The Translocator Protein YopD in Yersinia pseudotuberculosis: A Tool and a Weapon
- Author(s): Adams, Walter Isaiah
- Advisor(s): Auerbuch Stone, Victoria
- et al.
The aim of this dissertation is to enhance our fundamental understanding of host-pathogens interactions. I investigated these interactions by studying the relationship between the innate immune response and Yersinia pseudotuberculosis. Yersinia is a genus of Gram-negative bacteria that employ a type III secretion system (T3SS), an important virulence factor for Yersinia pathogenesis. The T3SS forms a pore in the membrane of host cells and injects Yersinia effector proteins into the host cell cytoplasm to impair the host immune response. YopD is a T3SS translocator protein involved in the post-transcriptional regulation of yop synthesis and pore formation in host cell membranes, both of which are directly linked to the effective translocation of effector proteins into the host cell cytosol. YopD is also engaged in interactions with several proteins including its chaperone LcrH/SycD, its fellow translocator proteins YopB and LcrV, the regulatory protein TyeA, the effector protein YopE, and it can even self-oligomerize. As YopD is a central player in key T3SS functions and associated protein interactions, there are many important questions that can be addressed through careful study of the YopD protein.
The first part of this dissertation utilizes YopD as a tool to understand how host cells detect T3SSs during Yersinia infection. Different models have been proposed to address how the host cell senses the presence of T3SSs. Some research indicates that the formation of a pore and subsequent K+ efflux itself leads to the induction of an immune response. A competing hypothesis is that pore formation alone is insufficient and that the translocation of T3SS cargo is required for the induction of this immune response. We constructed a Y. pseudotuberculosis mutant expressing YopD devoid of its predicted transmembrane domain (YopDdeltaTM) that formed pores in host cells but did not translocate T3SS cargo. Interestingly, this YopDdeltaTM mutant failed to induce significant production of pro-inflammatory cytokines, suggesting that host T3SS sensing requires either a translocation mediated event or the presence of a WT translocon.
The second part of my dissertation focuses on how YopD is required for the optimal performance of the T3SS weaponry. While the YopD amino and carboxy termini participate in pore formation and T3SS regulation, the role of the central region between amino acids 150-227 remains unknown. I assessed the contribution of the YopD central region by generating Y. pseudotuberculosis YopDdelta150-170 and YopDdelta207-227 mutants and analyzing their T3SS functions. These mutants formed robust pores in macrophages and displayed wildtype levels of Yop secretion in vitro. However, both mutants exhibited defects when translocating into neutrophils and macrophages, could not prevent phagocyte ROS production, and displayed a virulence defect in disseminated Yersinia infection in vivo. These findings suggest that the YopD central region facilitates optimal T3SS effector protein delivery into target host cells to effectively disarm innate immune defenses such as ROS production. Together these findings enhance our understanding of host-pathogen interactions, both in terms of how T3SSs are detected by the host and how specific components contribute to the optimal performance of T3SS function.