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United States Department of Agriculture

Agricultural Research Service

Research Project: PATHOGEN DETECTION AND INTERVENTION METHODS FOR SHELLFISH

Location: Food Safety and Intervention Technologies

Title: Vibrio parahaemolyticus ToxRS regulator is required for stress tolerance and colonization in a novel orogastric streptomycin-induced adult murine model

Authors
item Whitaker, Brian -
item Parent, Michelle -
item Aoife, Boyd -
item RICHARDS, GARY
item Boyd, Fidelma -

Submitted to: Infection and Immunity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 15, 2012
Publication Date: May 21, 2012
Citation: Whitaker, B.W., Parent, M.A., Aoife, B., Richards, G.P., Boyd, F.E. 2012. Vibrio parahaemolyticus ToxRS regulator is required for stress tolerance and colonization in a novel orogastric streptomycin-induced adult murine model. Infection and Immunity. 80:1834-1845.

Interpretive Summary: The leading cause of seafood-related bacterial illness in the United States is a naturally occurring marine bacterium known as Vibrio parahaemolyticus. The infectivity of V. parahaemolyticus is related to specific genes, such as the toxRS gene, which produce proteins known as ToxR and ToxS. We evaluated the influence of the toxRS gene on the ability of V. parahaemolyticus to survive stresses, like acid, bile salts, and chemical stress, as would be encountered within the stomach of an infected individual. To accomplish this, a mutant strain of V. parahaemolyticus lacking the toxRS gene was designed and compared with the normal strain containing the toxRS gene. We determined that the ToxR and ToxS proteins regulate an outer membrane protein referred to as OmpU, making the vibrios more tolerant of stresses, whereas mutants lacking the toxRS gene were less tolerant of a stressful environment. All V. parahemolyticus used in this study were resistant to the antibiotic streptomycin. Streptomycin resistance allowed us to develop a new model of infection using streptomycin-treated, adult mice. Antibiotic-treated mice experienced a persistent colonization of their intestinal tract by V. parahaemolyticus which contained the toxRS gene, but not when the toxRS gene was missing. This study demonstrated the usefulness of this adult, mouse model in monitoring the effects of the toxRS gene on bacterial invasiveness and will facilitate an evaluation of other genes that may be associated with vibrio invasiveness. Overall, we demonstrated that the toxRS gene is an important regulator of the stress response of V. parahaemolyticus and that the adult mouse model is a valuable new tool for studies to understand bacterial and host factors crucial for bacterial colonization of the gastrointestinal tract.

Technical Abstract: Vibrio parahaemolyticus, a marine bacterium, is the causative agent of gastroenteritis associated with the consumption of seafood. It contains a homologue of the toxRS operon that in V. cholerae is the key regulator of virulence gene expression. We examined a non-polar mutation in toxRS to determine the role of these genes in V. parahaemolyticus RIMD2210633, a O3:K6 isolate, and showed that the toxRS deletion mutant compared to the wild-type was significantly more sensitive to acid, bile salts and sodium dodecyl sulfate stresses. We demonstrated that ToxRS is a positive regulator of ompU expression and that complementation of the toxRS deletion mutant with ompU restored stress tolerance. Furthermore, we showed that ToxRS regulated type III secretion system genes in chromosome I independent of OmpU regulation. We examined the effect of the toxRS deletion mutant in vivo using a new orogastric adult murine model of colonization. We demonstrated that streptomycin-treated C57BL/6 adult mice experienced prolonged intestinal colonization along the entire intestinal tract by the streptomycin-resistant V. parahaemolyticus. In contrast, no colonization occurred in non-streptomycin-treated mice. A competition assay between the toxRS mutant and wild-type V. parahaemolyticus marked with the beta-galactosidase gene lacZ demonstrated that the toxRS deletion mutant was defective in colonization compared to wild-type. This defect was rescued by ectopically expressing ompU. Thus, the defect in stress tolerance and colonization in the toxRS mutant was solely due to OmpU. To our knowledge, the orogastric adult murine model reported here is the first showing sustained intestinal colonization by V. parahaemolyticus.

Last Modified: 8/19/2014
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