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

Abstract

The hallmarks of enteropathogenic Escherichia coli (EPEC) infection are formation of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals on cultured cells, both of which are dependent on the type III secretion system effector Tir. Following translocation into cultured cells and clustering by intimin, Tir Y474 is phosphorylated, leading to recruitment of Nck, activation of N-WASP, and actin polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no role in A/E lesion formation on mucosal surfaces following infection with the EPEC-like mouse pathogen Citrobacter rodentium. In this study, we investigated the roles of Tir segments located upstream of Y451 and downstream of Y471 in C. rodentium colonization and A/E lesion formation. We also tested the role that Tir residues Y451 and Y471 play in host immune responses to C. rodentium infection. We found that deletion of amino acids 382 to 462 or 478 to 547 had no impact on the ability of Tir to mediate A/E lesion formation, although deletion of amino acids 478 to 547 affected Tir translocation. Examination of enterocytes isolated from infected mice revealed that a C. rodentium strain expressing Tir_Y451A/Y471A recruited significantly fewer neutrophils to the colon and triggered less colonic hyperplasia on day 14 postinfection than the wild-type strain. Consistently, enterocytes isolated from mice infected with C. rodentium expressing Tir_Y451A/Y471A expressed significantly less CXCL1. These result show that Tir-induced actin remodeling plays a direct role in modulation of immune responses to C. rodentium infection. Enteropathogenic Escherichia coli (EPEC) strains are important human pathogens causing infantile diarrhea in low-income countries (1) Recently, the Global Enteric Multicenter Study (GEMS), designed to detect the cause of pediatric diarrheal disease in sub-Saharan Africa and south Asia, found that infection with typical EPEC is associated with increased risk of fatality in infants aged 0 to 11 months (2). Citrobacter rodentium is a mouse-specific pathogen, the etiological agent of transmissible colonic hyperplasia, and a model EPEC microorganism, as both pathogens share an infection strategy and virulence factors (3, 4). Host resistance to C. rodentium infection is mediated by diverse T cell effector responses, including T cell production of interferon gamma (IFN-␥) (5, 6), interleukin 17A (IL-17A) (7,8),. Expression of the proinflammatory cytokine IL-17A leads to recruitment of neutrophils (10), and the anti-inflammatory cytokine IL-22 upregulates expression of antimicrobial peptides (such as REGIII␤ and REGIII␥) in enterocytes (9, 11).While colonizing the gut mucosa, EPEC and C. rodentium induce attaching and effacing (A/E) lesions. These are characterized by extensive remodeling of the gut epithelium leading to elongation and effacement of the brush border (BB) microvilli, intimate bacterial attachment to the enterocyte apical...

Citrobacter rodentium Subverts ATP Flux and Cholesterol Homeostasis in Intestinal Epithelial Cells In Vivo

“…IECs isolated from uninfected mice were used as a control (five mice per group). Examination of IECs by microscopy and flow cytometry revealed that IEC preparations were enriched by over 90% ( Crepin et al., 2015 ). Immunofluorescence microscopy revealed that IECs extracted from uninfected mice exhibited typical columnar shape and projection of actin-rich BB microvilli.…”

Model of Host-Pathogen Interaction Dynamics Links In Vivo Optical Imaging and Immune Responses

“…Such successful predictions also serve to validate the model. The success of the current study opens the prospect of using a similar approach to analyze the influence of other effectors on the immune response, such as Tir, which can affect the recruitment of neutrophils (31). …”

“…Cells were stained for surface markers (such as CD4 + CD8 − T cells, B220 + CD3 − B cells, and CD11b + Ly6G + neutrophils) in PBS containing 1% bovine serum albumin with 0.5% sodium azide (PBA) for 30 min at 4°C and fixed with IC fixation buffer (eBioscience) as described previously (31). Nonviable cells were excluded using a fixable near-infrared dead cell staining kit with excitation at 633 or 635 nm (L10119; Invitrogen).…”

The Citrobacter rodentium type III secretion system effector EspO affects mucosal damage repair and antimicrobial responses

“…To this end, we compared the proteomes of IECs purified from mice infected with WT, Δ espO and Δ espO -p espO at 8 DPI. Assessing purity by flow cytometry revealed that the IEC preparations were enriched by over 90% [ 26 ]. For the proteomic analysis, we only considered changes to protein abundance between WT and Δ espO if they were fully restored upon Δ espO complementation.…”

“…Importantly, bone marrow transplantation from WT mice into Myd88-/- mice restored CCH [ 6 ]. In addition, the T3SS effector Tir, in a process dependent on Y451 and Y471, has been shown to modulate secretion of Cxcl-1 and recruitment of neutrophils 14 DPI [ 26 ]. Importantly, while exhibiting a similar level of shedding, mice infected with C .…”