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Tir-induced actin remodeling triggers expression of CXCL1 in enterocytes
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and neutrophil recruitment during Citrobacter rodentium infection
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Running title: Tir-mediated neutrophil recruitment
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Valerie F. Crepin*, Maryam Habibzay, Izabela Glegola-Madejska, Marianne Guenot, James
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W. Collins and Gad Frankel*
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MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial
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College, London, UK
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For Correspondence: Valerie Crepin, CMBI, Flowers Building, Imperial College, London
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SW7 2AZ. Telephone: +44 20 75943070; Email: v.crepin-sevenou@imperial.ac.uk &
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Telephone: +44 20 75945253; Email: g.frankel@imperial.ac.uk
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Abstract
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The hallmarks of enteropathogenic Escherichia coli (EPEC) infection are formation
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of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals
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on cultured cells, both dependent on the type III secretion system effector Tir.
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Following translocation into cultured cells and clustering by intimin, Tir Y474 is
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phosphorylated leading to recruitment of Nck, activation of N-WASP and actin
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polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization
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pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no
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role in A/E lesion formation on mucosal surfaces following infection with the EPEC-
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like mouse pathogen Citrobacter rodentium. In this study we investigated the roles of
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Tir segments located upstream of Y451 and downstream of Y471 in C. rodentium
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colonization and A/E lesion formation. We also tested the role Tir residues Y451 and
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Y471 play in host immune responses to C. rodentium infection. We found that
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deletion of amino acids 382-462 or 478-547 had no impact on the ability of Tir to
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mediate A/E lesion formation, although deletion of amino acids 478-547 affected Tir
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translocation. Examination of enterocytes isolated from infected mice revealed that a
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C. rodentium expressing Tir_Y451A/Y471A recruited significantly less neutrophils to
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the colon and triggered less colonic hyperplasia on day 14 post infection, compared to
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infection with the wild type strain. Consistently, enterocytes isolated from mice
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infected with C. rodentium expressing Tir_Y451A/Y471A expressed significantly
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less CXCL1. These result show that Tir-induced actin remodeling plays a direct role
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in modulation of immune responses to C. rodentium infection.
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Introduction
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Enteropathogenic Escherichia coli (EPEC) strains are important human pathogens
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causing infantile diarrhea in low-income countries (1) Recently, the Global Enteric
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Multicenter Study (GEMS), designed to detect the cause of paediatric diarrheal
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disease in sub-Saharan Africa and south Asia, found that infection with typical EPEC
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is associated with increased risk of fatality in infants aged 0-11 months (2).
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Citrobacter rodentium is a mouse-specific pathogen, the etiological agent of
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transmissible colonic hyperplasia, and a model EPEC microorganism, as both
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pathogens share an infection strategy and virulence factors (3, 4). Host resistance to
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C. rodentium infection is mediated by diverse T cell effector responses, including T
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cells production of interferon-γ (IFNγ) (5, 6), interleukin 17A (IL17A) (7, 8) or IL22
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(9). Expression of the pro-inflammatory cytokine IL-17A leads to recruitment of
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neutrophils (10), and the anti-inflammatory cytokine IL-22 up-regulates expression of
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antimicrobial peptides (such as REGIIIβ and REGIIIγ) in enterocytes (9, 11).
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While colonizing the gut mucosa EPEC and C. rodentium induce attaching and
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effacing (A/E) lesions. These are characterized by extensive remodeling of the gut
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epithelium leading to elongation and effacement of the brush border (BB) microvilli,
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intimate bacterial attachment to the enterocyte apical plasma membrane,
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accumulation of polymerized actin and formation of elevated pedestal-like structures
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(4, 12). Adhesion of EPEC (reviewed in (13)) and C. rodentium (14) to cultured cells
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triggers actin polymerization under attached bacteria.
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The ability to induce A/E lesions and actin polymerization is encoded within the locus
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of enterocyte effacement (LEE) (15), which encodes a type III secretion system
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(T3SS) (16), the outer membrane adhesin intimin (17), regulators, chaperones,
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translocator and effector proteins (reviewed in (18)). Following initial cell attachment,
3
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EPEC and C. rodentium use their T3SS to inject LEE- and non-LEE-encoded
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effectors that subvert multiple signaling pathways including apoptosis (the effectors
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NleH and NleB), endosomal trafficking (EspG and EspI), Rho GTPases (EspH and
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Map), innate immunity (NleC, NleD, NleE and NleF) and actin dynamics (Tir and
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EspF) (reviewed in (13)). In particular, following translocation, Tir, which contains
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two trans-membrane (TM) helixes, is integrated into the epithelial cell plasma
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membrane in a hairpin loop topology (19, 20), exposing an extracellular central
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domain that functions as an intimin receptor (21). Infection of cultured epithelial cells
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has shown that binding of intimin induces clustering of Tir, which leads to
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phosphorylation of a C-terminal tyrosine (20), Y474 in EPEC or Y471 in C.
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rodentium, by redundant tyrosine kinases, including Src, Fyn and Abl (22, 23). These
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in turn recruit Nck via its SH2 domain which activates the neural Wiskott–Aldrich
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syndrome protein (N-WASP) via its SH3 domain. This leads to recruitment of the
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Arp2/3 complex which triggers actin polymerization underneath the attached bacteria
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(reviewed in (24)). Campellone and Leong (25) have shown that TirEPEC can promote
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weak actin polymerization in an Nck-independent manner, involving the C-terminal
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Tir tyrosine residue Y454 (or Y451 in C. rodentium), which is present in the context
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of a conserved Asn-Pro-Tyr (NPY) motif (26). The NPY motif recruits the adaptor
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protein insulin receptor tyrosine kinase substrate (IRTKS) and/or the insulin receptor
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substrate protein of 53 kDa (IRSp53) (27, 28). In EPEC belonging to lineage 2, the
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weak Tir NPY-mediated actin polymerization pathway is amplified by the bacterial
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effector TccP2/EspFM, which also activates N-WASP (29).
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Although it was widely believed that the Tir-induced actin signaling pathways
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observed during infection of cultured cells were responsible for A/E lesion formation
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on mucosal surfaces, Deng et al (30) provided some initial indications that this might
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not be the case as complementation of a tir C. rodentium mutant with a plasmid
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encoding Tir Y471F restored A/E lesion formation in vivo. Moreover, infection of
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human in vitro organ cultures (IVOC) with EPEC expressing Tir_Y474F or
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Tir_Y454F/Y474F also resulted in A/E lesions (31). In addition, we have
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subsequently reported that incorporation of Y451A and Y471A double substitutions
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into C. rodentium chromosomal tir abrogated actin polymerization in cultured cells
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but had no effect on the level of colonization and A/E lesion formation in the mouse
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model (14). Importantly, Ritchie et al. (32) and Mallick et al. (33, 34) have shown
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that of A/E pathogens expressing tir mutant unable to trigger actin polymerization in
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vitro were attenuated in mucosal colonization in vivo. The aim of this study was to
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further investigate the role of the C-terminus of Tir during C. rodentium infection in
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vivo.
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Material and methods
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Bacterial strains and growth conditions
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The bacterial strains, plasmids and primers used in this study are listed in Table 1.
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Bacteria were grown in Luria–Bertani (LB) medium, M9 minimum media (35) or in
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Dulbecco's modified Eagle's medium (DMEM) supplemented with kanamycin
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(50 mg ml−1), ampicillin (100 mg ml−1) and nalidixic acid (50 mg ml−1) as required.
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Introduction of site-directed tir mutants into the C. rodentium chromosome
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We used the lambda red-based mutagenesis system (36) to introduce site-directed tir
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alterations into the endogenous chromosomal tir gene, together with a kanamycin
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cassette, in the tir-cesT intergenic region for 3′ mutagenesis as described before (14).
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Deletion of the DNA segment encoding amino acids 478-547 (478-547) within the
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tir gene was made by inverse-PCR on pICC433 (encoding Tir_Y451/Y471) and
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pICC438 (encoding Tir_Y451A/Y471A) templates, previously described in (14),
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using primer pair [Tir-P478DSV-stop-EcoRI-Rv] and [down-Tir-EcoRI-Fw]. The
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inverse-PCR product was then digested with EcoRI and the aphT gene cloned into the
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tir-cesT intergenic region to confer kanamycin resistance, resulting in plasmids
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pICC1842 and pICC1843, respectively (Table 1).
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Deletion of the DNA segment encoding amino acids 382-462 (382-462) within tir
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was made by overlapping-PCR. C. rodentium genomic DNA was used to amplify tir
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base pairs 664-1164 using primer pair [Tir-upTM1-Fw] and [Tir-down-TM2-Rv]. The
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primer pair [Tir-down-TM2-Y471-Fw] and [Tir-EcoRI-Rv] was used to amplify tir
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base pairs 1383-1644 from pICC433 (encoding Tir_Y471) and pICC438 (encoding
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Tir_Y471A) templates. The two PCR fragments ([664-1164] and [1383-1644/Y471])
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and ([664-1164] and [1383-1644/Y471A]) were PCR-overlapped, EcoRI digested and
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ligated to tir-cesT intergenic region (PCR-amplified as previously described in Crepin
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et al 2010, using primers [EcoRI-(tir-cesT)-Fw/NcesT-Rv]. The ligated PCR product
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was then re-amplified using primers [Tir-upTM1-Fw] and [NcesT-Rv] and cloned
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into pGEMT vector. The constructs were digested with EcoRI and the aphT gene
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cloned into the tir-cesT intergenic region to confer kanamycin resistance, resulting in
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plasmids pICC1844 and pICC1845, respectively (Table 1). All plasmid derivatives
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were checked by DNA sequencing.
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The various deletions 478-547_Y451/Y471 and 478-547_Y451A/Y471A; 382-
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462_Y471 and 382-462_Y471A were PCR amplified from pICC1842 and
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pICC1843, using primers [NcesT-Rv /Tir-Up-YY-Fw] and from pICC1844 and
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pICC1845, using primers [NcesT-Rv/Tir-upTM1-Fw], respectively. The PCR
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products were electroporated into wild type C. rodentium expressing the lambda red
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recombinase from pKD46 plasmid (36). The presence of the mutation was confirmed
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by PCR and DNA sequencing amongst the kanamycin resistant clones.
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Cell culture
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Swiss 3T3 cell line was grown, maintained and infected with the different C.
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rodentium strains, at a MOI of 50, as described (14). Cells were washed 6 h post
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infection with phosphate-buffered saline (PBS), fixed for 15 min in 4%
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paraformaldehyde, permeabilized with 0.1% Triton for 4 min. Phalloidin-Tetramethyl
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Rhodamine Iso-Thiocyanate (TRITC) (Sigma) was used to stain F-actin, while
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bacterial DNA was counterstained with Hoechst 33342. Tir was stained using rabbit
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anti-Tir EHEC (37, 38), recognizing the N-terminal domain, and carbocyanine-2-
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conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Europe) secondary
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antibody. Images were acquired using an AxioCam MRm monochrome camera and
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processed using AxioVision (Carl Zeiss MicroImaging GmbH, Germany).
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Tir translocation by C. rodentium was qualitatively assessed by calculating the ratio
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between the number of total visible bacterial nuclei and the number of bacteria which
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show Tir staining concentrated in a straight line at the interface between the
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bacterium. A minimum of 100 bacteria were counted by experiment and the
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experiment was performed twice.
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Oral infection of mice
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Pathogen-free female 18–20g C57Bl/6 mice were purchased from Charles River. All
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animals were housed in individually HEPA-filtered cages with sterile bedding and
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free access to sterilized food and water. All animal experiments were performed in
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accordance with the Animals Scientific Procedures (Act 1986) and were approved by
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the local Ethical Review Committee. Infections were performed twice using four to
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eight mice per group. Mice inoculated with mock mutant and nonsense mutant strains
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were included in every experiment. Mice inoculated with wild-type strain and
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uninfected mice were included in parallel with mutant strains.
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Mice were inoculated by oral gavage with 200μl of overnight LB-grown C. rodentium
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suspension in PBS (≈ 5 × 109 cfu). The number of viable bacteria used as inoculum
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was determined by retrospective plating onto LB agar containing antibiotics. Stool
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samples were recovered aseptically at various time points after inoculation and the
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number of viable bacteria per gram of stool was determined by plating onto LB agar
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(39). At day 7 and 14 post inoculum, the mice were culled and the colonic tissues
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were collected for further analyses.
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Sample collection and colonic crypt hyperplasia measurement
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Segments of the terminal colon (0.5cm) of each mouse were collected, flushed and
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fixed in 10% neutral buffered formalin. Formalin fixed tissues were then processed,
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paraffin-embedded, sectioned at 5μm and stained with haematoxylin and eosin (H&E)
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using standard techniques. H&E stained tissues were evaluated for colonic crypt
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hyperplasia microscopically without knowledge of the treatment condition used in the
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study and the length of at least 20 well-oriented crypts from each section from all of
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the mice per treatment group (n=4-6) were evaluated. H&E stained tissues were
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imaged with an Axio Lab.A1 microscope (Carl Zeiss MicroImaging GmbH,
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Germany), images were acquired using an Axio Cam ERc5s colour camera, and
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computer-processed using AxioVision (Carl Zeiss MicroImaging GmbH, Germany).
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Additional colonic segments were embedded in optimal cutting temperature (OCT)
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medium (Raymond A Lamb Limited, UK) and frozen in dry-ice/ethanol slush for
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further cryo-sectioning. Cryo-sections were then fixed in 3%-paraformaldehyde
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(PFA) in PBS as previously described (38, 40) and immuno-stained using primary
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antibodies at a 1/50 dilution, chicken anti-intimin (14), Ly-6G (RB6-8C5, Santa Cruz)
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and E-Cadherin (CD324, BD Biosciences). Secondary antibodies were used at 1/100
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dilution, Cy3 (103-165-175, Jackson Immunoresearch), Alexa 488 (712-546-150,
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Jackson Immunoresearch) and Cy5 (715-175-150, Jackson Immunoresearch),
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respectively. Images were acquired using an AxioCam MRm monochrome camera
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and processed using AxioVision (Carl Zeiss MicroImaging GmbH, Germany).
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Extraction of enterocytes and immunostaining
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Four cm segment of the terminal colon was cut longitudinally and placed in 4ml
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enterocyte dissociation buffer (1x Hanks’ balanced salt solution without Mg & Ca
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containing 10mM HEPES, 1mM EDTA and 5μl/ml 2-mercaptoethanol) and
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incubated at 37C, shaking, for 40 min. Left over tissue was removed by
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centrifugation (1900g for 5 min) before the samples for each group were pooled
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together and fixed with 1% formaldehyde. Fixed enterocytes (CD45-CD326+) were
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analyzed for purity by flow cytometry using leukocyte marker CD45 and epithelial
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cell marker CD326 (EpCAM). For immunofluorescence staining, fixed enterocytes
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were permeabilized with 0.1% Triton and blocked with 1% bovine serum albumin in
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PBS. Enterocytes were stained with polyclonal rabbit anti-Tir EHEC (GB1320,
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SK1786) for 20 mins followed by 30 min incubation with secondary donkey anti-
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rabbit IgG (H+L) Alexa488, phalloidin-TRITC (Sigma, P1951) was used for actin
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and DAPI (Invitrogen, D3571) to visualize the nucleus. Tir staining was visualized
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with an Axio Imager M1 microscope (Carl Zeiss MicroImaging GmbH, Germany),
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images were acquired using an AxioCam MRm monochrome camera, and computer-
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processed using AxioVision (Carl Zeiss MicroImaging GmbH, Germany).
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Isolation of mRNA and Q-RT-PCR
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mRNA of enterocytes was isolated using an RNeasy minikit according to the
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manufacturer’s instructions (Qiagen). Samples were treated with RQ1 DNase-1
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(Promega) at 37°C for 10 min, followed by 15 min at 72°C. Reverse transcription
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(RT)-PCR was carried out by adding RT M-MLV (Promega M170B), RT buffer
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(Promega), random primers (Promega), RNasin (Promega), dNTP (10mM) and
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RNase free water to the DNase treated RNA extract, incubated at 37°C for 1 hour
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followed by 10 min at 72°C and cooled samples were stored at -20°C. CXCL1 (KC)
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and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were amplified
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with primer pairs mCXCL1-F / mCXCL1-R and mGAPDH-F / mGAPDH-R (Table
10
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1), by Q-RT-PCR using the 7300 Applied Biosystems instrument under standard
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cycle conditions for Fast SYBR Green master mix. Changes in gene expression levels
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were analyzed relative to the control levels (PBS samples), with GAPDH as a
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standard, using the ΔΔCT method.
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Sample collection for flow cytometry
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Four cm segment of the terminal colon was cut, opened longitudinally and rinsed in
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sterile PBS and placed in 4ml of RPMI-1640 supplemented with 10% fetal bovine
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serum
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10104159001) and liberase (Roche, 540112001) in a C-Mac tube (Miltenyi Biotec)
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followed by tissue dissociation using gentleMACS dissociator (Miltenyi Biotec). The
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tissue was homogenized using ‘intestine’ setting followed by incubation at 37°C, 5%
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CO2 for 30 min in a shaking incubator and a final dissociation step was performed
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using ‘Lung 2’ setting. The digested preparation was disrupted to a single cell
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suspension by passage through a 70μm sieve (BD labware/falcon, USA Cat. No:
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352350) and suspended in RPMI-1640 supplemented with 10% FBS and P/S at 0.5-
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1x106 cells/ml.
(FBS),
penicillin/streptomycin
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(P/S),
GlutaMAX,
DNase
(Roche,
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Extracellular antigen analysis
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Cells were stained for surface markers as indicated in PBS containing 1% bovine
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serum albumin with 0.5% sodium azide (PBA) for 30 min at 4°C and fixed with IC
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fixation buffer (eBioscience). Prior to primary antibody staining, all cells were
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blocked for Fc receptors (FcR) using mouse FcR blocking reagent (Miltenyi biotec)
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for 10 min at 4°C. Antibodies were purchased from BD Pharmingen or eBioscience.
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Data acquired on a BD Fortessa III and 20,000 lymphocytes or myeloid events were
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analyzed with the FlowJO (Tress star) analysis program. Data is shown as a
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percentage of myeloid or lymphocyte gates. Myeloid and lymphocyte gates are
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determined by their position on the forward and side scatter plots generated by the
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cytometer. Fluorescence minus one (FMO) control was included for each fluorescent
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marker, the expression of a particular marker was calculated by subtracting FMO
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fluorescence values from fluorescent antibody levels.
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Total live cells were assessed by trypan blue exclusion. Forward and side scatter gates
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on Flowjo software were used to gate myeloid cells, the percentage of this gate was
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used to determine total number of myeloid cells. The myeloid cells gate was further
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analysed to finalise the percentage of neutrophils (CD11b+Ly6G+) and establish the
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total number of neutrophils in each mouse colon.
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Mouse intestinal in vitro organ cultures
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Mouse intestinal in vitro organ culture (mIVOC) model was used to assess A/E lesion
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formation caused by C. rodentium expressing Tir Y451A/Y471A/478-547 as
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described by Girard et al 2009 (41). Briefly, segments from the terminal colon were
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inoculated with 50l of the appropriate overnight bacterial culture, corresponding to
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approximately 107 colony forming units (cfu), and incubated at 37°C in 5% CO2
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atmosphere on a see-saw rocker (18 cycle min-1) for 8 h. Explants were gently rinsed
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with PBS and fixed in 2.5% glutaraldehyde for electron microscopy analysis.
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Electron microscopy
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Additional explants/tissue cultured cell samples were processed for electron
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microscopy, as previously described (38). Samples for scanning electron microscopy
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(SEM) were examined at an accelerating voltage of 25 kV using a JEOL JSM-5300
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scanning electron microscope (JEOL (UK) Ltd., Herts, United Kingdom). Samples
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for transmission electron microscopy (TEM) were observed using a Phillips 201
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transmission electron microscope at an accelerating voltage of 60kV (Philips, United
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Kingdom).
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Statistical analysis
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Results are presented as a line plot (colonization) with the mean and its standard
286
deviation. The non-parametric Mann–Whitney test and the non-parametric Kruskal–
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Wallis test with Bonferroni's corrected a posteriori comparisons were used to conduct
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pairwise and global statistical analysis, respectively, using commercially available
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GraphPad InStat v3.06 software (GraphPad Software, San Diego, CA, USA). Mann-
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Whitney compared to PBS controls (or as indicated in the figure) was used for data
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obtained by flow cytometry using GraphPad Prism software. A P = 0.05 was
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considered significant.
14
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Results
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Construction of the 3’ tir chromosomal deletion mutants
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Previously, using a method that allows expression of tir mutants from the C.
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rodentium chromosome, we have introduced point mutations which have shown that
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A/E lesion formation in vivo is independent of Tir residues Y451 and Y471 (14). In
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this study, we used this technique to introduce deletions at the 3’ end of tir (Fig. 1).
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While inserting a kanamycin cassette coupled to the mutated tir into the tir-cesT
300
intergenic region, we deleted Tir residues 382-462 (Tir382-462), removing the 80
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amino acids downstream of the distal TM helix and Tir residues 478-547 (Tir478-
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547), removing the entire segment downstream of Y471 phosphorylation site (Fig. 1).
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Tir382-462 was made in the context of either Y471 or Y471A (Tir382-462_Y471
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and Tir382-462_Y471A), while Tir478-547 was made in the context of either
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Y451/Y471
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547_Y451A/Y471A). As a positive control we used a mock mutant tir-cesT (TirC-
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ctrl) in which the kanamycin cassettes was introduced into the intergenic region
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which did not affect the tir coding sequence and as a negative control, a nonsense
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codon introduced at Tir position 33 (Tir1−33stop) (14). Growth curves in minimal
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and rich media confirmed that the mutants and parental wild-type strains had identical
311
growth rates (data not shown).
or
Y451A/Y471A
(Tir478-547
_Y451/Y471
and
Tir478-
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Testing the carboxy terminal Tir deletions during infection of cultured cells
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We characterized the behavior of the Tir derivatives in vitro, following infection of
315
Swiss 3T3 fibroblast cells with the C. rodentium mutants as described before (14).
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This revealed that, as expected, C. rodentium expressing either Tir382-462_Y471A
15
317
or Tir478-547_Y451A/Y471A failed to induce actin polymerization (data not
318
shown). In contrast, infection of Swiss 3T3 cells with C. rodentium expressing
319
Tir382-462_Y471 or Tir478-547_Y451/Y471 revealed robust actin polymerization
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under all the adherent bacteria that show Tir translocation (Fig. 2A). Importantly,
321
while Tir382-462 was translocated as efficiently as the wild type control Tir,
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Tir478-547 translocated in low efficiency, with Tir staining seen within 50% of the
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adherent bacteria (Fig. 2B). Interestingly, C. rodentium expressing Tir478-
324
547_Y451/Y471 produced longer pedestals. These results suggest that Tir residues
325
382-462 are dispensable for Tir translocation and actin polymerization, while the
326
carboxy terminus of Tir plays a role in translocation and hence indirectly in the
327
efficiency (both in terms of frequency and length) of actin pedestals formed in vitro.
328
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Testing the carboxy terminal Tir deletions during mouse infection
330
We next investigated the impact of the C-terminal Tir deletions on colonization of C.
331
rodentium in vivo by enumerating colony-forming units per gram of stools (cfu g-1)
332
collected daily following oral inoculation of C57BL/6 mice for 8 days. This has
333
shown that C. rodentium expressing either Tir382-462_Y471 or Tir382-
334
462_Y471A colonized the mice similarly to the control strain expressing TirC-ctrl
335
(Fig. 3A) (variations between groups seen on day 2 post infection are common during
336
C. rodentium infection and has not biological relevance; the differences seen on day 3
337
post infection are not significant). Transmission electron microscopy (TEM) revealed
338
typical A/E lesions in colons infected with C. rodentium expressing either Tir382-
339
462_Y471 or Tir382-462_Y471A (Fig. 3B). In contrast, C. rodentium expressing
340
Tir1−33stop was rapidly cleared and failed to initiate an infection, reaching
16
341
background level as soon as day 3 post infection (Fig. 3A). These results show that
342
Tir segment 382-462 is dispensable for colonization and that C. rodentium expressing
343
Tir382-462_Y471 or Tir382-462_Y471A are capable of forming A/E lesions in
344
vivo.
345
We then tested the phenotype of C. rodentium expressing Tir478-547. Consistent
346
with the in vitro data, this mutant exhibited an intermediate phenotype, reaching a
347
colonization level of 106/g of stool on day 3 post infection, which persisted in this
348
level until day 8 (Fig. 3A). The level of colonization was 100 fold lower than that
349
seen in mice infected with C. rodentium expressing TirC-ctrl or Tir382-462 but 104
350
fold higher than mice infected with Tir1−33stop. We observed no difference in
351
colonization between mice infected with Tir478-547_Y451/Y471 or Tir478-
352
547_Y451A/Y471A (Fig. 3A). However, as colonization was below the detection
353
level of TEM, we performed a mouse IVOC infection using C. rodentium expressing
354
Tir78-547_Y451A-Y471A. Examination of the samples by SEM revealed A/E
355
lesions, similar to those formed by the wild type strains, suggesting that Tir segment
356
478-547 is dispensable for A/E lesion formation on mucosal surfaces but does play a
357
role in colonization (Fig. 3B), probably due to its role in Tir translocation.
358
359
Tir-induces actin polymerization on enterocytes in vivo
360
As the results thus far have shown that C. rodentium expressing Tir deletions
361
(residues 382-462 and 478-547) and substitutions (Y451 and Y471) was able to form
362
A/E lesions, we tested whether Tir-induced actin polymerization on enterocytes in
363
vivo. For this, mice were infected with wild type C. rodentium or C. rodentium
364
expressing Tir_Y451A/Y471A (Fig. 1) and enterocytes were isolated at the peak of
17
365
colonization at day 7 post infection. Enterocytes isolated from naïve mice as a control
366
exhibited a typical ‘crown’ staining pattern of the BB microvilli (Fig. 4). Enterocytes
367
isolated from infected mice showed good level of C. rodentium adhesion, with
368
multiple bacteria on individual enterocytes. Tir was detected underneath both attached
369
wild type C. rodentium and C. rodentium expressing Tir_Y451A/Y471A (Fig. 4).
370
However, while intense actin staining was seen at the site of wild type C. rodentium
371
infection, enterocytes infected with C. rodentium expressing Tir_Y451A/Y471A
372
exhibited mainly weak actin polymerization at the site of bacterial attachment. This
373
suggests that Tir induces actin polymerization on enterocyte in a process involving
374
the tyrosine residues.
375
376
Recruitment of immune cells to the C. rodentium infection site
377
As actin remodeling during infection can trigger immune responses (42), we next
378
investigated if Tir Y451 and Y471 modulate host immune responses. For this, groups
379
of 4-6 mice were infected with wild type and mutant C. rodentium and recruitment of
380
immune cells were analyzed by flow cytometry of homogenized colons at days 7
381
(peak of colonization) and day 14 (peak of pathology) post infection. All the tested
382
strains were shed at equivalent levels (Fig. 5A). The flow cytometry analysis has
383
shown that in comparison to the PBS mock-infected control mice (baseline readout),
384
infection with either the wild type or mutant C. rodentium resulted in equivalent
385
recruitment of macrophages, CD4+ T cells, and B cells on day 7 (data not shown) and
386
14 (Fig. 5B-D) post infection. In contrast, significantly less neutrophils were recruited
387
to the colon following infection with C. rodentium expressing Tir_Y451A/Y471A
388
compared with the positive control mice infected with C. rodentium expressing wild
389
type Tir, or mice infected with the single Tir tyrosine mutants Y451A or Y471A (Fig.
18
390
5E-F). Similarly, although shed at equivalent levels (Fig. 5A), neutrophils recruitment
391
was observed following infection with C. rodentium expressing Tir382-462_Y471
392
but not after infection with C. rodentium expressing Tir382-462_Y471A (Fig. 5G-
393
H).
394
As C. rodentium and Tir mainly interact with enterocytes, we next determined if the
395
Tir tyrosine residues play a role in expression of pro-inflammatory chemokines. For
396
this we isolated colonic enterocytes from mice infected with the different C.
397
rodentium strains. The purity of the enterocytes preparation was confirmed by flow
398
cytometry analysis following staining with the leukocyte marker CD45 and epithelial
399
cell marker CD326 (EpCAM), revealing low level of contamination (Fig. 6A). Using
400
the purified enterocytes in Q-RT-PCR for the chemokines CXCL1 (KC) and CXCL2
401
(MIP2-alpha) revealed reduced expression in mice infected with C. rodentium
402
expressing TirY451A-Y471A (Fig. 6B). The attenuated inflammatory responses
403
triggered by C. rodentium expressing Tir_Y451A/Y471A were mirrored by a
404
significantly reduced colonic hyperplasia (Fig. 7A and B) as well as neutrophil
405
staining with Ly-6G antibodies (Fig. 7C). These results reveal a novel in vivo role for
406
the Tir tyrosine residues TirY451 and Y471, which are also implicated in actin
407
polymerization during C. rodentium infection (Fig. 4).
19
408
Discussion
409
In this study we found that C. rodentium expressing Tir382-462 colonized the
410
mouse gastrointestinal tract and produced A/E lesion whether in the context of Y471
411
or Y471A. This result shows that amino acids 382-462 are dispensable for Tir
412
activity. We also tested a chromosomal deletion of Tir residues 478-547. This has
413
shown that C. rodentium expressing either Tir478-547_Y451/Y471 or Tir478-
414
547_Y451A/Y471A behaved similarly, showing an intermediate colonization level
415
between the wild type and the Tir1-33stop strains. The fact that C. rodentium
416
expressing either Tir478-547_Y451/Y471 or Tir478-547_Y451A/Y471A showed
417
104 fold higher colonization level than mice infected with Tir1-33stop suggests that
418
Tir is at least partially active. Due to the low level of colonization we were unable to
419
determine if these strains could form A/E lesions in vivo using TEM but we could
420
confirm A/E lesions by SEM. A previous report demonstrated that a 6 amino acid
421
sequence (TYARLA) at position 519-524 within the carboxy-terminal region was
422
required for efficient secretion and translocation, but not for stability, of Tir-EHEC
423
(43). The carboxy terminus of Tir C. rodentium contain an equivalent 6 amino acid
424
sequence (TYALLA), which is consistent with the low translocation efficiency, seen
425
by immuno-fluorescence staining following infection of Swiss 3T3 cells with C.
426
rodentium expressing Tir478-547_Y451/471, and the intermediate in vivo phenotype
427
of this strain.
428
Immunostaining of enterocytes isolated from naïve mice revealed good preservation
429
of the BB microvilli. In contrast, individual enterocytes isolated from infected mice
430
were covered with adherent C. rodentium and exhibited effaced BB microvilli. Tir
431
was detected at equivalent intensity at the site of bacterial attachment, whether in the
432
context of wild type Tir or Tir_Y451A/Y471A. Importantly, actin staining was
20
433
considerably brighter under attached C. rodentium expressing wild type Tir. This
434
could potentially explain the competitive advantage of wild type C. rodentium over C.
435
rodentium expressing Tir_Y451A/Y471A during mixed infection (14). However, it is
436
important to note that while, as expected, no Nck was recruited to C. rodentium
437
expressing Tir_Y451A/Y471A in vivo (14) or to human intestinal biopsies infected
438
with EPEC expressing the Tir mutant (31), N-WASP was detected underneath the
439
attached mutant strains, which could explain the faint actin polymerization seen in the
440
enterocytes
441
Tir_Y451A/Y471A. Taken together these data suggest the existence of as yet
442
undetermined Tir actin polymerization pathway in mucosal surfaces.
443
If the Tir tyrosine residues do not play a role in A/E lesion formation, the question
444
remains, what function do they have during infection of mucosal surfaces? As EPEC
445
and C. rodentium interact intermediately with enterocytes, we hypothesized that Tir-
446
induced actin polymerization might contribute to signaling to the underlying immune
447
system. To test this, we infected mice with C. rodentium expressing wild type Tir and
448
Tir tyrosine mutants and compared recruitment of immune cells in homogenized
449
colons. This revealed no difference in recruitment of macrophages, T cells or B cells
450
at either 7 or 14 days post infection. In contrast, significantly reduced level of
451
recruited neutrophils was seen at day 14 following infection with C. rodentium
452
expressing Tir_Y451A/Y471A or Tir382-462_Y471A, compared to infection with
453
C. rodentium expressing wild type Tir or Tir382-462_Y471. Moreover, mice
454
infected with C. rodentium expressing Tir_Y451A/Y471A presented significantly
455
reduced levels of colonic hyperplasia. Importantly, infection with C. rodentium
456
expressing single tyrosine Tir mutant (Y451A or Y471A), resulted in neutrophil
457
recruitment equivalent to that seen following infection with wild type C. rodentium.
isolated
from
mice
infected
21
with
C.
rodentium
expressing
458
This phenotype was mirrored following testing for CXCL1 and CXCL2 expression by
459
Q-RT-PCR on enterocytes purified from C. rodentium infected mice. Similarly, (44)
460
reported that following EPEC infection both Tir Y454 and Y474 are needed for
461
efficient nuclear translocation of the transcription factor serum response factor (SRF)
462
co-factor MAL and transcription of SRF target genes. CXCL1 and CXCL2 signal via
463
CXCR2 to activate neutrophils and subsequently promote mucosal influx of
464
neutrophils (45). Importantly, although neutrophils contribute to host defense against
465
infection (10), wild type C. rodentium and C. rodentium expressing mutant Tir
466
colonized at equivalent levels. These results suggest that while Tir contains redundant
467
mechanisms leading to neutrophil recruitment, each relying of one of the two
468
tyrosines, the host immune response can compensate for the lack of neutrophils late
469
during infection and clear the pathogen.
470
Tir EPEC and C. rodentium have further two distal tyrosines (Y480_Y508 and
471
Y483_Y511, respectively), which comprise an immunoreceptor tyrosine-based
472
inhibition motif (ITIM). Smith et al. (46) reported that following EPEC infection Tir
473
residues Y483 and Y511 recruit the host inositol phosphatase SHIP2. Moreover, the
474
pedestals formed by EPEC expressing Tir Y483F_Y511F were significantly longer
475
than those formed by EPEC expressing wild type Tir, which is consistent with the
476
longer pedestals we observed following infection with C. rodentium expressing
477
Tir478-547_Y451/Y471, which lacks residues Y480_Y508. Recently Yan et al have
478
shown that infection with EPEC expressing Tir Y483F_Y511F resulted in elevated
479
levels of IL6 and TNF mRNA in splenic cells and enhanced bacterial clearance (47).
480
They have shown that phosphorylation of the Tir ITIM leads to the recruitment of
481
both SHP1 and SHP2 and inhibition of TRAF6 autoubiquitination, which helps the
482
bacteria to suppress and evade the host innate immune response (47, 48). This feature
22
483
of Tir could provide an alternative explanation for why C. rodentium expressing Tir
484
Tir478-547_Y451/Y471 or Tir478-547_Y451A/Y471A did not colonize the colon
485
at a wild type level.
486
Subversion of the actin cytoskeleton by bacterial virulence factors has been shown to
487
be an important mediator of immune signaling. For example, the Salmonella Rho
488
GTPase GEF SopE has been shown to activate the pattern recognition receptors
489
(PRRs) NOD1 (49), while NOD2 is regulated by Rac1 (50). NOD1 and NOD2, could
490
be found at the plasma membrane in association with F-actin, which is needed for
491
downstream activation of NF-kB signaling (42, 51). Recently, Bielig et al have shown
492
that the actin depolymerization factors (ADF)/cofilin phosphatase SSH1 is an
493
essential component of the NOD1 pathway, which plays a role in activation of NF-kB
494
and cell responses to Shigella infection (52). Indeed, depletion of SSH1 mRNA
495
resulted in reduced production of IL8 and IL6 following infection of HeLa cells with
496
S. flexneri strain M90T. Consistently, our data show that sensing Tir-induced actin
497
remodeling triggers host responses to C. rodentium infection.
498
C. rodentium translocates multiple effectors that contribute to coordinated
499
cytoskeleton remodeling (including Map that activates Cdc42 and Rac1 (53), EspM
500
that activates RhoA (54), EspT that activates Rac1 (55) and EspJ that inhibits Src
501
kinases (56) which play a role in Tir tyrosine phosphorylation) and subversion of
502
innate immune responses, including NF-kB (e.g. NleC, NleD, NleE, NleB and NleF
503
(13, 57-62)). Importantly, the difference in neutrophil recruitment seen at day 14
504
between wild type C. rodentium and C. rodentium expressing Tir_Y451A/Y471A is
505
at the time when both infections are close to being cleared. Future studies will aim at
506
unraveling the mechanism by which Tir induces expression of CXCL1 and CXCL2 in
507
enterocytes late during infection in the broader context of the other type III secreted
23
508
effectors that modulate inflammatory responses.
509
24
510
Acknowledgements
511
This study was supported by a grant from the Wellcome trust and the BBSRC.
512
25
513
References
514
515
1.
516
517
Chen HD, Frankel G. 2005. Enteropathogenic Escherichia coli: unravelling
pathogenesis. FEMS Microbiol Rev 29:83-98.
2.
Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH,
518
Panchalingam S, Wu Y, Sow SO, Sur D, Breiman RF, Faruque AS, Zaidi
519
AK, Saha D, Alonso PL, Tamboura B, Sanogo D, Onwuchekwa U, Manna
520
B, Ramamurthy T, Kanungo S, Ochieng JB, Omore R, Oundo JO,
521
Hossain A, Das SK, Ahmed S, Qureshi S, Quadri F, Adegbola RA,
522
Antonio M, Hossain MJ, Akinsola A, Mandomando I, Nhampossa T,
523
Acacio S, Biswas K, O'Reilly CE, Mintz ED, Berkeley LY, Muhsen K,
524
Sommerfelt H, Robins-Browne RM, Levine MM. 2013. Burden and
525
aetiology of diarrhoeal disease in infants and young children in developing
526
countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-
527
control study. Lancet 382:209-222.
528
3.
Collins JW, Keeney KM, Crepin VF, Rathinam VA, Fitzgerald KA,
529
Finlay BB, Frankel G. 2014. Citrobacter rodentium: infection, inflammation
530
and the microbiota. Nat Rev Microbiol 12:612-623.
531
4.
532
533
Mundy R, MacDonald TT, Dougan G, Frankel G, Wiles S. 2005.
Citrobacter rodentium of mice and man. Cell Microbiol 7:1697-1706.
5.
Higgins LM, Frankel G, Douce G, Dougan G, MacDonald TT. 1999.
534
Citrobacter rodentium infection in mice elicits a mucosal Th1 cytokine
535
response and lesions similar to those in murine inflammatory bowel disease.
536
Infect Immun 67:3031-3039.
26
537
6.
Shiomi H, Masuda A, Nishiumi S, Nishida M, Takagawa T, Shiomi Y,
538
Kutsumi H, Blumberg RS, Azuma T, Yoshida M. 2010. Gamma interferon
539
produced by antigen-specific CD4+ T cells regulates the mucosal immune
540
responses to Citrobacter rodentium infection. Infect Immun 78:2653-2666.
541
7.
Geddes K, Rubino SJ, Magalhaes JG, Streutker C, Le Bourhis L, Cho
542
JH, Robertson SJ, Kim CJ, Kaul R, Philpott DJ, Girardin SE. 2011.
543
Identification of an innate T helper type 17 response to intestinal bacterial
544
pathogens. Nat Med 17:837-844.
545
8.
546
547
Cua DJ, Tato CM. 2010. Innate IL-17-producing cells: the sentinels of the
immune system. Nat Rev Immunol 10:479-489.
9.
Zheng Y, Valdez PA, Danilenko DM, Hu Y, Sa SM, Gong Q, Abbas AR,
548
Modrusan Z, Ghilardi N, de Sauvage FJ, Ouyang W. 2008. Interleukin-22
549
mediates early host defense against attaching and effacing bacterial pathogens.
550
Nature Medicine 14:282-289.
551
10.
Spehlmann ME, Dann SM, Hruz P, Hanson E, McCole DF, Eckmann L.
552
2009. CXCR2-dependent mucosal neutrophil influx protects against colitis-
553
associated diarrhea caused by an attaching/effacing lesion-forming bacterial
554
pathogen. J Immunol 183:3332-3343.
555
11.
Manta C, Heupel E, Radulovic K, Rossini V, Garbi N, Riedel CU, Niess
556
JH. 2013. CX(3)CR1(+) macrophages support IL-22 production by innate
557
lymphoid cells during infection with Citrobacter rodentium. Mucosal
558
Immunol 6:177-188.
559
12.
Knutton S, Lloyd DR, McNeish AS. 1987. Adhesion of enteropathogenic
560
Escherichia coli to human intestinal enterocytes and cultured human intestinal
561
mucosa. Infect Immun 55:69-77.
27
562
13.
Wong AR, Pearson JS, Bright MD, Munera D, Robinson KS, Lee SF,
563
Frankel G, Hartland EL. 2011. Enteropathogenic and enterohaemorrhagic
564
Escherichia coli: even more subversive elements. Mol Microbiol 80:1420-
565
1438.
566
14.
Crepin VF, Girard F, Schuller S, Phillips AD, Mousnier A, Frankel G.
567
2010. Dissecting the role of the Tir:Nck and Tir:IRTKS/IRSp53 signalling
568
pathways in vivo. Mol Microbiol 75:308-323.
569
15.
McDaniel TK, Jarvis KG, Donnenberg MS, Kaper JB. 1995. A genetic
570
locus of enterocyte effacement conserved among diverse enterobacterial
571
pathogens. Proc Natl Acad Sci USA 92:1664-1668.
572
16.
Jarvis KG, Girón JA, Jerse AE, McDaniel TK, Donnenberg MS, Kaper
573
JB. 1995. Enteropathogenic Escherichia coli contains a putative type III
574
secretion system necessary for the export of proteins involved in attaching-
575
effacing lesions formation. Proc Natl Acad Sci USA 92:7996-8000.
576
17.
Jerse AE, Yu J, Tall BD, Kaper JB. 1990. A genetic locus of
577
enteropathogenic Escherichia coli necessary for the production of attaching
578
and effacing lesions on tissue culture cells. Proc Natl Acad Sci USA 87:7839-
579
7843.
580
18.
Garmendia J, Frankel G, Crepin VF. 2005. Enteropathogenic and
581
enterohemorrhagic Escherichia coli infections: translocation, translocation,
582
translocation. Infect Immun 73:2573-2585.
583
19.
Hartland EL, Batchelor M, Delahay RM, Hale C, Matthews S, Dougan G,
584
Knutton S, Connerton I, Frankel G. 1999. Binding of intimin from
585
enteropathogenic Escherichia coli to Tir and to host cells. Mol Microbiol
586
32:151-158.
28
587
20.
Kenny B. 1999. Phosphorylation of tyrosine 474 of the enteropathogenic
588
Escherichia coli (EPEC) Tir receptor molecule is essential for actin nucleating
589
activity and is preceded by additional host modifications. Mol Microbiol
590
31:1229-1241.
591
21.
Kenny B, DeVinney R, Stein M, Reinscheid DJ, Frey EA, Finlay BB.
592
1997. Enteropathogenic E. coli (EPEC) transfers its receptor for intimate
593
adherence into mammalian cells. Cell 91:511-520.
594
22.
Swimm A, Bommarius B, Li Y, Cheng D, Reeves P, Sherman M, Veach D,
595
Bornmann W, Kalman D. 2004. Enteropathogenic Escherichia coli use
596
redundant tyrosine kinases to form actin pedestals. Mol Biol Cell 15:3520-
597
3529.
598
23.
Swimm A, Bommarius B, Reeves P, Sherman M, Kalman D. 2004.
599
Complex kinase requirements for EPEC pedestal formation. Nat Cell Biol
600
6:795; author reply 795-796.
601
24.
Caron E, Crepin VF, Simpson N, Knutton S, Garmendia J, Frankel G.
602
2006. Subversion of actin dynamics by EPEC and EHEC. Curr Opin
603
Microbiol 9:40-45.
604
25.
Campellone KG, Leong JM. 2005. Nck-independent actin assembly is
605
mediated
606
Escherichia coli Tir. Mol Microbiol 56:416-432.
607
26.
Brady
by
MJ,
two
phosphorylated
Campellone
KG,
tyrosines
Ghildiyal
within
M,
enteropathogenic
Leong
JM.
2007.
608
Enterohaemorrhagic and enteropathogenic Escherichia coli Tir proteins
609
trigger a common Nck-independent actin assembly pathway. Cell Microbiol
610
9:2242-2253.
29
611
27.
Vingadassalom D, Kazlauskas A, Skehan B, Cheng HC, Magoun L,
612
Robbins D, Rosen MK, Saksela K, Leong JM. 2009. Insulin receptor
613
tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors
614
Tir and EspF(U) during pedestal formation. Proc Natl Acad Sci U S A
615
106:6754-6759.
616
28.
Weiss SM, Ladwein M, Schmidt D, Ehinger J, Lommel S, Stading K,
617
Beutling U, Disanza A, Frank R, Jansch L, Scita G, Gunzer F, Rottner K,
618
Stradal TE. 2009. IRSp53 links the enterohemorrhagic E. coli effectors Tir
619
and EspFU for actin pedestal formation. Cell Host Microbe 5:244-258.
620
29.
Whale AD, Hernandes RT, Ooka T, Beutin L, Schuller S, Garmendia J,
621
Crowther L, Vieira MA, Ogura Y, Krause G, Phillips AD, Gomes TA,
622
Hayashi T, Frankel G. 2007. TccP2-mediated subversion of actin dynamics
623
by EPEC 2 - a distinct evolutionary lineage of enteropathogenic Escherichia
624
coli. Microbiology 153:1743-1755.
625
30.
Deng W, Vallance BA, Li Y, Puente JL, Finlay BB. 2003. Citrobacter
626
rodentium translocated intimin receptor (Tir) is an essential virulence factor
627
needed for actin condensation, intestinal colonization and colonic hyperplasia
628
in mice. Mol Microbiol 48:95-115.
629
31.
Schuller S, Chong Y, Lewin J, Kenny B, Frankel G, Phillips AD. 2007. Tir
630
phosphorylation and Nck/N-WASP recruitment by enteropathogenic and
631
enterohaemorrhagic Escherichia coli during ex vivo colonization of human
632
intestinal mucosa is different to cell culture models. Cell Microbiol 9:1352-
633
1364.
634
635
32.
Ritchie JM, Brady MJ, Riley KN, Ho TD, Campellone KG, Herman IM,
Donohue-Rolfe A, Tzipori S, Waldor MK, Leong JM. 2008. EspFU, a type
30
636
III-translocated effector of actin assembly, fosters epithelial association and
637
late-stage intestinal colonization by E. coli O157:H7. Cell Microbiol 10:836-
638
847.
639
33.
Mallick EM, Garber JJ, Vanguri VK, Balasubramanian S, Blood T,
640
Clark S, Vingadassalom D, Louissaint C, McCormick B, Snapper SB,
641
Leong JM. 2014. The ability of an attaching and effacing pathogen to trigger
642
localized actin assembly contributes to virulence by promoting mucosal
643
attachment. Cell Microbiol 16:1405-1424.
644
34.
Mallick EM, McBee ME, Vanguri VK, Melton-Celsa AR, Schlieper K,
645
Karalius BJ, O'Brien AD, Butterton JR, Leong JM, Schauer DB. 2012. A
646
novel murine infection model for Shiga toxin-producing Escherichia coli. J
647
Clin Invest 122:4012-4024.
648
35.
Mundy R, Petrovska L, Smollett K, Simpson N, Wilson RK, Yu J, Tu X,
649
Rosenshine I, Clare S, Dougan G, Frankel G. 2004. Identification of a novel
650
Citrobacter rodentium type III secreted protein, EspI, and roles of this and
651
other secreted proteins in infection. Infect Immun 72:2288-2302.
652
36.
Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal
653
genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A
654
97:6640-6645.
655
37.
Batchelor M, Guignot J, Patel A, Cummings N, Cleary J, Knutton S,
656
Holden DW, Connerton I, Frankel G. 2004. Involvement of the
657
intermediate filament protein cytokeratin-18 in actin pedestal formation during
658
EPEC infection. EMBO Rep 5:104-110.
659
660
38.
Girard F, Dziva F, van Diemen P, Phillips AD, Stevens MP, Frankel G.
2007. Adherence of enterohemorrhagic Escherichia coli O157, O26, and
31
661
O111 strains to bovine intestinal explants ex vivo. Appl Environ Microbiol
662
73:3084-3090.
663
39.
Wiles S, Dougan G, Frankel G. 2005. Emergence of a 'hyperinfectious'
664
bacterial state after passage of Citrobacter rodentium through the host
665
gastrointestinal tract. Cell Microbiol 7:1163-1172.
666
40.
Girard F, Frankel G, Phillips AD, Cooley W, Weyer U, Dugdale AH,
667
Woodward MJ, La Ragione RM. 2008. Interaction of enterohemorrhagic
668
Escherichia coli O157:H7 with mouse intestinal mucosa. FEMS Microbiol
669
Lett 283:196-202.
670
41.
Girard F, Crepin VF, Frankel G. 2009. Modelling of infection by
671
enteropathogenic Escherichia coli strains in lineages 2 and 4 ex vivo and in
672
vivo by using Citrobacter rodentium expressing TccP. Infect Immun 77:1304-
673
1314.
674
42.
Kufer TA, Kremmer E, Adam AC, Philpott DJ, Sansonetti PJ. 2008. The
675
pattern-recognition molecule Nod1 is localized at the plasma membrane at
676
sites of bacterial interaction. Cell Microbiol 10:477-486.
677
43.
Allen-Vercoe E, Toh MC, Waddell B, Ho H, DeVinney R. 2005. A
678
carboxy-terminal domain of Tir from enterohemorrhagic Escherichia coli
679
O157:H7 (EHEC O157:H7) required for efficient type III secretion. FEMS
680
Microbiol Lett 243:355-364.
681
44.
Heath RJ, Leong JM, Visegrady B, Machesky LM, Xavier RJ. 2011.
682
Bacterial and host determinants of MAL activation upon EPEC infection: the
683
roles of Tir, ABRA, and FLRT3. PLoS Pathog 7:e1001332.
684
685
45.
Kolaczkowska E, Kubes P. 2013. Neutrophil recruitment and function in
health and inflammation. Nat Rev Immunol 13:159-175.
32
686
46.
Smith K, Humphreys D, Hume PJ, Koronakis V. 2010. Enteropathogenic
687
Escherichia coli recruits the cellular inositol phosphatase SHIP2 to regulate
688
actin-pedestal formation. Cell Host Microbe 7:13-24.
689
47.
Yan D, Wang X, Luo L, Cao X, Ge B. 2012. Inhibition of TLR signaling by
690
a bacterial protein containing immunoreceptor tyrosine-based inhibitory
691
motifs. Nat Immunol 13:1063-1071.
692
48.
Yan D, Quan H, Wang L, Liu F, Liu H, Chen J, Cao X, Ge B. 2013.
693
Enteropathogenic Escherichia coli Tir recruits cellular SHP-2 through ITIM
694
motifs to suppress host immune response. Cell Signal 25:1887-1894.
695
49.
Keestra AM, Winter MG, Auburger JJ, Frassle SP, Xavier MN, Winter
696
SE, Kim A, Poon V, Ravesloot MM, Waldenmaier JFT, Tsolis RM,
697
Eigenheer RA, Baumler AJ. 2013. Manipulation of small Rho GTPases is a
698
pathogen-induced process detected by NOD1. Nature 496:233-+.
699
50.
Legrand-Poels S, Kustermans G, Bex F, Kremmer E, Kufer TA, Piette J.
700
2007. Modulation of Nod2-dependent NF-kappaB signaling by the actin
701
cytoskeleton. J Cell Sci 120:1299-1310.
702
51.
703
704
Kufer TA. 2008. Signal transduction pathways used by NLR-type innate
immune receptors. Mol Biosyst 4:380-386.
52.
Bielig H, Lautz K, Braun PR, Menning M, Machuy N, Brugmann C,
705
Barisic S, Eisler SA, Andree M, Zurek B, Kashkar H, Sansonetti PJ,
706
Hausser A, Meyer TF, Kufer TA. 2014. The cofilin phosphatase slingshot
707
homolog 1 (SSH1) links NOD1 signaling to actin remodeling. PLoS Pathog
708
10:e1004351.
33
709
53.
Berger CN, Crepin VF, Jepson MA, Arbeloa A, Frankel G. 2009. The
710
mechanisms used by enteropathogenic Escherichia coli to control filopodia
711
dynamics. Cell Microbiol 11:309-322.
712
54.
Arbeloa A, Garnett J, Lillington J, Bulgin RR, Berger CN, Lea SM,
713
Matthews S, Frankel G. 2010. EspM2 is a RhoA guanine nucleotide
714
exchange factor. Cell Microbiol 12:654-664.
715
55.
Bulgin R, Arbeloa A, Goulding D, Dougan G, Crepin VF, Raymond B,
716
Frankel G. 2009. The T3SS effector EspT defines a new category of invasive
717
enteropathogenic E. coli (EPEC) which form intracellular actin pedestals.
718
PLoS Pathog 5:e1000683.
719
56.
Young JC, Clements A, Lang AE, Garnett JA, Munera D, Arbeloa A,
720
Pearson J, Hartland EL, Matthews SJ, Mousnier A, Barry DJ, Way M,
721
Schlosser A, Aktories K, Frankel G. 2014. The Escherichia coli effector
722
EspJ blocks Src kinase activity via amidation and ADP ribosylation. Nat
723
Commun 5:5887.
724
57.
Li S, Zhang L, Yao Q, Li L, Dong N, Rong J, Gao W, Ding X, Sun L,
725
Chen X, Chen S, Shao F. 2013. Pathogen blocks host death receptor
726
signalling by arginine GlcNAcylation of death domains. Nature 501:242-246.
727
58.
Pearson JS, Riedmaier P, Marches O, Frankel G, Hartland EL. 2011. A
728
type III effector protease NleC from enteropathogenic Escherichia coli targets
729
NF-kappaB for degradation. Mol Microbiol 80:219-230.
730
59.
Pearson JS, Giogha C, Ong SY, Kennedy CL, Kelly M, Robinson KS,
731
Lung TW, Mansell A, Riedmaier P, Oates CV, Zaid A, Muhlen S, Crepin
732
VF, Marches O, Ang CS, Williamson NA, O'Reilly LA, Bankovacki A,
733
Nachbur U, Infusini G, Webb AI, Silke J, Strasser A, Frankel G,
34
734
Hartland EL. 2013. A type III effector antagonizes death receptor signalling
735
during bacterial gut infection. Nature 501:247-251.
736
60.
Newton HJ, Pearson JS, Badea L, Kelly M, Lucas M, Holloway G,
737
Wagstaff KM, Dunstone MA, Sloan J, Whisstock JC, Kaper JB, Robins-
738
Browne RM, Jans DA, Frankel G, Phillips AD, Coulson BS, Hartland EL.
739
2010. The type III effectors NleE and NleB from enteropathogenic E. coli and
740
OspZ from Shigella block nuclear translocation of NF-kappaB p65. PLoS
741
Pathog 6:e1000898.
742
61.
Baruch K, Gur-Arie L, Nadler C, Koby S, Yerushalmi G, Ben-Neriah Y,
743
Yogev O, Shaulian E, Guttman C, Zarivach R, Rosenshine I. 2011.
744
Metalloprotease type III effectors that specifically cleave JNK and NF-
745
kappaB. EMBO J 30:221-231.
746
62.
Pallett MA, Berger CN, Pearson JS, Hartland EL, Frankel G. 2014. The
747
type III secretion effector NleF of enteropathogenic Escherichia coli activates
748
NF-kappaB early during infection. Infect Immun 82:4878-4888.
749
63.
Wiles S, Clare S, Harker J, Huett A, Young D, Dougan G, Frankel G.
750
2004. Organ specificity, colonization and clearance dynamics in vivo
751
following oral challenges with the murine pathogen Citrobacter rodentium.
752
Cell Microbiol 6:963-972.
753
64.
Galan JE, Ginocchio C, Costeas P. 1992. Molecular and functional
754
characterization of the Salmonella invasion gene invA: homology of InvA to
755
members of a new protein family. J Bacteriol 174:4338-4349.
756
65.
Raymond B, Crepin VF, Collins JW, Frankel G. 2011. The WxxxE effector
757
EspT triggers expression of immune mediators in an Erk/JNK and NF-
758
kappaB-dependent manner. Cell Microbiol 13:1881-1893.
35
759
Figure legends
760
Fig.1 Schematic representing the different Tir variants used in this study. The two
761
transmembrane helices, upstream and downstream of the intimin-binding domain, are
762
represented as black boxes and include amino acids 231-257 and 360-382,
763
respectively. Tyrosine residues Y451 and Y471 are shown in black and the
764
substitutions Y451A and Y471A in grey. Deletions are represented as dotted lines.
765
766
Fig. 2. (A) Immunofluoresence of Swiss 3T3 cells infected with C. rodentium. Tir
767
(green) was detected under adherent C. rodentium expressing either TirC-ctrl,
768
Tir382-462_Y451 or Tir478-547_Y451/Y471. Polymerized actin (red) was
769
observed under Tir staining of all adherent bacteria. Bar = 5 m. Infected cells were
770
also analyzed by SEM. Bar = 10 m. (B) Tir translocation was qualitatively assess by
771
determining the percentage of adherent bacteria showing translocated Tir staining.
772
While C. rodentium expressing either TirC-ctrl or Tir382-462_Y451 showed no
773
difference in their Tir translocation efficiency, C. rodentium expressing Tir478-
774
547_Y451/Y471 translocated Tir significantly less effectively.
775
776
Fig. 3. (A) Colonization dynamics to the peak of C. rodentium infection (day8 post
777
infection). C57Bl/6 mice inoculated with C. rodentium expressing TirC-ctrl, Tir382-
778
462_Y451 or Tir382-462_Y451A exhibit similar colonization dynamics. Mice
779
infected with C. rodentium expressing Tir478-547_Y451/Y471 or Tir478-
780
547_Y451A/Y471A showed a reduced colonization compared to strains expressing
781
TirC-ctrl, Tir382-462_Y451 or Tir382-462_Y451A. No difference was observed
782
whether the Tir variants were made in the context of either Y451/Y471 or
36
783
Y451A/Y471A. All strains colonized significantly better than mice infected with C.
784
rodentium expressing Tir1-33stop. ** P<0.005, *** P<0.001, Kruskal-Wallis test
785
comparing C. rodentium expressing TirC-ctrl and Tir478-547_Y451/Y471 or
786
Tir478-547_Y451A/Y471A. (B) Transmission and SEM of mice colonic epithelium
787
infected with C. rodentium expressing TirC-ctrl, Tir382-462_Y451A or Tir478-
788
547_Y451A/Y471A. Local effacement of the brush border microvilli and intimately
789
adherent bacteria (arrow), typical of A/E lesions, were observed following
790
inoculation of mice with any of the C. rodentium strains. Intact brush border
791
microvilli were observed in tissue extracted from uninfected mice. Bar = 1m or
792
5m.
793
794
Fig. 4. Colonic enterocytes were isolated from mice infected with wild-type C.
795
rodentium or C. rodentium expressing Tir_Y451A/Y471A, at day 7 post infection.
796
Enterocytes isolated from uninfected mice were used as controls. Tir (green) was
797
detected underneath attached C. rodentium bacteria; intense actin staining was seen at
798
the site of wild type C. rodentium infection, while weak staining was observed at the
799
attachment
800
Tir_Y451A/Y471A. Enterocytes isolated from uninfected mice showed typical brush
801
border actin staining (arrow). Bar = 5m.
site
of
enterocytes
infected
with
C.
rodentium
expressing
802
803
Fig. 5. At day 14 post infection, mice infected with C. rodentium expressing TirC-
804
ctrl, Tir_Y451A, Tir_Y471A, Tir_Y451A/Y471A, TirΔ382-462_Y471 or TirΔ382-
805
462_Y471A showed equivalent bacterial shedding (A). Colonic tissues from mice
806
infected with C. rodentium expressing TirC-ctrl, Tir_Y451A, Tir_Y471A,
37
807
Tir_Y451A/Y471A or PBS mock-infected mice were harvested 14 days post infection
808
and processed for FACS analyses. No difference in recruitment of (B) macrophages
809
(CD11b+Ly6G-F4/80+), (C) T helper cells (CD4+CD8-) and (D) B cells
810
(B220+CD3-) was observed in tissue infected with C. rodentium expressing the Tir
811
variants. Results are from two independent experiments with 5-6 mice per group. (E-
812
F) Neutrophils recruitment was significantly lower in tissue isolated from mice
813
infected with C. rodentium expressing Tir_Y451A/Y471A compared to Tir wild type
814
or single tyrosine mutant. (G-H) Neutrophils were also analyzed in colonic tissue of
815
mice infected with C. rodentium expressing either TirΔ382-462_Y471 or TirΔ382-
816
462_Y471A. Reduced neutrophils recruitment was observed specifically in tissue
817
infected with C. rodentium expressing TirΔ382-462_Y471A.
818
819
Fig. 6. (A) Enterocytes were isolated from colonic tissue of mice infected with C.
820
rodentium or mocked-infected with PBS and assessed for purity using flow
821
cytometry. Samples were stained for the leukocyte marker CD45 and the epithelial
822
cell marker CD326 (EpCAM). 80-90% of the cells were labelled CD326+CD45-,
823
which constituted the enterocyte population of the sample. Enterocytes were isolated
824
from colonic tissue of mice infected with C. rodentium expressing TirC-ctrl,
825
Tir_Y451A, Tir_Y471A or Tir_Y451A/Y471A and the expression of the neutrophil
826
chemoattractant CXCL1 (B) and CXCL2 (C) was measured by Q-RT-PCR (result is
827
from two independent enterocyte isolation experiments, n=4 per group). A reduced
828
level of CXCL1 and CXCL2 mRNA was observed in tissue infected with C.
829
rodentium expressing TirΔ382-462_Y471A. Data presented relative to GAPDH and
830
PBS control. *P<0.05, Students t test.
831
38
832
Fig. 7. (A) Representative H&E section of colonic tissue from mice (n=4) infected
833
with C. rodentium expressing TirC-ctrl or Tir_Y451A/Y471A and PBS mock-
834
infected mice, at day 14 post infection. (B) Measurements of crypt length reveal
835
significantly reduced level of colonic hyperplasia on day 14 post infection in mice
836
infected with C. rodentium expressing Tir_Y451A/Y471A compared to mice infected
837
with C. rodentium expressing TirC-ctrl, ***P<0.0001. (C) Frozen colonic sections
838
were stained with antibodies against E-Cadherin (tissue contrast), Ly-6G (neutrophils)
839
or hoechst (nuclei). Representative immunofluorescence showing more Ly-6G
840
positive neutrophils stain (pink) in colonic section of mice infected with C. rodentium
841
expressing
842
Tir_Y451A/Y471A or the PBS mock-infected control mice (Bar= 100m).
TirC-ctrl
than
mice
infected
843
39
with
C.
rodentium
expressing
844
845
Table 1. Strains, plasmids and primers used in this study
846
Description
Reference
ICC169
Wild type C. rodentium O152 serotype
(63)
ICC294
C. rodentium expressing TirC-ctrl
(14)
ICC295
C. rodentium expressing Tir1-33stop
(14)
ICC297
C. rodentium expressing Tir_Y451A
(14)
ICC298
C. rodentium expressing Tir_Y471A
(14)
ICC301
C. rodentium expressing Tir_Y451A/471A
(14)
ICC1168
C. rodentium expressing Tir Δ382-462_Y471
This study
ICC1169
C. rodentium expressing Tir Δ382-462_Y471A
This study
ICC1170
C. rodentium expressing Tir Δ478-547_Y451/Y471
This study
ICC1171
C. rodentium expressing Tir Δ478-547_Y451A/Y471A
This study
pGEMT
Cloning vector
Promega
pKD46
Coding for the lambda Red recombinase
(36)
pSB315
Plasmid coding for the kanamycin resistance aphT cassette
(64)
pICC433
pGEMT vector containing the 3’ end of tirCITRO (bp 1067-1644), the aphT
(14)
Strains
Plasmids
cassette, tir-cesT intergenic region and the 5’ end of cesT (bp 1-388)
pICC438
pICC433 containing tirCITRO Y451A/Y471A mutation
(14)
pICC1842
pGEMT vector containing the 3’ end of tirCITRO (bp 1067-1434), from which
This study
amino acids 478-547 have been deleted, the aphT cassette, tir-cesT intergenic
40
region and the 5’ end of cesT (bp 1-388)
pICC1843
pICC1842 containing tirCITRO Y451A/Y471A mutation
This study
pICC1844
pGEMT vector containing the 3’ end of tirCITRO (bp 664-1644), in which amino
This study
acids 382-462 have been deleted, the aphT cassette, tir-cesT intergenic region
and the 5’ end of cesT (bp 1-388)
pICC1845
pICC1844 containing tirCITRO Y471A mutation
Primer name
Nucleotide sequence
Tir-P478DSV-stop-EcoRI-Rv
5’-ccggaattcttaaacagaatcaggatccggagcgacttcatc-3’
This study
down-Tir-EcoRI-Fw
5’-ccggaattcatatataatgggtattttgttggggggg-3’
This study
Tir-down-TM2-Y471-Fw
5’-atgctccatagacgaaattcgcttctcgctccagaagag-3’
This study
Tir-EcoRI-Rv
5’-ccggaattcttagacgaaacgttcaactccc-3’
This study
Tir-upTM1-Fw
5’-acaacttcaagtgttcgttcag-3’
This study
Tir-down-TM2-Rv
5’-atttcgtctatggagcatagcc-3’
This study
EcoRI-(tir-cesT)-Fw
5’-gattatgtaataccaggtacagg-3'
(14)
NcesT-Rv
5’-gcagccctagcatcacaaacagacggcgcgacaag-3’
(14)
Tir-Up-YY-Fw
5’-tggatctctcatcaggtattgg-3’
This study
mCXCL1-F (KC-F)
5’-tggctgggattcacctcaagaaca-3’
(65)
mCXCL1-R (KC-R)
5’-tgtggctatgacttcggtttgggt-3’
(65)
mGAPDH-F
5’-tcaacagcaactcccactcttcca-3’
This study
mGAPDH-R
5’-accctgttgctgtagccgtattca-3’
This study
847
41
This study