SUMMARY Our laboratory studies the bacterial type III secretion system (T3SS) which is a sophisticated nanomachine that permits communication with eukaryotic cells to subvert normal cellular functions. T3SSs are essential virulence factors for a number of biomedically important human pathogens including Yersinia pestis (plague), Shigella spp. (dysentery), Salmonella spp. (diarrhea/enteric fever), Bordetella pertussis (whooping cough), and Pseudomonas aeruginosa (opportunistic/nosocomial infections). While these systems are diverse with regard to the effector proteins they deliver, the T3SS apparatus (T3SA) is well-conserved with respect to overall structure and architecture. The T3SA is comprised of: 1) a cytoplasmic sorting platform that recognizes secretion substrates and powers secretion; 2) a rigid basal body spanning the entire cell envelope; and 3) an exposed needle with tip complex that delivers cargo into host cells. The protein components of the sorting platform have been identified, but little was known about how they assemble until we published the first description of the in situ Shigella T3SA sorting platform. Since then, we have determined the structural details and dynamics of this platform, including the essential protein-protein contacts. Based on this background information, we hypothesize that key interfaces within the sorting platform can be targeted to block type III secretion in Shigella. Furthermore, we can extrapolate our findings to identify inhibitors of homologous interactions within distantly related T3SA sorting platforms such as that form P. aeruginosa. Here, we will target the interaction between inner membrane-imbedded MxiG (via its cytoplasmic domain, MxiGc) and its newly identified binding partner, MxiK, in Shigella. The homologous pair from P. aeruginosa (PscDc and PscK, respectively) will also be targeted. The specific aims of this pilot project application are to: 1) Complete high-throughput screens of extended chemical libraries to identify small molecules that interfere with the interaction of MxiGc (PscDc) with MxiK (PscK); and 2) Validate hits for inhibitors of the MxiG-MxiK and PscD-PscK interactions by performing ligand binding experiments and determining their effects on Shigella and Pseudomonas T3SS-related virulence functions. This high-risk, high-payoff potential of this exploratory project will employ specialized core laboratories in high-throughput screening, computational chemical biology and protein structure determination at the University of Kansas to generate a synergy that will allow an unprecedented level of understanding of the mechanism by which the T3SA sorting platform operates.