Genital Chlamydia spreading to the gastrointestinal tract promotes chlamydial pathogenicity in the upper genital tract Chlamydia trachomatis (CT) causes long-lasting tubal fibrosis/hydrosalpinx/infertility and is also frequently detected in human gastrointestinal (GI) tract. Most research efforts are focused on CT interactions with the genital but not GI tracts because CT is not associated with any significant pathology in the GI tract. However, it is not clear whether GI CT can impact pathogenicity of CT in the genital tract. Addressing this question directly in humans may require large-scale human studies with therapeutic interventions. Alternatively, we are proposing to use a C. muridarum (CM) mouse model for proof of concept studies. The CM mouse model has been used extensively for investigating chlamydial pathogenic mechanisms because CM can induce long- lasting tubal fibrosis/hydrosalpinx/infertility in mice. CM can also colonize the mouse GI tracts for long periods of time. Our in vivo imaging studies revealed CM spreading from the mouse genital to GI tracts via a hematogenous route. CM spreading to the GI tract may promote CM pathogenicity in the genital tract. First, our series of CM mutants are attenuated in both inducing hydrosalpinx and spreading to the GI tract although still maintaining robust infectivity in the genital tract. Second, co-inoculating a wild type CM into GI tract rescued a CM mutant to induce hydrosalpinx. GI CM does not autoinoculate or spread to the genital tract tissues even after oviduct surgery, indicating that the GI CM organisms must promote hydrosalpinx via an indirect mechanism. CD8+ T cell depletion significantly reduced hydrosalpinx induced with CM that is able to spread to the GI tract, suggesting that GI CM may promote hydrosalpinx by inducing the “pathogenic” CD8+ T cells. Thus, we propose a 2-hit model for chlamydial pathogenicity in the upper genital tract: CM ascends to the upper genital tract to cause the 1st hit that may be insufficient for driving long-term tubal fibrosis/hydrosalpinx in the absence of a 2nd hit. The 2nd hit is caused by recruiting pathogenic CD8+ T cells (induced by GI CM) to the oviduct to convert the 1st hit-induced tubal repairing response into long-lasting tubal fibrosis/hydrosalpinx. We will test the 2-hit hypothesis using 2 specific Aims by determining whether CM spreading to and colonizing the GI tract is both necessary and sufficient for promoting hydrosalpinx and whether the GI CM-induced CD8+ T cells are responsible for promoting hydrosalpinx. These proposed studies will lay a foundation for both future mechanistic and clinic investigations.