Friday, November 22, 2024
11/22/2024
Project Summary Coxiella burnetii is the highly infectious causative agent of Q fever, which can manifest as an acute or debil- itating chronic disease. This naturally obligate intracellular bacterium infects alveolar macrophages, replicating within a compartment that resembles a mature phagolysosome. A distinguishing characteristic of C. burnetii is its ability to enter these cells without activating detectable innate immune defenses or polarizing the cell to a proinflammatory phenotype. The virulence factors credited with this pathogenic paradigm are its unique anti- inflammatory lipopolysaccharide (LPS), and the effectors secreted by its Type IVb Secretion system (T4SS). To identify unappreciated virulence factors unrelated to LPS or T4SS of C. burnetii, a genetic screen employing TnSeq compared the replication of Coxiella transposon mutants in Vero cells to that in primary macrophages. The screen identified a series of mutants in genes related to cell wall function were required for replication only in the context of the primary macrophages. These data suggested that bacterial cell wall components may belong to an unappreciated class of Coxiella virulence factors. The long-term goal of this project is to describe the molecular mechanism(s) by which the identified cell wall components contribute to the intracellular pathogenesis of Coxiella. The central hypothesis is that the C. burnetii cell wall is specifically adapted to resist or evade the innate defenses that are present in its preferred cellular host, macrophages. The objectives of this application are to determine the 1) molecular functions and 2) virulence contributions of individual cell wall components that are required for Coxiella’s survival and proliferation within primary macrophages. The role(s) of cell wall compo- nents in Coxiella pathogenesis will be defined by pursuing the following specific aims: 1) determine the func- tions of the candidate cell wall-related virulence genes, and 2) determine the effect of these cell wall mutants on C. burnetii-host interactions. Specific aim 1 will focus on the molecular contribution of these candidate virulence genes to survival and resistance to stresses specifically encountered within macrophages. We will use multiple experimental approaches including complementation assays, biochemical analyses of pep- tidoglycan and lipids, developmental and morphological analyses, and in vitro functional assays to define the contribution of these cell wall components to Coxiella survival. Specific aim 2 will investigate the response of macrophages to infection with these interesting cell wall mutants. By employing RNAseq, enzymatic activity assays, and secretion assays, we will build a holistic view of the host response to these mutants. We anticipate that the proposed series of experiments will lay the foundation for a strong, long-lived research program that links cell wall integrity to Coxiella burnetii’s distinguishing environmental stability and incomparable ability to survive in a microbicidal niche.
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