Insights into the Chlamydial Niche: The Dynamic Roles of Inclusion Membrane (Inc) Proteins in Chlamydia trachomatis Development

Chlamydia trachomatis (Ctr) is the leading cause of bacterial sexually transmitted infections. Ctr, an obligate intracellular bacterium, develops within a membrane-bound vacuole called an inclusion. The inclusion membrane is modified by chlamydial inclusion membrane (Inc) proteins, the functions of which are poorly characterized. Bacterial two-hybrid analyses found some Incs (e.g., IncF) interacted with numerous Incs while others (e.g., IncA) did not. We hypothesize that some Incs organize the inclusion through Inc-Inc interactions whereas other Incs promote chlamydial-host interactions by binding eukaryotic proteins. To test our hypothesis, we implemented the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo. We transformed Ctr with inducible expression constructs containing incF-APEX2, incATM(transmembrane domain)-APEX2, incA-APEX2, and APEX2 alone. Affinity purification-mass spectrometry (AP-MS) of biotinylated proteins from chlamydial infected monolayers, followed by Significance Analysis of INTeractome (SAINT), was used to identify significant proteins that are proximal to the Inc-APEX2 constructs from five biological replicates. Consistent with our hypothesis, IncF-APEX2 biotinylated more chlamydial Inc proteins, whereas IncA-APEX2 biotinylated more unique eukaryotic proteins. We validated a SAINT significant eukaryotic protein, LRRF1, at the inclusion membrane by immunofluorescence and determined that the Inc CT226 interacts with LRRF1. Next, we compared our datasets with other AP-MS inclusion studies, finding only seven proteins that were similarly identified, likely a reflection of the different experimental approaches. Importantly, for the first time, we were able to directly compare two Inc-APEX2 studies, which also revealed fewer similarly identified proteins than expected. This led us to hypothesize that the overexpression of certain Incs may alter the organization of the inclusion membrane. Previously, we described defects in inclusion expansion and the production of progeny when IncF-APEX2 was expressed at high levels from transformed Ctr. To further investigate the role of Incs in chlamydial development, we transformed Ctr with inducible expression plasmids containing FLAG-tagged incF, ct813, and ct226. The expression of IncF and CT813, but not CT226, altered inclusion development, as observed by smaller inclusions and decreased IncE intensity in the inclusion membrane. These data suggest that coordinated Inc expression and insertion into the inclusion membrane is essential for optimal inclusion development