Enterohemorrhagic E. coli (EHEC) is an important subset of Shiga toxin-producing (Stx-producing) E. coli (STEC), pathogens that have been implicated in outbreaks of food-borne illness and can cause intestinal and systemic disease, including severe renal damage. Upon attachment to intestinal epithelium, EHEC generates "attaching and effacing" (AE) lesions characterized by intimate attachment and actin rearrangement upon host cell binding. Stx produced in the gut transverses the intestinal epithelium, causing vascular damage that leads to systemic disease. Models of EHEC infection in conventional mice do not manifest key features of disease, such as AE lesions, intestinal damage, and systemic illness. In order to develop an infection model that better reflects the pathogenesis of this subset of STEC, we constructed an Stx-producing strain of Citrobacter rodentium, a murine AE pathogen that otherwise lacks Stx. Mice infected with Stx-producing C. rodentium developed AE lesions on the intestinal epithelium and Stx-dependent intestinal inflammatory damage. Further, the mice experienced lethal infection characterized by histopathological and functional kidney damage. The development of a murine model that encompasses AE lesion formation and Stx-mediated tissue damage will provide a new platform upon which to identify EHEC alterations of host epithelium that contribute to systemic disease.

Enterohemorrhagic Escherichia coli (EHEC) adheres to intestinal epithelial cells, then stimulates the actin nucleation promoting factor N-WASP to induce localized actin assembly resulting in an actin “pedestal”, the function of which is poorly understood. EHEC also produces Shiga toxin (Stx), which penetrates the intestinal epithelium to cause a life-threatening renal and systemic disease. To assess the role of pedestal formation in colonization and disease, we utilized the murine pathogen Citrobacter rodentium, which also forms actin pedestals, and the genetically engineered C. rodentium (Φstx2dact), which additionally triggers Stx-mediated systemic disease. We found that an intestine-specific N-WASP-deficient (iNWKO) mouse suffered dramatically less colonization and disease than N-WASP-proficient littermate controls when infected with either strain. In addition, upon infection of wild type mice, mutants of C. rodentium or C. rodentium (Φstx2dact) that are specifically defective in pedestal formation demonstrated a relatively modest defect in cecal colonization and fecal shedding, but a more severe defect in colonization of the colonic mucosa. The C. rodentium (Φstx2dact) pedestal-defective mutant did not cause renal disease and, after normalizing for fecal bacterial load, was associated with a 16-fold lower risk of lethality. These findings suggest that the ability of an attaching and effacing pathogen to promote localized actin assembly contributes to virulence by promoting mucosal attachment.

Phenotypic diversity is critical to the lifestyles of many microbial species, enabling rapid responses to changes in environmental conditions. In the human fungal pathogen Candida albicans, cells exhibit heritable switching between two phenotypic states, white and opaque, which yield differences in mating, filamentous growth, and interactions with immune cells in vitro. Here, we address the in vivo virulence properties of the two cell states in a zebrafish model of infection. Multiple attributes were compared including the stability of phenotypic states, filamentation, virulence, dissemination, and phagocytosis by immune cells, and phenotypes equated across three different host temperatures. Importantly, we found that both white and opaque cells could establish a lethal systemic infection. The relative virulence of the two cell types was temperature dependent; virulence was similar at 25°C, but at higher temperatures (30 and 33°C) white cells were significantly more virulent than opaque cells. Despite the difference in virulence, fungal burden, and dissemination were similar between cells in the two states. Additionally, both white and opaque cells exhibited robust filamentation during infection and blocking filamentation resulted in decreased virulence, establishing that this program is critical for pathogenesis in both cell states. Interactions between C. albicans cells and immune cells differed between white and opaque states. Macrophages and neutrophils preferentially phagocytosed white cells over opaque cells in vitro, and neutrophils showed preferential phagocytosis of white cells in vivo. Together, these studies distinguish the properties of white and opaque cells in a vertebrate host, and establish that the two cell types demonstrate both important similarities and key differences during infection.

Upon binding to intestinal epithelial cells, enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium trigger formation of actin pedestals beneath bound bacteria. Pedestal formation has been associated with enhanced colonization, and requires intimin, an adhesin that binds to the bacterial effector translocated intimin receptor (Tir), which is translocated to the host cell membrane and promotes bacterial adherence and pedestal formation. Intimin has been suggested to also promote cell adhesion by binding one or more host receptors, and allelic differences in intimin have been associated with differences in tissue and host specificity. We assessed the function of EHEC, EPEC, or C. rodentium intimin, or a set of intimin derivatives with varying Tir-binding abilities in animal models of infection. We found that EPEC and EHEC intimin were functionally indistinguishable during infection of gnotobiotic piglets by EHEC, and that EPEC, EHEC, and C. rodentium intimin were functionally indistinguishable during infection of C57BL/6 mice by C. rodentium. A derivative of EHEC intimin that bound Tir but did not promote robust pedestal formation on cultured cells was unable to promote C. rodentium colonization of conventional mice, indicating that the ability to trigger actin assembly, not simply to bind Tir, is required for intimin-mediated intestinal colonization. Interestingly, streptomycin pre-treatment of mice eliminated the requirement for Tir but not intimin during colonization, and intimin derivatives that were defective in Tir-binding still promoted colonization of these mice. These results indicate that EPEC, EHEC, and C. rodentium intimin are functionally interchangeable during infection of gnotobiotic piglets or conventional C57BL/6 mice, and that whereas the ability to trigger Tir-mediated pedestal formation is essential for colonization of conventional mice, intimin provides a Tir-independent activity during colonization of streptomycin pre-treated mice.