Validation and subcellular localization of previously predicted type III secreted effector proteins in Chlamydia trachomatis.

The obligate intracellular bacterium Chlamydia trachomatis (C.t.) is the leading cause of bacterial sexually transmitted infections and infectious blindness worldwide. C.t. replicates within a specialized membrane-bound compartment known as the inclusion and employs a type III secretion system (T3SS) to deliver effector proteins into the host cell. These effectors reprogram host cellular processes to create an environment conducive to bacterial replication. Until recently, C.t. was genetically intractable, limiting efforts to identify effector proteins secreted during infection. As a result, early studies relied on heterologous expression in surrogate T3SS-component bacteria such as Yersinia pseudotuberculosis, Shigella flexneri, or Salmonella enterica serovar Typhimurium to assess secretion potential. With the advent of genetic tools for use in C.t., we are now able to directly assess whether previously identified effectors are translocated during C.t. infection. Using three complementary approaches-the adenylate cyclase assay, β-lactamase assay, and glycogen synthase kinase assay-we identified 11 secreted effectors and 10 potentially secreted effectors. Of these 21 secreted factors, 10 displayed unique localization patterns when ectopically expressed in mammalian cells. Together, these findings define a core set of secreted effectors C.t. effectors that warrant further functional characterization.

Importance: This study systematically identifies and validates Chlamydia trachomatis type III secreted effectors, confirming secretion for 11 proteins and identifying 12 additional candidates. Using multiple complementary translocation assays, we directly assess secretion in the native bacterial context, revealing key effectors that target specific host compartments. Notably, several effectors localize to the nucleus or cytoplasm, suggesting distinct roles in host cell manipulation. Our findings refine the known effector repertoire and provide critical insights into how C. trachomatis remodels host organelles to sustain infection. By emphasizing experimentally verified secretion rather than surrogate systems, this work strengthens the foundation for future mechanistic studies and therapeutic targeting.