Q fever is caused by the highly infectious zoonotic intracellular bacterial pathogen, Coxiella burnetii. C. burnetii is the leading cause of culture-negative endocarditis and the recommended antibiotic treatment for this disease regularly fails. Individuals with a history of arterial aneurysm or who have heart valve or vascular prostheses are at much greater risk of contracting chronic Q fever endocarditis. Thus, there is a critical need for new therapeutic approaches. Persistence of C. burnetii during chronic endocarditis has been attributed to the formation of the small cell variant (SCV), a phenotypically distinct variant with extreme intrinsic resistance and distinct cell envelope ultrastructure. In Mycobacterium tuberculosis, another intracellular pathogen, the host cell environment profoundly affects the cell envelope structure of the bacteria and its susceptibility to antibiotics. Our goal is to gain a detailed mechanistic understanding of the molecular processes governing SCV cell envelope structure in different hosts and cell types that are important to the infectious cycle. Recently, we showed that L,D-transpeptidases (Ldts) are critical for SCV formation. Ldts are a multifunctional family of cell envelope remodeling enzymes and the primary target of carbapenems, a class of broad-spectrum ßlactam antibiotics. C. burnetii encodes 10 ldts that are either known, or predicted to be important for SCV formation. Our preliminary data shows that the SCV cell envelope is differentially remodeled by Ldts depending on the culture environment. These data support the hypothesis that the host cell environment impacts SCV cell envelope structure through differential expression of ldts. In the proposed aims, we will develop comparative models of SCV development using cell lines that represent the following stages of the C. burnetii infectious process; zoonotic transmission (ovine placental trophoblast cells), acute disease (murine alveolar macrophages), and chronic disease (human macrophage-like cells), then test ldt expression, SCV cell envelope structure, and antibiotic susceptibility. We will then explore the metabolic differences in each cell line to determine how the host metabolic environment might be different in each model. These studies will provide a foundation for exploring existing cell envelope-targeting antibiotics as treatments for chronic Q fever, which could have immediate impacts in the clinic and significantly advance the C. burnetii field.