# Dual glycosylation of wall teichoic acid modulates the O‐antigen pattern and virulence in serovar 4b Listeria monocytogenes

## TL;DR

This study reveals how dual glycosylation of wall teichoic acid in Listeria monocytogenes serovar 4b affects its virulence and infection mechanisms.

## Contribution

The study identifies the synergistic roles of Glu-WTA and Gal-WTA in modulating bacterial pathogenesis and cell wall structure.

## Key findings

- Glu-WTA is essential for phage adsorption and enhances biofilm formation and resistance to antimicrobial peptides.
- Gal-WTA modulates surface protein anchoring and facilitates intracellular bacterial motility.
- Dual glycosylation of WTA enhances colonization and dissemination in mouse models.

## Abstract

Among the 14 serovars of Listeria monocytogenes (Lm), serovar 4b strains are the most predominant isolates linked to human listeriosis outbreaks‐a phenotype associated with their unique wall teichoic acid (WTA) decorated with galactose (Gal) and glucose (Glu). A wealth of knowledge is available for galactosylated‐WTA (Gal‐WTA) manipulating bacterial homeostasis and virulence, whereas the relationship between glucosylated‐WTA (Glu‐WTA) and Gal‐WTA in listerial physiology and pathogenesis remains unclear. Here, we find that Glu‐WTA and Gal‐WTA jointly constitute the O‐antigen pattern of serovar 4b Lm; however, Glu‐WTA specifically serves as the indispensable ligand for listeriophage LP4 adsorption. Moreover, the co‐operation between Glu‐ and Gal‐WTA increases biofilm formation and bacterial resistance to cationic antimicrobial peptide (CRAMP). We further demonstrate that Gal‐WTA modulates the anchoring of surface proteins, including IspC, Ami, and InlB. Additionally, dual glycosylated WTA interaction with ActA facilitates bacterial intracellular motility and dissemination. Consistently, Glu‐WTA significantly enhances bacterial colonization ability in the mesenteric lymph nodes (MLNs), ileum, liver, and brain of mouse, cooperating with Gal‐WTA to facilitate Lm dissemination to distant organs and tissues. In conclusion, we reveal the crucial roles of Glu‐WTA in synergizing with Gal‐WTA to modulate the integrity of the cell wall structure and exacerbate bacterial infection, providing a global understanding of the hypervirulence and pathogenicity of invasive serovar 4b Lm.

Serovar 4b Listeria monocytogenes (Lm), which is decorated with galactosylated and glucosylated wall teichoic acid (WTA), accounts for the majority of clinical listeriosis cases. Here, we confirm that both galactosylated‐WTA (Gal‐WTA) and glucosylated‐WTA (Glu‐WTA) are the major O‐antigen determinants of serovar 4b Lm. Importantly, both Glu‐ and Gal‐WTA promote ActA aggregation with host actin. Additionally, Gal‐WTA modulates the surface association of glycine–tryptophan (GW) proteins, thus playing crucial roles in mediating bacterial proliferation and translocation. Moreover, the absence of either Glu‐ or Gal‐WTA remarkably attenuates the ability of bacteria to colonize the intestine and spread to deep organs. This finding highlights the crucial roles of Glu‐WTA in synergy with Gal‐WTA in the pathogenesis of serovar 4b Lm.