UC San Diego

Pathogens utilize a variety of mechanisms to survive and reproduce within their host. The type III secretion (T3S) system is a pathogenic mechanism employed by many gram negative bacteria, including Salmonella, Pseudomonas, Escherichia coli, and Yersinia, the last of which is responsible for the bubonic plague (Y. pestis) as well as gastrointestinal infections (Y. enterocolitca and Y. pseudotuberculosis). The T3S system is made up of approximately 20-25 proteins which form a needle-like complex with similarity to the bacterial flagellum. Yersinia spp. utilize this system to translocate effectors into the host cell. These effectors enable Yersinia to evade host immune responses and ensure bacterial survival. This dissertation aims to answer questions regarding the mechanisms of effector secretion by the Yersinia T3S. In this dissertation, I present contributions I have made to published papers on the translocation of the effector YopE as well as structural and functional characteristics of an inner membrane protein of the T3S injectisome, YscD. I present my findings on the functional characteristics of an essential T3S component, YscO. I further show how protein-protein interactions between YscO and the T3S molecular ruler, YscP, dictate T3S function. In this dissertation, I also present methods for identifying potential binding partners to the secretion and translocation signals of the effector YopE. I present the construction of two vectors to use the RaPID and reverse- CLIP techniques for the identification of binding partners to the 5'mRNA signal of YopE. Both of these techniques utilize in vivo crosslinking and streptavidin affinity purification coupled with tandem mass spectrometry. Finally, I present potential binding partners to the first 80 amino acids of YopE which encompass the 5'/N-terminal signal as well as the chaperone binding (Cb) region. Using streptavidin affinity purification coupled with tandem mass spectrometry, I found that the inner rod protein YscI and the ATPase regulator YscL selectively bind wild-type YopE1-80 and a non-translocating mutant of YopE called 3- Ala1-80. I also found that the T3S gate-keeper YopN selectively binds 3-Ala1-80 and not wild-type YopE1-80