THE V-ANTIGEN OF YERSINIA-PESTIS REGULATES YOP VECTORIAL TARGETING ASWELL AS YOP SECRETION THROUGH EFFECTS ON YOPB AND LCRG

Yersinia pestis expresses a set of secreted proteins called Yops and t he bifunctional LcrV, which has both regulatory and antihost functions . Yops and LcrV expression and the activity of the type III mechanism for their secretion are coordinately regulated by environmental signal s such as Ca2+ concentration and eukaryotic cell contact. In vitro, Yo ps and LcrV are secreted into the culture medium in the absence of Ca2 + as part of the low-Ca2+ response (LCR), The LCR is induced in a tiss ue culture model by contact with eukaryotic cells that results in Yop translocation into cells and subsequent cytotoxicity. The secretion me chanism is believed to indirectly regulate expression of lcrV and yop operons by controlling the intracellular concentration of a secreted n egative regulator. LcrG, a secretion-regulatory protein, is thought to block secretion of Yops and LcrV, possibly at the inner face of the i nner membrane. A recent model proposes that when the LCR is induced, t he increased expression of LcrV yields an excess of LcrV relative to L crG, and this is sufficient for LcrV to bind LcrG and unblock secretio n, To test this LcrG titration model, LcrG and LcrV were expressed alo ne or together in a newly constructed lcrG deletion strain, a Delta lc rG2 mutant, of Y. pestis that produces low levels of LcrV and constitu tively expresses and secretes Yops. Overexpression of LcrG in this mut ant background was able to block secretion and depress expression of Y ops in the presence of Ca2+ and to dramatically decrease Yop expressio n and secretion in growth medium lacking Ca2+, Overexpression of both LcrG; and LcrV in the Delta lcrG2 strain restored wild-type levels of Yop expression and Ca2+ control of Yop secretion. Surprisingly, when H eLa cells were infected,vith the Delta lcrG2 strain, no cytotoxicity w as apparent and translocation of Yops was abolished. This correlated w ith an altered distribution of YopB as measured by accessibility to tr ypsin, These effects were not due to the absence of LcrG, because they were alleviated by restoration of LcrV expression and secretion alone . LcrV itself was found to enter HeLa cells in a nonpolarized manner. These studies supported the LcrG titration model of LcrV's regulatory effect at the level of Yop secretion and revealed a further role of Lc rV in the deployment of YopB, which in turn is essential for the vecto rial translocation of Yops into eukaryotic cells.