Dissecting the role of the Inclusion membrane protein IncE, a master multi-tasking scaffolding protein, in the pathogenesis of Chlamydia trachomatis infections - Abstract Chlamydia trachomatis (Ct) infections are important causes of human disease for which no vaccine exists. An important gap in our knowledge is how this obligate intracellular vacuolar bacterium establishes a privileged niche--a membrane bound compartment termed the inclusion. Chlamydia encode a distinctive family of secreted effectors, the Incs (Inclusion membrane proteins), which are translocated from the bacteria through the type III secretion system and inserted into the inclusion membrane. We have discovered that an early expressed Inc, IncE, binds to two different host cell protein complexes that are involved in trafficking, sorting nexins (SNX)5 and 6 as well as two closely related Q-SNARES, Syntaxin(STX)7 and STX12, which are involved in late and early endosome trafficking, respectively. Our results support a model wherein IncE directly binds SNX5/6, which redirects the ESCPE-1 complex (SNX5/6 together with SNX1/2) to the inclusion membrane, potentially disrupting ESCPE-1-mediated trafficking. In preliminary experiments, we have defined the regions of IncE that are necessary for binding to STX7/12 and discovered that IncE encodes an additional distinct short motif separate from the SNX5/6 cargo binding motif that mimics the zero layer motif (ZLM) of the VAMP3 family of R-SNARE proteins, known binding partners of STX7/12. This exciting observation suggests that IncE may modulate STX7/12 vesicle fusion with the inclusion, either by stimulating it, inhibiting it, or functioning as a tether. Using RNAi, we have discovered that STX7 and STX12 serve distinct non-overlapping roles in Ct infection. We have created an IncE null mutant and complemented it with informative IncE variants. This heroic application of Ct genetics will allow us to separate IncE binding to SNX5/6 from its binding to STX7/12, and to test the role of a single Inc in an animal model of Ct genital tract infection. In this grant, we propose to employ a combination of Ct genetics, cell biology, advanced microscopy, and biochemistry to: Aim 1. Determine the role of IncE in the pathogenesis of Ct infections. We will characterize the phenotypes of the IncE in-frame deletion mutant strain and informative isogenic complemented derivatives using (A) cell-based assays and (B) a mouse model of upper genital tract infection. Aim 2. Understand the molecular mechanism and consequences of IncE:STX7/12 interactions. Using informative IncE mutant strains and proteins, we will (A) determine whether IncE:STX7/12 interactions modulate fusion, using live cell microscopy that employs novel fluorescent lipid probes (B) determine if IncE, and specifically its ZLM, is sufficient to modulate STX7 vesicle fusion, using an in vitro liposome fusion assay; (C) test the hypothesis that the IncE ZLM binds directly to STX7 and/or STX12, using purified proteins; ad (D) Test whether IncE can bind simultaneously to STX7/12 and SNX5/6; Aim 3. Decode the roles of STX7/12 in regulating the Ct intracellular lifecycle. We will investigate (A) how STX12 contributes to homotypic inclusion fusion; (B) how STX7 contributes inclusion formation and production of infectious progeny. Our studies will increase our knowledge of host cell biology and reveal how microbes subvert these processes.
