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

Among the 14 serovars of <i>Listeria monocytogenes</i> (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.