The life-threatening diarrheal disease cholera is caused by toxigenic strains of the Gram-negative organism Vibrio cholerae. In addition to the well-characterized ADP-ribosylating cholera toxin (CT), V. cholerae secretes a novel toxin that is the founding member of new family of Multifunctional, Autoprocossin RTX toxins (MARTX). The MARTX toxin of V. cholerae (MARTX-Vc) contributes to virulence in mice and may be an important factor contributing to persistent colonization both in cholera patients and non-symptomatic carriers.. MARTX-Vc is of intrinsic interest due to its novel biochemical properties and mode of action. At a predicted size of >480 kDa, the toxin is a multifunctional toxin. Thus far, we have described three activities associated with this toxin. We have demonstrated that this toxin depolymerizes actin stress fibers and covalently crosslinks actin into oligomers. We have demonstrated that this toxin causes rounding of cells due to inactivation of RhoGTPases by an unknown mechanism. Finally, this toxin has a cysteine protease domain with autoprocessing activity that may also have cellular targets resulting in cytotoxicity. Each of these activities has been mapped to a specific domain of the toxin. A fourth domain homologous to the alpha-beta hydrolase family of proteins is also a putative activity domain. In this grant proposal, we will pursue the multiple catalytic activities of the toxin by studying the mechanism of action of the independent activity domains. A structure function analysis of the actin crosslinking domain will be performed and the regions essential for catalysis and actin binding will be identified. The target of the RhoGTPase inactivating domain will be identified and characterized. The requirements for autocleavage site recognition and inositol hexakisphosphate binding will be investigated and other potential sites of cleavage identified. In all, this work will further our understanding of the mechanism of action of this toxin and provide insight into its role in disease and will also provide information about related toxins that share the novel functional domains carried by this unique multifunctional toxin.

Public Health Relevance

The life-threatening diarrheal disease cholera is caused the Gram-negative organism Vibrio cholerae. In addition to the well-characterized ADP-ribosylating cholera toxin (CT), V. cholerae secretes a novel toxin that is the founding member of new family of the RTX toxins, the Multifunctional-Autoprocessing RTX toxins (MARTX). The MARTX toxin of V. cholerae contributes to virulence in mice and may be an important factor contributing to persistent colonization both in cholera patients and non-symptomatic carriers. This proposal seeks to understand the cellular mechanism of action of this toxin that has three known enzymatic activities: actin crosslinking, Rho-inactivation, and autoprocessing. Completion of this research will impact not only our understanding of cholera pathogenesis, but also the function of other uncharacterized toxins produced by other human pathogens that share these unique enzymatic activities.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI051490-10
Application #
8230475
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Hall, Robert H
Project Start
2002-04-01
Project End
2013-08-28
Budget Start
2012-03-01
Budget End
2013-08-28
Support Year
10
Fiscal Year
2012
Total Cost
$369,988
Indirect Cost
$124,963
Name
Northwestern University at Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
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Antic, Irena; Biancucci, Marco; Satchell, Karla J F (2014) Cytotoxicity of the Vibrio vulnificus MARTX toxin effector DUF5 is linked to the C2A subdomain. Proteins 82:2643-56
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Ahrens, Sebastian; Geissler, Brett; Satchell, Karla J F (2013) Identification of a His-Asp-Cys catalytic triad essential for function of the Rho inactivation domain (RID) of Vibrio cholerae MARTX toxin. J Biol Chem 288:1397-408
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Kwak, Jayme S; Jeong, Hee-Gon; Satchell, Karla J F (2011) Vibrio vulnificus rtxA1 gene recombination generates toxin variants with altered potency during intestinal infection. Proc Natl Acad Sci U S A 108:1645-50
Geissler, Brett; Tungekar, Rehman; Satchell, Karla J F (2010) Identification of a conserved membrane localization domain within numerous large bacterial protein toxins. Proc Natl Acad Sci U S A 107:5581-6

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