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


Other title
Vibrio cholerae
type VI secretion system
Type of item
Degree grantor
University of Alberta
Author or creator
Miyata, Sarah, T
Supervisor and department
Stefan Pukatzki Dept of Medical Microbiology and Immunology
Examining committee member and department
Rebecca Case, Dept of Biological Sciences
Francis Nano, Dept of Biochemistry and Microbiology (University of Victoria)
Bart Hazes, Dept of Medical Microbiology and Immunology
Tracy Raivio, Department of Biological Sciences
Department of Medical Microbiology and Immunology
Date accepted
Graduation date
Doctor of Philosophy
Degree level
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.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Miyata, S.T., Kitaoka, M., Brooks, T.M., McAuley, S.B., and Pukatzki, S. (2011) Vibrio cholerae Requires the Type VI Secretion System Virulence Factor VasX to Kill Dictyostelium discoideum. Infection and Immunity, 779(7):2941-9.MacIntyre, D., Miyata, S.T., Kitaoka, M., and Pukatzki, S. (2010) The Vibrio cholerae type VI secretion system displays antimicrobial properties. Proceedings of the National Academy of Sciences U.S.A, 107(45):19520-4.Unterweger, D., Kitaoka, M., Miyata, S.T., Bachmann, V., Brooks, T.M., Moloney, J., Sosa, O., Silva, D., Duran-Gonzalez, J., Provenzano, D. and Pukatzki, S. (2012) Constitutive type VI secretion system expression gives Vibrio cholerae intra- and inter-specific competitive advantages. PLoS One, 7(10): e48320.

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