Listeria monocytogenes wall teichoic acid decoration in virulence and cell‐to‐cell spread

Abstract

Wall teichoic acid (WTA) comprises a class of glycopolymers covalently attached to the peptidoglycan of gram positive bacteria. In Listeria monocytogenes, mutations that prevent addition of certain WTA decorating sugars are attenuating. However, the steps required for decoration and the pathogenic process interrupted are not well described. We systematically examined the requirement for WTA galactosylation in a mouse oral-virulent strain by first creating mutations in four genes whose products conferred resistance to a WTA-binding bacteriophage. WTA biochemical and structural studies indicated that galactosylated WTA was directly required for bacteriophage adsorption and that mutant WTA lacked appreciable galactose in all except one mutant - which retained a level ca. 7% of the parent. All mutants were profoundly attenuated in orally infected mice and were impaired in cell-to-cell spread in vitro. Confocal microscopy of cytosolic mutants revealed that all expressed ActA on their cell surface and formed actin tails with a frequency similar to the parent. However, the mutant tails were significantly shorter - suggesting a defect in actin based motility. Roles for the gene products in WTA galactosylation are proposed. Identification and interruption of WTA decoration pathways may provide a general strategy to discover non-antibiotic therapeutics for gram positive infections. © 2016 John Wiley & Sons Ltd.

Use of bacteriophage to target bacterial surface structures required for virulence: a systematic search for antibiotic alternatives

“…Subsequently, a collection of P35h4 resistant mutants was systematically assembled. All mutants (each having a lesion in one of four genes required for phage attachment) were attenuated in orally inoculated mice and defective in their ability to spread from cell-to-cell in mouse enterocyte monolayers (Spears et al 2016). With the help of the phage, we found that each mutant’s wall teichoic acid (WTA; a glycopolymer covalently attached to the cell wall) was modified: each repeat unit was missing a single substituent sugar (galactose).…”

“…Nevertheless, in the host cell cytosol, the mutants all exhibited reduced actin polymerization, and decreased cytosolic motility. The enzymatic steps required to add the WTA decoration have been proposed (Spears et al 2016). Currently, those enzymes provide targets for inhibition by small secondary metabolite derivatives.…”

“…Exactly how the absence of a WTA decorating sugar decreases actin-based motility is unclear, but we suspect that the modified WTA composition results in a more elongated polymer (Doyle et al 1974), and that this may impede the access of cytosolic unpolymerized actin to the listerial cell surface protein (ActA) that nucleates the polymerization process (Spears et al 2016). A similar elongation of lipopolysaccharide (LPS; a glycopolymer anchored in the outer membrane of gram negative bacteria) is proposed to result from a lack of specific glucosylation of the Shigella flexneri LPS glycopolymeric repeat units (West et al 2005).…”

“…Also, if a multistep pathway is required to produce the phage receptor, the initial target identified could reveal additional potential targets in the receptor biosynthesis pathway using metabolomic data (Nicholson and Wilson 2003). Targets identified in this manner allow the application of in silico docking simulations to be performed that can pre-screen a random small molecule library—reducing and refining the number of candidate inhibitors (Fourches et al 2013; Spears et al 2016). …”

“…An ideal situation would arise in cases where the pathway for phage receptor production is known before beginning the inhibitor library screen (Brown et al 2012; Spears et al 2016; Xia et al 2010). This would allow the in silico studies to precede a random library screen—reducing the compounds actually tested in the laboratory.…”