Figure 1.
V. parahaemolyticus colonizes the small intestine of infant rabbits and induces a destructive enteritis.
(A) Diarrhea and mortality in V. parahaemolyticus-infected rabbits over time (n = 15 rabbits). (B) Representative images of the intact intestine of V. parahaemolyticus-infected and mock-infected rabbits. (C) Recovery of V. parahaemolyticus (cfu g−1) from tissue homogenates from indicated intestinal section. Points represent individual rabbits; bars represent geometric means. Open boxes represent the limit of detection for samples from which no cfu were detected. (D–G) H&E-stained sections from the distal small intestine of infected (D, F–G) and mock infected (E) rabbits. In (D), the long arrow points to heterophils, arrowheads indicate sloughed or detaching host cells and the star indicates attached bacteria. In (F), the arrowhead indicates a cluster of bacteria and the arrow indicates heterophils traversing the epithelial barrier, whereas in (G), the arrowheads indicate bacteria and the arrow indicates degranulated heterophils. Scale bars are 100 µm and 10 µm in D–E and F–G, respectively. (H) Relative levels (mean ± average deviation) of transcripts for cytokines in homogenates of the distal small intestine from wild type (WT), the triple mutant (Δtdh ΔvcrD1 ΔvcrD2) and POR-2 (Δtdh ΔvscN1) -infected rabbits. In B-H samples were collected at 38 hr PI.
Table 1.
Diarrhea in rabbits following oro-gastric inoculation of V. parahaemolyticus or one of its isogenic derivatives.
Figure 2.
Kinetics of fluid accumulation and pathology in V. parahaemolyticus-infected infant rabbits.
(A) Concentration (cfu g−1) of V. parahaemolyticus recovered in distal small intestine homogenates at different times after inoculation. Data points represent individual rabbits; bars represent geometric means. Open boxes represent the limit of detection for samples from which no cfu were isolated. Groups were compared using one-way ANOVA with Boneffoni's multiple comparison post-test (*** P<0.001). (B) Fluid accumulation ratio (mean ± SEM) in the distal small intestine of rabbits at various times PI. Data were compared to uninfected rabbits using one-way ANOVA with Boneffoni's multiple comparison post-test (*** P<0.001). (C–D and F–G) Representative H&E-stained sections from the distal small intestine of infected rabbits at 18 hr (C), 28 hr (D, F) and 38 hr PI (G) showing the frequency and size of attached bacterial clusters as well as histological changes. Arrowheads in C and D point to bacterial microcolonies and long arrows indicate luminal heterophils; in F, arrowheads point to extruding epithelial cells with V. parahaemolyticus at their base, and in G, arrowheads point to extruding cells and long arrows point to cytoplasmic fragments. Scale bars are 100 µm and 20 µm in C–D and F–G, respectively. (E) Small intestinal tissue sections from rabbits infected with V. parahaemolyticus expressing GFP (green) counterstained with phalloidin (red) and DAPI (blue) to detect F-actin and nuclei acid, respectively.
Figure 3.
Representative electron micrographs of V. parahaemolyticus attached to the intestinal epithelium at 28 hr PI.
(A) Scanning electron micrograph of the small intestine of V. parahaemolyticus-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 V. parahaemolyticus 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 V. parahaemolyticus 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) V. parahaemolyticus (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 V. parahaemolyticus. Scale bar = 2 µm.
Figure 4.
Extruding epithelial cells show redistribution of some tight junction proteins but lack markers of apoptosis.
(A–C) Immunofluorescence micrographs of small intestinal sections from mock and infected rabbits stained for (A) ZO-1, (B) occludin-1 and (C) claudin-1. Note altered localization of ZO-1 and occludin-1 but not claudin-1 in extruding epithelial cells with adjacent V. parahaemolyticus (arrowheads). Actin cytoskeleton was stained with phalloidin-Alexa 568 (yellow), bacteria constitutively expressed green fluorescent protein (green) and nuclei were stained with DAPI (blue). (D) Tissue sections from infected rabbits were stained for apoptotic cells using the TUNEL assay. TUNEL-positive cells were detected using a fluorescein-based detection system thus both bacteria and apoptotic host cells appear green, but can be distinguished based on their relative size and shape. Examples of TUNEL-positive (arrowhead) and TUNEL-negative (arrows) host cells are indicated. Sections were counterstained with phalloidin-Alexa 568 (yellow) and DAPI (blue) as described above.
Figure 5.
V. parahaemolyticus compromises epithelial barrier function in the small intestine.
Penetration of biotin (red) into the lamina propria in mock-infected (A) or V. parahaemolyticus-infected rabbits (B) at 25 hr PI. Tissues were counterstained with DAPI (blue) and phalloidin-Alexa 568 (yellow) to stain nucleic acid and F-actin, respectively. Note that biotin is located within the villi in sections from infected rabbits, but not in control rabbits. Adherent bacteria are marked with an arrowhead.
Figure 6.
Colonization and pathological changes induced by wild type V. parahaemolyticus and isogenic mutants at 38 hr PI.
(A–C) Concentration (cfu g−1) of bacteria recovered from tissue homogenates of intestinal samples from rabbits infected with wild type V. parahaemolyticus or from mutants lacking one or more putative virulence factors. Data points represent individual rabbits. Open symbols represent the limit of detection for samples from which no cfu were isolated. Bars show the geometric mean. Statistical analysis was performed using one way ANOVA and Bonferroni's multiple comparison post-test. *P≤0.05, **P≤0.01 and ***P≤0.001. (D–F) Pathologic scores for abnormalities in the small intestine of infected or mock-infected rabbits. Data points represent individual rabbits. Statistical analysis was performed using Kruskal-Wallis statistic with Dunn's post-test for multiple. *P≤0.05, **P≤0.01 and ***P≤0.001.
Figure 7.
Schematic of kinetics of V. parahaemolyticus-induced damage to the intestinal epithelial surface.
Following initial attachment, V. parahaemolyticus 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 V. parahaemolyticus 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 V. parahaemolyticus.