The foodborne pathogen Listeria monocytogenes (Lm) is a highly heterogeneous species and currently comprises of 4 evolutionarily distinct lineages. Here, we characterize isolates from severe ovine listeriosis outbreaks that represent a hybrid sub-lineage of the major lineage II (HSL-II) and serotype 4h. HSL-II isolates are highly virulent and exhibit higher organ colonization capacities than well-characterized hypervirulent strains of Lm in an orogastric mouse infection model. The isolates harbour both the Lm Pathogenicity Island (LIPI)-1 and a truncated LIPI-2 locus, encoding sphingomyelinase (SmcL), a virulence factor required for invasion and bacterial translocation from the gut, and other non-contiguous chromosomal segments from another pathogenic species, L. ivanovii. HSL-II isolates exhibit a unique wall teichoic acid (WTA) structure essential for resistance to antimicrobial peptides, bacterial invasion and virulence. The discovery of isolates harbouring pan-species virulence genes of the genus Listeria warrants global efforts to identify further hypervirulent lineages of Lm.

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen. L. monocytogenes is a Gram-positive, food-borne, intracellular pathogen that causes severe infection in susceptible individuals. Interestingly, almost all infections are caused by a subset of strains belonging to certain serovars featuring a complex glycosylation pattern on their cell surface. Using an engineered bacteriophage that specifically recognizes these modifications we selected for mutants that lost these sugars. We found that the resulting strains are severely deficient in invading host cells as we observed that a major virulence factor mediating host cell entry requires galactose decoration of the cell surface for its function. Without this galactose decoration, the strain represents a serovar not associated with disease. Altogether, we show a complex interplay between bacteriophages, bacteria, and the host, demonstrating that cellular invasiveness is dependent upon a serovar-defining structure, which also serves as a phage receptor.

The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Technologies for mapping the spatial relationships of cell surface receptors and their extracellular signaling synapses would unlock theranostic opportunities to target protein communities and the possibility to engineer extracellular signaling. Here, we develop an optoproteomic technology termed LUX-MS that enables the targeted elucidation of acute protein interactions on and in between living cells using light-controlled singlet oxygen generators (SOG). By using SOG-coupled antibodies, small molecule drugs, biologics and intact viral particles, we demonstrate the ability of LUX-MS to decode ligand receptor interactions across organisms and to discover surfaceome receptor nanoscale organization with direct implications for drug action. Furthermore, by coupling SOG to antigens we achieved light-controlled molecular mapping of intercellular signaling within functional immune synapses between antigen-presenting cells and CD8⁺ T cells providing insights into T cell activation with spatiotemporal specificity. LUX-MS based decoding of surfaceome signaling architectures thereby provides a molecular framework for the rational development of theranostic strategies.

Wall teichoic acids (WTAs) are the most abundant glycopolymers found on the cell wall of many Gram-positive bacteria, whose diverse surface structures play key roles in multiple biological processes. Despite recent technological advances in glycan analysis, structural elucidation of WTAs remains challenging due to their complex nature. Here, we employed a combination of UPLC-MS/MS and NMR to determine the structural complexity of WTAs from Listeria species. We unveiled more than 10 different types of WTA polymers that vary in their linkage and repeating units. Disparity in GlcNAc to ribitol connectivity, as well as variable O-acetylation and glycosylation of GlcNAc contribute to the structural diversity of WTAs. Notably, SPR analysis indicated that constitution of WTA determines the recognition by bacteriophage endolysins. Collectively, these findings provide detailed insight into Listeria cell wall-associated carbohydrates, and will guide further studies on the structure-function relationship of WTAs.