Antimicrobial resistance is a global challenge that is compounded by the limited number of available targets. Glycocins are antimicrobial glycopeptides that are believed to have novel targets. Previous studies have shown that the mechanism of action of the glycocin sublancin 168 involves the glucose uptake system. The phosphoenolpyruvate:sugar phosphotransferase system (PTS) phosphorylates the C6 hydroxyl group on glucose during import. Since sublancin carries a glucose on a Cys on an exposed loop, we investigated whether phosphorylation of this glucose might be involved in its mechanism of action by replacement with xylose. Surprisingly, the xylose analog was more active than wild-type sublancin and still required the glucose PTS for activity. Overexpression of the individual components of the PTS rendered cells more sensitive to sublancin, and their resistance frequency was considerably decreased. These observations suggest that sublancin is activated in some form by the glucose PTS or that sublancin imparts a deleterious gain-of-function on the PTS. Superresolution microscopy studies with fluorescent sublancin and fluorescently labeled PTS proteins revealed localization of both at the poles of cells. Resistant mutants raised under conditions that would minimize mutation of the PTS revealed mutations in FliQ, a protein involved in the flagellar protein export process. Overexpression of FliQ lead to decreased sensitivity of cells to sublancin. Collectively, these findings enforce a model in which the PTS is required for sublancin activity, either by inducing a deleterious gain-of-function or by activating or transporting sublancin.
Keywords: S-glycosylation; antibiotic; antimicrobial; glucose PTS; microscopy; resistance frequency.