Characterization of a Vibrio cholerae Type VI Secretion System Toxin/Immunity Pair

  • Author / Creator
    Miyata, Sarah, T
  • Vibrio cholerae is the Gram-negative bacterium responsible for the diarrheal disease cholera. In addition to the well-characterized virulence factors – cholera toxin and the toxin co-regulated pilus – used by this organism to cause watery diarrhea, V. cholerae uses the type VI secretion system (T6SS) to mediate host-pathogen and inter-bacterial interactions. The T6SS is the most-recently described mechanism by which Gram-negative bacteria export toxins across their cell envelope. This macromolecular structure assembles in the cytoplasm and consists of an outer sheath and inner tube. The inner tube is thought to be capped with three or more proteins possessing both structural and toxic functions. Upon contraction of the outer sheath, the inner tube and cap are ejected out of the bacterium and into neighboring prokaryotic or eukaryotic cells. I discovered VasX – a T6SS toxin required for V. cholerae virulence toward the host model Dictyostelium discoideum and for killing Gram-negative bacteria. My Ph.D. work showed that VasX associates with structural T6SS proteins and has homology to pore-forming colicins. Secretion of VasX depends on certain structural T6SS proteins and VasW – a proposed VasX chaperone protein encoded upstream of vasX. Upon insertion into the cytoplasmic membrane of target cells, VasX disrupts the integrity of the membrane leading to a loss of the cell’s membrane potential, permeability to propidium iodide, and lysis of the cell in the presence of detergent. Thus, I propose that VasX acts similar to pore-forming colicins by insertion into the cytoplasmic membrane followed by pore-formation, ultimately leading to the death of the target cell. The VasX immunity protein TsiV2 mediates protection from an oncoming VasX attack generated by neighboring sister cells. TsiV2 localizes to the bacterial membrane and generates protection against VasX even when TsiV2 is present in the cell at low protein levels. Expression of tsiV2 is influenced by a dual regulatory mechanism where one promoter is located upstream of the TsiV2-encoding operon and another promoter is located within the upstream gene (i.e. vasX). This dual regulation ensures tsiV2 is constitutively expressed at basal levels when expression of other T6SS genes is turned off, mediating protection against T6SS attack from kin bacteria. Taken together, this thesis characterizes the function of a T6SS toxin and the mechanism by which V. cholerae protects itself from being killed by sister cells.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • 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
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Medical Microbiology and Immunology
  • Specialization
    • Bacteriology
  • Supervisor / co-supervisor and their department(s)
    • Stefan Pukatzki Dept of Medical Microbiology and Immunology
  • Examining committee members and their departments
    • Tracy Raivio, Department of Biological Sciences
    • Rebecca Case, Dept of Biological Sciences
    • Francis Nano, Dept of Biochemistry and Microbiology (University of Victoria)
    • Bart Hazes, Dept of Medical Microbiology and Immunology