Project Summary. Gram-negative bacterial pathogens interact with mammalian cells by using a specialized ‘type III secretion system’ (T3SS) to inject proteins directly into infected host cells. Many of these injected protein ‘effectors’ are enzymes that modify the structure and function of human proteins by catalyzing the addition of unusual post- translational modifications. T3SS effectors play essential roles in bacterial virulence and are important targets for anti-virulence compounds that can be used to replace or augment traditional antibiotic regimens. The NleB (E. coli) and SseK (Salmonella enterica) T3SS effectors are glycosyltransferases that modify protein substrates on arginine residues. This modification is especially interesting because it occurs on the guanidinium groups of arginines, which are poor nucleophiles. These enzymes are extremely important to pathogen virulence. NleB- deficient Citrobacter rodentium (a mouse pathogen used as a model organism for studying pathogenic E. coli) do not cause mortality to mice. NleB is also a signature of enterohemorrhagic E. coli (EHEC) strains with the ability to cause foodborne outbreaks and the often-fatal hemolytic uremic syndrome (HUS) in humans. Preliminary data are available to show 1) crystallization of the NleB/SseK orthologs; 2) determination of the mechanism by which these proteins glycosylate host substrates; 3) development and optimization of a preliminary high-throughput screening (HTS) assay to identify EHEC NleB1 inhibitors; 4) characterization of two compounds that inhibit NleB1 with IC50s of ~200 nM; and 5) validation that neither inhibitor blocks the activity of the essential human O-GlcNAc-transferase (OGT) that glycosylates serine and threonine residues. The following specific aims are proposed to respond to PAR-17-438: 1) Conduct a larger HTS assay to identify and optimize NleB/SseK inhibitors with increased potency.; 2) Characterize the mechanisms by which the small molecules inhibit NleB/SseK activity; 3) Provide proof of concept that NleB inhibitors reduce C. rodentium replication in mouse models of disease. The proposed experiments will provide novel insight into how NleB/SseK catalyze the glycosylation of the poorly nucleophilic guanidinium group of arginines, will provide novel probes to monitor the activity of these enzymes, and will also advance the development of anti-virulence compounds against important human pathogens.