Characterizing the virulence factor YapE in Yersinia pestis.

Yersinia pestis is the causative agent of bubonic plague and is primarily transmitted by fleas. Upon infection, the bacteria rapidly travel to the regional lymph nodes causing inflammation and cellulitis in these tissues (referred to as buboes). Two outer membrane proteins, YapE and Pla, have been implicated to have roles in dissemination to the lymph nodes. Their adhesive properties have shown that they are able to interact with host macrophages thereby increasing their ability to disseminate to regional lymph nodes. More recently, we have shown that YapE is cleaved by another virulence factor important for lymph node colonization, Pla, to become an active adhesin. To further understand the biology of YapE, I used a transposon mutagenesis screen to identify possible regulators. While the current approach did not identify novel regulators, I discuss potential modifications to screening conditions that may allow for increased success in future attempts. I also investigated the role that Pla may play in the activation or degradation of other outer membrane proteins. 2-DIGE analysis identified 76 Y. pestis proteins that may be processed by Pla and I confirmed Pla-dependent degradation on HmsF at 37°C. To further investigate the role outer membrane proteins may play in intracellular survival, I infected macrophages with Y. pestis strains lacking pla, yapE, ail or ompA. Before performing these intracellular assays, I developed an optimized gentamicin protection assay to monitor intracellular growth of Y. pestis. To this end, I determined that the concentration of gentamicin used for Y. pestis should not exceed 8 µg/ml for 1 hour. Using this assay, I verified that OmpA was required for Y. pestis intracellular survival in macrophages. While Y. pestis lacking Pla or YapE had minor defects in intracellular growth, an ail mutant showed no phenotype difference compared to WT. Future studies with mutants lacking multiple adhesins will help to define the potential contributions of these proteins to intracellular survival