated proteins, each having a signal peptide according to the Pfam annotation. However, the current model is that DltD would function intracellularly, providing binding sites for DltA and Dcp on the cytoplasmic leaflet, while DltB would form a channel for the secretion of D-alanyl-Dcp towards the cell wall, where D-alanylation occurs (130). This pathway still needs clarification.
As described above, mature proteins exhibiting a specific C-terminal sorting signal are processed by sortases, while lipoproteins are processed by Lgt (Fig. 5) (13, 16, 17, 62). Proteins that will remain at the surface inserted in the membrane will be processed by components that catalyze efficient lateral transport into or across the plasma membranes. These include the conserved YidC/Oxa1/Alb3 proteins family that are believed to function as membrane insertases, possibly functioning also as membrane chaperones (reviewed in references 139 and 182). Two L. monocytogenes lipoproteins, Lmo1379 (OxaA) and Lmo2854 (OxaB), share homology with YidC/Oxa1 and may thus target membrane-bound surface proteins (43). Future work is needed to validate this hypothesis.
The majority of proteins that are translocated across the cytoplasmic membrane are delivered to the membrane-cell wall interface essentially unfolded. They must then be folded into their native configuration without blocking the translocation machinery in the membrane, by forming inadequate interactions with the cell wall or insoluble aggregates. Misfolded or aberrant proteins have to be cleared off the translocase and the cell wall. Bacteria encode membrane- and cell wall-associated foldases and proteases that act as folding catalysts and quality control machines (159). Several genes in the L. monocytogenes genome encode proteins likely to be involved in folding and degradation of polypeptides at the bacterial surface (Fig. 5). PrsA (Lmo1444) and PrsB (Lmo2219) are lipoproteins related to the PrsA protein of B. subtilis. PrsA is a parvulin-type peptidyl-prolyl cis/trans isomerase, which assists posttranslocational folding of exported proteins and their stabilization at the bacterial surface. Interestingly, lmo2219 is preceded by a PrfA box, in contrast to lmo1444 (67), and is regulated by PrfA, the major regulator of Listeria virulence gene expression (13, 126). In addition, lmo2219 is upregulated during the intracytosolic phase of the Listeria cellular infectious process (39). We therefore suggest that PrsB could play a specific role in bacterial adaptation to the host.
lmo0292 (degP) encodes an HtrA-like serine protease related to B. subtilis YycK (172). HtrA is a protease that can also function as a chaperone. The listerial HtrA-like protease is essential for optimal growth under stress conditions and is involved in listerial pathogenesis (172, 195). Interestingly, like the gene encoding PrsB, the htrA gene is upregulated intracellularly (39). In addition to its N-terminal protease domain (Pfam PF00089), HtrA possesses a C-terminal PDZ domain. PDZ domains consist of 80 amino acids compactly arranged in a globular structure thought to function in protein or peptide binding (Pfam PF00595). They are found in diverse signaling proteins, in both bacteria and eukaryotes. We found two other proteins with protease and PDZ domains in the L. monocytogenes genome, Lmo1851 and Lmo1318 (Fig. 5). Lmo1851 carries a peptidase domain of the S41 family (Pfam PF03572), to which the ClpP protease belongs. Lmo1318 is a hypothetical zinc metalloprotease of the S2P/M50 peptidase family (Pfam PF02163), such as the membrane-bound E. coli protease EcfE (90). Related to Lmo1318 is Lmo2563, which is also predicted to be a S2P/M50 peptidase albeit without a PDZ domain and sharing similarities with the B. subtilis sporulation factor SpoIVFB (35). Interestingly, S2P/M50 proteases, by catalyzing cleavage of membrane-bound proteins, seem to be involved in a variety signal transduction pathways, some off which play roles in host-pathogen interaction (117). Whether Lmo1851, Lmo1318, and Lmo2563 function as cell surface proteases will require further investigation.
Sequence comparisons with previously characterized proteins suggest that some domains found in listerial proteins may play a role in adhesion to host cells.
Collagen binding domain. Five LPXTG proteins (Lmo0159, Lmo0160, Lmo0880, Lmo2158, and Lmo2576) are predicted to have a collagen binding domain (Pfam PF05737) (Fig. 2), which is found in the collagen binding adhesins of several other gram-positive pathogens (137, 199). The prototype member of this family is the collagen binding adhesin Cna of S. aureus. Cna participates in the infectious process of pathogenic S. aureus (52), suggesting that the ability to interact with collagen provides a general advantage to the bacteria in pathogenesis. Cna is composed of an N-terminal A region that carries the collagen binding activity and a C-terminal region of B repeats (CnaB-like repeats) (see below) (137). The Cna A region is composed of three subdomains, N1, N2, and N3 (199). Crystal structure determination of the N1 and N2 domains suggest a model in which both domains, each taking an Ig-like fold, cooperate to wrap around and hug the collagen triple helix (199). The collagen binding domain present in listerial proteins shows similarity to N2, which is the minimal collagen binding domain of Cna. Further work is required to explore the function of these listerial proteins, especially their possible binding to collagen.
Fibronectin binding domain. FbpA (Lmo1829) is a Listeria surface fibronectin binding protein required for intestinal and liver colonization of L. monocytogenes following oral infection (48). FbpA has strong homology to atypical fibronectin binding proteins, such as PavA of S. pneumoniae (81) and Fbp54 of S. pyogenes (40). FbpA binds to immobilized human fibronectin in a dose-dependent and saturable manner and increases adherence of wild-type L. monocytogenes to HEp-2 cells in the presence of exogenous fibronectin. Like FbpA, Lmo0721 is another reported listerial fibronectin binding protein that lacks conventional secretion/anchoring signals (66). Whether it is exposed at the bacterial surface has not been formally demonstrated.
Proteoglycan binding domain. As mentioned above, InlB GW repeats bind not only to LTAs present at the bacterial surface but also to GAGs. GW repeats binding to GAGs releases InlB from the bacterial surface (87). Furthermore, the presence of GAGs on the cell surface significantly increases InlB-dependent invasion, suggesting that binding of GW repeats to cellular GAGs could enhance interaction of InlB with its cellular receptor, Met. It is not known whether GW modules present in autolysins also bind GAGs. However, the purified GW domain of Ami binds eukaryotic cells, suggesting that a similar interaction may explain the role of Ami in listerial adhesion to host cells (127).
RGD motif. Three listerial surface proteins contain an arginine-glycine-aspartate (RGD) motif: ActA, the LPXTG protein Lmo1666, and the lipoprotein Lmo0460. The RGD motif is found in various proteins of the extracellular matrix and is known to interact with integrins. Although the RGD motif in ActA does not seem to contribute to Listeria adhesion to host cells (3), it may be a potential integrin binding site in Lmo1666 and Lmo0460.
Mucin binding domains.
Twelve LPXTG proteins (Fig. 2) and one protein with a membrane anchor domain, Lmo0576, contain a MucBP (mucin binding protein) domain (Pfam PF06458). Identified in MUB, an extracellular mucus binding protein of Lactobacillus reuteri 1063, this domain is arranged as tandem repeats that were shown to trigger adhesion to mucus material (152). MucBP repeats are present in 1 to 14 copies in listerial proteins. According to the Pfam database, MucBP repeats are present only in Lactobacillales and Listeria bacterial species. Sequence analysis indicates that the MucBP repeats vary in size, for instance, ranging from 60 amino acids in listerial proteins to almost 200 amino acids in MUB proteins of L. reuteri (19, 152). The difference in size between Listeria MucBP and Lactobacillus MUB repeats can be explained by the presence of a distinct N-terminal region in the MUB domain. MUB repeats and MucBP repeats may therefore be different functional units (19). At present, there is no experimental evidence for a role of listerial MucBP binding to mucins.
Ig-like fold domains. The Ig fold is an important structure, playing essential roles in the vertebrate immune response, cell adhesion, and many other processes. Novel three-dimensional structures of domains from distantly related proteins have revealed that unrelated sequences may take a similar Ig fold, which is characterized by the presence of ß sheets. Thus, nearly all Listeria LPXTG proteins possess Ig-like domains that fall into one of five distinct sequence families: the Cna collagen binding domain described above, the Cna protein B-type domain, the LRR-adjacent domain, the PKD repeat domain, and the bacterial Ig-like domain (Big 3). Two main functions may be attributed to these domains. They may be directly involved in adhesion by interacting with carbohydrates on the bacterial or host cell surface. Alternatively, they may serve as a stalk that projects a ligand binding region from the bacterial surface, thus facilitating bacterial interaction with host cells. For instance, the receptor binding domain of intimin in enteropathogenic Escherichia coli, invasin in Yersinia, or internalin in Listeria is presented at the tip of an elongated structure, in which the repeat region is used as a spacer domain or a stabilizer for the whole structure (133).
(i) CnaB-like repeat domain. As mentioned above, staphylococcal Cna has a nonrepetitive collagen binding A region followed by a repetitive B region. The B region has one to four 23-kDa repeat units [B(1) to B(4)], depending on the strain of origin. Each B repeat has two domains, D1 and D2, each exhibiting an inverse Ig-like fold. Modeling studies suggest an accordion structure in which the B repeat units could project the A region from the cell surface and aid in binding to collagen (41). Six listerial LPXTG proteins contain repeats related to the D domain of CnaB repeats (Pfam PF05738) (Fig. 2), among which four possess a collagen binding domain (Lmo0159, Lmo0160, Lmo2178, and Lmo2576).
(ii) LRR-adjacent Ig-like domain. Eleven LPXTG proteins of the internalin family contain the LRR-adjacent Ig-like domain (Pfam PF08191) (Fig. 2). It is a small beta-strand domain fused to the C-terminal end of the LRR region (133, 164). This domain belongs to the family of Ig-like domains in that it consists of two sandwiched beta sheets. The beta strands in one of the sheets is, however, much smaller than those in most standard Ig-like domains. The function of this domain appears to be mainly structural, forming a common rigid entity with the LRR. It is thought to facilitate the presentation of the adjacent LRR domain for protein-protein-interactions.
(iii) PKD repeat domain.
Eleven LPXTG proteins contain the PKD repeat domain (Pfam PF00801) (Fig. 2). The PKD repeat domain was first identified in the human polycystic kidney disease 1 (PKD1) protein as 16 Ig-like repeats, each of 80 amino acid in length. PKD1 is involved in adhesive protein-protein and protein-carbohydrate interactions. PKD1 Ig-like repeats II to XVI are involved in strong calcium-independent homophilic interactions in vitro (84), suggesting that PKD1 may be involved in cell-cell adhesion through its cluster of Ig-like repeats. However, the function of PKD1 repeats in listerial proteins remains totally unknown. For instance, InlI, the longest internalin-like protein (Fig. 2), possesses eight PKD repeats, but no detectable role could be attributed to InlI in vitro or in virulence assays (156).
(iv) Big 3 domain.
Four LPXTG proteins contain a domain with an Ig-like fold called the Big 3 domain (Pfam PF07523) (Fig. 2). This domain is present in one copy in the internalin-like Lmo2026, Lmo0732, and Lmo0171 proteins and is repeated seven times in the Lmo0842 protein. Interestingly, Lmo0842 repeats are related to those found in the Alp family in streptococci, which comprises alpha, Rib, R28, and Alp2 proteins, which are important surface proteins known to confer protective immunity (110). Alp repeats are themselves related to repeats of 80 amino acids found in biofilm-associated proteins (Bap) in staphylococci and in Esp in enterococci (102), as well as to HYR (hyalin repeat) domains identified in several eukaryotic proteins (33). By BLAST analysis, we found that PKD repeats present in Listeria LPXTG proteins are also weakly similar to the Bap family repeats. Together, these data suggest an evolutionary relationship between all these repeated domains.
Flagellin from L. monocytogenes stimulates TLR5 (76), which is expressed by intestinal epithelial cells, monocytes, and dendritic cells. TLR5 activation induces the production of tumor necrosis factor (TNF) alpha, an important cytokine in host resistance to enteric listeriosis. In addition, flagella seem to facilitate the adhesion to and invasion of eukaryotic host cells by L. monocytogenes (45). However, the relationship between flagellin and virulence is not clear. On one hand, no differences in virulence, bacterial clearance, or induction of L. monocytogenes-specific T-lymphocyte responses between L. monocytogenes strain 10403S and an isogenic flaA deletion strain were observed in the mouse infection model, suggesting that flagellin is not essential for pathogenesis (192). On the other hand, inactivation of the regulator of flaA expression, MogR, induces overexpression of FlaA and causes defects in bacterial division, intracellular spread, and virulence in mice (166). Finally, virulence of flaA mutants is diminished relative to that of the wild-type strain in tumor necrosis factor alpha receptor-p55–/– mice, suggesting that flagella participate in the triggering of tumor necrosis factor secretion, leading to protection against infection (45).
Intracellular movement. The well-known listerial surface protein ActA mediates polymerization of host actin to propel the bacterium through the cytoplasm of its host (for recent reviews, see references 68 and 73). ActA is the first bacterial protein identified to promote actin nucleation, by mimicking its eukaryotic counterparts, the Wiskott-Aldrich syndrome (WASP) family of proteins. The N-terminal region of ActA has homology to the C-terminal region of WASP, and both ActA and the WASP proteins act as nucleation-promoting factors for the Arp2/3 comp
