Virulence Factor Discovery

C. burnetii infects macrophages, which are cells of the human immune system that are adapted specifically to detect, engulf, and destroy invading pathogens. In order to set up shop inside these cells and replicate without being degraded, the bacteria must be able to evade the host cell's pathogen detection systems, but they also actively manipulate the host to their own benefit.  Our lab is actively searching for undiscovered or unappreciated virulence factors that allow C. burnetii to invade and replicate in a cell that otherwise would kill it.

One of the ways we are approaching virulence factor discovery is to ask what genes Coxiella requires to survive in the specific context of a macrophage. We used a panel of random Coxiella mutants to infect epithelial cells and primary macrophages in parallel, and determined which mutant strains could survive in those epithelial cells (Vero), but not in the macrophage. The screen used DNA sequencing technology to measure replication of these mutants in the two different cell types. We predicted that if a specific mutant strain failed to survive in the macrophage, but could survive or replicate in a Vero cell, the single disrupted gene in that mutant strain may encode a macrophage-specific virulence factor. 

The screen revealed mutations in genes across multiple different functional categories are required for survival in macrophages: membrane and cell wall integrity, nutrient acquisition or utilization, cellular signalling. Some genes we identified have no attributed function yet. We are now beginning the work of determining how these genes contribute to Coxiella pathogenesis in the context of macrophages.

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