EspFU, a type III-translocated effector of actin assembly, fosters epithelial association and late-stage intestinal colonization by E. coli O157:H7

Abstract

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 induces filamentous actin-rich 'pedestals' on intestinal epithelial cells. Pedestal formation in vitro requires translocation of bacterial effectors into the host cell, including Tir, an EHEC receptor, and EspF(U), which increases the efficiency of actin assembly initiated by Tir. While inactivation of espF(U) does not alter colonization in two reservoir hosts, we utilized two disease models to explore the significance of EspF(U)-promoted actin pedestal formation. EHECDeltaespF(U) efficiently colonized the rabbit intestine during co-infection with wild-type EHEC, but co-infection studies on cultured cells suggested that EspF(U) produced by wild-type bacteria might have rescued the mutant. Significantly, EHECDeltaespF(U) by itself was fully capable of establishing colonization at 2 days post inoculation but unlike wild type, failed to expand in numbers in the caecum and colon by 7 days. In the gnotobiotic piglet model, an espF(U) deletion mutant appeared to generate actin pedestals with lower efficiency than wild type. Furthermore, aggregates of the mutant occupied a significantly smaller area of the intestinal epithelial surface than those of the wild type. Together, these findings suggest that, after initial EHEC colonization of the intestinal surface, EspF(U) may stabilize bacterial association with the epithelial cytoskeleton and promote expansion beyond initial sites of infection.

Figure 1. Efficient trans-complementation by EspF_U but not Tir during mixed infection on HeLa cells

Infected monolayers were examined microscopically after staining with DAPI to visualize bacteria, anti-O157 antiserum to specifically detect EHEC strains and fluorescent phalloidin to detect F-actin. For merged images, DAPI staining is shown in blue, bacteria in green, F-actin in red, and foci of co-localized bacteria and F-actin in yellow. HeLa cells were infected singly or in combination with the indicated strains. In rows 4 or 7, large arrowheads on merged images indicate TUV93-0 or its derivatives; arrows indicate efficient (row 4) or inefficient (row 7) trans-complementation, i.e., sites of actin assembly associated with KC12 (anti-O157 negative bacteria).

Figure 2. EspF_U is associated with increased efficiency of colonization at day 7 post-infection of infant rabbits

905 or 905ΔespF_U were recovered from intestinal segments or stool samples from infected rabbits at 2 and 7 days post-inoculation. Each point represents an individual rabbit and bars represent the geometric mean for each strain. The level of wild type colonization differed significantly between day 2 and day 7 in the ileum (P≤0.01), cecum (P≤0.01), mid colon (P≤0.0001) and stool (P≤0.01), whereas the level of mutant colonization only differed significantly (P≤0.05) in the ileum, when analyzed by the Student’s t-test (two-way).

Figure 3. EspF_U may increase the efficiency of, but is not absolutely required for A/E lesion formation during piglet infection

Electron micrographs of the ileum of piglets infected with wild type (A), ΔespF_U (B) orΔespF_U / pespF_U (C). Arrows show areas of electron-dense material. Bars equal 1μm.

Figure 4. EspF_U is associated with a larger size of clusters of attached bacteria

O157-labeled bacteria adherent to the cecum of piglets infected with wild type (A), ΔespF_U (B), and ΔespF_U / pespF_U (C) –infected piglets. Bacteria are stained green and F-actin is stained red. Note the relatively small clusters of attached bacteria in (B). Scale bars are 10μm in each panel. Panel (D): Area of tissue colonized by O157-labeled bacteria divided by area visible with phalloidin staining expressed as a percentage. ^aStrains are derivatives of TUV93-0. ^bValues are mean ± standard deviation. Data were compared using the Student’s t-test (two-way) and considered significantly different (*) at P≤0.05.