Development of Two Animal Models To Study the Function of Vibrio parahaemolyticus Type III Secretion Systems

Abstract

Vibrio parahaemolyticus is an emerging food-and waterborne pathogen that encodes two type III secretion systems (T3SSs). Previous studies have linked type III secretion system 1 (T3SS1) to cytotoxicity and T3SS2 to intestinal fluid accumulation, but animal challenge models needed to study these phenomena are limited. In this study we evaluated the roles of the T3SSs during infection using two novel animal models: a model in which piglets were inoculated orogastrically and a model in which mice were inoculated in their lungs (intrapulmonarily). The bacterial strains employed in this study had equivalent growth rates and beta-hemolytic activity based on in vitro assays. Inoculation of 48-h-old conventional piglets with 10 11 CFU of the wild-type strain (NY-4) or T3SS1 deletion mutant strains resulted in acute, self-limiting diarrhea, whereas inoculation with a T3SS2 deletion mutant strain failed to produce any clinical symptoms. Intrapulmonary inoculation of C57BL/6 mice with the wild-type strain and T3SS2 deletion mutant strains (5 ؋ 10 5 CFU) induced mortality or a moribund state within 12 h (80 to 100% mortality), whereas inoculation with a T3SS1 deletion mutant or a T3SS1 T3SS2 double deletion mutant produced no mortality. Bacteria were recovered from multiple organs regardless of the strain used in the mouse model, indicating that the mice were capable of clearing the lung infection in the absence of a functional T3SS1. Because all strains had a similar beta-hemolysin phenotype, we surmise that thermostable direct hemolysin (TDH) plays a limited role in these models. The two models introduced herein produce robust results and provide a means to determine how different T3SS1 and T3SS2 effector proteins contribute to pathogenesis of V. parahaemolyticus infection.Vibrio parahaemolyticus is a Gram-negative food-and waterborne pathogen that is recognized worldwide as a causative agent of gastroenteritis associated with the consumption of undercooked seafood (22). Gastrointestinal infection is characterized by acute self-limiting diarrhea, abdominal cramps, nausea, and vomiting. In approximately 5% of cases, V. parahaemolyticus gastrointestinal infection can progress to septicemia and may be fatal for immunocompromised patients, including those with leukemia, liver disease, and patients infected by human immunodeficiency virus (14, 28).The thermostable direct hemolysin (TDH) is a widely recognized virulence factor of V. parahaemolyticus. This hemolysin is associated with the Kanagawa phenomenon, which is a beta-hemolysis reaction on a defined blood agar (10). TDH is encoded by 1 or 2 genes (tdhA and tdhS), and the protein increases permeability in human erythrocytes (19), increases chloride secretion in an intestinal cell line (29), and is also thought to be responsible for enterotoxigenicity in a rabbit small intestine model (5, 23). Early studies demonstrated that deletion of tdhS and tdhA will significantly reduce fluid accumulation in a rabbit ileal-loop model, but the phenotype is not completely abrogated, sug...

Loss of Sigma Factor RpoN Increases Intestinal Colonization of Vibrio parahaemolyticus in an Adult Mouse Model

“…T3SS-2 plays only a minor role in cytotoxicity but is the major contributing factor toward enterotoxicity (17,22,23,27). Studies have demonstrated that fluid accumulation in the rabbit ileal loop model as well as virulence in the infant rabbit and infant pig models are dependent upon a functional 22,23,27).…”

“…The T3SS-2 gene cluster is flanked by a copy of the tdh gene (tdhA and tdhS) within an 80-kb pathogenicity island named VPaI-7, a region found only in clinical isolates (16,(24)(25)(26). T3SS-2 plays only a minor role in cytotoxicity but is the major contributing factor toward enterotoxicity (17,22,23,27). Studies have demonstrated that fluid accumulation in the rabbit ileal loop model as well as virulence in the infant rabbit and infant pig models are dependent upon a functional T3SS-2 (17,22,23,27).…”

The Vibrio parahaemolyticus ToxRS Regulator Is Required for Stress Tolerance and Colonization in a Novel Orogastric Streptomycin-Induced Adult Murine Model

“…More recently, a third gene cluster, encoding a T3SS, has been identified in a tdh-negative, trh-positive V. parahaemolyticus strain (56). Several functional T3SS effector proteins have been identified (2,21,34,45,60,65,76), including the T3SS-1 effectors VopQ (VP1680), VopR (VP1683), VopS (VP1686), VP1659, and VPA0450. It has been found that the production of these effector proteins by V. parahaemolyticus induces autophagy, cell rounding, cell lysis, and cell death in vitro in a variety of different eukaryotic cell types (7-9, 36, 39, 76).…”

“…It has been found that the production of these effector proteins by V. parahaemolyticus induces autophagy, cell rounding, cell lysis, and cell death in vitro in a variety of different eukaryotic cell types (7-9, 36, 39, 76). Each T3SS in V. parahaemolyticus has been shown to be functional, although data suggest that the contribution of each to virulence differs markedly (21,61,65,85). The regulation of the T3SS-1 system was shown to fall under the control of the ExsACDE system, with ExsA and ExsD acting as positive and negative regulators of T3SS-1 gene expression, respectively.…”

Virulence gene expression of Vibrio parahaemolyticus in the viable but nonculturable state

“…T3SS1 appeared to be responsible for the cytotoxicity to several mammalian cell lines (Burdette et al, 2008;Hiyoshi et al, 2010;Nydam et al, 2014;Zhou et al, 2009) and mortality in a mouse model Pineyro et al, 2010).…”