Chlamydia trachomatis (C.t.) is the leading cause of non-congenital blindness and the most prevalent sexually transmitted bacterial infection in the world and thus, has a major impact on global health. C.t. replicates in a membrane-bound vacuole, termed the inclusion, that is extensively modified early in infection through the incorporation of over 50 type III secreted effector proteins termed inclusion membrane proteins (Incs). From within the confines of the inclusion, the bacterium must engage select host organelles to obtain key nutrients such as lipids and iron for bacterial replication and inclusion expansion. Despite the importance of nutrient acquisition to chlamydial infection, a major gap in our knowledge exists regarding how C.t. acquires these essential substrates from the host cell. Recent work from our group shows that the Inc protein CT229 binds and recruits Rab GTPases and specific Rab effectors to the inclusion. Using cutting-edge genetics, we showed that the absence of CT229 results in defects in intracellular replication and inclusion development. We hypothesize that CT229-Rab interactions direct post-Golgi vesicles and recycling endosomes to the inclusion for the acquisition of essential substrates needed for proliferation and inclusion expansion.
In Aim 1, we will determine how CT229-Rab interactions promote lipid acquisition by hijacking host vesicular trafficking pathways and iron acquisition by promoting the recruitment of transferrin-positive vesicles to the inclusion. We will evaluate the lipid composition of the bacterial and inclusion membranes and evaluate the importance of lipid acquisition for incorporation into the inclusion and bacterial membranes. We will also answer a major long-standing question in the field by determining whether transferrin indeed serves as a major source of iron for chlamydia and furthermore mechanistically probe the mechanisms by which chlamydia may extract iron from this pathway. It is unlikely that CT229 alone controls vesicle hijacking from start to finish, therefore in Aim 2 we will identify the complex of host and bacterial proteins that are formed at the inclusion during CT229-Rab interactions. Using cellular, genetic, and molecular techniques we will evaluate the impact of Inc-Inc interactions on Rab-dependent vesicle transport to the inclusion. Detailed characterization of the bacterial and host proteins that co-opt host vesicular trafficking during C.t. infection will provide a holistic view of how intracellular pathogens coordinate the capture and fusion of host vesicles that are necessary for bacterial infection.
Chlamydia trachomatis is the etiological agent of blinding trachoma and a sexually transmitted infection that can result in serious complications including pelvic inflammatory disease (PID), ectopic pregnancy, and infertility. The molecular mechanisms used by C. trachomatis to co-opt host vesicular trafficking for the acquisition of essential nutrients are poorly understood since until recently chlamydia was intractable to genetic manipulation. This proposal will define how the essential C. trachomatis Inc protein, CT229, coordinates interactions with host and bacterial factors to acquire essential nutrients required for proliferation and inclusion stability, which will ultimately identify novel targets for therapeutic intervention and greatly expand our understanding of host-pathogen interactions.
