To be specifically localized at the cell surface, proteins from gram-positive bacteria need to translocate across the membrane and then associate with a cell surface component. Surface localization of proteins relies on the presence of specific domains or motifs known to mediate secretion and attachment to the cell envelope. The first step of transport across the membrane occurs by two main pathways, both of which require a specific N-terminal secretion signal: the general secretory Sec pathway and, to a lesser extent, the Tat pathway (176
). However several alternative pathways have been discovered, such as the SecA2 pathway (27
) or specialized pathways for secretion of specific proteins, such as the pseudopilin export pathway, various ABC transporter pathways, the holin systems (176
), and the ESAT-6/WXG100 secretion system (135
). The presence of these secretion systems in L. monocytogenes
has been extensively reviewed recently by Desvaux and Hebraud (43
). In this review, we focus only on proteins exported through the Sec and SecA2 pathways, as well as flagellin, which is secreted by the specialized flagellar export machinery. The numerous nonsecreted integral membrane proteins that presumably also expose specific domains on the outside of the membrane bilayer are not discussed in this review.
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. ).
The different types of surface proteins found in L. monocytogenes. A prototype of each family is given.
Proteins Covalently Linked to the Peptidoglycan: Sortase Substrates
LPXTG sorting signal.
The best known class of L. monocytogenes
surface proteins is that of the LPXTG proteins, which in strain EGDe comprises 41 members. They are characterized by a short C-terminal sorting signal predicted to direct covalent attachment to the peptidoglycan (Fig. ) (Pfam PF00746, http://www.sanger.ac.uk/Software/Pfam
). Originally characterized in Streptococcus pyogenes
protein M and Staphylococcus aureus
protein A, this signal consists of an LPXTG motif, followed by an hydrophobic domain of about 20 amino acids and a tail of positively charged residues (60
). The sorting signal is the substrate of sortase, a membrane-bound transpeptidase, which cleaves the LPXTG motif between the threonine and glycine residues and catalyzes the formation of an amide link between the carboxyl group of the threonine and cell wall precursors (123
). For a thorough analysis of the sorting mechanism, we refer the reader to an excellent recent review on sortases (121
). The most studied LPXTG protein in L. monocytogenes
is internalin (InlA), which promotes bacterial entry into epithelial cells (61
). InlA harbors an LPTTG motif and is anchored covalently by the sortase SrtA to meso
-diaminopimelic acid residues of the peptidoglycan (17
). The recently discovered virulence factor LPXTG protein Vip is also localized on the bacterial surface by an SrtA-dependent pathway (32
). Using a novel nongel proteomic method, Pucciarelli et al. (146
) were able to identify 13 SrtA substrate proteins (Fig. ), through comparison of the peptidoglycan contents from wild-type EGDe and sortase-defective mutants grown in rich medium. The remaining LPXTG proteins predicted by the genome may not be produced under the growth conditions used in that study.
FIG. 2. Sortase substrates. A schematic representation of L. monocytogenes LPXTG and NXXTX proteins is shown. The numbers within domains indicate the number of repeats. Nineteen proteins are absent in L. innocua and are indicated in green letters. Proteins detected (more ...)
The L. monocytogenes
genome encodes the highest number of LPXTG proteins among all gram-positive bacteria whose genome sequences are known. InlA and 18 other family members remarkably display an N-terminal leucine-rich repeat (LRR) domain and belong to the so-called “internalin family” (Fig. ) (30
), which also includes the GW protein InlB, the WxL protein Lmo0549 (see below), and four proteins without any obvious surface anchoring motif, among which is InlC (73
). The LRR domain consists of tandemly arranged repeats of 22 amino acids each. In InlA, InlB, and 10 other surface internalins, the LRR domain is flanked at its C terminus by a conserved LRR-adjacent domain (Fig. ; also see below). InlA, InlB, InlG, InlH, InlE, and InlF also possess a region of B repeats. The crystal structures of the LRR domains of InlA, InlB, InlH, and InlE have been solved, revealing a curved structure ideally shaped for protein-protein interaction (120
). However, while the LRR domains of InlA and InlB are structurally related, they bind to very different mammalian receptors, namely, E-cadherin (Ecad) and Met (125
). Therefore, one cannot assume that members of the internalin family share similar functions solely on the basis of their structural classification. Of the internalin-like proteins, only InlA and InlB have so far been identified as invasins (see below).
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
). 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
). 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
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
). IsdC and Lmo2186 each contain one NEAT (nea
ransporter) domain, and Lmo2185 contains three NEAT domains that are expected to play a role in iron transport (Fig. ) (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.
Proteins with Noncovalent Association to the Cell Wall
A second group of cell surface proteins in gram-positive bacteria comprises those that are thought to bind to the cell wall by noncovalent interactions, most often via repeated domains (Table ) (for extensive recent reviews, see references 42
, and 165
L. monocytogenes EGDe proteins predicted to carry motifs promoting noncovalent association to the cell wall
The first characterized protein of this type in L. monocytogenes
was InlB, a protein of the internalin family required for L. monocytogenes
entry into many eukaryotic cell types (15
). The InlB C-terminal domain comprises three highly conserved tandem repeats of ~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
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 ; Fig. ). Among the autolysins are Ami and Auto, which also are implicated in interactions with eukaryotic cells (32
). 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.
FIG. 3. Surface proteins predicted to be involved in cell wall metabolism. (A) Schematic representation of L. monocytogenes surface proteins potentially involved in cell wall synthesis, hydrolysis, or modification. AA, amino acids. Most of the enzymatic activities (more ...)
Association of the GW domain with LTA occurs whether the protein is produced within the bacterium or added externally (25
). It displays specificities, as GW modules of InlB do not bind to the surface of L. innocua
or to that of Streptococcus pneumoniae
). In addition, the strength of the association seems to increase with the number of GW modules. Thus, an InlB variant bearing the eight GW modules of the autolysin Ami binds more efficiently to the cell surface (25
). The InlB GW modules are structurally related to Src homology 3 (SH3) domains found in many eukaryotic adaptor proteins, but the binding site for proline-rich ligands of bona fide SH3 domains is obstructed (119
). Interestingly, two non-GW surface proteins, the autolysin P60 and Lmo0394 (Fig. ), also contain an SH3-related domain (Pfam PF08239). Whether this domain interacts with cell wall polymers is unknown. A related bacterial SH3 domain (Pfam PF08460) mediates binding to the cell wall of lysostaphin, a bacteriocin secreted by Staphylococcus simulans
GW modules like those in Listeria
promote surface localization of several staphylococcal surface autolysins, such as AtlC from Staphylococcus caprae
), AtlE from Staphylococcus epidermidis
), and Aas from Staphylococcus saprophyticus
). 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
) and PspA (197
), or in clostridial proteins, such as CspA of Clostridium acetobutylicum
) or the toxin ToxB of Clostridium difficile
). These modules are responsible for binding of the protein to choline residues that decorate the TAs and LTAs in these species (29
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
). 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. ). 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.
FIG. 4. The two Csc Clusters in L. monocytogenes. The first line shows a characteristic four-component Csc cluster, with the predicted surface-anchoring domain of Csc proteins indicated below. The two L. monocytogenes Csc clusters are schematically represented, (more ...) LysM domain.
Six listerial proteins, including P60 and MurA, carry one to four copies of a domain of ~40 amino acids called the ly
otif (LysM, Pfam PF01476) domain (Table ). 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. ) (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 ) (Fig. ). 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.
FIG. 5. Surface proteins predicted to be involved in protein processing, folding, and anchoring at the cell surface. A schematic representation of L. monocytogenes surface proteins potentially involved in modifications or degradation of proteins at the surface (more ...)
Proteins with hydrophobic tails.
Proteins carrying a signal peptide can be retained in the membrane bilayer by hydrophobic segments generally present at the N- or C-terminal part of the protein. Proteins associated with the membrane by their C terminus contain a carboxyl-terminal stretch of hydrophobic residues followed by few charged residues thought to serve as a stop-transfer signal. This class includes the surface protein ActA, which promotes Listeria
intracellular motility (94
); two CscA-like proteins as mentioned above (Fig. ); and four proteins of unknown function (Table ). It also includes Lm0058, an ortholog of EssA, a component of the Wss(WGX100) specific secretion pathway (43
). Finally, it includes two proteins, Lmo0528 and Lmo0529, which are similar to a UDP-glucose dehydrogenase and UDP-glucose glycosyltransferase, respectively. Whether these proteins play a role in exopolysaccharide production at the bacterial surface is an interesting possibility that has to be tested.
L. monocytogenes EGDe proteins with a carboxyl-terminal hydrophobic tail
Proteins may also be tethered to the cell membrane by an amino-terminal hydrophobic stretch, which may be the signal peptide itself if it remains uncleaved (176
). Sortases SrtA and SrtB, as well as several proteins involved in protein folding or in cell wall synthesis, such as penicillin binding proteins (PBPs), belong to this class of proteins (Fig. and ). Whether they remain attached to the bacterial surface needs to be experimentally verified. Nevertheless, some of them have been detected in the L. monocytogenes
membrane fraction by proteomics (193
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 Δlgt
). 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 ).
Lipoproteins of L. monocytogenes strain EGDea
Nonconventional Secreted Surface Proteins
Proteomic studies identified proteins that have no predicted signal sequence, and are assumed to have cytoplasmic functions, in the L. monocytogenes
cell wall or supernatant fractions (160
). Among them are glycolytic enzymes, chaperones, and heat shock proteins, as well as proteins involved in detoxification and adaptation to atypical conditions, nucleic acid metabolism, transcription, and translation. Although these cytosolic proteins may be recovered in these fractions due to cell lysis, increasing evidence suggests that some of them may be specifically transported to the surface by alternative pathways. In L. monocytogenes
the auxiliary secretory protein SecA2 is involved in export of a subset of listerial proteins, some of which lack recognizable Sec sequences (107
). Recent reports identified superoxide dismutase as a protein dependent on SecA2 for secretion and surface association in L. monocytogenes
). Superoxide dismutase is also secreted via the SecA2 pathway in Mycobacterium tuberculosis
). Another study reports the unambiguous presence at the bacterial surface of FbpA (Lmo1829), a protein that does not possess any characteristic of a surface-exposed protein. FbpA is also exported via the SecA2-dependent secretion pathway (48
). Orthologs of FbpA, including PavA of S. pneumoniae
and Fbp54 of S. pyogenes
, are also cell surface proteins (40
). Together these data suggest that SecA2-dependent export is a new type of secretion pathway that is partly responsible for L. monocytogenes
The autolysin-like protein Lmo0129 (Fig. ) 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
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.