Project Summary Coxiella burnetii (Cb) is an obligate intracellular bacterium in nature and the causative agent of human acute Q fever as well as chronic disease. Upon infection, Cb is trafficked along a bacterially modified endocytic pathway and establishes a parasitophorous vacuole (PV) that retains many of the features of mature phagolysosomes. Throughout the infectious cycle within a host cell, Cb face an array of physiological needs and stresses unique to life within an acidified intracellular vesicle. The pathogen manipulates a plethora of host cell pathways and processes from within the PV via a bacterial Type 4 Secretion System (T4BSS) in order to survive and replicate. However, axenic media supports Cb replication in an environment where the bacterial genes required for many of its adaptations to intracellular growth are no longer needed. Deletion and insertion mutants have demonstrated some of Cb host cell specific gene requirements, however, an understanding of the breadth of the bacteria’s genes, and/or their expression, that will change when cellular stresses are removed is still lacking. This is a fundamental gap in our understanding of how Cb subverts host cell processes during infection and will lead to a broader understanding of Cb biology and pathogenesis. The long-term goal of our research is to elucidate molecular mechanisms employed by Cb to orchestrate parasitism of the host cell during infection. Our hypotheses are 1- Cb changes its gene expression within the first few passages upon transition from intracellular to axenic media growth, and 2- Cb will progressively acquire and accumulate DNA mutations upon transition from intracellular to axenic media growth after repetitive passages since certain bacterial genes/proteins are no longer required for successful growth. We will test our hypotheses by 1- Identifying Cb gene expression changes that occur during early and long term passages after inoculating axenic growth media with host cell propagated Cb, and 2- Identifying Cb genetic mutations/changes that occur/accumulate over multiple passages after inoculating axenic growth media with host cell propagated Cb. Understanding the virulence mechanisms employed by this unique pathogen to survive within the harsh environment of the host cell phagosome and cause disease will enable us to develop countermeasures to this poorly understood bacteria.