Type III secretion system (T3SS) effector proteins are primarily recognized for binding host proteins to subvert host immune response during infection. Besides their known host target proteins, several T3SS effectors also interact with endogenous bacterial proteins. Here we demonstrate that the Salmonella T3SS effector glycosyltransferase SseK1 glycosylates the bacterial two-component response regulator OmpR on two arginine residues, R15 and R122. Arg-glycosylation of OmpR results in reduced expression of ompF, a major outer membrane porin gene. Glycosylated OmpR has reduced affinity to the ompF promoter region, as compared to the unglycosylated form of OmpR. Additionally, the Salmonella ΔsseK1 mutant strain had higher bile salt resistance and increased capacity to form biofilms, as compared to WT Salmonella, thus linking OmpR glycosylation to several important aspects of bacterial physiology.

The type III secretion system effector proteins NleB and SseK are glycosyltransferases that glycosylate protein substrates on arginine residues. We conducted high-throughput screening assays on 42,498 compounds to identify NleB/SseK inhibitors. Such small molecules may be useful as mechanistic probes and may have utility in the eventual development of anti-virulence therapies against enteric bacterial pathogens. We observed that YM155 (sepantronium bromide) inhibits the activity of Escherichia coli NleB1, Citrobacter rodentium NleB, and both Salmonella enterica SseK1 and SseK2. YM155 was not toxic to mammalian cells, nor did it show cross-reactivity with the mammalian O-linked N-acetylglucosaminyltransferase (OGT). YM155 reduced Salmonella survival in mouse macrophage-like cells but had no direct impact on bacterial growth rates, suggesting YM155 may have utility as a potential anti-virulence inhibitor.

We are interested in identifying and characterizing small molecule inhibitors of bacterial virulence factors for their potential use as anti-virulence inhibitors. We identified from high-throughput screening assays a potential activity for avasimibe, a previously characterized acyl-coenzyme A: cholesterol acyltransferase inhibitor, in inhibiting the NleB and SseK arginine glycosyltransferases from Escherichia coli and Salmonella enterica, respectively. Avasimibe inhibited the activity of the Citrobacter rodentium NleB, E. coli NleB1, and S. enterica SseK1 enzymes, without affecting the activity of the human serine/threonine N-acetylglucosamine (O-GlcNAc) transferase. Avasimibe was not toxic to mammalian cells at up to 200 µM and was neither bacteriostatic nor bactericidal at concentrations of up to 125 µM. Doses of 10 µM avasimibe were sufficient to reduce S. enterica abundance in RAW264.7 macrophage-like cells, and intraperitoneal injection of avasimibe significantly reduced C. rodentium survival in mice, regardless of whether the avasimibe was administered pre- or post-infection. We propose that avasimibe or related derivates created using synthetic chemistry may have utility in preventing or treating bacterial infections by inhibiting arginine glycosyltransferases that are important to virulence.