B-299. Small Molecule Compounds that Inhibit Translocation of Yersinia Yops

D. E. Harmon, C. Castillo, A. J. Davis, J. Mecsas;
Tufts Univ., Boston, MA.

Type three secretion systems (TTSS) are used by a wide variety of gram negative bacterial pathogens and symbionts. These systems transport effector proteins from bacterial cytoplasm to host cell. In Yersinia, the TTSS is encoded on a 70kb virulence plasmid, along with other proteins called Yersinia outer proteins (Yops) that are translocated into the host cell to subvert the innate immune response. To begin to dissect the translocation process, we screened for small molecules that inhibit translocation of Yops. The screen was based on an assay that uses a fragment of β-lactamase, TEM1, which lacks secretory signal of it’s own but maintains its catalytic activity towards a dye called CCF2-AM, a coumarin-cephalosphorin-fluorescein conjugate, which is readily taken up by mammalian cells. TEM is fused to the secretion and translocation signals of YopE which permit the chimeric protein to be translocated into cells when cells are infected with Yersinia. In the uncleaved state, excitation of CCF2-AM results in a FRET signal emitting a green fluorescent signal. In the presence of YopE-TEM1 CCF2-AM loses the FRET signal and fluoresces blue. Defects in translocation were determined by analyzing the 447/520 ratio (blue versus green) of fluorescence. Over 100,000 small molecules were screened and 200 compounds appeared to reduce TEM activity. Forty-five compounds were tested in secondary screens to test for specific defects in translocation. After several secondary screens, we identified 10 compounds reduce the amount of Yop translocation. Several of these altered the pore forming ability of Yersinia in sheep red blood cells. Specifically, three compounds had significantly reduced levels of YopE translocation and lower levels of hemolysis of sheep red blood cells than control untreated Yersinia, whereas one compound had poor translocation but greater hemolysis. Since Yop secretion was normal in the presence of these compounds, our working hypothesis is that the compounds target the translocon.