Galactosylated wall teichoic acid, but not lipoteichoic acid, retains InlB on the surface of serovar 4b Listeria monocytogenes

Abstract

Listeria monocytogenes is a Gram‐positive, intracellular pathogen harboring the surface‐associated virulence factor InlB, which enables entry into certain host cells. Structurally diverse wall teichoic acids (WTAs), which can also be differentially glycosylated, determine the antigenic basis of the various Listeria serovars. WTAs have many physiological functions; they can serve as receptors for bacteriophages, and provide a substrate for binding of surface proteins such as InlB. In contrast, the membrane‐anchored lipoteichoic acids (LTAs) do not show significant variation and do not contribute to serovar determination. It was previously demonstrated that surface‐associated InlB non‐covalently adheres to both WTA and LTA, mediating its retention on the cell wall. Here, we demonstrate that in a highly virulent serovar 4b strain, two genes gtlB and gttB are responsible for galactosylation of LTA and WTA respectively. We evaluated the InlB surface retention in mutants lacking each of these two genes, and found that only galactosylated WTA is required for InlB surface presentation and function, cellular invasiveness and phage adsorption, while galactosylated LTA plays no role thereof. Our findings demonstrate that a simple pathogen‐defining serovar antigen, that mediates bacteriophage susceptibility, is necessary and sufficient to sustain the function of an important virulence factor.

Glycotyping and Specific Separation of Listeria monocytogenes with a Novel Bacteriophage Protein Tool Kit

“…To identify suitable phage RBPs for complementing the glycotyping scheme, putative baseplate-associated proteins of various Listeria phages with distinct host ranges were subjected to in silico analyses using HHpred (38), BLASTp (39), and Jpred secondary structure predictions (40). Based on the conserved synteny of Listeria siphoviral structural genes (23), four tail spike RBPs from bacteriophages A006 (gp17), A500 (gp19) (16), PSA (gp15) (41), and B025 (gp18) were selected to expand the repertoire of GFP-tagged phage proteins, aiming at SV-specific glycotype identification. We produced and purified recombinant His-tagged versions of these proteins (see Fig.…”

“…The pB025_gp18-GFP vector was created by inserting a gfp followed by a SacI restriction site between the NdeI and BamHI restriction sites of the pET302 NT-His vector (Invitrogen, Carlsbad, CA, USA); subsequently, a commercially ordered DNA fragment of the full-length B025_gp18 sequence (GeneArt; Thermo Fisher Scientific, Waltham, USA) was inserted between SacI and BamHI, as previously described (36). The pA500_gp19-GFP vector was created by ligating the full-length A500_gp19 sequence (amplified by primers A500_gp19-F/R from whole purified phage) into the SacI-SalI restriction sites of the pHGFP vector (16,37). The pA006_gp17-biotin vector was created by inserting the truncated A006_gp17 sequence (amplified from whole A006 phage) via Gibson assembly (69) into the p165-His/Avi-tagged vector.…”

“…These findings suggested that the decorations themselves contribute to virulence since SVs 3, 7, and 4d are not known to cause listeriosis. Indeed, we and others have recently shown that these galactose and rhamnose decorations contribute directly to phage susceptibility, resistance to osmotic pressure, growth fitness, and the maintenance of major virulence factors, such as InlB and Ami (15)(16)(17)(18)(19). These revelations are now leading to a renewed focus on SV identity and show that phenotypic evaluations may hold more merit in certain situations over whole-genome sequencing (WGS), which otherwise offers far greater discerning power.…”

Structure and function of Listeria teichoic acids and their implications

“…It has been shown that autolysins that possess such GW domains mediate their surface presentation through direct binding to glycosylated WTAs (Cabanes, Dussurget, Dehoux, & Cossart, 2004). WTA rhamnosylation in an SV 1/2a background, and galactosylation in a 4b strain were shown to be responsible for retaining and displaying InlB (Carvalho, Sousa, & Cabanes, 2018;Sumrall et al, 2019;Sumrall et al, 2020). Previously, LTA had also been implicated as the binding partner for GW-domain containing proteins (Jonquieres, Bierne, Fiedler, Gounon, & Cossart, 1999).…”

“…Such knowledge allows the construction of mutants with specifically modified TA decoration. Recent studies have highlighted the roles of TAs in Listeria physiology, antigenic determination, susceptibility to antibacterials, virulence, and in the interactions between bacteria, bacteriophages and eukaryotic host cells (Abachin et al, 2002;Carvalho et al, 2015;Spears et al, 2016;Sumrall et al, 2019;Sumrall et al, 2020;Yin et al, 2019).…”