Allele- and Tir-Independent Functions of Intimin in Diverse Animal Infection Models

Abstract

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.

Expression of Enteropathogenic Escherichia coli Map Is Significantly Different than That of Other Type III Secreted Effectors In Vivo

“…Tir is required for ex vivo colonization of both EPEC and EHEC in human intestinal mucosa in in vitro organ culture experiments (41). However, intimin but not Tir is required for colonization of C. rodentium in C57BL/6 mice pretreated with streptomycin (40). EspF, EspH, and EspG are all required for EHEC colonization in infant rabbits (38).…”

“…In addition to Map, intimin (40) and many other effectors, such as Tir (41), EspF (38), EspH (38), and EspG (38), promote colonization of A/E pathogens. Tir is required for ex vivo colonization of both EPEC and EHEC in human intestinal mucosa in in vitro organ culture experiments (41).…”

A novel murine infection model for Shiga toxin–producing Escherichia coli

“…To create an antibiotic-marked Stx2-expressing Φ1720a-02 bacteriophage, a chloramphenicol resistance marker (cat, Cm R ) was inserted into the nonessential gene Rz (115) using lambda red recombination. Briefly, a 707-bp fragment containing sequence approximately 5 × 10 9 of overnight culture of C. rodentium in 100 μl PBS, an inoculum that minimizes the potential variability in colonization kinetics (119). Inoculum concentrations were confirmed by serial dilution plating.…”

“…Filamentous actin staining (FAS) assays using C. rodentium were done as previously described (119). Briefly, a single colony from each strain was grown in 1 ml medium (with or without antibiotic) for 8 hours.…”

Enteropathogenic and enterohemorrhagic E. coli: ecology, pathogenesis and evolution

“…This has also been explored as a source of EPEC/EHEC tissue tropism in the past (Fitzhenry et al, 2002). Here a Tir-independent role for intimin in the colonization of streptomycin-treated mice provides more evidence that in some experimental systems, intimin promotes intestinal colonization independently of its role in Tir-binding (Mallick et al, 2012).…”