Other Cell Wall Polyanionic Polymers Teichoic Acids and Lipoteichoic Acids

As most gram-positive bacteria, L. monocytogenes contains two different polyan-ionic polymers decorating the cell wall: the teichoic acids (TA), covalently bound to the peptidoglycan and the lipoteichoic acids (LTAs), amphipathic molecules that are embedded into the plasma membrane by a diacylglycerolipid (Navarre and Schneewind 1999; Neuhaus and Baddiley 2003). These polymers represent as much as 50-60 % of the total content of isolated dry cell walls of L. monocytogenes (Fiedler 1988) and play important functions in metal cation homeostasis, anchoring of surface proteins, and transport of ions, nutrients, and proteins. TA and LTA, which are synthesized by noninterconnecting metabolic pathways (Neuhaus and Baddiley 2003), are main determinants of surface hydrophobicity and immunogenicity. In fact, both TA and LTA confer the basis of the serotype diversity known in L. monocytogenes.

Two main types of TA exist in L. monocytogenes. The first is formed by a polymer consisting of repeating units (~20 to ~45) of 1,5-phopshodiester-linked ribitol residues (Fiedler 1988; Uchikawa et al. 1986a). These ribitol-P units bear variable substitutions. Thus, in serotypes 3a, 3b, and 3c, a GlcNAc residue is linked at position C-4 of the ribitol-P repeating unit whereas in serotypes 1/2a, 1/2b, and 1/2c there is an additional rhamnose residue at position C-2. Remarkably, the serotype 7 has no substitutions in the ribitol-P. The second type of TA includes more complex structures in which the GlcNAc residue incorporates as a part of the poly-ribitol-P chain (Fiedler 1988; Uchikawa et al. 1986b). Thus, the C-1 of GlcNAc binds to hydroxyl groups present at positions C-4 (serotypes 4a and 6) or C-2 (serotypes 4b, 4d, and 4f) of the ribitol-P. The C-4 position of GlcNAc then links to the phosphate of the adjacent ribitol-P unit. In addition, the GlcNAc units are decorated with glucose and/or galactose in some serotypes (case of 4b, 4d, and 4f) (Uchikawa et al. 1986a). A TA structure similar to that of L. monocytogenes 4b serotype has been identified in a few L. innocua strains. It is possible that L. innocua may have acquired from L. monocytogenes serotype 4b, the set of genes responsible for these modifications, a hypothesis supported by comparative genome analysis (Doumith et al. 2004). The genome sequences obtained from four L. monocytogenes strains, two of serotype 1/2a (EGD-e andF6854) and two of serotype4b (F2365 andH7858), have revealed the presence of 1/2a serotype-specific genes involved in rhamnose biosynthetic pathway (Nelson et al. 2004). The existence of these serotype-specific genes related to TA biosynthesis was recently confirmed upon genome content analysis of 93 L. monocytogenes strains of diverse serotypes (Doumith et al. 2004). Thus, serotypes 1/2,3, and 7 carry genes involved in TA biosynthesis that are absent in serotype 4. Inversely, a gene annotated with function putatively related to TA synthesis (ORF2372) is present exclusively in serotypes 4b, 4d, and 4e (Doumith et al. 2004). The exact function of ORF2372 has not been elucidated. Another gene named gtcA was initially claimed as a 4b serotype-specific gene involved in the decoration (glycosylation) of TA, concretely in the incorporation of galactose and glucose to the GlcNAc residues (Promadej et al. 1999). However, gtcA ortholog genes exist in the genome of 1/2a strains (Autret et al. 2001). In fact, insertions in the gtcA gene of the serotype 1/2a strain EGD-e impair virulence and its product has been proposed to mediate incorporation of rhamnose to the TA (Autret et al. 2001). Further work is required to unravel whether the role of GtcA in the decoration of TA differs in serotypes 1/2a and 4b. It was later shown that mutants in the gltA-gltB gene cassette, which is a truly specific locus of serotype 4b, display a severe reduction or total loss of incorporation of galactose to the GlcNAc residue of TA (Lei et al. 2001). These mutants have unaltered the amount of glucose in the TA, which suggests that GltA and GltB are specifically involved in the linkage of galactose to GlcNAc independently of the glucose substitution. The contribution of GltA and GltB to L. monocytogenes pathogenicity has not been yet tested.

The ribitol-P polymer of the L. monocytogenes TA is covalently bound to the peptidoglycan by a linkage unit formed by two disaccharides bound by a molecule of phosphoglycerol. This linkage unit has the structure Glc(^1^3)-Glc(P 1 ^ 1 /3)Gro-P-(3/4)ManNAc((3 1 ^4)GlcNAc (Kaya et al. 1985). The disac-charide formed by the two molecules of Glc is bound to the ribitol-P polymer whereas that containing the acetylated amino sugars binds via a 1,6-phosphodiester linkage to the MurNAc residue of the peptidoglycan. The L. monocytogenes linkage unit of the TA is more complex than that of the closely related bacteria B. subtilis and Staphylococcus aureus, which contain only one disaccharide (Navarre and Schneewind 1999; Neuhaus and Baddiley 2003).

Unlike TA, the LTA polymer of L. monocytogenes is formed by repeating units of glycerol-P bound by 1,3 linkages. In some strains, the C-2 position of the glycerol-P is decorated with galactose (Fischer et al. 1990). The glycerol-P polymer attaches to the C-6 position of a nonreducing sugar molecule carried by a glycolipid molecule that embeds the LTA into the membrane. In L. monocytogenes, the glycolipid molecule has the structure of Gal(a1^2)Glu-1(3),2-diacylglycerol (Fiedler 1988; Fischer et al. 1990). A substitution of a phosphatidic acid in the C-6 position of Glu has been reported in some strains.

A common modification found in TA and LTA of many gram-positive bacteria is D-alanine-esterification at carbons of their respective repeating units (Neuhaus and Baddiley 2003). This modification is accomplished by a unique D-Ala incorporation system encoded in the dlt operon. D-Ala esterification has a profound effect on the electromechanical properties of the cell wall since it reduces its global negative charge. This modification modulates distinct cellular functions as the activities of autolysins, the maintenance of cation-homeostasis, and the assimilation of metal cations (Neuhaus and Baddiley 2003). The existence of a dtl operon in the genome does not necessarily imply that the TA are D-Ala-esterified. Thus, Streptococcus pneumoniae strain R6 harbours an entire dlt operon but contains phosphorylcholine-esters instead of D-Ala-esters in both TA and LTA.

The L. monocytogenes dlt operon consists of four genes, dltA, dltB, dltC, and dltD, encoding all components required for D-Ala esterification (Abachin et al. 2002). D-Ala-esterification is important for L. monocytogenes pathogenicity. Thus, a dltA mutant displays enhanced sensitivity to antimicrobial cationic peptides and virulence attenuation in the mouse-infection model (Abachin et al. 2002). D-Ala-esterification occurs in laboratory conditions in ~20% of the glycerol-P residues of LTA and is not detected in LTA of the dltA mutant. No study has reported the rate of D-Ala-esterification in TA of L. monocytogenes. Interestingly, the defect in D-Ala-esterification of LTA displayed by the dltA mutant does not alter the relative amount of surface proteins as internalin-A (InlA), InlB, and ActA that are extracted from the cell surface. Based on this result, D-Ala-esterification was postulated to be important for certain surface proteins to reach a functional folding state (Abachin et al. 2002), although it does not discard a potential role of D-Ala-esterification in modulating the anchoring of surface proteins to the cell wall. Noteworthily, VirR, a new response regulator implicated in L. monocytogenes virulence, controls among other functions the expression of the dlt operon (Mandin et al. 2005). This observation reinforces the idea that D-Ala-esterification is a cell-wall modification playing a prominent role in the L. monocytogenes infection process.

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