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Characterization of the Cpx Response in Vibrio cholerae

  • Author / Creator
    Acosta Amador, Paula N
  • The gram negative bacterial cell envelope is composed of the outer membrane, the periplasm and the inner membrane. These compartments are exposed directly to changes in the environment that are sensed and adapted to through different signaling transduction pathways. This often occurs through two-component signal transduction systems (TCS), which are broadly distributed among different bacterial species. The Cpx pathway is a TCS that employs the sensor histidine kinase CpxA and the response regulator CpxR, and regulates crucial adaptations to envelope stress response that affects many functions, including antibiotic resistance, across bacterial species. This system has also been implicated in the regulation of a number of envelope localized virulence determinants across bacterial species. The first goal of this thesis was to characterize the Cpx regulon members in the human pathogen Vibrio cholerae when the Cpx pathway is activated. For this purpose I characterized the transcriptional profile of the pandemic V. cholerae El Tor strain C6706 upon overexpression of cpxR, and the inducing cues that lead to the activation of the Cpx pathway. My data shows that the Cpx regulon of V. cholerae is enriched for genes encoding membrane localized and transport proteins, including a large number of genes known or predicted to be iron-regulated. The V. cholerae Cpx regulon included three strongly Cpx-regulated, putative ferric reductases that are likely directly regulated by CpxR. I present evidence that the function of these ferric reductases is likely tied to the up-regulation of iron-related genes by the Cpx response. Activation of the Cpx pathway also led to the expression of TolC, the major outer membrane pore, and components of two resistance-nodulation-division (RND) efflux systems in V. cholerae. I found that iron chelation, toxic compounds, or deletion of specific RND efflux components lead to Cpx pathway activation. Further, mutations that eliminated the Cpx response or members of its regulon resulted in growth phenotypes in the presence of these inducers that, together with Cpx pathway activation, are partially suppressed by iron. Cumulatively, these results suggest that a major function of the Cpx response in V. cholerae is to mediate adaptation to envelope perturbations caused by toxic compounds and the depletion of iron. A second goal of this thesis was to characterize the effect of Cpx pathway activation on V. cholerae virulence factors. I found that activation of the Cpx pathway lead to a decrease in expression of the major virulence factors in this organism, cholera toxin (CT), and the toxin-coregulated pilus (TCP). The Cpx response controls virulence factor expression by repressing expression of the ToxT and TcpP regulators. I showed that the effect of the Cpx response on ctxB and tcpA expression is mostly abrogated in a cyclic adenosine monophosphate (cAMP) receptor protein (CRP) mutant, although expression of the crp gene is unaltered. These observations indicate that CRP function is affected by Cpx response activation. Altogether, the data presented here suggest a model whereby the Cpx response reduces production of CT and TCP by controlling the expression and function of regulators that function on the ToxR regulon in V. cholerae.

  • Subjects / Keywords
  • Graduation date
    2015-06
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3JH3DF3C
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Biological Sciences
  • Specialization
    • Microbiology and Biotechnology
  • Supervisor / co-supervisor and their department(s)
    • Raivio, Tracy (Biological Sciences)
  • Examining committee members and their departments
    • Szymanski, Christine (Biological Sciences)
    • Pukatzki, Stefan (Medical Microbiology & Immunology)
    • Feldman, Mario (Biological Sciences)
    • DiRita, Victor (Microbiology & Immunology, University of Michigan)