Vibrio cholerae is an aquatic Gram-negative bacterium that causes the diarrheal disease cholera. There is relatively little known about the role ofthe accessory toxins of V. cholerae, and whether these toxins interact with the innate immune system to establish colonization. An adult mouse model of intestinal colonization by V. cholerae has recently been developed, and will be utilized in the proposed study. The MARTX toxin of V. cholerae, known to have actin cross-linking and Rho inactivation domains, is hypothesized to target neutrophils recruited to the site of infection to evade phagocytosis.
Specific Aim 1. Demonstrate that the accessory toxins of Vibrio cholerae target neutrophils to establish intestinal colonization in an adult mouse model. Neutrophil replete and neutropenic mice will be inoculated with wild-type V. cholerae and a multi-toxin deficient strain to demonstrate whether the action of neutrophils is important to clearing early infection with V. cholerae, and whether accessory toxins target neutrophils to establish intestinal colonization.
Specific Aim 2. Determine which catalytic function of MARTX -- Rho Inactivation or actin cross-linking - is essential for the ability of V. cholerae to promote persistent colonization. The Rho inactivation and actin cross-linking domains of MARTX will be individually mutated on the V. cholerae chromosome to inactive each domain. V. cholerae strains with these mutations will be administered to neutrophil replete and neutropenic mice to determine which domain is essential to the in vivo effects of MARTX.
Specific Aim 3. Establish whether neutrophils are a direct target of MARTX. Primary human neutrophils will be incubated with MARTX in vitro, and assayed for evidence of Rho inactivation, actin cross-linking, and defective phagocytosis.
The diarrheal disease cholera is caused by the bacterium Vibrio cholerae. This bacterium secretes several toxins that are important to its success in causing disease. We hypothesize that one of these toxins, MARTX, targets neutrophils to establish colonization in the small intestine.
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