Abstract An obligate intracellular bacterium, Chlamydia trachomatis (Ct), is the leading sexually transmitted bacterial infection worldwide. Chronic Ct infection leads to serious reproductive complications in women, including pelvic inflammatory disease, pelvic pain, tubal infertility, and ectopic pregnancy. Recent compelling molecular data support historical studies showing that Ct can establish chronic infections even with repeated antimicrobial treatments. Yet, the mechanisms underpinning Ct adaptation, survival, and persistence during this onslaught and host immune-imposed antimicrobial insults are unknown. To evaluate antimicrobial responses of intracellular bacteria, it is desirable to establish quantitative relations between the fitness of growing or persisting bacteria and the target host cells. These important relations are challenging to study during Chlamydia infection due to the current limitations in techniques. To overcome this barrier, we have created a robust endogenous promoter- fluorescent protein reporter system. Building on our recent success in probing the central aspect of a unique Ct developmental cycle in situ, we hypothesize that Ct adapts to, and modulates, host cell metabolic pathways to maintain viability during exposure to external and host immune-imposed antimicrobial insults. To test this hypothesis, we propose two Aims: 1. To develop a novel in situ method using fluorescent protein expression levels to quantify interchangeable Ct developmental cycle in live cells. This powerful tool will allow us to quantitatively probe divergent chlamydial forms in a single Ct-containing vacuole and in a culture composed of physiologically and phenotypically different cells; and 2. To characterize key signaling pathways, in Ct and its host cells, that are vital to Ct adaptation and survival, in the presence of antimicrobial insults. Our innovative studies will be achieved using advanced technologies, including gene reporter assays, florescence activated cell sorting (FACS), and dual RNA sequencing (FACS-dual-RNAseq). Greater understanding of how Ct adapts, survives, and persists in the presence of host immunity or antimicrobial agents, as well as reactivates from persistence may provide important new insights into chlamydial pathogenesis. The findings from these studies will provide the foundation to develop new treatment strategies targeting both acute and the chronic, transmissible reservoirs of Ct infection.