Hybiske, KevinDickinson, Mary Shelton2019-05-022019-05-022019Dickinson_washington_0250E_19669.pdfhttp://hdl.handle.net/1773/43728Thesis (Ph.D.)--University of Washington, 2019Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection, responsible for millions of infections each year. Despite this high prevalence, the elucidation of the molecular mechanisms of Chlamydia pathogenesis has been difficult due to limitations in genetic tools and its intracellular developmental cycle. Within a host epithelial cell, chlamydiae replicate within a vacuole called the inclusion. Many Chlamydia–host interactions are thought to be mediated by the Inc family of type III secreted proteins that are anchored in the inclusion membrane, but their array of host targets are largely unknown. To investigate how the inclusion membrane proteome changes over the course of an infected cell, we have adapted the APEX system of proximity-dependent biotinylation. APEX is capable of specifically labeling proteins within a 20 nm radius in living cells. We transformed C. trachomatis to express the enzyme APEX fused to known inclusion membrane proteins, allowing biotinylation and purification of inclusion-associated proteins. Using quantitative mass spectrometry against APEX labeled samples, we identified over 400 proteins associated with the inclusion membrane at early, middle, and late stages of epithelial cell infection. This system was sensitive enough to detect inclusion interacting proteins early in the developmental cycle, at 8 hours post infection, a previously intractable time point. A spreadsheet of the proteins identified by APEX at each time point is included as the supplemental file Table S21. Mass spectrometry analysis revealed a novel, early association between C. trachomatis inclusions and endoplasmic reticulum exit sites (ERES), functional regions of the ER where COPII-coated vesicles originate. Pharmacological disruption of ERES function severely restricted early chlamydial growth and the development of infectious progeny. Depletion of certain ERES proteins with RNA interference also reduced formation of infectious progeny. Most ERES proteins tested localized to small punctae that sometimes colocalized with the inclusion membrane, but two ERES proteins that were identified by APEX, PDCD6 and TFG, accumulated on the inclusion membrane in certain conditions. When intracellular calcium was elevated, PDCD6 strongly associated with the inclusion membrane. A truncated form of TFG accumulated on the inclusion membrane. Unlike other ERES proteins tested, depletion of PDCD6 caused enhanced C. trachomatis infectious progeny formation. The work in this dissertation shows that APEX is a powerful in situ approach for identifying critical protein interactions on the membranes of pathogen-containing vacuoles. Furthermore, the data derived from proteomic mapping of Chlamydia inclusions has illuminated an important functional role for ERES in promoting chlamydial developmental growth.application/pdfen-USCC BY-NC-SAMicrobiologyPathobiologyThesis