| Bordetella are Gram negative respiratory pathogens. B. pertussis and B. parapertussishu are restricted to human host and are the causative agents of pertussis, while B. bronchiseptica infects an unusually broad range of four-legged mammals. The genomes of B. pertussis, B. parapertussis hu, and B. bronchiseptica contain nearly identical set of genes encoding a type III secretion system (TTSS), which is used by Gram negative bacteria to deliver effector proteins directly into the cytosol of eukaryotic cells to alter host functions. The hallmark of the B. bronchiseptica TTSS is the efficient induction of death in a broad range of cells in vitro. However, in vivo, the B. bronchiseptica TTSS appears to promote persistent colonization, possibly by modulating host immunity. It has been previously shown that the Bordetella TTSS is transcriptionally regulated by the BvgAS phosphorelay signal transduction system via the sigma factor BtrS. The secretion process itself requires a partner switching module consisting of serine phosphatase BtrU, serine kinase BtrW, and antagonist BtrV. The first two sections of this dissertation characterize the mechanism of Bordetella TTSS regulation by the partner switching module. Biochemical analysis of the wild type and mutant forms of BtrU, BtrW and BtrV has shown that BtrW binds BtrV and phosphorylates its conserved serine residue S55, whereas BtrU dephosphorylates BtrV∼P. We also demonstrated that BtrU phosphatase activity and BtrW kinase activity are essential for the proper functioning of the TTSS in Bordetella. In addition, both BtrV and BtrU are required for maintaining the steady state level of BtrW. The third section of the dissertation focuses on the alternative sigma factor, sigma 54. The presence of a putative sigma54 binding site at the btrS promoter suggested the involvement of sigma 54 in TTSS transcription regulation. The sigma54 deletion mutant was assessed in vitro for defects in TTSS, motility and growth rate and analyzed in vivo using a rat model of respiratory tract infection. While sigma54 was not involved in regulating TTSS, we demonstrated a role for sigma54 in early tracheal colonization. |
