We have developed a simple non-surgical oral infection model of V. parahaemolyticus-induced intestinal pathology and diarrhea. This experimental model enabled us to define several previously unknown but key features of the pathogenesis of the disease elicited by this common agent of seafood-borne gastroenteritis. First, we discovered that this organism chiefly colonizes the distal small intestine, the region of the intestine that is also the major site of V. parahaemolyticus-induced damage, increased permeability of the epithelial barrier and inflammation. Together, these observations strongly suggest that disease in this region of the gastrointestinal tract accounts for most, if not all, of the diarrhea that accompanies V. parahaemolyticus infection. Second, we found that the V. parahaemolyticus T3SS2 is essential for the pathogen to colonize the small intestine; prior to our work no V. parahaemolyticus intestinal colonization factors had been definitively identified because of the absence of a robust animal model. Third, we observed that V. parahaemolyticus causes marked disruption of the villous epithelial surface in the small intestine. Effacement of microvilli, re-distribution of cytoskeletal and tight junction proteins, and extrusion of epithelial cells in the small intestine all appear to contribute to villus disruption and the breakdown of epithelial barrier function. Furthermore, the pathogen induces remarkable elongation of microvilli in epithelial cells adjacent to attached V. parahaemolyticus. Finally, early in the infection, before widespread damage to the epithelium becomes evident, V. parahaemolyticus induces both proliferation of intestinal epithelial cells and recruitment of inflammatory cells. Thus, our observations suggest that V. parahaemolyticus elicits disease via a previously undescribed sequence of events that, to our knowledge, differs from those outlined for other enteric pathogens.
, like V. cholerae
, preferentially colonizes the distal small intestine. However, the manner in which these two pathogenic vibrios associate with the host epithelial surface differs. Prototypical V. cholerae
O1 colonizes as layers of cells embedded in mucin-rich material that covers much of the epithelial surface of the villi as well as the crypts 
. In contrast, V. parahaemolyticus
colonizes as more discrete clusters of bacteria (i.e.
microcolonies) that predominantly localize to the upper half of the villi; furthermore, unlike V. cholerae
, V. parahaemolyticus
does not induce goblet cell degranulation. The mechanism(s) that hold the V. parahaemolyticus
microcolonies together are not known. However, since the microcolonies appear to form during contact with the epithelial surface, it is tempting to speculate that V. parahaemolyticus
lateral flagella, which can be induced by surface contact 
, may promote microcolony formation. These lateral cell appendages have been reported to form linkages between neighboring bacteria and the surface 
. Induction of peritrichous flagella is associated with conversion of V. parahaemolyticus
from small (2–3 µm), polarly flagellated swimmer cells to swarmer cells, which are elongated (5–20 µm) as well as peritrichously flagellated 
. Interestingly, we observed V. parahaemolyticus
cells up to 10 µm in length in electron micrographs of infected tissues (Figure S3D
, asterisk), consistent with the idea that these cells have switched from the swimmer to the swarmer cell state.
The consequences of V. cholerae
O1 and V. parahaemolyticus
colonization of the small intestine are also fundamentally different. V. cholerae
O1 does not damage the surface of the intestinal epithelium; instead, the cholera pathogen grows on the luminal side of the microvilli which remain largely intact (
and see Figure S3C
). In marked contrast, V. parahaemolyticus
damages the epithelial surface, leading to villus disintegration. Our histologic analyses of samples from 12 to 38 hr PI suggest that there is a characteristic sequence of steps through which V. parahaemolyticus
proceeds to cause villus disruption in the small intestine ( and ). Initial attachment of V. parahaemolyticus
to the epithelial surface is associated with effacement of microvilli and apparent depletion of cytoplasmic contents; thus, even at 18 hr PI, attached V. parahaemolyticus
was observed situated below the epithelial surface (). Both marked depletion of epithelial cell cytoplasmic contents as well as epithelial cell extrusion contribute to the formation of these V. parahaemolyticus
-filled cavities in the epithelial surface. It is not clear if V. parahaemolyticus
penetration of the paracellular space to reach the base of the extruding cell is a required step for extrusion, though this was often observed ( and ). The benefit of the epithelial cavities for the pathogen is not known, but it seems plausible that the cavities may provide the pathogen increased access to nutrients, or serve as a niche, offering protection from peristaltic flow.
Schematic of kinetics of V. parahaemolyticus-induced damage to the intestinal epithelial surface.
The molecular mechanisms by which V. parahaemolyticus
elicits epithelial cell extrusion are not known. The process bears some similarity to the normal process that leads to shedding of apoptotic epithelial cells 
. V. parahaemolyticus
-induced cell extrusion, as in physiologic cell shedding, is accompanied by redistribution of tight junction-associated proteins, including ZO-1 and occludin-1, towards the basolateral membrane where they co-localized with actin to form a funnel-like structure. However, claudin-1 did not follow this pattern in extruding cells of infected tissues, as occurs in physiologic and pathologic shedding 
. Furthermore, while paracellular barrier integrity is maintained during physiological cell shedding, there is an increase in paracellular permeability observed in infected rabbits. V. parahaemolyticus
-induced cell shedding could be a direct effect of the pathogen (e.g., perhaps a consequence of the activities of translocated T3SS effectors on tight junction complexes), or an indirect consequence of infection. For example, certain pro-inflammatory cytokines including TNF (whose production is stimulated by V. parahaemolyticus
) have been shown to elicit cell shedding 
. Regardless of the mechanisms leading to epithelial cell extrusion, V. parahaemolyticus
-induced break down in epithelial barrier function, which has also been observed in a polarized epithelial monolayer 
, likely contributes to the loss of intestinal fluid (diarrhea) caused by V. parahaemolyticus
Pathogen-induced epithelial cell extrusion has also recently been detected for other enteric pathogens. For example, EHEC induces extrusion of cells from polarized monolayers and during infection of calves 
. Tissue-culture-based studies have revealed that the EHEC T3SS effector EspM, which interferes with the RhoA-signaling pathways that regulate actin cytoskeleton dynamics in eukaryotic cells, is sufficient to cause extrusion 
. The purpose of pathogen-induced cell extrusion is difficult to ascertain, particularly since it can have benefits for both the pathogen and the host. For Salmonella
, intestinal epithelial cell extrusion has been shown to promote the pathogen's spread within, and escape from, the intestinal tract 
. Cell extrusion could also promote the egress of V. parahaemolyticus
from the intestine since extruded cells and debris often had adherent V. parahaemolyticus
. However, it is also possible that extrusion aids the host, by enabling shedding of adherent bacteria. In support of this idea, it has been found that several bacterial pathogens (e.g., Shigella
) produce factors that appear to counteract epithelial shedding 
Some of the ultrastructural changes V. parahaemolyticus
elicits in intestinal epithelial cells are reminiscent of phenotypes previously described for EPEC, a member of the A/E family of pathogens 
. Similar to EPEC-induced changes in small intestine explants, we observed long spaghetti-like protrusions from epithelial cells surrounding the edges of the V. parahaemolyticus
clusters, effacement of microvilli, and the close apposition of individual V. parahaemolyticus
cells to the effaced epithelial surface within cup-like structures (see ). The mechanism(s) that mediate these dramatic alterations in host epithelial cell morphology remain to be determined. However, it seems likely that, similar to EPEC, the activities of some of the effectors translocated by one or both of the V. parahaemolyticus
T3SS manipulate the host cytoskeleton and thereby alter cell morphology. Indeed, several type III translocated proteins of V. parahaemolyticus
and the related pathogen V. cholerae
, including the recently described VopV, have been shown to alter actin dynamics in cultured cells 
. Furthermore, AM-19226, a non-O1, non-O139 V. cholerae
strain that encodes a T3SS similar to the V. parahaemolyticus
T3SS2 causes villus destruction in the small intestine of infant rabbits, suggesting that common effectors translocated by these systems may contribute to the pathology 
. However, the steps leading to the destruction of intestinal villi by AM-19226 have not been elucidated, and the pathologic features of AM-19226-induced disease differ from those caused by V. parahaemolyticus
Besides damaging the villous epithelium in the small intestine, V. parahaemolyticus
also causes elevated proliferation of cells in the crypts. Several other enteric pathogens, as well as the microbiota, have been reported to alter intestinal epithelial dynamics 
. In some cases, pathogen-induced changes in epithelial homeostasis are thought to promote bacterial colonization. Similarly, since V. parahaemolyticus
damages the epithelium, promoting epithelial renewal could enhance colonization. However, the increased cell proliferation occurs early during V. parahaemolyticus
infection, suggesting that proliferation is not a direct response to epithelial damage. Identification of the V. parahaemolyticus
factor(s) that lead to elevated proliferation, and the manner by which they are antagonized by TDH, may yield insight into mechanisms that normally govern turnover of intestinal stem cells.
Our findings suggest that each of the three previously proposed V. parahaemolyticus
virulence-linked loci – TDH and the two T3SSs – modulate the organism's pathogenicity. Our results confirm and extend earlier ileal-loop-based studies 
indicating that T3SS2 is the major virulence factor contributing to V. parahaemolyticus'
enterotoxicity. We observed that T3SS2 is not only required for intestinal fluid accumulation, but it is also essential for colonization of the small intestine. It will be interesting to explore how T3SS2 promotes colonization and investigate if one (or more) translocated effector(s) act in a similar fashion as the EPEC/EHEC translocated intimin receptor, Tir 
, to enable V. parahaemolyticus
adherence. Alternatively, do the T3SS2 effectors modulate host cell processes, including those of the immune response, to generate a niche permissive for V. parahaemolyticus
proliferation? For example, increased access to nutrients may occur as a consequence of epithelial disruption.
Unexpectedly, we found that TDH negatively impacts colonization of the upper regions of the small intestine and appears to dampen some aspects of V. parahaemolyticus
-induced disease. This result contrasts with findings from studies using ligated ileal loops 
, where TDH was found to contribute to fluid accumulation. Differences between the experimental systems may explain these contradictory results; ligated ileal loops are closed systems where intestinal peristalsis is reduced and infections are of limited duration (typically 18 hr).
Collectively, our findings suggest that infant rabbits will be a very useful experimental model to shed light on the pathogen and host factors, and mechanisms that explain the pathogenesis of V. parahaemolyticus
-induced intestinal disease. It is important to note however, that while many of the features of V. parahaemolyticus
-induced disease resemble those reported in humans, rabbits do not exhibit all the signs of V. parahaemolyticus
infection that have been reported. For example, infected individuals can have occult blood in their stool and occasionally present with grossly bloody stools 
. The presence of blood in the stool appears to correlate with epithelial damage consisting of ‘superficial ulcerations’ in the lower intestine of patients 
; no pathology was detected within the colons of infected rabbits and neither gross nor occult blood was observed in fecal material obtained from the rabbits. Nevertheless, infant rabbits reproduce the inflammatory enteritis and watery diarrhea that are the chief signs of disease in most infected individuals. Thus, studies using this model host should enable dissection of the complex interplay of pathogen and host factors that result in disease as well as testing of new therapeutics to combat and/or prevent infection.