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Tir-induced actin remodeling triggers expression of CXCL1 in enterocytes
and neutrophil recruitment during Citrobacter rodentium infection
Running title: Tir-mediated neutrophil recruitment
Valerie F. Crepin*, Maryam Habibzay, Izabela Glegola-Madejska, Marianne Guenot, James
W. Collins and Gad Frankel*
MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial
College, London, UK
For Correspondence: Valerie Crepin, CMBI, Flowers Building, Imperial College, London
SW7 2AZ. Telephone: +44 20 75943070; Email: [email protected] &
Telephone: +44 20 75945253; Email: [email protected] k
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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 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 Tir residues Y451 and
Y471 play in host immune responses to C. rodentium infection. We found that
deletion of amino acids 382-462 or 478-547 had no impact on the ability of Tir to
mediate A/E lesion formation, although deletion of amino acids 478-547 affected Tir
translocation. Examination of enterocytes isolated from infected mice revealed that a
C. rodentium expressing Tir_Y451A/Y471A recruited significantly less neutrophils to
the colon and triggered less colonic hyperplasia on day 14 post infection, compared to
infection with 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.
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Introduction
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 paediatric 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 0-11 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
cells production of interferon-γ (IFNγ) (5, 6), interleukin 17A (IL17A) (7, 8) or IL22
(9). Expression of the pro-inflammatory cytokine IL-17A leads to recruitment of
neutrophils (10), and the anti-inflammatory cytokine IL-22 up-regulates expression of
antimicrobial peptides (such as REGIIIβ and REGIIIγ) 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 plasma membrane, accumulation
of polymerized actin and formation of elevated pedestal-like structures (4, 12).
Adhesion of EPEC (reviewed in (13)) and C. rodentium (14) to cultured cells triggers
actin polymerization under attached bacteria.
The ability to induce A/E lesions and actin polymerization is encoded within the locus
of enterocyte effacement (LEE) (15), which encodes a type III secretion system
(T3SS) (16), the outer membrane adhesin intimin (17), regulators, chaperones,
translocator and effector proteins (reviewed in (18)). Following initial cell attachment,
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EPEC and C. rodentium use their T3SS to inject LEE- and non-LEE-encoded
effectors that subvert multiple signaling pathways including apoptosis (the effectors
NleH and NleB), endosomal trafficking (EspG and EspI), Rho GTPases (EspH and
Map), innate immunity (NleC, NleD, NleE and NleF) and actin dynamics (Tir and
EspF) (reviewed in (13)). In particular, following translocation, Tir, which contains
two trans-membrane (TM) helixes, is integrated into the epithelial cell plasma
membrane in a hairpin loop topology (19, 20), exposing an extracellular central
domain that functions as an intimin receptor (21). Infection of cultured epithelial cells
has shown that binding of intimin induces clustering of Tir, which leads to
phosphorylation of a C-terminal tyrosine (20), Y474 in EPEC or Y471 in C.
rodentium, by redundant tyrosine kinases, including Src, Fyn and Abl (22, 23). These
in turn recruit Nck via its SH2 domain which activates the neural Wiskott–Aldrich
syndrome protein (N-WASP) via its SH3 domain. This leads to recruitment of the
Arp2/3 complex which triggers actin polymerization underneath the attached bacteria
(reviewed in (24)). Campellone and Leong (25) have shown that TirEPEC can promote
weak actin polymerization in an Nck-independent manner, involving the C-terminal
Tir tyrosine residue Y454 (or Y451 in C. rodentium), which is present in the context
of a conserved Asn-Pro-Tyr (NPY) motif (26). The NPY motif recruits the adaptor
protein insulin receptor tyrosine kinase substrate (IRTKS) and/or the insulin receptor
substrate protein of 53 kDa (IRSp53) (27, 28). In EPEC belonging to lineage 2, the
weak Tir NPY-mediated actin polymerization pathway is amplified by the bacterial
effector TccP2/EspFM, which also activates N-WASP (29).
Although it was widely believed that the Tir-induced actin signaling pathways
observed during infection of cultured cells were responsible for A/E lesion formation
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
encoding Tir Y471F restored A/E lesion formation in vivo. Moreover, infection of
human in vitro organ cultures (IVOC) with EPEC expressing Tir_Y474F or
Tir_Y454F/Y474F also resulted in A/E lesions (31). In addition, we have
subsequently reported that incorporation of Y451A and Y471A double substitutions
into C. rodentium chromosomal tir abrogated actin polymerization in cultured cells
but had no effect on the level of colonization and A/E lesion formation in the mouse
model (14). Importantly, Ritchie et al. (32) and Mallick et al. (33, 34) have shown
that of A/E pathogens expressing tir mutant unable to trigger actin polymerization in
vitro were attenuated in mucosal colonization in vivo. The aim of this study was to
further investigate the role of the C-terminus of Tir during C. rodentium infection in
vivo.
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Material and methods
Bacterial strains and growth conditions
The bacterial strains, plasmids and primers used in this study are listed in Table 1.
Bacteria were grown in Luria–Bertani (LB) medium, M9 minimum media (35) or in
Dulbecco's modified Eagle's medium (DMEM) supplemented with kanamycin
(50 mg ml−1), ampicillin (100 mg ml−1) and nalidixic acid (50 mg ml−1) as required.
Introduction of site-directed tir mutants into the C. rodentium chromosome
We used the lambda red-based mutagenesis system (36) to introduce site-directed tir
alterations into the endogenous chromosomal tir gene, together with a kanamycin
cassette, in the tir-cesT intergenic region for 3′ mutagenesis as described before (14).
Deletion of the DNA segment encoding amino acids 478-547 (478-547) within the
tir gene was made by inverse-PCR on pICC433 (encoding Tir_Y451/Y471) and
pICC438 (encoding Tir_Y451A/Y471A) templates, previously described in (14),
using primer pair [Tir-P478DSV-stop-EcoRI-Rv] and [down-Tir-EcoRI-Fw]. The
inverse-PCR product was then digested with EcoRI and the aphT gene cloned into the
tir-cesT intergenic region to confer kanamycin resistance, resulting in plasmids
pICC1842 and pICC1843, respectively (Table 1).
Deletion of the DNA segment encoding amino acids 382-462 (382-462) within tir
was made by overlapping-PCR. C. rodentium genomic DNA was used to amplify tir
base pairs 664-1164 using primer pair [Tir-upTM1-Fw] and [Tir-down-TM2-Rv]. The
primer pair [Tir-down-TM2-Y471-Fw] and [Tir-EcoRI-Rv] was used to amplify tir
base pairs 1383-1644 from pICC433 (encoding Tir_Y471) and pICC438 (encoding
Tir_Y471A) templates. The two PCR fragments ([664-1164] and [1383-1644/Y471])
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
et al 2010, using primers [EcoRI-(tir-cesT)-Fw/NcesT-Rv]. The ligated PCR product
was then re-amplified using primers [Tir-upTM1-Fw] and [NcesT-Rv] and cloned into
pGEMT vector. The constructs were digested with EcoRI and the aphT gene cloned
into the tir-cesT intergenic region to confer kanamycin resistance, resulting in
plasmids pICC1844 and pICC1845, respectively (Table 1). All plasmid derivatives
were checked by DNA sequencing.
The various deletions 478-547_Y451/Y471 and 478-547_Y451A/Y471A; 382-
462_Y471 and 382-462_Y471A were PCR amplified from pICC1842 and
pICC1843, using primers [NcesT-Rv /Tir-Up-YY-Fw] and from pICC1844 and
pICC1845, using primers [NcesT-Rv/Tir-upTM1-Fw], respectively. The PCR
products were electroporated into wild type C. rodentium expressing the lambda red
recombinase from pKD46 plasmid (36). The presence of the mutation was confirmed
by PCR and DNA sequencing amongst the kanamycin resistant clones.
Cell culture
Swiss 3T3 cell line was grown, maintained and infected with the different C.
rodentium strains, at a MOI of 50, as described (14). Cells were washed 6 h post
infection with phosphate-buffered saline (PBS), fixed for 15 min in 4%
paraformaldehyde, permeabilized with 0.1% Triton for 4 min. Phalloidin-Tetramethyl
Rhodamine Iso-Thiocyanate (TRITC) (Sigma) was used to stain F-actin, while
bacterial DNA was counterstained with Hoechst 33342. Tir was stained using rabbit
anti-Tir EHEC (37, 38), recognizing the N-terminal domain, and carbocyanine-2-
conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Europe) secondary
antibody. Images were acquired using an AxioCam MRm monochrome camera and
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processed using AxioVision (Carl Zeiss MicroImaging GmbH, Germany).
Tir translocation by C. rodentium was qualitatively assessed by calculating the ratio
between the number of total visible bacterial nuclei and the number of bacteria which
show Tir staining concentrated in a straight line at the interface between the
bacterium. A minimum of 100 bacteria were counted by experiment and the
experiment was performed twice.
Oral infection of mice
Pathogen-free female 18–20g C57Bl/6 mice were purchased from Charles River. All
animals were housed in individually HEPA-filtered cages with sterile bedding and
free access to sterilized food and water. All animal experiments were performed in
accordance with the Animals Scientific Procedures (Act 1986) and were approved by
the local Ethical Review Committee. Infections were performed twice using four to
eight mice per group. Mice inoculated with mock mutant and nonsense mutant strains
were included in every experiment. Mice inoculated with wild-type strain and
uninfected mice were included in parallel with mutant strains.
Mice were inoculated by oral gavage with 200μl of overnight LB-grown C. rodentium
suspension in PBS (≈ 5 × 109 cfu). The number of viable bacteria used as inoculum
was determined by retrospective plating onto LB agar containing antibiotics. Stool
samples were recovered aseptically at various time points after inoculation and the
number of viable bacteria per gram of stool was determined by plating onto LB agar
(39). At day 7 and 14 post inoculum, the mice were culled and the colonic tissues
were collected for further analyses.
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
fixed in 10% neutral buffered formalin. Formalin fixed tissues were then processed,
paraffin-embedded, sectioned at 5μm and stained with haematoxylin and eosin (H&E)
using standard techniques. H&E stained tissues were evaluated for colonic crypt
hyperplasia microscopically without knowledge of the treatment condition used in the
study and the length of at least 20 well-oriented crypts from each section from all of
the mice per treatment group (n=4-6) were evaluated. H&E stained tissues were
imaged with an Axio Lab.A1 microscope (Carl Zeiss MicroImaging GmbH,
Germany), images were acquired using an Axio Cam ERc5s colour camera, and
computer-processed using AxioVision (Carl Zeiss MicroImaging GmbH, Germany).
Additional colonic segments were embedded in optimal cutting temperature (OCT)
medium (Raymond A Lamb Limited, UK) and frozen in dry-ice/ethanol slush for
further cryo-sectioning. Cryo-sections were then fixed in 3%-paraformaldehyde
(PFA) in PBS as previously described (38, 40) and immuno-stained using primary
antibodies at a 1/50 dilution, chicken anti-intimin (14), Ly-6G (RB6-8C5, Santa Cruz)
and E-Cadherin (CD324, BD Biosciences). Secondary antibodies were used at 1/100
dilution, Cy3 (103-165-175, Jackson Immunoresearch), Alexa 488 (712-546-150,
Jackson Immunoresearch) and Cy5 (715-175-150, Jackson Immunoresearch),
respectively. Images were acquired using an AxioCam MRm monochrome camera
and processed using AxioVision (Carl Zeiss MicroImaging GmbH, Germany).
Extraction of enterocytes and immunostaining
Four cm segment of the terminal colon was cut longitudinally and placed in 4ml
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
incubated at 37C, shaking, for 40 min. Left over tissue was removed by
centrifugation (1900g for 5 min) before the samples for each group were pooled
together and fixed with 1% formaldehyde. Fixed enterocytes (CD45-CD326+) were
analyzed for purity by flow cytometry using leukocyte marker CD45 and epithelial
cell marker CD326 (EpCAM). For immunofluorescence staining, fixed enterocytes
were permeabilized with 0.1% Triton and blocked with 1% bovine serum albumin in
PBS. Enterocytes were stained with polyclonal rabbit anti-Tir EHEC (GB1320,
SK1786) for 20 mins followed by 30 min incubation with secondary donkey anti-
rabbit IgG (H+L) Alexa488, phalloidin-TRITC (Sigma, P1951) was used for actin and
DAPI (Invitrogen, D3571) to visualize the nucleus. Tir staining was visualized with
an Axio Imager M1 microscope (Carl Zeiss MicroImaging GmbH, Germany), images
were acquired using an AxioCam MRm monochrome camera, and computer-
processed using AxioVision (Carl Zeiss MicroImaging GmbH, Germany).
Isolation of mRNA and Q-RT-PCR
mRNA of enterocytes was isolated using an RNeasy minikit according to the
manufacturer’s instructions (Qiagen). Samples were treated with RQ1 DNase-1
(Promega) at 37°C for 10 min, followed by 15 min at 72°C. Reverse transcription
(RT)-PCR was carried out by adding RT M-MLV (Promega M170B), RT buffer
(Promega), random primers (Promega), RNasin (Promega), dNTP (10mM) and RNase
free water to the DNase treated RNA extract, incubated at 37°C for 1 hour followed
by 10 min at 72°C and cooled samples were stored at -20°C. CXCL1 (KC) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were amplified with
primer pairs mCXCL1-F / mCXCL1-R and mGAPDH-F / mGAPDH-R (Table 1), by
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Q-RT-PCR using the 7300 Applied Biosystems instrument under standard cycle
conditions for Fast SYBR Green master mix. Changes in gene expression levels were
analyzed relative to the control levels (PBS samples), with GAPDH as a standard,
using the ΔΔCT method.
Sample collection for flow cytometry
Four cm segment of the terminal colon was cut, opened longitudinally and rinsed in
sterile PBS and placed in 4ml of RPMI-1640 supplemented with 10% fetal bovine
serum (FBS), penicillin/streptomycin (P/S), GlutaMAX, DNase (Roche,
10104159001) and liberase (Roche, 540112001) in a C-Mac tube (Miltenyi Biotec)
followed by tissue dissociation using gentleMACS dissociator (Miltenyi Biotec). The
tissue was homogenized using ‘intestine’ setting followed by incubation at 37°C, 5%
CO2 for 30 min in a shaking incubator and a final dissociation step was performed
using ‘Lung 2’ setting. The digested preparation was disrupted to a single cell
suspension by passage through a 70μm sieve (BD labware/falcon, USA Cat. No:
352350) and suspended in RPMI-1640 supplemented with 10% FBS and P/S at 0.5-
1x106 cells/ml.
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Extracellular antigen analysis
Cells were stained for surface markers as indicated 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). Prior to primary antibody staining, all cells were
blocked for Fc receptors (FcR) using mouse FcR blocking reagent (Miltenyi biotec)
for 10 min at 4°C. Antibodies were purchased from BD Pharmingen or eBioscience.
Data acquired on a BD Fortessa III and 20,000 lymphocytes or myeloid events were
analyzed with the FlowJO (Tress star) analysis program. Data is shown as a
percentage of myeloid or lymphocyte gates. Myeloid and lymphocyte gates are
determined by their position on the forward and side scatter plots generated by the
cytometer. Fluorescence minus one (FMO) control was included for each fluorescent
marker, the expression of a particular marker was calculated by subtracting FMO
fluorescence values from fluorescent antibody levels.
Total live cells were assessed by trypan blue exclusion. Forward and side scatter gates
on Flowjo software were used to gate myeloid cells, the percentage of this gate was
used to determine total number of myeloid cells. The myeloid cells gate was further
analysed to finalise the percentage of neutrophils (CD11b+Ly6G+) and establish the
total number of neutrophils in each mouse colon.
Mouse intestinal in vitro organ cultures
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 2009 (41). Briefly, segments from the terminal colon were
inoculated with 50l of the appropriate overnight bacterial culture, corresponding to
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
with PBS and fixed in 2.5% glutaraldehyde for electron microscopy analysis.
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Electron microscopy
Additional explants/tissue cultured cell samples were processed for electron
microscopy, as previously described (38). Samples for scanning electron microscopy
(SEM) were examined at an accelerating voltage of 25 kV using a JEOL JSM-5300
scanning electron microscope (JEOL (UK) Ltd., Herts, United Kingdom). Samples for
transmission electron microscopy (TEM) were observed using a Phillips 201
transmission electron microscope at an accelerating voltage of 60kV (Philips, United
Kingdom).
Statistical analysis
Results are presented as a line plot (colonization) with the mean and its standard
deviation. The non-parametric Mann–Whitney test and the non-parametric Kruskal–
Wallis test with Bonferroni's corrected a posteriori comparisons were used to conduct
pairwise and global statistical analysis, respectively, using commercially available
GraphPad InStat v3.06 software (GraphPad Software, San Diego, CA, USA). Mann-
Whitney compared to PBS controls (or as indicated in the figure) was used for data
obtained by flow cytometry using GraphPad Prism software. A P = 0.05 was
considered significant.
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Results
Construction of the 3’ tir chromosomal deletion mutants
Previously, using a method that allows expression of tir mutants from the C.
rodentium chromosome, we have introduced point mutations which have shown that
A/E lesion formation in vivo is independent of Tir residues Y451 and Y471 (14). In
this study, we used this technique to introduce deletions at the 3’ end of tir (Fig. 1).
While inserting a kanamycin cassette coupled to the mutated tir into the tir-cesT
intergenic region, we deleted Tir residues 382-462 (Tir382-462), removing the 80
amino acids downstream of the distal TM helix and Tir residues 478-547 (Tir478-
547), removing the entire segment downstream of Y471 phosphorylation site (Fig. 1).
Tir382-462 was made in the context of either Y471 or Y471A (Tir382-462_Y471
and Tir382-462_Y471A), while Tir478-547 was made in the context of either
Y451/Y471 or Y451A/Y471A (Tir478-547 _Y451/Y471 and Tir478-
547_Y451A/Y471A). As a positive control we used a mock mutant tir-cesT (TirC-
ctrl) in which the kanamycin cassettes was introduced into the intergenic region which
did not affect the tir coding sequence and as a negative control, a nonsense codon
introduced at Tir position 33 (Tir1−33stop) (14). Growth curves in minimal and rich
media confirmed that the mutants and parental wild-type strains had identical growth
rates (data not shown).
Testing the carboxy terminal Tir deletions during infection of cultured cells
We characterized the behavior of the Tir derivatives in vitro, following infection of
Swiss 3T3 fibroblast cells with the C. rodentium mutants as described before (14).
This revealed that, as expected, C. rodentium expressing either Tir382-462_Y471A
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or Tir478-547_Y451A/Y471A failed to induce actin polymerization (data not
shown). In contrast, infection of Swiss 3T3 cells with C. rodentium expressing
Tir382-462_Y471 or Tir478-547_Y451/Y471 revealed robust actin polymerization
under all the adherent bacteria that show Tir translocation (Fig. 2A). Importantly,
while Tir382-462 was translocated as efficiently as the wild type control Tir,
Tir478-547 translocated in low efficiency, with Tir staining seen within 50% of the
adherent bacteria (Fig. 2B). Interestingly, C. rodentium expressing Tir478-
547_Y451/Y471 produced longer pedestals. These results suggest that Tir residues
382-462 are dispensable for Tir translocation and actin polymerization, while the
carboxy terminus of Tir plays a role in translocation and hence indirectly in the
efficiency (both in terms of frequency and length) of actin pedestals formed in vitro.
Testing the carboxy terminal Tir deletions during mouse infection
We next investigated the impact of the C-terminal Tir deletions on colonization of C.
rodentium in vivo by enumerating colony-forming units per gram of stools (cfu g-1)
collected daily following oral inoculation of C57BL/6 mice for 8 days. This has
shown that C. rodentium expressing either Tir382-462_Y471 or Tir382-
462_Y471A colonized the mice similarly to the control strain expressing TirC-ctrl
(Fig. 3A) (variations between groups seen on day 2 post infection are common during
C. rodentium infection and has not biological relevance; the differences seen on day 3
post infection are not significant). Transmission electron microscopy (TEM) revealed
typical A/E lesions in colons infected with C. rodentium expressing either Tir382-
462_Y471 or Tir382-462_Y471A (Fig. 3B). In contrast, C. rodentium expressing
Tir1−33stop was rapidly cleared and failed to initiate an infection, reaching
background level as soon as day 3 post infection (Fig. 3A). These results show that
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Tir segment 382-462 is dispensable for colonization and that C. rodentium expressing
Tir382-462_Y471 or Tir382-462_Y471A are capable of forming A/E lesions in
vivo.
We then tested the phenotype of C. rodentium expressing Tir478-547. Consistent
with the in vitro data, this mutant exhibited an intermediate phenotype, reaching a
colonization level of 106/g of stool on day 3 post infection, which persisted in this
level until day 8 (Fig. 3A). The level of colonization was 100 fold lower than that
seen in mice infected with C. rodentium expressing TirC-ctrl or Tir382-462 but 104
fold higher than mice infected with Tir1−33stop. We observed no difference in
colonization between mice infected with Tir478-547_Y451/Y471 or Tir478-
547_Y451A/Y471A (Fig. 3A). However, as colonization was below the detection
level of TEM, we performed a mouse IVOC infection using C. rodentium expressing
Tir78-547_Y451A-Y471A. Examination of the samples by SEM revealed A/E
lesions, similar to those formed by the wild type strains, suggesting that Tir segment
478-547 is dispensable for A/E lesion formation on mucosal surfaces but does play a
role in colonization (Fig. 3B), probably due to its role in Tir translocation.
Tir-induces actin polymerization on enterocytes in vivo
As the results thus far have shown that C. rodentium expressing Tir deletions
(residues 382-462 and 478-547) and substitutions (Y451 and Y471) was able to form
A/E lesions, we tested whether Tir-induced actin polymerization on enterocytes in
vivo. For this, mice were infected with wild type C. rodentium or C. rodentium
expressing Tir_Y451A/Y471A (Fig. 1) and enterocytes were isolated at the peak of
colonization at day 7 post infection. Enterocytes isolated from naïve mice as a control
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exhibited a typical ‘crown’ staining pattern of the BB microvilli (Fig. 4). Enterocytes
isolated from infected mice showed good level of C. rodentium adhesion, with
multiple bacteria on individual enterocytes. Tir was detected underneath both attached
wild type C. rodentium and C. rodentium expressing Tir_Y451A/Y471A (Fig. 4).
However, while intense actin staining was seen at the site of wild type C. rodentium
infection, enterocytes infected with C. rodentium expressing Tir_Y451A/Y471A
exhibited mainly weak actin polymerization at the site of bacterial attachment. This
suggests that Tir induces actin polymerization on enterocyte in a process involving the
tyrosine residues.
Recruitment of immune cells to the C. rodentium infection site
As actin remodeling during infection can trigger immune responses (42), we next
investigated if Tir Y451 and Y471 modulate host immune responses. For this, groups
of 4-6 mice were infected with wild type and mutant C. rodentium and recruitment of
immune cells were analyzed by flow cytometry of homogenized colons at days 7
(peak of colonization) and day 14 (peak of pathology) post infection. All the tested
strains were shed at equivalent levels (Fig. 5A). The flow cytometry analysis has
shown that in comparison to the PBS mock-infected control mice (baseline readout),
infection with either the wild type or mutant C. rodentium resulted in equivalent
recruitment of macrophages, CD4+ T cells, and B cells on day 7 (data not shown) and
14 (Fig. 5B-D) post infection. In contrast, significantly less neutrophils were recruited
to the colon following infection with C. rodentium expressing Tir_Y451A/Y471A
compared with the positive control mice infected with C. rodentium expressing wild
type Tir, or mice infected with the single Tir tyrosine mutants Y451A or Y471A (Fig.
5E-F). Similarly, although shed at equivalent levels (Fig. 5A), neutrophils recruitment
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was observed following infection with C. rodentium expressing Tir382-462_Y471
but not after infection with C. rodentium expressing Tir382-462_Y471A (Fig. 5G-
H).
As C. rodentium and Tir mainly interact with enterocytes, we next determined if the
Tir tyrosine residues play a role in expression of pro-inflammatory chemokines. For
this we isolated colonic enterocytes from mice infected with the different C.
rodentium strains. The purity of the enterocytes preparation was confirmed by flow
cytometry analysis following staining with the leukocyte marker CD45 and epithelial
cell marker CD326 (EpCAM), revealing low level of contamination (Fig. 6A). Using
the purified enterocytes in Q-RT-PCR for the chemokines CXCL1 (KC) and CXCL2
(MIP2-alpha) revealed reduced expression in mice infected with C. rodentium
expressing TirY451A-Y471A (Fig. 6B). The attenuated inflammatory responses
triggered by C. rodentium expressing Tir_Y451A/Y471A were mirrored by a
significantly reduced colonic hyperplasia (Fig. 7A and B) as well as neutrophil
staining with Ly-6G antibodies (Fig. 7C). These results reveal a novel in vivo role for
the Tir tyrosine residues TirY451 and Y471, which are also implicated in actin
polymerization during C. rodentium infection (Fig. 4).
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Discussion
In this study we found that C. rodentium expressing Tir382-462 colonized the
mouse gastrointestinal tract and produced A/E lesion whether in the context of Y471
or Y471A. This result shows that amino acids 382-462 are dispensable for Tir
activity. We also tested a chromosomal deletion of Tir residues 478-547. This has
shown that C. rodentium expressing either Tir478-547_Y451/Y471 or Tir478-
547_Y451A/Y471A behaved similarly, showing an intermediate colonization level
between the wild type and the Tir1-33stop strains. The fact that C. rodentium
expressing either Tir478-547_Y451/Y471 or Tir478-547_Y451A/Y471A showed
104 fold higher colonization level than mice infected with Tir1-33stop suggests that
Tir is at least partially active. Due to the low level of colonization we were unable to
determine if these strains could form A/E lesions in vivo using TEM but we could
confirm A/E lesions by SEM. A previous report demonstrated that a 6 amino acid
sequence (TYARLA) at position 519-524 within the carboxy-terminal region was
required for efficient secretion and translocation, but not for stability, of Tir-EHEC
(43). The carboxy terminus of Tir C. rodentium contain an equivalent 6 amino acid
sequence (TYALLA), which is consistent with the low translocation efficiency, seen
by immuno-fluorescence staining following infection of Swiss 3T3 cells with C.
rodentium expressing Tir478-547_Y451/471, and the intermediate in vivo phenotype
of this strain.
Immunostaining of enterocytes isolated from naïve mice revealed good preservation
of the BB microvilli. In contrast, individual enterocytes isolated from infected mice
were covered with adherent C. rodentium and exhibited effaced BB microvilli. Tir
was detected at equivalent intensity at the site of bacterial attachment, whether in the
context of wild type Tir or Tir_Y451A/Y471A. Importantly, actin staining was
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considerably brighter under attached C. rodentium expressing wild type Tir. This
could potentially explain the competitive advantage of wild type C. rodentium over C.
rodentium expressing Tir_Y451A/Y471A during mixed infection (14). However, it is
important to note that while, as expected, no Nck was recruited to C. rodentium
expressing Tir_Y451A/Y471A in vivo (14) or to human intestinal biopsies infected
with EPEC expressing the Tir mutant (31), N-WASP was detected underneath the
attached mutant strains, which could explain the faint actin polymerization seen in the
enterocytes isolated from mice infected with C. rodentium expressing
Tir_Y451A/Y471A. Taken together these data suggest the existence of as yet
undetermined Tir actin polymerization pathway in mucosal surfaces.
If the Tir tyrosine residues do not play a role in A/E lesion formation, the question
remains, what function do they have during infection of mucosal surfaces? As EPEC
and C. rodentium interact intermediately with enterocytes, we hypothesized that Tir-
induced actin polymerization might contribute to signaling to the underlying immune
system. To test this, we infected mice with C. rodentium expressing wild type Tir and
Tir tyrosine mutants and compared recruitment of immune cells in homogenized
colons. This revealed no difference in recruitment of macrophages, T cells or B cells
at either 7 or 14 days post infection. In contrast, significantly reduced level of
recruited neutrophils was seen at day 14 following infection with C. rodentium
expressing Tir_Y451A/Y471A or Tir382-462_Y471A, compared to infection with
C. rodentium expressing wild type Tir or Tir382-462_Y471. Moreover, mice
infected with C. rodentium expressing Tir_Y451A/Y471A presented significantly
reduced levels of colonic hyperplasia. Importantly, infection with C. rodentium
expressing single tyrosine Tir mutant (Y451A or Y471A), resulted in neutrophil
recruitment equivalent to that seen following infection with wild type C. rodentium.
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This phenotype was mirrored following testing for CXCL1 and CXCL2 expression by
Q-RT-PCR on enterocytes purified from C. rodentium infected mice. Similarly, (44)
reported that following EPEC infection both Tir Y454 and Y474 are needed for
efficient nuclear translocation of the transcription factor serum response factor (SRF)
co-factor MAL and transcription of SRF target genes. CXCL1 and CXCL2 signal via
CXCR2 to activate neutrophils and subsequently promote mucosal influx of
neutrophils (45). Importantly, although neutrophils contribute to host defense against
infection (10), wild type C. rodentium and C. rodentium expressing mutant Tir
colonized at equivalent levels. These results suggest that while Tir contains redundant
mechanisms leading to neutrophil recruitment, each relying of one of the two
tyrosines, the host immune response can compensate for the lack of neutrophils late
during infection and clear the pathogen.
Tir EPEC and C. rodentium have further two distal tyrosines (Y480_Y508 and
Y483_Y511, respectively), which comprise an immunoreceptor tyrosine-based
inhibition motif (ITIM). Smith et al. (46) reported that following EPEC infection Tir
residues Y483 and Y511 recruit the host inositol phosphatase SHIP2. Moreover, the
pedestals formed by EPEC expressing Tir Y483F_Y511F were significantly longer
than those formed by EPEC expressing wild type Tir, which is consistent with the
longer pedestals we observed following infection with C. rodentium expressing
Tir478-547_Y451/Y471, which lacks residues Y480_Y508. Recently Yan et al have
shown that infection with EPEC expressing Tir Y483F_Y511F resulted in elevated
levels of IL6 and TNF mRNA in splenic cells and enhanced bacterial clearance (47).
They have shown that phosphorylation of the Tir ITIM leads to the recruitment of
both SHP1 and SHP2 and inhibition of TRAF6 autoubiquitination, which helps the
bacteria to suppress and evade the host innate immune response (47, 48). This feature
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of Tir could provide an alternative explanation for why C. rodentium expressing Tir
Tir478-547_Y451/Y471 or Tir478-547_Y451A/Y471A did not colonize the colon
at a wild type level.
Subversion of the actin cytoskeleton by bacterial virulence factors has been shown to
be an important mediator of immune signaling. For example, the Salmonella Rho
GTPase GEF SopE has been shown to activate the pattern recognition receptors
(PRRs) NOD1 (49), while NOD2 is regulated by Rac1 (50). NOD1 and NOD2, could
be found at the plasma membrane in association with F-actin, which is needed for
downstream activation of NF-kB signaling (42, 51). Recently, Bielig et al have shown
that the actin depolymerization factors (ADF)/cofilin phosphatase SSH1 is an
essential component of the NOD1 pathway, which plays a role in activation of NF-kB
and cell responses to Shigella infection (52). Indeed, depletion of SSH1 mRNA
resulted in reduced production of IL8 and IL6 following infection of HeLa cells with
S. flexneri strain M90T. Consistently, our data show that sensing Tir-induced actin
remodeling triggers host responses to C. rodentium infection.
C. rodentium translocates multiple effectors that contribute to coordinated
cytoskeleton remodeling (including Map that activates Cdc42 and Rac1 (53), EspM
that activates RhoA (54), EspT that activates Rac1 (55) and EspJ that inhibits Src
kinases (56) which play a role in Tir tyrosine phosphorylation) and subversion of
innate immune responses, including NF-kB (e.g. NleC, NleD, NleE, NleB and NleF
(13, 57-62)). Importantly, the difference in neutrophil recruitment seen at day 14
between wild type C. rodentium and C. rodentium expressing Tir_Y451A/Y471A is
at the time when both infections are close to being cleared. Future studies will aim at
unraveling the mechanism by which Tir induces expression of CXCL1 and CXCL2 in
enterocytes late during infection in the broader context of the other type III secreted
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effectors that modulate inflammatory responses.
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Acknowledgements
This study was supported by a grant from the Wellcome trust and the BBSRC.
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Figure legends
Fig.1 Schematic representing the different Tir variants used in this study. The two
transmembrane helices, upstream and downstream of the intimin-binding domain, are
represented as black boxes and include amino acids 231-257 and 360-382,
respectively. Tyrosine residues Y451 and Y471 are shown in black and the
substitutions Y451A and Y471A in grey. Deletions are represented as dotted lines.
Fig. 2. (A) Immunofluoresence of Swiss 3T3 cells infected with C. rodentium. Tir
(green) was detected under adherent C. rodentium expressing either TirC-ctrl,
Tir382-462_Y451 or Tir478-547_Y451/Y471. Polymerized actin (red) was
observed under Tir staining of all adherent bacteria. Bar = 5 m. Infected cells were
also analyzed by SEM. Bar = 10 m. (B) Tir translocation was qualitatively assess by
determining the percentage of adherent bacteria showing translocated Tir staining.
While C. rodentium expressing either TirC-ctrl or Tir382-462_Y451 showed no
difference in their Tir translocation efficiency, C. rodentium expressing Tir478-
547_Y451/Y471 translocated Tir significantly less effectively.
Fig. 3. (A) Colonization dynamics to the peak of C. rodentium infection (day8 post
infection). C57Bl/6 mice inoculated with C. rodentium expressing TirC-ctrl, Tir382-
462_Y451 or Tir382-462_Y451A exhibit similar colonization dynamics. Mice
infected with C. rodentium expressing Tir478-547_Y451/Y471 or Tir478-
547_Y451A/Y471A showed a reduced colonization compared to strains expressing
TirC-ctrl, Tir382-462_Y451 or Tir382-462_Y451A. No difference was observed
whether the Tir variants were made in the context of either Y451/Y471 or
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Y451A/Y471A. All strains colonized significantly better than mice infected with C.
rodentium expressing Tir1-33stop. ** P<0.005, *** P<0.001, Kruskal-Wallis test
comparing C. rodentium expressing TirC-ctrl and Tir478-547_Y451/Y471 or
Tir478-547_Y451A/Y471A. (B) Transmission and SEM of mice colonic epithelium
infected with C. rodentium expressing TirC-ctrl, Tir382-462_Y451A or Tir478-
547_Y451A/Y471A. Local effacement of the brush border microvilli and intimately
adherent bacteria (arrow), typical of A/E lesions, were observed following inoculation
of mice with any of the C. rodentium strains. Intact brush border microvilli were
observed in tissue extracted from uninfected mice. Bar = 1m or 5m.
Fig. 4. Colonic enterocytes were isolated from mice infected with wild-type C.
rodentium or C. rodentium expressing Tir_Y451A/Y471A, at day 7 post infection.
Enterocytes isolated from uninfected mice were used as controls. Tir (green) was
detected underneath attached C. rodentium bacteria; intense actin staining was seen at
the site of wild type C. rodentium infection, while weak staining was observed at the
attachment site of enterocytes infected with C. rodentium expressing
Tir_Y451A/Y471A. Enterocytes isolated from uninfected mice showed typical brush
border actin staining (arrow). Bar = 5m.
Fig. 5. At day 14 post infection, mice infected with C. rodentium expressing TirC-ctrl,
Tir_Y451A, Tir_Y471A, Tir_Y451A/Y471A, TirΔ382-462_Y471 or TirΔ382-
462_Y471A showed equivalent bacterial shedding (A). Colonic tissues from mice
infected with C. rodentium expressing TirC-ctrl, Tir_Y451A, Tir_Y471A,
Tir_Y451A/Y471A or PBS mock-infected mice were harvested 14 days post infection
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and processed for FACS analyses. No difference in recruitment of (B) macrophages
(CD11b+Ly6G-F4/80+), (C) T helper cells (CD4+CD8-) and (D) B cells
(B220+CD3-) was observed in tissue infected with C. rodentium expressing the Tir
variants. Results are from two independent experiments with 5-6 mice per group. (E-
F) Neutrophils recruitment was significantly lower in tissue isolated from mice
infected with C. rodentium expressing Tir_Y451A/Y471A compared to Tir wild type
or single tyrosine mutant. (G-H) Neutrophils were also analyzed in colonic tissue of
mice infected with C. rodentium expressing either TirΔ382-462_Y471 or TirΔ382-
462_Y471A. Reduced neutrophils recruitment was observed specifically in tissue
infected with C. rodentium expressing TirΔ382-462_Y471A.
Fig. 6. (A) Enterocytes were isolated from colonic tissue of mice infected with C.
rodentium or mocked-infected with PBS and assessed for purity using flow
cytometry. Samples were stained for the leukocyte marker CD45 and the epithelial
cell marker CD326 (EpCAM). 80-90% of the cells were labelled CD326+CD45-,
which constituted the enterocyte population of the sample. Enterocytes were isolated
from colonic tissue of mice infected with C. rodentium expressing TirC-ctrl,
Tir_Y451A, Tir_Y471A or Tir_Y451A/Y471A and the expression of the neutrophil
chemoattractant CXCL1 (B) and CXCL2 (C) was measured by Q-RT-PCR (result is
from two independent enterocyte isolation experiments, n=4 per group). A reduced
level of CXCL1 and CXCL2 mRNA was observed in tissue infected with C.
rodentium expressing TirΔ382-462_Y471A. Data presented relative to GAPDH and
PBS control. *P<0.05, Students t test.
Fig. 7. (A) Representative H&E section of colonic tissue from mice (n=4) infected
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with C. rodentium expressing TirC-ctrl or Tir_Y451A/Y471A and PBS mock-
infected mice, at day 14 post infection. (B) Measurements of crypt length reveal
significantly reduced level of colonic hyperplasia on day 14 post infection in mice
infected with C. rodentium expressing Tir_Y451A/Y471A compared to mice infected
with C. rodentium expressing TirC-ctrl, ***P<0.0001. (C) Frozen colonic sections
were stained with antibodies against E-Cadherin (tissue contrast), Ly-6G (neutrophils)
or hoechst (nuclei). Representative immunofluorescence showing more Ly-6G
positive neutrophils stain (pink) in colonic section of mice infected with C. rodentium
expressing TirC-ctrl than mice infected with C. rodentium expressing
Tir_Y451A/Y471A or the PBS mock-infected control mice (Bar= 100m).
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Table 1. Strains, plasmids and primers used in this study
Description Reference
Strains
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 382-462_Y471Δ This study
ICC1169 C. rodentium expressing Tir 382-462_Y471AΔ This study
ICC1170 C. rodentium expressing Tir 478-547_Y451/Y471Δ This study
ICC1171 C. rodentium expressing Tir 478-547_Y451A/Y471AΔ This study
Plasmids
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
cassette, tir-cesT intergenic region and the 5’ end of cesT (bp 1-388)
(14)
pICC438 pICC433 containing tirCITRO Y451A/Y471A mutation (14)
pICC1842 pGEMT vector containing the 3’ end of tirCITRO (bp 1067-1434), from which This study
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845
amino acids 478-547 have been deleted, the aphT cassette, tir-cesT intergenic
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
acids 382-462 have been deleted, the aphT cassette, tir-cesT intergenic region
and the 5’ end of cesT (bp 1-388)
This study
pICC1845 pICC1844 containing tirCITRO Y471A mutation This study
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
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