FLI1 localization to the chlamydial inclusion involves multiple mechanisms

Following entry into a host cell, the obligate intracellular pathogen, Chlamydia trachomatis, establishes an intracellular niche within a membrane derived vacuole called the chlamydial inclusion. The resulting inclusion membrane is modified by the pathogen and is a hybrid host-chlamydial structure. From within this intracellular niche, C. trachomatis must orchestrate numerous host-pathogen interactions to surreptitiously acquire nutrients from its host and to limit detection by the host innate immune system. C. trachomatis mediates many of these interactions with the host, in part, by using a family of type III secreted membrane proteins, termed inclusion membrane proteins (Incs). Incs are embedded within the inclusion membrane, and some function to recruit host proteins to the inclusion. Two such recruited host proteins are leucine rich repeat Flightless-1 interacting protein 1 (LRRF1/LRRFIP1) and its binding partner Flightless 1 (FLI1/FLII). LRRF1 interacts with Inc protein Ct226. However, interactions of FLI1 with candidate Incs or with LRRF1 during infection have not been defined. We hypothesized that FLI1 recruitment to the inclusion would be dependent on LRRF1 localization. To test this hypothesis, we used siRNA targeting lrrf1 or fli1, revealing that FLI1 can localize to the inclusion independently of LRRF1. Therefore, to further characterize FLI1 localization, we developed and characterized a series of CRISPRi knockdown and complementation strains in C. trachomatis serovar L2 that target ct226 and co-transcribed candidate Incs, ct225 and ct224, to understand the mechanisms of FLI1 and LRRF1 localization to the inclusion. Our results indicate that FLI1 is recruited to the inclusion by multiple mechanisms. IMPORTANCEChlamydia trachomatis is a leading cause of both preventable infectious blindness and bacterial sexually transmitted infections worldwide. Since C. trachomatis must grow and replicate within human host cells, it has evolved several ways of manipulating the host to establish a successful infection. As such, it is important to describe the interactions between host proteins and chlamydial proteins to understand which strategies C. trachomatis uses to shape its intracellular environment. This study looks in detail at such interactions of two host proteins, FLI1 and LRRF1, during chlamydial infection. Importantly, the series of knockdown and complement strains developed in this study suggest these proteins have both independent and overlapping mechanisms for localization, which ultimately will dictate how these proteins function during chlamydial infection.