The Serine Protease Autotransporter Pic Modulates Citrobacter rodentium Pathogenesis and Its Innate Recognition by the Host.

The Serine Protease Autotransporter Pic Modulates Citrobacter rodentium Pathogenesis and Its Innate Recognition by the Host Kirandeep Bhullar,a Maryam Zarepour,a Hongbing Yu,a Hong Yang,a Matthew Croxen,b Martin Stahl,a B. Brett Finlay,b Stuart E. Turvey,a Bruce A. Vallancea Department of Pediatrics, Division of Gastroenterology, Child and Family Research Institute, Vancouver, British Columbia, Canadaa; Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canadab

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itrobacter rodentium is a natural attaching and effacing (A/E) mouse pathogen that is widely used to model the pathogenesis of the human-specific bacterial pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). C. rodentium has been widely used to explore the innate and adaptive immune responses elicited during A/E bacterial infections (1–4). For example, studies by our group and others have shown that C. rodentium-based activation of Toll-like receptors 2 (TLR2) and 4 (TLR4) drives much of the inflammation and tissue pathology seen during infection (5, 6). Studies of C. rodentium infection also have helped characterize the in vivo role of type III secretion system (T3SS) effector proteins such as translocated intimin receptor (Tir) in pathogenesis, since there is strong homology between the translocated effector proteins produced by EPEC/EHEC and those produced by C. rodentium (7). While the impact of less defined virulence factors, such as serine protease autotransporters of Enterobacteriaceae (SPATEs), has received less attention, recent identification of three SPATEs (one class 1 SPATE and two class 2 SPATEs) as well as 17 other autotransporters encoded by the C. rodentium genome has opened the potential for investigating the role of these proteins in vivo (8). For example, a recent study examining the role of C. rodentium’s class 1 SPATE crc1 showed it played an unexpectedly important immunomodulatory role. Deletion of crc1 resulted in a hyperinflammatory phenotype during infection, leading to exaggerated colitis, as measured by increased pathology, cytokine levels, and inflammatory cell infiltration within the infected colon (9). Pic (protein involved in intestinal colonization) is a class 2 SPATE produced by several enteric pathogens, including Shigella flexneri, enteroaggregative E. coli (EAEC), and uropathogenic E. coli (UPEC) (10–14). A homologue of this protein, which we have

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termed PicC, recently was found to be encoded by the C. rodentium genome (YP_003368482.1) (8). So far, several in vitro or ex vivo functions have been attributed to Pic, including mucinolytic activity, thought to be important for enteric pathogens to colonize the intestines of their hosts by helping them penetrate the mucus layer that coats the intestinal epithelium (10, 15). Curiously, several homologues of Pic also have been shown to induce rapid and exaggerated mucus secretion in rat ileal loop models, suggesting that Pic also can act as a mucus secretagogue (16). More recent studies also have shown that class 2 SPATEs, like Pic, possess immunomodulatory properties by cleaving the O-glycosylated molecules found on the surface of immune cells, chemokines, and complement proteins (17, 18). Despite these varied functions, the potential impact of Pic in bacterial virulence in vivo has yet to be defined. Considering that C. rodentium is a nonmotile pathogen yet

Received 8 January 2015 Returned for modification 6 February 2015 Accepted 9 April 2015 Accepted manuscript posted online 20 April 2015 Citation Bhullar K, Zarepour M, Yu H, Yang H, Croxen M, Stahl M, Finlay BB, Turvey SE, Vallance BA. 2015. The serine protease autotransporter Pic modulates Citrobacter rodentium pathogenesis and its innate recognition by the host. Infect Immun 83:2636 –2650. doi:10.1128/IAI.00025-15. Editor: A. J. Bäumler Address correspondence to Bruce A. Vallance, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.00025-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/IAI.00025-15

Infection and Immunity

July 2015 Volume 83 Number 7

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Bacterial pathogens produce a number of autotransporters that possess diverse functions. These include the family of serine protease autotransporters of Enterobacteriaceae (SPATEs) produced by enteric pathogens such as Shigella flexneri and enteroaggregative Escherichia coli. Of these SPATEs, one termed “protein involved in colonization,” or Pic, has been shown to possess mucinase activity in vitro, but to date, its role in in vivo enteric pathogenesis is unknown. Testing a pic null (⌬picC) mutant in Citrobacter rodentium, a natural mouse pathogen, found that the C. rodentium ⌬picC strain was impaired in its ability to degrade mucin in vitro compared to the wild type. Upon infection of mice, the ⌬picC mutant exhibited a hypervirulent phenotype with dramatically heavier pathogen burdens found in intestinal crypts. ⌬picC mutant-infected mice suffered greater barrier disruption and more severe colitis and weight loss, necessitating their euthanization between 10 and 14 days postinfection. Notably, the virulence of the ⌬picC mutant was normalized when the picC gene was restored; however, a PicC point mutant causing loss of mucinase activity did not replicate the ⌬picC phenotype. Exploring other aspects of PicC function, the ⌬picC mutant was found to aggregate to higher levels in vivo than wild-type C. rodentium. Moreover, unlike the wild type, the C. rodentium ⌬picC mutant had a red, dry, and rough (RDAR) morphology in vitro and showed increased activation of the innate receptor Toll-like receptor 2 (TLR2). Interestingly, the C. rodentium ⌬picC mutant caused a degree of pathology similar to that of wild-type C. rodentium when infecting TLR2-deficient mice, showing that despite its mucinase activity, PicC’s major role in vivo may be to limit C. rodentium’s stimulation of the host’s innate immune system.

Role of Pic in Citrobacter rodentium Pathogenesis

MATERIALS AND METHODS Construction of C. rodentium mutant strains. To generate the C. rodentium ⌬picC strain, the C. rodentium picC gene (ROD_p1411) was replaced using the lambda red system (19). Briefly, primer pair ROD_p1411 Fwd (5=-GTGAATAAAATATACTCGCTGTGTAGGCTGGAGCTGCT TC-3=) and ROD_p1441 Rev (5=-TCAGAACATATAACGGAAGTTCGC ATTCCGGGGATCCGTCGACC-3=) was used to amplify the FLP recombination target (FRT)-flanked kanamycin resistance gene (kan) from the plasmid pKD13 with PFU Turbo (Promega) (19). These two primers included sequences homologous to the flanking regions of picC. The resulting PCR fragment was electroporated (Bio-Rad Micropulser) into wildtype C. rodentium DBS100 carrying the pSIM9 recombineering plasmid (20). The recombinant genes carried by the plasmid pSIM9 catalyzed the exchange of picC with the FRT-flanked kan gene. Recombinants were selected on LB agar containing kanamycin (50 ␮g/ml) by incubation overnight (O/N) at 37°C, followed by removal of the kan gene from kanamycin-resistant clones with the pCP20 plasmid as described previously (19, 21). To verify the deletion of picC, colony PCR was performed using primers external to picC (ROD_p1411comp Fwd and ROD_p1411comp Rev; see below). Construction of plasmids to complement the C. rodentium ⌬picC strain. Primer pair ROD p1411comp Fwd (5=-TGACCTCGAGACACAT ACACCGCGGAAATAG-3=) and ROD_p1411comp Rev (5=-AGCTGGA TCCGTCCTTTGATAACGCCCTGAC-3=) was used to amplify the region containing the native promoter and coding sequence of picC. The resulting PCR product was cloned into the XhoI/BamHI restriction sites of pZA31MCS (Expressys, Ruelzheim, Germany) (22) to generate the plasmid pPic containing wild-type picC. This plasmid also was used as a template to construct a site-directed catalytic mutant named pPicS258I. Using a QuikChange site-directed mutagenesis kit (Agilent Technologies), site-directed mutagenesis was performed with primer pair Fwd (5=CGGCGTCCCCGGAGACATTGGTTCCCCTCTTTTTG-3=) and Rev (5=-CAAAAAGAGGGGAACCAATGTCTCCGGGGACGCCG-3=). Plasmids pPic and pPicS258I then were electroporated into the C. rodentium


⌬picC strain to produce C. rodentium ⌬picC ⫹ pPicC (complemented) and C. rodentium ⌬picC(pPicS258I) strains, respectively. Mice. C57BL/6 and Tlr2⫺/⫺ mice were bred in-house at the Child and Family Research Institute. Mice were kept in sterilized, filter-topped cages, handled in tissue culture hoods, and fed autoclaved food and water under specific-pathogen-free conditions (Child and Family Research Institute). The protocols used in the study were approved by the University of British Columbia’s Animal Care Committee and were in direct accordance with guidelines provided by the Canadian Council on the Use of Laboratory Animals. Bacterial strains, their growth, and mouse infections. Wild-type C. rodentium, C. rodentium ⌬picC, and C. rodentium ⌬picC ⫹ pPicC (complemented) strains and the C. rodentium ⌬picC(pPicS258I) point mutant were used in this study. All bacterial strains used in this study had similar in vitro growth rates. WT C. rodentium and the C. rodentium ⌬picC mutant were grown with shaking overnight at 200 rpm at 37°C in LuriaBertani (LB) broth. Complemented C. rodentium ⌬picC ⫹ pPicC and ⌬picC(pPicS258I) mutants were grown in LB containing 30 ␮g/ml chloramphenicol. Mice (6 to 8 weeks old) were infected with 0.1 ml of the O/N cultures (⬃2.5 ⫻ 108 CFU) by oral gavage. To visualize the RDAR (red, dry, aggregative) phenotype, an indicator of the production of extracellular matrix components, such as curli and cellulose, and 5 ␮l of O/N bacterial cultures were inoculated onto LB-agar (no salt) plates containing 40 ␮g/ml of Congo red dye and 20 ␮l/ml of Coomassie blue R-250. Plates were incubated at 25°C for 5 days. Biofilm formation in vitro was assessed using the crystal violet assay. Briefly, bacteria were grown at 25°C for 5 days in LB media (no salt) in a 96-well plate. Cultures were decanted and washed 3⫻ with water. Crystal violet dye (0.1%) was added to the plate and eluted with 100% ethanol after 1 h of incubation. The optical density at 595 nm (OD595) was used to measure biofilm formation. Cellulose production assays using Congo red and calcofluor were performed as previously described (23). Mucinase activity assays. WT, ⌬picC, complemented ⌬picC ⫹ pPicC, and ⌬picC(pPicS258I) C. rodentium strains were grown O/N in 5 ml LB and cultured as described above. Bacterial cultures were spun down at 4,000 rpm for 30 min to remove bacterial cells. Supernatants were filtered through Amicon Ultra 15 (100-kDa cutoff; Millipore) filters to concentrate the samples to a final volume of ⬃50 ␮l. Crude supernatants (30 ␮l) were incubated for 24 h at 37°C in a medium containing 5 ␮l of bovine submaxillary mucin (BSM; Sigma) and 15 ␮l of water. Treated (BSM and crude supernatants) and untreated (BSM alone) mucin samples were electrophoresed on an 8% SDS-PAGE gel. The gel was developed using a Pierce glycoprotein staining kit (Thermo Scientific). Tissue collection. Mice were monitored for mortality and morbidity throughout the course of infection and were euthanized when they showed ⬎15% body weight loss compared to their starting body weight. Uninfected and infected mice were anesthetized with isoflurane and euthanized through cervical dislocation. For enumeration of bacterial counts within the tissue (adherently attached) and luminal content, ceca and colon were opened longitudinally, and luminal contents (stool) were transferred into preweighted 2.0-ml tubes containing 1.0 ml of phosphate-buffered saline, pH 7.2 (PBS), and steel beads (Qiagen). Cecal and colonic tissues were washed 2⫻ in PBS and placed into tubes. Systemic tissues (liver, spleen, and mesenteric lymph nodes [MLN]) also were collected. All samples were weighed and homogenized with a MixerMill 301 homogenizer (Retsch, Newtown, PA). CFU were determined by serial dilutions of homogenized samples and plated on MacConkey agar plates. Plates were incubated O/N at 37°C. For assessing commensal translocation to MLN, reinforced clostridial agar (Oxoid, Thermo Scientific) and anaerobe basal agar (Oxoid, Thermo Scientific) were used. MLN were harvested and transferred into 1 ml sterile PBS. CFU were determined by serial dilutions of homogenized samples and plating on the above-mentioned media. Plates were incubated in an Oxoid Anaerojar, where a CampyGen 2.5-liter atmosphere generation system sachet was used to create an


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crosses the intestinal mucus layer to infect the underlying epithelium of its murine hosts, we were interested in testing whether PicC possessed mucinolytic activity and if it played a critical role in C. rodentium pathogenesis. We constructed a PicC mutant (termed the ⌬picC mutant) that demonstrated that much of the in vitro mucinolytic activity of C. rodentium was dependent on the presence of PicC. Surprisingly, upon infection of mice, the ⌬picC mutant exhibited a hypervirulent phenotype, with infected mice carrying dramatically heavier intestinal pathogen burdens and suffering more severe colitis than mice infected with wild-type (WT) C. rodentium. Notably, the virulence of the ⌬picC mutant was normalized when the picC gene was restored; however, a PicC point mutant causing the loss of mucinase activity did not replicate the ⌬picC phenotype. Further evaluation of the ⌬picC mutant revealed that in vitro, it showed an increased propensity to develop an aggregative, red, dry, and rough (RDAR) morphology, while it was found to form mixed pathogen-commensal microcolony-like structures in vivo. Interestingly, compared to the wild-type, the C. rodentium ⌬picC mutant showed increased in vitro activation of the innate receptor TLR2 but not TLR4. Moreover, wild-type C. rodentium and the ⌬picC mutant caused comparable damage in TLR2-deficient mice. While this in part reflected increased damage caused by wild-type bacteria, the ability of the ⌬picC mutant to cause greater damage than wild-type C. rodentium was lost in the TLR2-deficient mice. Thus, despite its mucinase activity, PicC’s major roles in vivo may be to limit C. rodentium aggregation as well as limit its recognition by the host’s innate immune system.

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treated) and infected (6 days postinfection [dpi] with C. rodentium WT or the ⌬picC mutant) C57BL/6 mice were injected intraperitoneally with 20 ␮Ci of [3H]glucosamine (Amersham) and left for 4 h for the metabolic labeling of mucins in the intestine. After 4 h, mice were euthanized and colon sections were scraped with a glass slide to remove the mucin pool in PBS. Collected scrapings were vortexed for 10 min, and the supernatants were spun down (1,000 ⫻ g for 10 min) to remove any cell/tissue debris. The glycoproteins were precipitated using 10% trichloroacetic acid (TCA) and 1% phosphotungstic acid (PTA) (1:1 by volume). Precipitated glycoproteins were suspended in the column buffer and neutralized to a pH of 7.0. Five milliliters of scintillation fluid (UniverSol) was added to the samples, and a scintillation counter was used to measure the total radioactivity in the samples. To separate the high-molecular-weight mucin pool (glycoproteins) from the nonmucin pool, mucin secretions were loaded onto a Sepharose 4B column calibrated with blue dextran (2,000 kDa), thyroglobulin (669 kDa), and BSA (67 kDa) (Amersham). Approximately 1-ml fractions were collected (column volume, 40 ml), and the radioactivity of each fraction was measured using a scintillation counter. The results were analyzed using GraphPad Prism, version 5.0. Profiling T3SS effectors in C. rodentium WT and ⌬picC strains. As previously described (27, 28), bacterial strains were grown in 5 ml LB (with shaking O/N at 37°C). The O/N cultures were subcultured (1:40 dilution) into Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) and grown statically for 6 h at 37°C with 5% CO2. Cultures then were pelleted by centrifugation (13,200 rpm, 4°C, 10 min), and the supernatants were filtered through a 0.2-␮m filter. Proteins secreted from the supernatants were precipitated with 10% TCA (Sigma) O/N at 4°C. Precipitated proteins were pelleted by centrifugation (13,200 rpm, 4°C, 10 min) and resuspended in Laemmli SDS-PAGE buffer. Samples were resolved by SDS–15% PAGE and visualized by Coomassie R-250 blue staining. Cell adhesion assay. CMT-93 (mouse rectal epithelial) cells (ATCC CCL-223) were seeded at a density of 1 ⫻ 105 cells/well (12-well plate) and grown until ⬎90% confluence in DMEM supplemented with 10% fetal bovine serum (FBS; Life Technologies) (37°C, 5% CO2). Before bacterial infection, cells were washed twice with prewarmed DMEM supplemented with 2% FBS (2⫻). Cells were infected with an overnight culture of C. rodentium WT and ⌬picC strains (multiplicity of infection [MOI] of 1:10 and 1:100) for 4 h. Cell monolayers then were washed three times with prewarmed Dulbecco’s PBS (Life Technologies) to remove any nonadherent bacteria. To look at adherent bacteria, 500 ␮l PBS was added to the wells and cells were scraped off (using a cell scraper) and thoroughly mixed by pipetting. Samples were serially diluted in PBS and plated onto MacConkey plates (O/N at 37°C). Commensal enumeration by DAPI DNA staining. Stool samples were collected from uninfected mice (baseline) and mice infected with the C. rodentium WT or ⌬picC strain (days 0, 2, 4, 6, and 8). Samples were homogenized and transferred to 10% neutral buffered formalin to a final concentration of 3.7%. Stool samples were further diluted (1/100 in PBS) and filtered through Anodisc 25 filters (Whatman International Ltd.) with a pore size of 0.2 ␮m. Filter discs were thoroughly dried and stained using Gold antifade reagent containing DAPI (Molecular Probes). DAPI-positive microbes (average of 6 chosen fields/slide) were counted (⫻630 magnification). The percentage of commensals remaining following infection was calculated based on the commensal numbers recovered from infected mice divided by the numbers recovered from uninfected controls. Measuring in vitro TLR2 and TLR4 activation through TLR reporter cells and colorimetric assay. HEK TLR reporter cell lines HEK-Blue hTLR2 and HEK-Blue hTLR4 were purchased from InvivoGen (San Diego, CA, USA). HEK-Blue hTLR2 cells were obtained by cotransfection of hTLR2 and hCD14 coreceptor genes into HEK 293 cells, while HEK-Blue hTLR4 cells were obtained by cotransfection of hTLR4 and hMD-2/CD14 coreceptor genes. These cells also were transfected with the secreted embryonic alkaline phosphatase (SEAP) gene and stably express SEAP under the control of a promoter inducible by NF-␬B and activator protein 1




oxygen-depleted environment for 2 days. Colony counts were normalized to the weight of the tissue collected to obtain CFU/gram. For histology, distal colon tissues were fixed in 10% neutral buffered formalin (Fisher Scientific) overnight and then transferred into 70% ethanol. Tissues were stained with hematoxylin and eosin (H&E) according to standard techniques by the University of British Columbia Histology Laboratory (Vancouver, BC, Canada). To preserve the mucus layer, sections of cecal tissue were fixed in water-free ethanolCarnoy’s fixative (60% ethanol, 30% chloroform, and 10% acetic acid; all reagents were purchased from Fisher Scientific), and after 3 h of storage at 4°C, samples were transferred into 100% ethanol for subsequent processing. Fixed tissues were embedded in paraffin and cut into 5-␮m sections. Tissue pathology scoring. H&E-stained distal colons (n ⫽ 12 for each group) were examined for pathological damage infections by 2 blinded observers as previously described (5, 6, 24). Briefly, tissue sections were assessed for submucosal edema (0, no edema; 3, profound edema), epithelial hyperplasia (scored based on the percent change in crypt height compared to that of the control crypts; 0, no change; 1, 1 to 50% change; 2, 51 to 100%; 3, ⬎100%), mononuclear cell infiltration (0, none; 3, severe), and epithelial integrity (0, no damage; 1, ⬍10 epithelial cells shedding per lesion; 2, 11 to 20 epithelial cells shedding per lesion; 3, maximum damage to epithelial surface as noted by crypt destruction and epithelial ulceration). The maximum possible score was 12. RNA extraction and qPCR. Immediately after euthanization of mice, the distal colon was collected and stored in RNAlater buffer (Qiagen) at ⫺80°C. Total RNA was extracted using a Qiagen RNeasy kit per the manufacturer’s instructions. Total RNA was quantified using a NanoDrop spectrophotometer (ND1000). One microgram of RNA was reverse transcribed using an Omniscript reverse transcription (RT) kit (Qiagen). For quantitative real-time PCR (qPCR), cDNA was diluted 1:5 in RNase- and DNase-free water, and 5 ␮l of diluted cDNA was added to a PCR mixture (10 ␮l of Bio-Rad SYBR green supermix; primers were at a final concentration of 300 nM; final reaction volume, 20 ␮l). 18S rRNA and ␤-actin were used as housekeeping genes. Quantitative PCR was carried out using a Bio-Rad MiniOpticon or Opticon 2 system. The PCR cycling conditions used were denaturation at 94°C for 30 s and then annealing for 30 s at 55°C for tumor necrosis factor alpha (TNF-␣), interleukin-1␤ (IL-1␤), and 18S RNA genes and at 60°C for gamma interferon (IFN-␥), IL-17A, IL-10, IL-6, monocyte chemoattractant protein 1 (MCP-1) and KC genes, followed by an extension step at 72°C for 45 s, for a total of 30 cycles as previously described (24, 25). Data were analyzed/quantified using Gene Expression Macro OM 3.0 software (Bio-Rad). Immunohistochemistry. For immunohistochemical detection of Muc2 and for periodic acid-Schiff/alcian blue (PAS/AB) staining, Carnoy’s fixed tissues were used. For immunostaining, deparaffinized sections were boiled for 20 min in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for antigen retrieval. The sections then were blocked for 20 min using blocking buffer (2% goat serum, 1% bovine serum albumin [BSA], 0.1% Triton X-100, 0.05% Tween 20 in 0.1 M PBS, pH 7.2) to prevent nonspecific antibody binding. Sections were incubated overnight at 4°C with rabbit anti-Muc2 (1:200; Santa Cruz Biotechnologies), C. rodentium-specific Tir (1:5,000; gift from W. Deng), anti-F4/80 (1:200; AbD Serotec), and anti-CD3 (1:100; Abcam). The following secondary antibodies were used: Alexa Fluor 568-conjugated goat anti-rabbit IgG (1:2,000 dilution; Molecular Probes) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:2,000 dilution; Molecular Probes). Fluorescence in situ hybridization (FISH) staining was carried out as previously described (24). Tissues were mounted using ProLong gold antifade (Molecular Probes) containing 4=,6-diamidino-2-phenylindole (DAPI) for DNA staining. Images were captured using a Zeiss AxioImager microscope equipped with an AxioCam HRm camera operating through AxioVision software. Mucin quantification studies. Mucin quantification studies were conducted as previously described (24, 26). Uninfected (only LB


RESULTS

Characterization of C. rodentium mucinase activity: involvement of the Pic homolog PicC. Bioinformatic analysis of the C. rodentium genome revealed the presence of a gene closely related to that encoding Shigella flexneri Pic. Sequence alignment showed the presence of a conserved GDSGS serine protein domain, N-terminal sequences, and high sequence homology (⬃80% to S. flexneri Pic), so we named this homolog PicC (see Fig. S1 in the supplemental material). Since previously reported Pic homologs have been shown to possess mucinase activity (10–12), concentrated


FIG 1 (A) Characterization of mucinase activity of picC in C. rodentium. Zones of mucin (bovine submaxillary mucin [BSM]) clearance are visible in the stacking region of the SDS-PAGE gel (boxed area), indicative of mucinase activity. Deletion of picC (the C. rodentium ⌬picC mutant) results in loss of mucinase activity, which is restored after complementation of picC (C. rodentium ⌬picC ⫹ pPicC). Incubation of samples with PMSF significantly reduced mucinase activity, with the exception of the C. rodentium ⌬picC mutant (no mucinase activity). (B) Reduced mucinase activity also was seen with a C. rodentium strain expressing a PicC protein containing a mutation in the serine-protease active site, S258I. Lanes labeled BSM represent untreated mucin. The gels are stained with PAS.

supernatants from C. rodentium WT, ⌬picC, and complemented ⌬picC ⫹ pPicC strains were tested for potential mucinase activity. The samples were incubated with bovine submaxillary mucin (BSM), loaded onto 7% SDS-PAGE gels, and stained using a PAS glycoprotein staining kit. As shown in Fig. 1A, while the lane carrying untreated BSM showed high-molecular-weight bands indicating intact mucin, these bands were absent following treatment with WT C. rodentium supernatant, suggesting that the supernatant contained significant mucinase activity. Notably, when BSM was treated with C. rodentium ⌬picC supernatant, high-molecular-weight bands still were evident, suggesting that the majority of the mucinase activity observed following treatment with C. rodentium WT supernatant could be attributed to the presence of Pic. Correspondingly, restoration of Pic in supernatants collected from the C. rodentium ⌬picC ⫹ pPicC strain again led to the loss of high-molecular-weight mucin bands, similar to that seen with WT C. rodentium, indicating a restoration of mucinase activity. Interestingly, the mucinase activity of PicC was significantly attenuated upon treatment with the protease inhibitor phenylmethylsulfonyl fluoride (PMSF), suggesting that the serine protease motif was involved in the mucinolytic digestion of the mucin. To further assess the role of the serine protease motif in the mucinase activity of PicC, we generated an S258I mutant (catalytic residue serine changed to isoleucine through site-directed mutagenesis) and confirmed that the mutation caused the loss of mucinase activity in vitro (Fig. 1B). C. rodentium ⌬picC mutant is highly virulent in infected mice. Since most bacterial pathogens that express pic homologs (e.g., S. flexneri, EAEC, and UPEC) (11, 12) are human specific,


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(AP-1). Thus, stimulation of hTLR2 and hTLR4 leads to the production of extracellular SEAP in the culture medium that is proportional to the level of NF-␬B/AP-1 activation. Cells were grown in high-glucose DMEM (HyClone, Logan, UT, USA) with 2 mM L-glutamine, 10% heat-inactivated FBS (HyClone), 100 ␮g/ml Normocin (InvivoGen), and selective antibiotics (1⫻ HEK-Blue selection; InvivoGen) according to the manufacturer’s instructions. The activation of TLR2 and TLR4 was assessed by measuring SEAP activity using the Quanti-Blue (InvivoGen) colorimetric assay. Reporter cells were seeded in a 96-well plate (BD Bioscience, Mississauga, ON, Canada) at a density of 5 ⫻ 104 cells per well in 100 ␮l medium. The next day, cells were treated with fresh media (without selective antibiotics) containing C. rodentium WT and ⌬picC strains for 4 h. Cells treated with culture medium only, TLR2 ligand Pam3CSK4 (10 ng/ml; InvivoGen), or TLR4 ligand lipopolysaccharide (LPS; Escherichia coli K-12; 100 ng/ml; InvivoGen) served as the negative and positive controls for each corresponding TLR stimulation. The experiments were carried out three times independently, and all conditions in each experiment were tested in triplicate. After 4 h of incubation, culture media were collected and centrifuged to remove bacteria. The supernatants (20 ␮l) then were incubated with Quanti-Blue solution (180 ␮l) in a 96-well flat-bottom plate at 37°C for 16 to 18 h. The color change of the substrate solution was quantified by optical density (at 655 nm) measurement using a SpectraMax 384 plus plate reader (Molecular Devices, Sunnyvale, CA, USA), which corresponds to the activation of NF-␬B/AP-1 by specific TLR stimulation. In vivo FITC-dextran intestinal permeability assay. As previously described (24, 25), mice used in the study were administered 150 ␮l of 80 mg/ml fluorescein isothiocyanate (FITC)-dextran (FD4; Sigma-Aldrich) by oral gavage 4 h prior to euthanizing mice. Mice were anesthetized using isoflurane, and blood samples (⬃500 ␮l/mouse) were collected using cardiac puncture. The collected samples were immediately transferred to 3% acid-citrate dextrose (ACD) containing 20 mM citric acid, 110 nM sodium citrate, 5 mM dextrose (protocol provided by Harald Schulze, Shivdasani Laboratory, DFCI). The serum concentration of the FITC-dextran was measured using a fluorometer (PerkinElmer Life Sciences) (excitation wavelength of 490 nm and emission wavelength of 530 nm). Transmission experiments. To examine the ability of WT and ⌬picC strains of C. rodentium to transmit between hosts, index mice were infected with 0.1 ml of the O/N cultures (⬃2.5 ⫻ 108 CFU) by oral gavage. On 6 dpi, index mice were transferred to a clean cage and secondary (naive) mice were added. After 48 h of cohousing, all mice were euthanized and C. rodentium counts in the formerly naive mice were enumerated in the distal colon (primary site for C. rodentium infection). Distal colon colonization was used to assess the transmission success of each strain from index mice to naive mice. Statistical analysis. All of the results shown in this study are plotted as the mean values with standard errors of the means (SEM). Statistical analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA) using nonparametric MannWhitney t tests. A P value of ⱕ0.05 was indicative of statistical significance. Statistical analysis on survival curves was performed using the logrank (Mantel-Cox) test.

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mutant. (A) Weight loss of WT- and ⌬picC mutant-infected mice, plotted as a percentage of initial body weight and normalized to day 0 body weight. Error bars represent SEM, and asterisks indicate significant differences (***, P ⬍ 0.0005) by the Mann-Whitney test. (B) Survival curve of C57BL/6 mice following WT and ⌬picC mutant infection. P values (0.0253) are from the logrank test and indicate a statistically significant difference between the survival curves. Results are representative of 3 independent experiments (12 mice per group).

the lack of appropriate animal models has, until now, prevented the investigation of the in vivo biological role of Pic. Since C. rodentium is a natural mouse pathogen, we decided to investigate the biological role of PicC, anticipating that the reduced mucinase activity of the ⌬picC C. rodentium strain would dramatically impair its pathogenesis. Surprisingly, when we compared C57BL/6 mice orally infected with WT C. rodentium to mice infected with the C. rodentium ⌬picC mutant and monitored their body weights and survival rates over a 2-week infection period, we discovered that the C. rodentium ⌬picC mutant was strikingly virulent in infected mice. Interestingly, C. rodentium ⌬picC mutant-infected mice steadily lost weight starting by 8 dpi, and their weight loss continued until 14 dpi (Fig. 2A). In contrast, WT C. rodentium-infected mice displayed only a modest weight loss between 2 and 4 dpi, following which their weights stabilized and remained stable throughout the remaining infection time course. While all mice survived WT C. rodentium infection until they were euthanized at 14 dpi, the C. rodentium ⌬picC mutant proved much more virulent. Depending on the infection, 50 to 80% of C. rodentium ⌬picC mutant-infected mice required early euthanization between 10 and 13 dpi due to significant weight loss as well as other signs of morbidity, such as hunched posture, inactivity, and piloerection of the fur (Fig. 2B). Based on the severe disease suffered by C. rodentium

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FIG 2 C57BL/6 mice exhibit dramatic susceptibility to the C. rodentium ⌬picC

⌬picC mutant-infected mice from 10 dpi onward, further studies focused on 6 and 8 dpi. C. rodentium ⌬picC mutant colonizes the mouse intestine more heavily than WT C. rodentium. To better define the basis for the exaggerated lethality of the ⌬picC C. rodentium strain, we enumerated pathogen burdens in the colon and cecal tissues (considered adherent or directly infecting the epithelium) as well as those of C. rodentium found in the luminal contents of both mouse groups at 8 dpi, as this time point was just prior to the exaggerated morbidity seen in the C. rodentium ⌬picC mutantinfected mice. We found that C. rodentium ⌬picC mutant-infected mice carried significantly greater pathogen burdens in their cecal and distal colon tissues (5- to 20-fold higher) than mice infected with WT C. rodentium. Interestingly, the luminal pathogen burdens were not significantly different between groups, suggesting that the increased C. rodentium ⌬picC mutant burdens were predominantly tissue adherent (Fig. 3A). Since C. rodentium infection is most prominent in the distal colon, we examined pathogen localization at this site by staining for the translocated intimin receptor (Tir). Tir is a T3SS effector that is injected into infected epithelial cells; therefore, it is a selective marker of C. rodentium infection (7). As expected, WT C. rodentium colonization was found predominantly localized to the mucosal surface of the distal colon, with a patchy localization. In contrast, staining for Tir in mice infected with the C. rodentium ⌬picC mutant revealed more positive staining that was continuously distributed over a larger mucosal surface. Interestingly, the C. rodentium ⌬picC mutant also showed deeper penetration into colonic crypts than WT C. rodentium (Fig. 3B and C), with significant staining not only directly adherent to the epithelium but also within the crypt lumen. Thus, despite losing the mucinase activity of PicC, the C. rodentium ⌬picC mutant appeared to reach the intestinal epithelium and colonize the intestinal crypts of infected mice even better than the wild type. C. rodentium ⌬picC mutant-infected mice exhibit exaggerated mucosal damage and inflammation. We next investigated the intestinal pathology that developed in mice infected with the C. rodentium ⌬picC mutant compared to that in mice infected with WT C. rodentium. We noted that C. rodentium ⌬picC mutant-infected mice suffered significantly higher intestinal pathology scores than WT C. rodentium-infected mice, as reflected by increased crypt epithelial cell hyperplasia and inflammatory cell infiltration and greater damage to the epithelial surface in the distal colon (Fig. 4A and B). As expected, WT C. rodentium infection led to elevated gene transcript levels of several proinflammatory cytokines (IL-17A, IFN-␥, IL-10, TNF-␣, IL-6, and IL-1␤) and chemokines (MCP-1 and KC) in the distal colon; however, consistent with the worsened pathology, the transcript levels for these genes were significantly higher in tissues from C. rodentium ⌬picC mutant-infected mice, suggesting that C. rodentium ⌬picC mutant-infected mice developed exaggerated intestinal inflammation compared to WT C. rodentium-infected mice (Fig. 4C). Upon complementation of picC into the C. rodentium ⌬picC mutant (yielding the ⌬picC ⫹ pPicC strain), the WT C. rodentium infectious phenotype was restored for body weight loss, mortality, pathogen burdens/location, and tissue pathology (see Fig. S2 in the supplemental material), suggesting that the deletion mutation was in frame and the observed phenotype in the deletion mutant was not caused by a polar effect. It also confirmed the specific role of PicC protein in the observed phenotypes.


burdens at 8 dpi. (B) Representative images from distal colon showing C. rodentium localization as seen via Tir (C. rodentium-specific effector; red) and DAPI (counterstain; blue) staining. Original magnification, ⫻200. WT C. rodentium is seen mostly on the epithelial surface (inset, top row, ⫻630), whereas the C. rodentium ⌬picC mutant penetrates deeper into the crypts (inset, bottom row, ⫻630). (C) Quantitative analysis looking at Tir-positive crypts/100 crypts. Analysis was done at the original magnification, ⫻200, and represents averages for distal colons from at least 9 different mice/group. Error bars represent SEM, and asterisks indicate significant difference (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.0005) by the Mann-Whitney test.

C. rodentium-infected mice display exaggerated epithelial barrier disruption and increased translocation of pathogenic and commensal bacteria. To explore the basis for the exaggerated inflammation, pathology, and increased morbidity/mortality seen in mice infected by the C. rodentium ⌬picC mutant, we assayed epithelial barrier function in the two groups of infected mice. FITC-dextran was orally administered to C. rodentium WT and ⌬picC mutant-infected mice at 6 dpi, with the levels of FITCdextran translocated into the serum measured. C. rodentium ⌬picC mutant-infected mice had significantly higher levels of FITC-dextran in their serum than WT C. rodentium-infected mice, indicating increased damage to the intestinal epithelial barrier following infection with the C. rodentium ⌬picC mutant (Fig. 5A). To assess the impact of the exaggerated barrier disruption suffered by C. rodentium ⌬picC mutant-infected mice, we examined pathogen burdens at systemic tissue sites (liver, spleen, and MLN). While there was a trend toward higher burdens in the spleens of mice infected with the C. rodentium ⌬picC mutant than in the spleens of mice infected with WT C. rodentium, significantly more C. rodentium organisms were recovered from the liver and MLN


of C. rodentium ⌬picC mutant-infected mice (Fig. 5B). We also assessed whether infection with the C. rodentium ⌬picC mutant affected the translocation of commensal microbes to the MLN of infected mice. We collected MLN from mice at 8 dpi and enumerated the commensal CFU by plating on commensal-specific media under controlled anaerobic conditions. Notably, there were significantly higher commensal numbers recovered from the MLN of C. rodentium ⌬picC mutant-infected mice (Fig. 5C). Collectively, our data indicate that infection with the C. rodentium ⌬picC mutant not only leads to increased epithelial barrier dysfunction but also results in higher systemic translocation of both pathogenic and commensal bacteria, potentially contributing to the increased mortality rates in these mice. The in vivo impact of PicC on C. rodentium virulence does not reflect its effects on mucins. While the mucinase activity of PicC did not appear to be required for C. rodentium to colonize the murine intestine, we decided to clarify whether the loss of its serine protease activity was the basis for the exaggerated pathology suffered during infection by the C. rodentium ⌬picC mutant. We confirmed that the S258I mutation caused the loss of mucinase activity in vitro (Fig. 1A). However, when we tested the point


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FIG 3 C. rodentium ⌬picC mutant-infected mice carry heavier pathogen burdens. (A) Adherent (distal and cecal tissues) and nonadherent luminal C. rodentium

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mutant in vivo, we noted a phenotype similar to that seen with WT C. rodentium, with roughly similar pathogen burdens (Fig. 6A and B), tissue pathology (Fig. 6C and D), and epithelial barrier dysfunction (Fig. 6E). These results indicate that the exaggerated pathology seen in the absence of PicC reflects actions of Pic other than its serine protease activity. Aside from mucinase activity, Pic proteins from several pathogens (EAEC, UPEC, and Shigella flexneri) act as mucin secretagogues (16). To test whether the loss of PicC from C. rodentium affected mucus secretion during infection, we measured mucin production/release during infection. As shown in Fig. 7A and B, while C. rodentium infection was associated with an increase in mucin secretion, the quantitative levels did not differ significantly between mice infected with WT C. rodentium and mice infected with the ⌬picC mutant (Fig. 7C), indicating that in our model system, PicC did not exhibit significant mucin secretagogue activity. C. rodentium ⌬picC mutant shows increased aggregation in vivo. Considering our data indicating that PicC modulates C. rodentium pathogenesis but not through its impact on the host, at

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least in terms of mucin degradation or release, we considered literature indicating that autotransporters also can affect the structure and function of the bacteria that express them (29–32). Therefore, we tested whether PicC altered the structure and/or function of C. rodentium, starting with a functional assessment of its type III secretion system. The secretion-deficient C. rodentium ⌬escN mutant was used as a control. Interestingly, lysates of C. rodentium WT and ⌬picC strains looked similar, as did the profiles of the T3SS effectors secreted by these strains when grown in DMEM (see Fig. S3A in the supplemental material). We also tested if there were any differences in epithelial adherence of C. rodentium WT and ⌬picC strains using the CMT-93 mouse rectal epithelial cell line. Confluent CMT-93 cells were infected (MOI of 10 and 100) with the above-mentioned strains, and adherent bacterial counts were enumerated through plating. No differences were seen between C. rodentium WT and ⌬picC strains, suggesting that there were no intrinsic differences in in vitro infectivity of these strains that could account for the increased virulence displayed by the C. rodentium ⌬picC mutant (see Fig. S3B).




FIG 4 Heightened histopathological damage and increased proinflammatory cytokines in C. rodentium ⌬picC mutant-infected mice. (A) Representative H&E-stained distal colon (original magnification, ⫻100) at 8 dpi for WT- and ⌬picC mutant-infected mice. Asterisks represent damage to the intestinal epithelial surface, as seen by ruffling and loss of crypt structure and/or epithelial integrity. Arrows point to increased edema and infiltration of immune/inflammatory cells. (B) Cumulative tissue pathology damage scores from distal colon of WT- and ⌬picC mutant-infected mice. Scoring was done by blinded observers and represents averages from 9 mice/group. (C) Quantitative PCR of proinflammatory cytokine and chemokine genes in the distal colon of the WT- and ⌬picC mutant-infected mice at 8 dpi. Note that WT C. rodentium infection resulted in increased gene transcript levels of tested chemokines and cytokines; however, there was a greater induction seen with C. rodentium ⌬picC mutant infection, indicative of hyperinflammatory responses. Errors bars represent SEM from three independent experiments and at least 9 mice/group. P ⬍ 0.01 (**) and P ⬍ 0.05 (*) by the Mann-Whitey test.


We next investigated the status of the C. rodentium ⌬picC mutant in vivo. It was notable that despite the heavier adherent burdens seen in C. rodentium ⌬picC mutant-infected mice, luminal burdens were not increased to a similar degree. Upon examination of infected tissues using immunostaining for Tir and Muc2, the ⌬picC strain was found abnormally clustered within the adherent mucus layer and in the colonic crypts, as though they were aggregating together to form microcolonies. In contrast, WT C. rodentium organisms were observed primarily directly adhering to the mucosal surface without any signs of aggregation (Fig. 8A). To investigate if the C. rodentium ⌬picC mutant-infected mouse tissues also show abnormal pathogen-commensal interactions, we conducted dual FISH staining on infected tissues. We used a Texas red-conjugated EUB338 probe that recognizes the majority (99%) of gut bacteria and an Alexa Fluor 488-conjugated GAM42a probe that detects Gammaproteobacteria, which, in infected tissues, are almost entirely C. rodentium (24). Consistent with Tir and Tir/Muc2 dual staining, there was patchy distribution of WT C. rodentium on the epithelial surface (yellow, EUB338⫹ GAM42a⫹), whereas commensal microbes (red, EUB338⫹ GAM42a⫺) could be seen in the lumen, distant from the epithelial surface. In contrast, dual FISH staining on C. rodentium ⌬picC mutant-infected tissues revealed multispecies microcolonies comprising commensal (non-C. rodentium) bacteria (stained red) mixed with C. rodentium (yellow) in close proximity to the mucosal surface (Fig. 8B). Based on this finding, we assessed commensal microbe numbers in the colons of WT and ⌬picC mutant-


infected mice. As previously shown (33), commensal microbes were rapidly depleted from the colon during infection by WT C. rodentium. In contrast, this depletion was significantly impaired in mice infected with the C. rodentium ⌬picC mutant (see Fig. S3D in the supplemental material), with the increased commensal numbers potentially explaining the increased translocation of commensal microbes seen in these mice (Fig. 5C). Taken together, these results indicate that PicC affects bacterium-bacterium interactions, such that in its absence, there is increased aggregation of C. rodentium with itself as well as with commensal microbes. C. rodentium ⌬picC mutant is impaired in transmitting infection to naive mice. Interestingly, as mentioned before, despite heavier intestinal burdens, mice infected with the C. rodentium ⌬picC mutant showed reduced pathogen numbers in their stool (see Fig. S3C in the supplemental material). Speculating that the heightened aggregation seen with the C. rodentium ⌬picC mutant impacts the ability to exit the intestine and spread to new hosts, we next analyzed the ability of the C. rodentium ⌬picC mutant to transmit to new hosts using a strategy previously outlined by Wickham et al. (34). To examine this, transmission experiments were conducted (as described in Materials and Methods). Transmission was defined as the percentage of naive mice that were infected/colonized upon exposure to infected index mice. Interestingly, we noted that the C. rodentium ⌬picC mutant’s ability to transmit to new hosts (secondary mice) was much lower than that of WT C. rodentium (⬃11% versus ⬃67%, respectively) (Fig. 9A).


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FIG 5 C. rodentium ⌬picC mutant-infected mice have impaired epithelial barrier integrity and increased translocation of pathogenic and commensal bacteria. (A) C. rodentium ⌬picC mutant-infected mice display greater FITC-dextran flux across their intestinal barrier on 6 dpi than WT C. rodentium-infected mice. The bar graph shows the quantity of FD4 in serum and represents an average of 9 mice/group. UN, uninfected C57BL/6 mice. (B) Quantification of C. rodentium burdens recovered from systemic sites (liver, spleen, and MLN) on 8 dpi. (C) Recovery of viable commensal bacteria from MLN under controlled anaerobic conditions and commensal-specific media. Errors bars represent SEM from 3 independent experiments. P ⬍ 0.0005 (***), P ⬍ 0.01 (**), and P ⬍ 0.05 (*) by the Mann-Whitney test.

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and MLN, for WT C. rodentium- and S258I C. rodentium (catalytic mutant with no mucinase activity)-infected mice. (B) WT and S258I mutant burdens recovered from distal colon, cecum, and lumen at 8 dpi. (C) Representative H&E-stained distal colon (original magnification, ⫻100) at 8 dpi. (D) Histologic damage scores from distal colon of WT- and S258I mutant-infected mice. Pathology scoring included submucosal edema, hyperplasia, goblet cell depletion, damage to epithelial integrity, and PMN infiltration. Scores were determined by 2 independent observers under blinded conditions. (E) FITC-dextran intestinal permeability assay for WT- and S258I mutant-infected C57BL/6 mice. The baseline FITC-dextran flux for uninfected C57BL/6 mice also is shown. Uninfected and infected mice were gavaged with FITC-dextran, and serum was collected using cardiac puncture. Serum FD4 levels were measured. Asterisks represent statistically significant differences (*, P ⬍ 0.05 by the Mann-Whitney test); ns, not significant. Errors bars show SEM from 3 independent infections and 9 mice/group.

Furthermore, secondary mice that were colonized by the C. rodentium ⌬picC mutant carried consistently lower pathogen burdens than mice exposed to WT C. rodentium (Fig. 9B). Examining distal colon colonization, we found that 5 out of 8 naive (secondary) mice exposed to WT C. rodentium-infected index mice showed distal colon colonization (⬃106 CFU/g), whereas only 1 out of 8 secondary mice exposed to a C. rodentium ⌬picC mutant-infected index mouse was colonized. Overall, these findings suggested that the C. rodentium ⌬picC mutant is impaired in successfully transmitting to new hosts. C. rodentium ⌬picC mutant exhibits a pronounced RDAR morphotype in vitro. To further explore the impact of PicC on C. rodentium structure and function, we tried growing the C. rodentium strains on Congo red plates (LB-agar plates lacking salt but supplemented with Congo red dye). This medium allows the assessment of bacterial growth under limiting nutrient conditions

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and the production of what is termed the RDAR morphotype. This phenotype reflects an aggregative and resistant physiology that has been linked to survival in nutrient-limited environments and previously was shown to indicate the production of extracellular components, such as curli fimbriae and cellulose (35–38). Notably, the C. rodentium ⌬picC mutant exhibited a more pronounced RDAR morphotype than the WT strain (Fig. 8C). While this phenotype is best known and studied outside of hosts, its expression within the gut could explain the aggregative nature of the C. rodentium ⌬picC mutant in vivo. We also examined in vitro biofilm formation and cellulose production in WT and ⌬picC strains of C. rodentium and found that the C. rodentium ⌬picC mutant displayed moderately but significantly higher biofilm formation (crystal violet dye assay) and cellulose production (as assessed by Congo red binding assay and the calcofluor binding assay) than WT C. rodentium (see Fig. S4 in the supplemental




FIG 6 Mucinase activity of PicC is not essential for intestinal colonization. (A) C. rodentium burdens enumerated at 8 dpi from systemic sites, i.e., liver, spleen,


material). While there are other bacterial products, such as curli, that could be differentially expressed by the C. rodentium ⌬picC mutant that could explain the aggregative nature, rather than examining each one, we decided to instead focus on the effects such changes might have on the ability of the host’s immune system to recognize C. rodentium. C. rodentium ⌬picC mutant induces significantly greater TLR2 activation than WT C. rodentium. Based on the aberrant in vivo and in vitro behavior of the C. rodentium ⌬picC mutant, and since SPATEs have been shown to modulate inflammatory responses, we tested whether the exaggerated inflammatory response elicited against the C. rodentium ⌬picC mutant reflected an abnormal activation of the host immune system. To investigate this hypothesis, we used HEK293 cell lines expressing either TLR2 or TLR4, as these are the primary innate receptors that recognize C. rodentium. These cell lines then were exposed to diluted over-


night cultures of WT C. rodentium (contains PicC) and the C. rodentium ⌬picC mutant (no PicC), and the activation of these TLRs was measured using a SEAP (secreted alkaline phosphatase) reporter assay. Interestingly, we noted that while there was no difference in activation of TLR4 by the two strains (Fig. 10A), TLR2 showed significantly more activation with the C. rodentium ⌬picC mutant than with WT C. rodentium (Fig. 10B). Notably, preincubating an exogenous TLR2 ligand, Pam3CSK4, with WT C. rodentium and the C. rodentium ⌬picC mutant had no effect on the ligand’s ability to activate TLR2 signaling (data not shown). Interestingly, the differences in TLR2 activation between WT and ⌬picC strains of C. rodentium were lost upon lysing the bacteria (Fig. 10B) or when cell-free bacterial supernatants were tested (data not shown). Overall, these results suggest that PicC modulates the surface structure of C. rodentium in a manner that typically limits TLR2 activation.


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FIG 7 C. rodentium PicC is not a potent mucus secretagogue in vivo. (A) Representative dual immunofluorescence staining in Carnoy’s fixed distal colon at 8 dpi. Mucin Muc2 (green), Tir (red; shows the association of C. rodentium with Muc2-positive crypts), and DAPI (nuclei; counterstain) are shown. The white arrowhead indicates secreted/luminal mucus, and the yellow arrowhead shows representative Muc2-filled goblet cells. (B) Representative PAS/AB staining of Carnoy’s fixed distal colon collected from WT- and ⌬picC mutant-infected mice. Arrows point to the secreted mucus. (C) Quantification of total mucus secretion in WT- and ⌬picC mutant-infected mice at 6 dpi. Plot shows the counts per minute (cpm) in individual column fractions containing radioactive 3H activity after total secretions were subjected to gel filtration chromatography (Sepharose 4B column). This plot represents averages from 6 mice/group and 2 independent infections. The inset shows a bar graph representation of total counts per minute in void (Vo) volume fractions (points 10 to 20, large, mucin glycoproteins) plotted as a percentage of uninfected samples. Vt represents fractions eluting nonmucin glycoproteins.

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The exaggerated colitis caused by the C. rodentium ⌬picC mutant is largely TLR2 dependent. The higher (in vitro) stimulation of TLR2 by the C. rodentium ⌬picC mutant than by WT C. rodentium suggested a mechanism by which the C. rodentium ⌬picC mutant could cause exaggerated colitis. To further investigate this finding in vivo, we infected Tlr2⫺/⫺ mice with WT C. rodentium and the ⌬picC mutant to clarify whether the exaggerated pathology caused by the C. rodentium ⌬picC mutant would be reduced in the absence of TLR2. While Tlr2⫺/⫺ mice already have been shown to be moderately more susceptible to WT C. rodentium infection than C57BL/6 mice, the susceptibility tends to be exhibited only at later stages of infection. Notably, at 8 dpi we found that C. rodentium ⌬picC mutant-infected Tlr2 ⫺/⫺ mice

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showed magnitudes of histological and macroscopic damage similar to that of Tlr2 ⫺/⫺ mice infected with WT C. rodentium. Weight loss and histologic measures of colonic pathology also were similar between WT and ⌬picC mutant C. rodentium infection (Fig. 10C, D, and E). Taken together, these findings indicate that the autotransporter PicC impacts C. rodentium virulence primarily through its role in controlling innate recognition of C. rodentium rather than its actions as a mucinase. DISCUSSION

SPATEs are large extracellular proteases secreted by Gram-negative bacteria that have been implicated in bacterial pathogenesis. At present, more than 25 SPATEs with phylogenetically diverse




FIG 8 C. rodentium ⌬picC mutant forms microcolony-like structures in vivo. (A) Representative Muc2/Tir dual staining profile in distal colon. White arrowheads show localization of WT C. rodentium mostly on the epithelial surface in a single layer. Yellow arrowheads point to clustering of the C. rodentium ⌬picC mutant close to epithelial surfaces and within the colonic crypts. Original magnification, ⫻630. Blue, DAPI counterstain. (B) Dual FISH staining in distal colon using EUB338 DNA probe (stains all bacteria red) and GAM42 DNA probe (stains Gammaproteobacteria green; C. rodentium belongs to this family). Yellow, C. rodentium; red, commensals; L, lumen. White arrows point to microcolony-like structures on the mucosal surface in C. rodentium ⌬picC mutant sections not seen in WT C. rodentium sections. Original magnification, ⫻200. The last panel is a magnified image (⫻630) of the white boxed area showing a microcolony intermixed with commensals and the C. rodentium ⌬picC mutant. (C) RDAR colony morphology of WT C. rodentium (top) and the C. rodentium ⌬picC mutant (bottom) on Congo red plates incubated at 25°C for 5 days. Note the differences in colony morphology between WT C. rodentium and the C. rodentium ⌬picC mutant and the dry, rough phenotype localized in the central region of the C. rodentium ⌬picC mutant colonies.


functions have been identified. In the last few decades, numerous studies have performed in vitro characterization of SPATEs secreted by enteric pathogens such as Shigella, EPEC, EAEC, and UPEC (10–12). While SPATEs have been shown to impact bacterial virulence through cytotoxic effects on intestinal epithelial cells (class 1 SPATEs) and immunomodulatory properties (class 2 SPATEs), virtually all of these studies have been performed under in vitro, in situ, or ex vivo conditions (14, 39, 40). There is now significant interest in understanding how these proteases interact with the host, but the lack of appropriate animal models has limited our understanding of how SPATEs can modulate bacterial pathogenesis in vivo. C. rodentium is widely used as an in vivo model for studying A/E infections and is a natural mouse pathogen. Recently, a study was published reporting 19 putative autotransporters in C. rodentium. Five of these were SPATES showing close homology to previously reported/characterized SPATES expressed by pathogenic E. coli strains (8). A putative homolog of Pic, a class 2 SPATE, also was reported (ROD_p1411; YP_003368482.1), confirming our bioinformatics analysis. Pic, a class 2 SPATE, has been shown to target extracellular targets that include glycoproteins in the mucus layer and leukocyte surface glycoproteins involved in diverse immune functions, suggesting that class 2 SPATEs, such as Pic, are bacterial virulence factors with immunomodulatory properties (17, 18). Our initial interest in studying PicC was in regard to its predicted mucinolytic activity. Previous studies have shown that C. rodentium can bind to intestinal mucins, potentially as an early



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FIG 9 C. rodentium ⌬picC mutant shows reduced transmission to new hosts. (A) Bar graph showing percent transmission (i.e., percentage of naive secondary mice getting colonized from exposure to index mice infected with the WT or C. rodentium ⌬picC mutant). The C. rodentium ⌬picC mutant displayed reduced transmission to new hosts compared to WT C. rodentium. (B) Viable C. rodentium (CFU) recovered from distal colon of secondary mice. Note that index mice were mixed with naive (secondary) mice on 6 dpi and cohoused for 48 h, after which all mice were euthanized. Results shown are representative of 2 independent infections. The asterisk indicates statistical significance (*, P ⬍ 0.05 by the Mann-Whitney test).

step in its colonization of host intestines (41). While it is clear that C. rodentium is able to cross the mucus layer to reach and infect the underlying intestinal epithelium, we previously showed that the presence of the mucin Muc2, the main constituent of the intestinal mucus, significantly delayed the rate at which C. rodentium reached and infected the intestinal epithelium. Moreover, Muc2⫺/⫺ mice were highly susceptible to C. rodentium infection, carrying higher pathogen burdens and suffering exaggerated intestinal pathology and epithelial barrier damage (24). Thus, Muc2 is a protective host barrier that C. rodentium must overcome; however, little is known about how this and other bacterial pathogens accomplish this task. While motile microbes like Salmonella (42) and Campylobacter species (43) likely swim through mucus to reach the epithelium, nonmotile pathogens, such as Shigella and C. rodentium, potentially use proteases to degrade the mucus layer (44). We demonstrated that WT C. rodentium is able to cleave bovine submaxillary mucin, whereas the loss of PicC significantly reduced this ability. In order to determine the role of PicC in C. rodentium pathogenesis, we infected C57BL/6 mice with WT and ⌬picC strains of C. rodentium. Surprisingly, the ⌬picC C. rodentium mutant was far more virulent than the WT, heavily colonizing the host’s intestine and causing exaggerated inflammation and tissue damage that often required early euthanization of infected mice. These results indicate that C. rodentium does not require PicC to colonize, suggesting either that other proteases can compensate in the absence of PicC to degrade the intestinal mucus barrier or that C. rodentium is able to colonize without overtly degrading the mucus layer. At present, we cannot rule out either possibility. Another striking finding was the heavier pathogen burdens seen in the ⌬picC C. rodentium mutant-infected mice. Notably, these mice only showed increased tissue-adherent pathogen burdens, with the luminal burdens being similar to WT levels. Upon further investigation, it was found that the tissue-adherent burdens reflected aggregates of the ⌬picC C. rodentium mutant on the epithelial surface as well as in the overlying mucus layer and deep in colonic crypts. While showing no overt defect in infecting the intestinal epithelium, the aggregation of the ⌬picC C. rodentium mutant appeared to impair the pathogen’s ability to leave the mucus layer and reach the lumen. Moreover, the ⌬picC C. rodentium mutant showed a severe impairment in its ability to transmit from an infected host to new hosts, suggesting that PicC plays an important role in pathogen transmission/shedding. An increased propensity of the ⌬picC C. rodentium mutant to aggregate in vivo may be linked to its in vitro ability to produce the RDAR morphotype. As previously suggested, the RDAR phenotype represents an aggregative state due in part to the production of extracellular matrix components, such as curli and cellulose (35–38). The C. rodentium ⌬picC mutant’s RDAR morphotype (potentially linked to its ability to produce higher levels of cellulose and a modest increase in its ability to form biofilms in vitro) may be analogous to the pathogen’s ability to form in vivo microcolony-like structures associated with the intestinal mucus layer, thereby decreasing the ability of the pathogen to shed into the lumen. It should be noted, however, that our current understanding of the RDAR morphotype is far from complete, and almost nothing is known about how it may be expressed in an in vivo situation. While the exaggerated colitis suffered during ⌬picC C. rodentium mutant infection may in part reflect the heavier pathogen

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exposed to supernatants from either live or heat-killed (HK) C. rodentium WT and ⌬picC mutant for 4 h. The C. rodentium ⌬picC mutant significantly increased the stimulation of TLR2 compared to WT C. rodentium. HK bacteria maxed out the TLR4 and TLR2 stimulation under tested conditions. Error bars indicate SEM. P ⬍ 0.0005 (***) and P ⬍ 0.01 (**) by Mann-Whitney test; ns, not significant. Values represent the means from three independent experiments. (C) Body weights of Tlr2⫺/⫺ mice infected with WT C. rodentium and the C. rodentium ⌬picC mutant, plotted as a percentage of starting body weight. (D) H&E-stained representative images showing histological damage to distal colon of WT- and ⌬picC mutant-infected Tlr2⫺/⫺ mice at 8 dpi (original magnification, ⫻200). (E) Pathology scores comparing damage to distal colon in Tlr2⫺/⫺ mice infected with WT C. rodentium and the C. rodentium ⌬picC mutant. Each bar represents an average score from 6 tissues, scored under blinded conditions. Error bars represent SEM, and results from 3 independent experiments are pooled.

burdens, the dramatic weight loss and high mortality rates suffered by infected mice suggested that other factors are at play. In previous studies, using in vitro reporter assays, we showed that like other Gram-negative bacteria, C. rodentium predominantly activates TLR2 and TLR4. These findings were supported by in vivo studies, where TLR4 signaling was highly procolitic during C. rodentium infection (5), whereas TLR2 signaling drove both proinflammatory as well as tissue-protective effects, including promoting the expression of the cytokine IL-6 (6). Notably, we found that the C. rodentium ⌬picC mutant hyperstimulates the innate receptor TLR2 but induces normal activation of TLR4. Moreover, the ability of the C. rodentium ⌬picC mutant to cause greater damage than wild-type C. rodentium was lost in the TLR2-deficient mice, suggesting that TLR2 signaling plays a role in regulating the pathology seen during C. rodentium ⌬picC mutant infection. In fact, it is possible that exaggerated TLR2 activation plays a role in lim-

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iting the survival of the C. rodentium ⌬picC mutant within the lumens of infected C57BL/6 mice. This finding suggests that PicC directly or indirectly helps C. rodentium evade the host immune system. In fact, we recently used a novel mouse model of Campylobacter jejuni infection to show that the capsule that surrounds this pathogen played an important role in limiting the innate recognition of C. jejuni, potentially as a means to evade host inflammatory/antimicrobial responses. While the ⌬kpsM (capsule) mutant strain also triggered exaggerated gut inflammation, it stimulated both TLR2 and TLR4 reporter cell lines to a significantly higher degree than the wild-type 81-176 strain, since LPS as well as lipoproteins and other TLR2 ligands were more exposed (45). The C. rodentium ⌬picC mutant shows enhanced activation of TLR2 only, which suggests that the ⌬picC mutant shows increased expression of TLR2 ligands. Interestingly, the RDAR morphotype is associated with increased ex-




FIG 10 PicC plays a role in innate immune recognition through TLR2. (A and B) TLR reporter assay. HEK-Blue hTLR4 (A) and HEK-Blue hTLR2 (B) were



ACKNOWLEDGMENTS We thank Caixia Ma and Tina Huang for their technical assistance. This study was supported by operating grants from the Canadian Institute of Health Research (CIHR) (B.A.V. and B.B.F.) and Crohn’s and Colitis Canada (CCC) (B.A.V.). K.B. was funded by Vanier Canada Graduate Scholarships (VGS). M.C. was funded by a Canadian Association of Gastroenterology/CIHR/Ferring Pharmaceuticals postdoctoral fellowship. B.A.V. is the Children with Intestinal and Liver Disorders (CHILD) Foundation Research Chair in Pediatric Gastroenterology.

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pression of curli and cellulose, and bacterial curli have been shown to activate TLR2 (46–49). While it remains unclear how the loss of PicC promotes the RDAR phenotype, secreted SPATEs like EspP and EspC have been reported to be associated with macroscopic rope-like structures (extracellular matrix) that promote aggregative phenotypes (50). Furthermore, a genome-wide transposon mutagenesis study revealed a link between EspP and biofilm formation (51). Taken together, these studies suggest an intriguing link between the ability of secreted SPATEs to interact with components of extracellular matrix and the ability of the microbes secreting them to aggregate. We speculate that the loss of PicC affects the surface structure of C. rodentium through direct interactions with bacterial extracellular components (e.g., curli fibrils and cellulose) or indirectly by affecting other autotransporters that are directly involved in bacterial aggregation. Thus, our findings indicate a previously unsuspected role for class 2 SPATEs like Pic in affecting host immune responses by altering bacterial surface structures and modulating TLR2 activation. However, ascertaining a definitive link between SPATEs and the surface structure of bacteria in the future will require significant in-depth investigation. It is notable that several class 2 SPATEs (Pic and Tsh/Hbp) have been described to have immunomodulatory properties due to their ability to cleave a broad range of human leukocyte Olinked glycoproteins. Pic homologs from Shigella, UPEC, and EAEC also have been hypothesized to suppress inflammation through the cleavage of leukocyte surface signaling glycoproteins (17, 18). However, these studies have been performed only in vitro, limiting our understanding of whether these actions occur in vivo. Interestingly, a recent study examined the role of a class 1 SPATE in C. rodentium (9), finding that C. rodentium ⌬crc1 mutant infection led to a hyperinflammatory phenotype, similar to what we noted with the C. rodentium ⌬picC mutant. Unfortunately, the basis for the exaggerated inflammatory response elicited against the C. rodentium ⌬crc1 mutant was not defined, but it is tempting to speculate that different SPATEs play common roles in regulating host recognition of the microbes that produce them, perhaps by controlling their surface structures. Taken together, it appears that PicC ultimately modulates both the function of C. rodentium and its ability to activate the host’s immune system in ways that benefit the pathogen and its host. For C. rodentium infection, it has been shown previously that virulence is positively selected by successful transmission to new hosts (fecal-oral route of transmission) (34). During C. rodentium ⌬picC mutant infection, increased host mortality, as well as reduced transmission to new hosts, confers significant disadvantages to this pathogen. From an evolutionary perspective, host-to-host transmission of an enteric pathogen ensures its successful propagation. This suggests that Pic acts as an antivirulence factor, modulating C. rodentium’s morphotype so that it does not aggregate or hyperstimulate the host immune system. These results shed light on novel functions of SPATEs during enteric infections. Moreover, the lack of a distinct impairment in the C. rodentium ⌬picC mutant’s ability to colonize the mouse intestine suggests either that C. rodentium uses other mucinases to cross the mucus barrier or that this pathogen uses other as-yet undiscovered mechanisms to cross the intestinal mucus barrier.

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