A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

Gut pathogens such as Salmonella enterica serovar Typhimurium target intestinal epithelial cells for adhesion and type-3-secretion-system-dependent invasion, while also invading multiple other cell types as the infection progresses. Mechanistic studies have uncovered virulence factors involved in this process, but host cell determinants affecting Salmonella cell surface targeting remain less deeply explored. Furthermore, cell surface protein expression and glycosylation patterns differ dramatically between epithelial and blood-derived cell types, and even across maturation states of the same cell type. Here, we explored bottom-up reconstruction of the host cell surface, using simplistic suspension-growing K562 cells, to determine the contribution of individual cell surface constituents during Salmonella targeting. Combined with flow cytometry and a stringently tunable gene expression system, this model enabled high-throughput analysis and combined genetic manipulations in both pathogen and host cells. Transcriptomic and proteomic data along with lectin characterization revealed minimal K562 surface glycosylation at baseline. Chemical manipulations substantiated the role of cell membrane cholesterol in promoting Salmonella targeting, whereas ectopic expression of glycoproteins such as transmembrane mucins introduced a size-dependent steric barrier towards invading bacteria. Strikingly, even established glycoprotein receptors for Salmonella adhesins hampered rather than promoted invasion, suggesting that adhesins are required to overcome cellular glycocalyces in vivo , while a bare host cell membrane would be the pathogen’s preferred interaction surface.