Surface Proteins Anchored Covalently

to the Peptidoglycan 5.3.1.1. The LPXTG Protein Family

Pioneering work performed with the S. aureus protein A demonstrated that this surface protein is anchored covalently to the peptidoglycan by an enzyme

Srtb Gene Svpa Listeria

Figure 5.1. Global view of the distinct interactions of the Listeria monocytogenes surface proteins with the cell wall. Listed in the upper part are the proteins identified by proteomics in purified peptidoglycan material (in bold); in extracts obtained from the cell wall upon incubation of bacteria with high concentration of salts (asterisk, *); or, in the extracellular medium as components of the "secretome" (double asterisk, **). Lmo0320 (Vip), although unidentified in these studies, was detected on the cell surface by immunological assays (Cabanes et al. 2005). Other nonlisted surface proteins with mode of association to the cell wall unknown but identified in proteomic analysis include: Lmo1892 (PbpA) in purified peptidoglycan material and Lmo2504, Lmo2522, and Lmo2754 (PBP5) in the extracellular medium. TA: teichoic acid; LTA: lipoteichoic acid.

Figure 5.1. Global view of the distinct interactions of the Listeria monocytogenes surface proteins with the cell wall. Listed in the upper part are the proteins identified by proteomics in purified peptidoglycan material (in bold); in extracts obtained from the cell wall upon incubation of bacteria with high concentration of salts (asterisk, *); or, in the extracellular medium as components of the "secretome" (double asterisk, **). Lmo0320 (Vip), although unidentified in these studies, was detected on the cell surface by immunological assays (Cabanes et al. 2005). Other nonlisted surface proteins with mode of association to the cell wall unknown but identified in proteomic analysis include: Lmo1892 (PbpA) in purified peptidoglycan material and Lmo2504, Lmo2522, and Lmo2754 (PBP5) in the extracellular medium. TA: teichoic acid; LTA: lipoteichoic acid.

named sortase (Mazmanian et al. 1999). Protein A contains an N-terminal signal peptide and a C-terminal domain consisting of an LPXTG motif followed by a hydrophobic region of about 20 amino acids that ends in a tail of mostly positively charged residues (Ton-That et al. 2004). This C-terminus configuration, named "sorting signal", is conserved in many surface proteins of gram-positive bacteria (Navarre and Schneewind 1999; Ton-That et al. 2004).

The in-silico genome analysis of the L. monocytogenes strain EGD-e unravelled 41 genes-encoding surface proteins bearing an LPXTG motif (Glaser et al. 2001). To date, this number of LPXTG proteins is the highest among all the gram-positive bacteria with genome sequence known. A high number of genes-encoding LPXTG proteins, in the average of 45, were also identified in the genome of other three L. monocytogenes strains (Nelson et al. 2004). Despite this bulk of information, very few of these proteins have been characterized at the biochemical and/or functional level. The LPXTG protein most extensively studied is InlA, which promotes entry of L. monocytogenes into epithelial cells. InlA harbours an LPTTG motif and is anchored covalently by the sortase SrtA to m-Dap residues of the peptidoglycan (Bierne et al. 2002; Dhar et al. 2000; Garandeau et al. 2002). In S. aureus, the primary acceptor molecule in the anchoring reaction catalyzed by SrtA is the lipid-II precursor (Perry et al. 2002). Whether the L. monocytogenes sortase SrtA uses the same mechanism has not yet been formally demonstrated.

The large set of LPXTG proteins of L. monocytogenes clusters in two subfamilies that differentiate by the presence in their N-terminal half of a variable number of leucine-rich repeats (LRR) containing 20-22 amino acids each (Cabanes et al. 2002). This domain is a feature shared by all proteins belonging to the "internalin family". The LRR domain is thought to mediate protein-protein interactions, and in the case of InlA is necessary and sufficient to promote bacterial uptake. The L. monocytogenes strain EGD-e has 19 LPXTG proteins containing the LRR domain (Cabanes et al. 2002). Of these, only eight are present in the nonpathogenic species L. innocua. A similar number of internalins bearing LPXTG motifs (from 14 to 17 proteins) has been reported in the other three L. monocyto-genes strains with genome sequence known (Nelson et al. 2004). Besides InlA, a few LPXTG proteins of the internalin family have been recently characterized in the EGD-e strain. These include InlH (Lmo0263), InlI(Lmo0333), andInlJ (Lmo2821) (Sabet et al. 2005; Schubert et al. 2001) (see Section 5.4.1).

Proteins containing diverse non-LRR repeat regions preceding the C-terminal sorting region form the second class of LPXTG proteins. The EGD-e strain has 22 LPXTG proteins in this class, of which 14 have orthologs in L. innocua (Cabanes et al. 2002; our unpublished data). To date, only one protein of this group, Vip (Lmo0320), has been characterized at a functional level (Cabanes et al. 2005) (see Section 5.4.1).

Genomic comparison studies have revealed that six LPXTG proteins of L. monocytogenes serotypes responsible for most cases of listeriosis are absent in the rest of Listeria species (Doumith et al. 2004). LPXTG proteins displaying this narrow distribution in the Listeria genus are currently investigated for their role in pathogenesis (see Section 5.4.1).

5.3.1.2. Non-LPXTG Proteins Bearing Cell-Wall Sorting Signals

Staphylococcus aureus has a second sortase, SrtB, which recognizes a sorting motif different than LPXTG. This sortase specifically cleaves an NPQTN motif in a surface protein involved in iron transport, IsdC (Mazmanian et al. 2002). L. monocytogenes has also an alternative SrtB sortase (Bierne et al. 2004). Like in S. aureus, the L. monocytogenes srtB gene maps in an operon containing two genes encoding the Lmo2185 (formerly SvpA) and Lmo2186 (SvpB) proteins, which share homology to S. aureus IsdC. Lmo2185 and Lmo2186 bear as putative sorting motifs NAKTN and NKVTN (NPKSS), respectively. Although a direct proof of their covalent anchoring to the peptidoglycan has not been yet shown, both proteins are detected in highly purified peptidoglycan material (Calvo et al. 2005) (see Sect. 5.5.1). Furthermore, Lmo2185 displays a unique migration on gels when extracted from peptidoglycan material (Bierne et al. 2004), suggesting that this species may correspond to the processed form covalently anchored to the peptidoglycan. Detection of Lmo2185 at the cell surface is also abolished in an srtB mutant (Bierne et al. 2004). Interestingly, the L. monocytogenes operon containing the lmo2185, lmo2186, and srtB genes is induced in iron-deficient conditions, but neither Lmo2185 nor Lmo2186 are required for haemin, haemoglobin, or ferrichrome utilization (Newton et al. 2005). The exact function of these two surface proteins, which are conserved in all Listeria species, remains therefore elusive.

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