Interaction of Chlamydia trachomatis inclusion membrane protein CT226 with host proteins human flightless homolog-1 and Tropomodulin-3

Chlamydia trachomatis is a gram-negative, obligate intracellular pathogen transmitted sexually in humans. It is estimated that it results in more than 3 million STI infections in the USA, including pelvic inflammatory disease, ectopic pregnancy, and reduced fertility. This bacterium has a distinctive biphasic life cycle; its reproductive stage creates a parasitophorous vacuole, also called an inclusion. The elementary bodies (EBs) are the pathogen's metabolic inactive and infective form, whereas the reticulate bodies (RBs) are the metabolic active and replicative form. Chlamydia secretes inclusion membrane proteins (Incs; T3SS effector proteins) that decorate the inclusion membrane and mediate host-pathogen interactions. C. trachomatis secretes 50 putative Inc proteins. Previous studies have identified that the Inc CT226 interacts with the host actin-remodelling protein Tropomodulin-3 (TMOD3) and the host inflammasome-associated protein Human flightless homolog-1 (FLII). We hypothesize that Inc CT226 interacts with host proteins FLII and TMOD3 using the leucine-rich regions present in all three proteins. This study aimed to understand which domains of FLII and TMOD3 interact with CT226. We used two approaches: a bacterial two-hybrid system to understand the interaction in a prokaryotic environment and co-immunoprecipitation to understand interaction in a eukaryotic environment. We generated several variants of FLII and TMOD3 to test them for interaction with CT226 and define their interaction domains. CT226 interacted with full-length TMOD3 (TMOD3 FL) and the TMOD3 C-terminus (leucine-rich region) in both prokaryotic and eukaryotic environments, but no interaction was observed between CT226 and the TMOD3 N-terminus. In a prokaryotic environment, CT226 interacted with the FLII gelsolin-like domain (Gel) and leucine-rich region (LRR), whereas no interaction was observed between CT226 and full-length FLII FL or a construct containing the LRR and 160 additional amino acids C-terminal to the LRR (termed LRR+). We were unable to express the FLII proteins in a eukaryotic environment.