Inflammation and Disintegration of Intestinal Villi in an Experimental Model for Vibrio parahaemolyticus-Induced Diarrhea

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis in many parts of the world, but there is limited knowledge of the pathogenesis of V. parahaemolyticus-induced diarrhea. The absence of an oral infection-based small animal model to study V. parahaemolyticus intestinal colonization and disease has constrained analyses of the course of infection and the factors that mediate it. Here, we demonstrate that infant rabbits oro-gastrically inoculated with V. parahaemolyticus develop severe diarrhea and enteritis, the main clinical and pathologic manifestations of disease in infected individuals. The pathogen principally colonizes the distal small intestine, and this colonization is dependent upon type III secretion system 2. The distal small intestine is also the major site of V. parahaemolyticus-induced tissue damage, reduced epithelial barrier function, and inflammation, suggesting that disease in this region of the gastrointestinal tract accounts for most of the diarrhea that accompanies V. parahaemolyticus infection. Infection appears to proceed through a characteristic sequence of steps that includes remarkable elongation of microvilli and the formation of V. parahaemolyticus-filled cavities within the epithelial surface, and culminates in villus disruption. Both depletion of epithelial cell cytoplasm and epithelial cell extrusion contribute to formation of the cavities in the epithelial surface. V. parahaemolyticus also induces proliferation of epithelial cells and recruitment of inflammatory cells, both of which occur before wide-spread damage to the epithelium is evident. Collectively, our findings suggest that V. parahaemolyticus damages the host intestine and elicits disease via previously undescribed processes and mechanisms

Inflammation and disintegration of intestinal villi in an experimental model for Vibrio parahaemolyticus-induced diarrhea.

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis in many parts of the world, but there is limited knowledge of the pathogenesis of V. parahaemolyticus-induced diarrhea. The absence of an oral infection-based small animal model to study V. parahaemolyticus intestinal colonization and disease has constrained analyses of the course of infection and the factors that mediate it. Here, we demonstrate that infant rabbits oro-gastrically inoculated with V. parahaemolyticus develop severe diarrhea and enteritis, the main clinical and pathologic manifestations of disease in infected individuals. The pathogen principally colonizes the distal small intestine, and this colonization is dependent upon type III secretion system 2. The distal small intestine is also the major site of V. parahaemolyticus-induced tissue damage, reduced epithelial barrier function, and inflammation, suggesting that disease in this region of the gastrointestinal tract accounts for most of the diarrhea that accompanies V. parahaemolyticus infection. Infection appears to proceed through a characteristic sequence of steps that includes remarkable elongation of microvilli and the formation of V. parahaemolyticus-filled cavities within the epithelial surface, and culminates in villus disruption. Both depletion of epithelial cell cytoplasm and epithelial cell extrusion contribute to formation of the cavities in the epithelial surface. V. parahaemolyticus also induces proliferation of epithelial cells and recruitment of inflammatory cells, both of which occur before wide-spread damage to the epithelium is evident. Collectively, our findings suggest that V. parahaemolyticus damages the host intestine and elicits disease via previously undescribed processes and mechanisms

Representative electron micrographs of <i>V. parahaemolyticus</i> attached to the intestinal epithelium at 28 hr PI.

<p>(A) Scanning electron micrograph of the small intestine of <i>V. parahaemolyticus</i>-infected infant rabbits. Scale bar = 50 µm. (B) Higher magnification image of the boxed area in (A) showing elongated microvilli (arrowheads) and ‘blebs’ of material being lost from the epithelial surface (long arrow). Scale bar = 2 µm. (C) Transmission electron micrograph of <i>V. parahaemolyticus</i> colonizing the epithelial surface, surrounded by a tangle of elongated microvilli-like projections. Note the organized pattern of microvilli (seen in cross-section) on adjacent uninfected cells (arrowheads) compared to the disorganized mix of microvilli sections, cytoplasmic debris and bacteria that extends much further into the luminal space (long arrows). Scale bar = 2 µm. (D) Higher magnification image of the boxed area in (C) showing the defined membranes and presence of internal filaments (dark staining) in cross-sections (long arrow) and longitudinal sections (arrowhead) of elongated microvilli. Vp = bacterium. Scale bar = 100 nm. (E) Close contact between bacteria and host epithelial cell membranes (arrowheads) in the absence of normal microvilli and pedestal formation. Scale bar = 1 µm. (F) Clusters of <i>V. parahaemolyticus</i> located in a cavity below the normal level of the surrounding epithelium. Note the intact brush border of adjacent cells. Scale bar = 2 µm. (G) <i>V. parahaemolyticus</i> (arrowheads) located at the base of an extruding epithelial cell (ex. cell), with more bacteria in the intestinal lumen. Scale bar = 2 µm. (H) Membrane-bound cytoplasm ‘bleb’ (B) extruding from the epithelium, surrounded by <i>V. parahaemolyticus</i>. Scale bar = 2 µm.</p

Schematic of kinetics of <i>V. parahaemolyticus</i>-induced damage to the intestinal epithelial surface.

<p>Following initial attachment, <i>V. parahaemolyticus</i> induces erosion of microvilli and depletion of cytoplasmic contents resulting in the formation of bacterial clusters located just below the level of the surrounding epithelium. Continued depletion of epithelial cell contents either by cytoplasmic ‘blebbing’, whole cell extrusion and microvilli elongation around the edge of the cluster, leaves <i>V. parahaemolyticus</i> clusters situated within deeper cavities in the epithelium. Eventually, this leads to disintegration of normal villus structure and the generation of large amounts of luminal debris. These pathological changes appear to be attributed to T3SS2 as a similar pathology was observed in rabbits infected with mutants lacking TDH or T3SS1. The purple rods represent <i>V. parahaemolyticus</i>.</p