The role of IncV as a tether of endoplasmic reticulum-Chlamydia inclusion membrane contact sites

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections and can lead to infertility if left untreated. C. trachomatis infects epithelial cells and replicate exclusively within a membrane-bound vacuole called the inclusion. C. trachomatis Inc proteins are translocated through a type III secretion system, then embedded into the inclusion membrane where they interact with host molecules to support the establishment of the C. trachomatis intracellular niche. The inclusion establishes direct contact with the endoplasmic reticulum (ER-Inclusion Membrane Contact Sites (MCS)), and these contacts have been proposed to be important for the C. trachomatis developmental cycle by facilitating lipid acquisition.
Our lab has demonstrated both bacterial and host factors that localize to ER-Inclusion MCS. The Inc protein IncD recruits the host ceramide transfer protein CERT to the inclusion membrane. This forms an IncD-CERT-VAP complex which has been pro-posed to function in facilitating the transfer of host lipids that become incorporated into inclusion and bacterial membranes. Additionally, the host ER calcium sensor protein STIM1 localizes to ER-Inclusion MCS, though its interacting partner and function there is still unknown. While we had learned about a possible function of ER-Inclusion MCS in lipid acquisition, how they are formed and maintained was unclear.
I showed that Inc protein IncV plays a structural role at ER-Inclusion MCS through its interaction with VAP via the molecular mimicry of two eukaryotic FFAT motifs. VAP interacts with proteins containing FFAT motifs, which contain a 7-residue core and flanking acidic regions called the acidic tract. IncV contains two FFAT motif cores and mutating conserved core residues abolished the IncV-VAP interaction. IncV is sufficient, but not required, for the formation of ER-Inclusion MCS, suggesting IncV plays a sup-porting role in their formation and/or maintenance.
In eukaryotic cells, phosphorylation has been shown to regulate protein interactions with VAP and the cytosolic tail of IncV is highly enriched in phosphorylatable residues. I demonstrated that IncV is modified by a host factor and that IncV expressed by C. trachomatis in infected cells is phosphorylated. Phosphorylation of IncV is both necessary and sufficient for the IncV-VAP interaction in vitro and during infection. VAP recruitment to the inclusion is dependent on the host serine/threonine kinase Protein Kinase CK2 activity.
Altogether, my studies support a model in which CK2 phosphorylates IncV to promote the IncV-VAP interaction. I hypothesize that this this phosphorylation event supports the maintenance of ER-Inclusion MCS. In this dissertation, I present the characterization of two novel components of ER-Inclusion MCS, the host CK2 and the C. trachomatis Inc protein IncV, and identify their roles in the formation of these contacts. I dis-cuss how my findings add to our understanding of ER-Inclusion MCS formation and maintenance, as well as the MCS field, in general. Identifying molecules involved in the formation and maintenance of ER-Inclusion MCS could uncover potential targets for vac-cine or therapeutic drug development.