Inhibitors of Type III Secretion and Translocation in yersinia

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Many gram negative bacterial pathogens, including Yersinia, Salmonella, Pseudomonas, Shigella, enteropathogenic E. coli, Chylamydia, and Burkholderia, use type III secretion systems (TTSS) to translocate effector proteins from the bacterial cytosol into mammalian cells. Translocated effector proteins, called Yops in Yersinia, subvert normal host processes to promote the survival of the pathogen, and thus TTSS play an essential role in the infectious process and virulence of these bacteria. TTSS are comprised of a base, which spans the inner and outer membranes of the bacteria, a needle, which extends from the base to the host cell, and a translocon, which is inserted into host cell membranes and through which Yops are thought to travel to reach host cell cytoplasm. In Yersinia, the needle is primarily composed of one 7kD protein, YscF, which polymerizes to form a long tube. The needle is thought to be a conduit for the passage of Yops from the bacteria to the translocon and is thought to conduct signals from the host cell membrane to the TTSS base when the pathogen comes in contact with a host cell. These signals trigger Yop translocation. A tip protein, LcrV, is found at the distal end of the needle. One function of LcrV is to position, assemble, and/or insert the translocon, into plasma membranes, which is essential for Yop delivery into cells. Two proteins, YopB and YopD comprise the translocon. The overall goals of this proposal are to understand the molecular interactions between TTSS components that are required for translocating Yops upon contact with mammalian cells. We have identified 3 dominant negative YscF alleles and 8 small molecules that inhibit Yop translocation. These unique mutants and compounds will be studied intensively as probes to understand the required molecular interactions for Yop translocation into host cells. Our working hypothesis is that our inhibitors interfere with critical interactions between YscF, LcrV, YopB and/or YopD. Understanding the mechanism of translocation and needle assembly will support the development of new strategies that interfere with these processes and thus neutralize the bacteria's pathogenecity.