Early Expression of Type III Secretion is Critical for Bacterial Survival in Acute Pseudomonas aeruginosa Pneumonia

The Pseudomonas aeruginosa type III secretion system has been associated with poor outcomes for both animal models and human patients. Despite a large number of studies exploring the regulation of type III secretion in vitro, little is known about the timing of secretion during mammalian infection. The work described in this dissertation demonstrates that the exoU gene, which encodes the highly cytotoxic type III effector ExoU, is induced early during acute P. aeruginosa pneumonia. Immunofluorescence microscopy indicated that the amount of ExoU protein in the lung also increased over time. The importance of early expression was examined using a strain of P. aeruginosa with inducible production of ExoU. Delays in expression as short as 3 hours led to reduced bacterial burdens in the lungs of mice and improved survival. My results demonstrate that early expression of exoU is critical for bacterial survival during pneumonia and suggest that therapeutic interventions that delay ExoU secretion for even short periods of time may be efficacious. In addition, this document describes the development of a novel technique for visualizing immune cell trafficking to the lungs of infected mice using bioluminescence imaging. The goal of these experiments was to determine whether ExoU promotes bacterial persistence in the lungs of mice during acute pneumonia by killing recruited neutrophils. I demonstrated that loss of luminescence is a valid way to measure neutrophil death in vitro. I also showed that transferred neutrophils were rapidly recruited to the lungs of mice infected with either ExoU+ or Exo-- P. aeruginosa. I have demonstrated that this technique has potential to be used for examining the fate of phagocytic cells upon arrival in the lungs of mice during acute pneumonia, but still requires optimization. Taken together, these studies add to our understanding of the contribution of ExoU to the pathogenesis of P. aeruginosa. During the early stages of pneumonia, neutrophils are rapidly recruited to the lungs in response to signals indicating the presence of bacteria. P. aeruginosa responds to the influx of inflammatory cells by quickly inducing expression of type III secretion genes, specifically exoU, and injects ExoU protein into these cells upon contact. Upon intoxication with ExoU, neutrophils rapidly succumb to the cytotoxic activity of this protein, resulting in the ability of bacteria to persist, multiply, and disseminate into the bloodstream. However, ExoU, and thus P. aeruginosa, is vulnerable to interventions capable of inhibiting the early expression and/or secretion of this toxin.