Modelling of Infection by EnteropathogenicEscherichia coliStrains in Lineages 2 and 4 Ex Vivo and In Vivo by UsingCitrobacter rodentiumExpressing TccP

Abstract

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are diarrheal pathogens which colonize the gut epithelium via attaching-and-effacing (A/E) lesion formation (reviewed in references 9 and 10). A/E lesions are characterized by effacement of the brush border microvilli and intimate bacterial attachment to the mammalian cell plasma membrane (24). The genes required for A/E lesion formation are carried on the locus of enterocyte effacement (26), which encodes transcriptional regulators (21), the adhesin intimin (20), a type III secretion system (T3SS) (19), chaperones, translocators, and several effector proteins (reviewed in references 10 and 12).While infecting cultured cells in vitro, EPEC and EHEC trigger strong actin polymerization at the site of bacterial attachment. The principal T3SS effector protein needed for A/E lesion formation on mucosal surfaces and actin polymerization in vitro is Tir (23, 33). Once translocated, Tir is integrated into the plasma membrane of the mammalian cell in a hairpin loop topology (18). The extracellular loop, presented above the plasma membrane, serves as an intimin receptor (reviewed in reference 11). In EPEC, actin polymerization in vitro is initiated once clustering of Tir by intimin (34) leads to phosphorylation of a Tir tyrosine (Y) residue at position 474 in the prototype EPEC strain E2348/69 (22), which is present in the context of a consensus binding site (Y P DEP/D/V) for the mammalian adaptor protein Nck (5, 17). Binding of Nck to phosphorylated Tir leads to recruitment and activation of the neuronal Wiskott-Aldrich syndrome protein (N-WASP) and actin polymerization via the actin-related protein 2/3 complex (reviewed in reference 7). Tir from E2348/69 can also trigger weak actin polymerization in the absence of Nck recruitment (6). This Nck-independent pathway is dependent on a universally conserved NPY Tir motif (2).Studying the interaction of EHEC O157:H7 with cultured cells revealed that activation of the actin polymerization cascade occurs by a mechanism that is distinct from that of E2348/69 (reviewed in reference 9) since in contrast to EPEC Tir, EHEC O157:H7 Tir lacks a Y474 equivalent and hence cannot assemble the Tir-Nck signaling complex. Instead, EHEC O157:H7 employs the T3SS effector TccP (also known as EspF U ) (3, 13), which mimics Nck in terms of linking Tir to the N-WASP actin polymerization machinery. Recruitment of TccP to Tir is dependent on the conserved NPY Tir motif (1, 4).Recently, while screening for the presence of tccP in clinical EPEC isolates, we unexpectedly found that strains belonging to EPEC-2 (represented by EPEC O111:NM strain B171) and EPEC-4 (represented by EPEC O119:H6 strain ICC199) (25, 36) encode TccP and TccP2, respectively, which are functionally interchangeable (36). In contrast, strains belonging to the EPEC-1 lineage (represented by EPEC O127:H6 strain E2348/ 69) are tccP/tccP2 gene negative (25,36). As such, strains belonging to EPEC-1, EPEC-2, and EPEC-4 are able to trigger strong acti...

The ability of an attaching and effacing pathogen to trigger localized actin assembly contributes to virulence by promoting mucosal attachment

“…In fact, we found that the 11‐ to 16‐fold defect in colonic colonization was associated with only an approximately fourfold defect in faecal shedding, possibly due to luminal bacteria released from the (efficiently colonized) caecum. Given that many studies utilize faecal shedding as a convenient serial readout of intestinal colonization (Mundy et al ., ; Marches et al ., ; Ritchie and Waldor, ; Vlisidou et al ., ; Girard et al ., ; Crepin et al ., ), our finding that pedestal formation may play an essential role in mucosal colonization for only a subset of intestinal segments could partially explain varying apparent defects in colonization by pedestal‐defective mutants in other studies.…”

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

“…This IVOC system has been used mainly to study the mechanisms of virulence of ETEC (839), EPEC (840)(841)(842)(843)(844)(845)(846)(847)(848)(849)(850)(851), EHEC (852)(853)(854)(855)(856)(857), EAEC (507,858), and Salmonella (392) and the action of Stx toxins (641). Other IVOCs have been used for studies on C. jejuni (859), EPEC (692,860,861), EAEC (645,(862)(863)(864), and Shigella (865) pathogenesis. Moreover, IVOCs from colonic biopsy specimens from CD patients have been used to show the adhesion of AIEC (444,866).…”

Tir Triggers Expression of CXCL1 in Enterocytes and Neutrophil Recruitment during Citrobacter rodentium Infection

“…The mouse intestinal in vitro organ culture (mIVOC) model was used to assess A/E lesion formation caused by C. rodentium expressing Tir Y451A/Y471A/⌬478-547 as described by Girard et al (41). Briefly, segments from the terminal colon were inoculated with 50 l of the appropriate overnight bacterial culture, corresponding to approximately 10 7 CFU, and incubated at 37°C in 5% CO 2 atmosphere on a seesaw rocker (18 cycles min Ϫ1 ) for 8 h. Explants were gently rinsed with PBS and fixed in 2.5% glutaraldehyde for electron microscopy analysis.…”