The Bacterial Virulence Factor Lymphostatin Compromises Intestinal Epithelial Barrier Function by Modulating Rho GTPases
2009; Elsevier BV; Volume: 174; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.080640
ISSN1525-2191
AutoresBrian A. Babbin, Maiko Sasaki, Kirsten W. Gerner-Schmidt, Asma Nusrat, Jan‐Michael A. Klapproth,
Tópico(s)Vibrio bacteria research studies
ResumoLymphocyte inhibitory factor A (lifA) in Citrobacter rodentium encodes the large toxin lymphostatin, which contains two enzymatic motifs associated with bacterial pathogenesis, a glucosyltransferase and a protease. Our aim was to determine the effects of each lymphostatin motif on intestinal epithelial-barrier function. In-frame mutations of C. rodentium lifA glucosyltransferase (CrGlM21) and protease (CrPrM5) were generated by homologous recombination. Infection of both model intestinal epithelial monolayers and mice with C. rodentium wild type resulted in compromised epithelial barrier function and mislocalization of key intercellular junction proteins in the tight junction and adherens junction. In contrast, CrGlM21 was impaired in its ability to reduce barrier function and influenced the tight junction proteins ZO-1 and occludin. CrPrM5 demonstrated decreased effects on the adherens junction proteins β-catenin and E-cadherin. Analysis of the mechanisms revealed that C. rodentium wild type differentially influenced Rho GTPase activation, suppressed Cdc42 activation, and induced Rho GTPase activation. CrGlM21 lost its suppressive effects on Cdc42 activation, whereas CrPrM5 was unable to activate Rho signaling. Rescue experiments using constitutively active Cdc42 or C3 exotoxin to inhibit Rho GTPase supported a role of Rho GTPases in the epithelial barrier compromise induced by C. rodentium. Taken together, our results suggest that lymphostatin is a bacterial virulence factor that contributes to the disruption of intestinal epithelial-barrier function via the modulation of Rho GTPase activities. Lymphocyte inhibitory factor A (lifA) in Citrobacter rodentium encodes the large toxin lymphostatin, which contains two enzymatic motifs associated with bacterial pathogenesis, a glucosyltransferase and a protease. Our aim was to determine the effects of each lymphostatin motif on intestinal epithelial-barrier function. In-frame mutations of C. rodentium lifA glucosyltransferase (CrGlM21) and protease (CrPrM5) were generated by homologous recombination. Infection of both model intestinal epithelial monolayers and mice with C. rodentium wild type resulted in compromised epithelial barrier function and mislocalization of key intercellular junction proteins in the tight junction and adherens junction. In contrast, CrGlM21 was impaired in its ability to reduce barrier function and influenced the tight junction proteins ZO-1 and occludin. CrPrM5 demonstrated decreased effects on the adherens junction proteins β-catenin and E-cadherin. Analysis of the mechanisms revealed that C. rodentium wild type differentially influenced Rho GTPase activation, suppressed Cdc42 activation, and induced Rho GTPase activation. CrGlM21 lost its suppressive effects on Cdc42 activation, whereas CrPrM5 was unable to activate Rho signaling. Rescue experiments using constitutively active Cdc42 or C3 exotoxin to inhibit Rho GTPase supported a role of Rho GTPases in the epithelial barrier compromise induced by C. rodentium. Taken together, our results suggest that lymphostatin is a bacterial virulence factor that contributes to the disruption of intestinal epithelial-barrier function via the modulation of Rho GTPase activities. Enteric Gram-negative bacteria are a significant cause of intestinal inflammation and diarrhea worldwide. Pathogens such as enteropathogenic Escherichia coli (EPEC) or enterohemorrhagic E. coli (EHEC) are associated with significant morbidity and mortality in humans,1Clarke SC Diarrhoeagenic Escherichia coli—an emerging problem?.Diagn Microbiol Infect Dis. 2001; 41: 93-98Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar leading to dehydration and occasionally extraintestinal manifestations, including renal failure.2Noël JM Boedeker EC Enterohemorrhagic Escherichia coli: a family of emerging pathogens.Dig Dis. 1997; 15: 67-91Crossref PubMed Scopus (26) Google ScholarCitrobacter rodentium, a related mouse pathogen, functions as a model for human Gram-negative infection.3Schauer DB Falkow S Attaching and effacing locus of a Citrobacter freundii biotype that causes transmissible murine colonic hyperplasia.Infect Immun. 1993; 61: 2486-2492Crossref PubMed Google Scholar Like EPEC and EHEC, C. rodentium harbors a large pathogenicity island, termed locus for enterocyte effacement,4Deng W Li Y Vallance BA Finlay BB Locus of enterocyte effacement from Citrobacter rodentium: sequence analysis and evidence for horizontal transfer among attaching and effacing pathogens.Infect Immun. 2001; 69: 6323-6335Crossref PubMed Scopus (169) Google Scholar encoding for numerous effector proteins. After oral infection and adhesion to epithelial cells, Gram-negative enteric pathogens induce dense actin accumulation underneath the site of bacterial attachment and loss of microvilli, termed attaching and effacing lesions (A/E).5Nougayrède JP Fernandes PJ Donnenberg MS Adhesion of enteropathogenic Escherichia coli to host cells.Cell Microbiol. 2003; 5: 359-372Crossref PubMed Scopus (144) Google Scholar Furthermore, EPEC activates sophisticated mechanisms to breach the intestinal epithelial barrier.6Philpott DJ McKay DM Sherman PM Perdue MH Infection of T84 cells with enteropathogenic Escherichia coli alters barrier and transport functions.Am J Physiol. 1996; 270: G634-G645PubMed Google ScholarPreviously, we have described a large immunomodulatory virulence factor in EPEC, termed lymphostatin, which suppresses cytokine expression in vitro.7Klapproth JM Donnenberg MS Abraham JM Mobley HL James SP Products of enteropathogenic Escherichia coli inhibit lymphocyte activation and lymphokine production.Infect Immun. 1995; 63: 2248-2254PubMed Google Scholar Lymphostatin is encoded for by the lifA gene, which is present in Chlamydia psittaci, Chlamydia muridarum,8Xie G Bonner CA Jensen RA Dynamic diversity of the tryptophan pathway in chlamydiae: reductive evolution and a novel operon for tryptophan recapture.Genome Biol. 2002; 3 (research0051)Crossref Google Scholar EPEC, EHEC,9Klapproth JM Scaletsky IC McNamara BP Lai LC Malstrom C James SP Donnenberg MS A large toxin from pathogenic Escherichia coli strains that inhibits lymphocyte activation.Infect Immun. 2000; 68: 2148-2155Crossref PubMed Scopus (151) Google Scholar and C. rodentium.10Klapproth JM Sasaki M Sherman M Babbin B Donnenberg MS Fernandes PJ Scaletsky IC Kalman D Nusrat A Williams IR Citrobacter rodentium lifA/efa1 is essential for colonic colonization and crypt cell hyperplasia in vivo.Infect Immun. 2005; 73: 1441-1451Crossref PubMed Scopus (55) Google Scholar Lymphostatin expression has been associated with high virulence,11Jores J Wagner S Rumer L Eichberg J Laturnus C Kirsch P Schierack P Tschape H Wieler LH Description of a 111-kb pathogenicity island (PAI) encoding various virulence features in the enterohemorrhagic E. coli (EHEC) strain RW1374 (O103:H2) and detection of a similar PAI in other EHEC strains of serotype 0103:H2.Int J Med Microbiol. 2005; 294: 417-425Crossref PubMed Scopus (22) Google Scholar and has the strongest statistical association with diarrhea caused by atypical EPEC strains.12Afset JE Bruant G Brousseau R Harel J Anderssen E Bevanger L Bergh K Identification of virulence genes linked with diarrhea due to atypical enteropathogenic Escherichia coli by DNA microarray analysis and PCR.J Clin Microbiol. 2006; 44: 3703-3711Crossref PubMed Scopus (122) Google ScholarlifA consists of 9669 bp in EPEC and 9627 bp in C. rodentium, and encodes for three enzymatic motifs: a glucosyltransferase, a protease, and an aminotransferase motif. Infection with C. rodentium identified lymphostatin as an important bacterial effector protein regulating large bowel colonization and development of transmissible murine colonic hyperplasia.10Klapproth JM Sasaki M Sherman M Babbin B Donnenberg MS Fernandes PJ Scaletsky IC Kalman D Nusrat A Williams IR Citrobacter rodentium lifA/efa1 is essential for colonic colonization and crypt cell hyperplasia in vivo.Infect Immun. 2005; 73: 1441-1451Crossref PubMed Scopus (55) Google ScholarEpithelial barrier function is maintained by two distinct structural protein complexes at apical intercellular junctions: tight junctions (TJ) and subjacent adherens junctions (AJ),13Anderson JM Van Itallie CM Fanning AS Setting up a selective barrier at the apical junction complex.Curr Opin Cell Biol. 2004; 16: 140-145Crossref PubMed Scopus (185) Google Scholar which are collectively referred to as the apical junctional complex (AJC). The AJC consists of transmembrane and cytoplasmic plaque proteins that associate with the actin cytoskeleton and play a pivotal role in regulating epithelial paracellular permeability.14Gumbiner BM Cell adhesion: the molecular basis of tissue architecture and morphogenesis.Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2913) Google Scholar The paracellular permeability is tightly controlled in diverse physiological and pathological states by signaling molecules that include diacylglycerol,15Balda MS Gonzalez-Mariscal L Matter K Cereijido M Anderson JM Assembly of the tight junction: the role of diacylglycerol.J Cell Biol. 1993; 123: 293-302Crossref PubMed Scopus (350) Google Scholar PKC,16Turner JR Angle JM Black ED Joyal JL Sacks DB Madara JL PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase.Am J Physiol. 1999; 277: C554-C562PubMed Google Scholar protein kinase C,17Fasano A Fiorentini C Donelli G Uzzau S Kaper JB Margaretten K Ding X Guandalini S Comstock L Goldblum SE Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.J Clin Invest. 1995; 96: 710-720Crossref PubMed Scopus (296) Google Scholar Ca2+,18Kassab Jr, F Marques RP Lacaz-Vieira F Modeling tight junction dynamics and oscillations.J Gen Physiol. 2002; 120: 237-247Crossref PubMed Scopus (7) Google Scholar and small GTPases such as the Rho family of GTPases.19Saha C Nigam SK Denker BM Involvement of Galphai2 in the maintenance and biogenesis of epithelial cell tight junctions.J Biol Chem. 1998; 273: 21629-21633Crossref PubMed Scopus (74) Google Scholar The Rho family of small GTPases encompasses three members, RhoA, Cdc42, and Rac1, which not only regulate AJC function but are also targeted by bacterial virulence factors.20Hopkins AM Li D Mrsny RJ Walsh SV Nusrat A Modulation of tight junction function by G protein-coupled events.Adv Drug Deliv Rev. 2000; 41: 329-340Crossref PubMed Scopus (56) Google Scholar, 21Just I Gerhard R Large clostridial cytotoxins.Rev Physiol Biochem Pharmacol. 2004; 152: 23-47Crossref PubMed Scopus (181) Google ScholarWe demonstrate that C. rodentium is able to breach the intestinal epithelial barrier in vivo and disseminate systemically. Mutation of lymphostatin significantly impaired the ability of C. rodentium to colonize distant organs after intestinal infection. Our study suggests that lymphostatin contributes to disease pathogenesis and compromises the intestinal epithelial barrier in vitro and in vivo by modulating Rho GTPase activity and AJC structure.Materials and MethodsIn Vivo ExperimentsAll in vivo studies involving mice had prior approval by the Emory University Institutional Animal Care and Use Committee. Female, 4- to 6-week-old, pathogen-free C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Groups of five animals for each time point (days 2, 8, 14, and 20) were orally infected with a 100-μl suspension of ∼5 × 108 CFU C. rodentium wild type and EID3; control mice received phosphate-buffered saline (PBS) only.lifA MutationsMutation of lifA in a region that does not encode for a known motif (EID3) has been previously described.10Klapproth JM Sasaki M Sherman M Babbin B Donnenberg MS Fernandes PJ Scaletsky IC Kalman D Nusrat A Williams IR Citrobacter rodentium lifA/efa1 is essential for colonic colonization and crypt cell hyperplasia in vivo.Infect Immun. 2005; 73: 1441-1451Crossref PubMed Scopus (55) Google Scholar Because motifs encoded by lifA could not be expressed as a recombinant protein, we generated new glucosyltransferase and protease motif mutations, both of which have been implicated in bacterial pathogenesis.21Just I Gerhard R Large clostridial cytotoxins.Rev Physiol Biochem Pharmacol. 2004; 152: 23-47Crossref PubMed Scopus (181) Google Scholar, 22Shao F Merritt PM Bao Z Innes RW Dixon JE A Yersinia effector and a Pseudomonas avirulence protein define a family of cysteine proteases functioning in bacterial pathogenesis.Cell. 2002; 109: 575-588Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar Previously generated mutants GlM12 and PrM31 contained a stop codon (TAG) in position 2 of the scar sequence.23Datsenko KA Wanner BL One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.Proc Natl Acad Sci USA. 2000; 97: 6640-6645Crossref PubMed Scopus (10979) Google Scholar To replace the stop codon, 5′ primers Klapp-440 and -441 were designed to encode for leucine, used for polymerase chain reaction (PCR) amplification with downstream primers Klapp-167 and Klapp-170, respectively, and pKD4 as template.10Klapproth JM Sasaki M Sherman M Babbin B Donnenberg MS Fernandes PJ Scaletsky IC Kalman D Nusrat A Williams IR Citrobacter rodentium lifA/efa1 is essential for colonic colonization and crypt cell hyperplasia in vivo.Infect Immun. 2005; 73: 1441-1451Crossref PubMed Scopus (55) Google Scholar PCR-amplified DNA (2 μg) was electroporated (2.5 kV) into CrpKD46, recovered in SOC/10 mmol/L arabinose, and incubated at 42°C on LB agar with kanamicin. Insertion mutations were cured with 100 ng of pFT-A and heat-inactivated chlortetracycline (Sigma Chemicals Co., St. Louis, MO). Clones grown on plates without antibiotics were subjected to PCR and sequencing analysis. Scar sequences for clones CrGlM21 and CrPrM5 were determined to be in frame and encoded for TTG in codon 2 when sequenced in both directions.Bacterial Proliferation, Epithelial Cell Adhesion, and Induction of A/E LesionsC. rodentium wild type, CrGlM21, and CrPrM5 were diluted 1:100, OD600 measured, and serial dilutions plated on LB agar plates for up to 15 hours. CFU were enumerated the following day and the mean with SEM was calculated. For bacterial adhesion, confluent Caco-2 cells were infected with C. rodentium strains at MOI 1:10, washed after 3 hours, and lysed in 1% Triton X-100/PBS.24Zobiack N Rescher U Laarmann S Michgehl S Schmidt MA Gerke V Cell-surface attachment of pedestal-forming enteropathogenic E. coli induces a clustering of raft components and a recruitment of annexin 2.J Cell Sci. 2002; 115: 91-98Crossref PubMed Google Scholar, 25Nicholls L Grant TH Robins-Browne RM Identification of a novel genetic locus that is required for in vitro adhesion of a clinical isolate of enterohaemorrhagic Escherichia coli to epithelial cells.Mol Microbiol. 2000; 35: 275-288Crossref PubMed Scopus (209) Google Scholar Serial dilutions were propagated on LB agar plates and enumerated the following day. Data are expressed as mean with SEM.A/E lesions10Klapproth JM Sasaki M Sherman M Babbin B Donnenberg MS Fernandes PJ Scaletsky IC Kalman D Nusrat A Williams IR Citrobacter rodentium lifA/efa1 is essential for colonic colonization and crypt cell hyperplasia in vivo.Infect Immun. 2005; 73: 1441-1451Crossref PubMed Scopus (55) Google Scholar were examined in 3T3 fibroblast cultures infected with EPEC E2348/69, C. rodentium wild type, and mutant strains at MOI 10:1. Cell cultures were fixed with 3.7% paraformaldehyde/PBS, permeabilized with 0.2% Saponin (Sigma), and blocked in 3% bovine serum albumin/0.2% Saponin/PBS. F-actin was labeled with phalloidin/Alexa 488 (1:1000; Invitrogen, Carlsbad, CA) and bacteria stained with 4′,6-diamidino-2-phenylindole. Stained 3T3 cultures were mounted in Prolong Gold (Invitrogen) and visualized with an Axioskope 2 plus scope (Zeiss, Jena, Germany).Assessment of Epithelial Barrier FunctionTransepithelial electrical resistance (TEER) was measured by EVOM (World Precision Instruments, Sarasota, FL) and paracellular permeability with fluoresceinated dextran (FD-3, MW 3000; Molecular Probes, Eugene, OR).26Sanders SE Madara JL McGuirk DK Gelman DS Colgan SP Assessment of inflammatory events in epithelial permeability: a rapid screening method using fluorescein dextrans.Epithelial Cell Biol. 1995; 4: 25-34PubMed Google Scholar Monolayers were washed in Hanks' balanced salt solution (HBSS+/+), 10 mmol/L HEPES, and equilibrated at 37°C, 10 minutes. After 5 hours of infection with MOI 10:1 C. rodentium strains, epithelial monolayers were loaded apically with 10 μg/ml of FD-3 and fluorescence intensity analyzed in aliquots from the lower chamber 60 minutes later (CytoFluor 2350 fluorescence measurement system; Millipore, Cambridge, MA). Control monolayer epithelial cell cultures were treated identically and instead of bacteria, 10 μl of PBS was added apically. TEER data are expressed as an average percentage change from baseline value with SEM. For fluorescein isothiocyanate (FITC)-dextran flux, numerical values from individual experiments were pooled and are expressed as mean with SEM.Immunofluorescence Confocal MicroscopyCaco-2 cells were grown on polycarbonate membrane supports (Corning Inc., Corning, NY) to TEER ∼200 Ωs/cm2. C. rodentium strains were added to the apical compartment of the Caco-2 monolayers, fixed/permeabilized in ice-cold 100% ethanol after 5 hours, and blocked with 3% bovine serum albumin/PBS at 4°C. F-actin was visualized with Alexa Fluor 488-conjugated phalloidin (Invitrogen), junctional proteins with primary antibodies at 1:1000 dilution (Invitrogen), and C. rodentium with Citrobacter koseri antibody (Cell Sciences, Canton, MA), 1:3000 dilution. Alexa Fluor 546- and 694-conjugated secondary goat antibodies, diluted 1:1000, were used for labeling of junctional proteins and bacteria. Samples were mounted in ProLong Gold (Invitrogen), images taken with Zeiss 510 confocal microscope and acquired with LSM image browser software (Carl Zeiss MicroImaging, Thornwood, NY). Images from three independent experiments were taken at identical detector gain settings for pixel intensity analysis, which was performed using the LSM 510 image analysis software (V 4.03).Activation Status of Small GTPasesTo determine Rac1, Cdc42, and Rho activity (Upstate, Charlottesville, VA), lysed monolayers were normalized for protein concentrations, and incubated with either recombinant PAK1-GST (Rac1, Cdc42) or Rhotekin-GST (RhoA, -B, -C) coupled to agarose beads (45 minutes, 4°C). This time point was chosen because it represented a 50% reduction in epithelial TEER from baseline. For Western blot analysis, rabbit polyclonal anti-Rac1, anti-Cdc42, or anti-Rho antibody beads (Santa Cruz Biotechnology, Santa Cruz, CA) were resuspended in sodium dodecyl sulfate sample buffer. Rho antibodies react with RhoA, -B, and -C and thus the term Rho is used to describe our results. Results of three independent Western blot analyses were subjected to densitometry and results expressed as mean with SEM.Small GTPase Rescue ExperimentsActivation of Cdc42-mediated signaling was achieved using a recombinant adenoviral vector encoding N-terminal myc-tagged constitutively active Cdc42.27Kuhn TB Brown MD Wilcox CL Raper JA Bamburg JR Myelin and collapsin-1 induce motor neuron growth cone collapse through different pathways: inhibition of collapse by opposing mutants of rac1.J Neurosci. 1999; 19: 1965-1975Crossref PubMed Google Scholar Caco-2 cells were incubated in S-MEM/10% FBS (<10 μmol/L extracellular Ca++) overnight and then placed into complete media with 5 × 105 plaque-forming U/ml viral particles for 2 days. Infection efficiency averaged ∼70% (data not shown). Epithelial cell cultures were infected with C. rodentium wild type, MOI 1:10 and TEER determined by EVOM. Inhibition of Rho activity was performed using C3 exotoxin from Clostridium botulinum. Polarized Caco-2 cells were pre-incubated with 100 ng/ml of C3 exotoxin for 2 hours before infection with C. rodentium wild type in triplicate. TEER was measured throughout a 15-hour time course. Data are expressed as the mean with SEM of the percent change from baseline TEER from three independent experiments.In Vivo Immunofluorescence MicroscopyFor each condition and time point (days 8 and 14), three C57BL/6 female mice at 4 weeks of age were infected with 5 × 108 CFU of C. rodentium wild type, CrGlM21, and CrPrM5 in 100 μl of PBS via oral gavage. Control animals received 100 μl of PBS alone. On days 8 and 14 the distal half of the colon was resected and fecal matter removed. Colon specimens were embedded in OCT medium (Sakura Finetechnical Company, Tokyo, Japan) and cut into 6-μm frozen sections with a cryostat (Leica Microsystems, Nussloch, Germany). Samples were fixed with 100% ethanol for 5 minutes at −20°C. Slides were blocked in 3% bovine serum albumin, 0.1% saponin in PBS for 1 hour at room temperature. Junctional proteins were labeled with polyclonal antibodies directed against β-catenin (Sigma) and occludin (Invitrogen) at 1:250 dilution for 1 hour followed by secondary goat anti-rabbit Alexa 488 antibody (1:1000 dilution, Invitrogen) for 30 minutes at room temperature. Junctional proteins were visualized using an AxioCam MRc microscope (Zeiss) loaded with Axiovision MRc and microscopic images analyzed using Axiovision (Rel. 4.6).Statistical AnalysisNonparametric analysis was performed by Student's t-test for all data. Differences were considered significant if P was ≤0.05.ResultsLymphostatin Contributes to the Ability of C. rodentium to Compromise the Intestinal Epithelial Barrier in Vivo and Systemically DisseminateAlthough infection of mice with C. rodentium is generally thought to be confined to the mucosal surface of the colon, some studies have identified dissemination of this pathogen to extra-intestinal sites.28Brennan PC Fritz TE Flynn RJ Poole CM Citrobacter freundii associated with diarrhea in a laboratory mice.Lab Anim Care. 1965; 15: 266-275PubMed Google Scholar Similarly, our in vivo studies revealed colonization of extra-intestinal sites by C. rodentium. C. rodentium wild type was recovered from splenic tissues at 996 CFU/organ on day 2 after oral administration of the pathogen, which increased to 32,152 CFU/organ by day 8 after infection. By days 14 and 20 splenic colonization was decreased to 712 CFU/organ and 196 CFU/organ, respectively (Figure 1). Interestingly, infectious counts for a lymphostatin mutant, lifA EID3, were significantly lower at all time points investigated: day 2, 368 CFU/organ; days 8 and 14, 72 CFU/organ; day 20, 8 CFU/organ (P < 0.05). Results were similar for mesenteric lymph nodes and liver (data not shown). Thus, these findings suggest that lymphostatin plays a role in the ability of C. rodentium to cross the intestinal epithelial barrier and colonize extra-intestinal organs.Given the implication of lymphostatin in mediating the C. rodentium-induced compromise in intestinal epithelial barrier function and systemic infection, we sought to determine whether C. rodentium infection results in disruption of the AJC and if lymphostatin contributes to such an effect. For these studies, we generated two new C. rodentium lymphostatin mutants with in frame mutations of lifA glucosyltransferase motif (CrGlM21) and protease motif (CrPrM5), both of which have been implicated in bacterial pathogenesis (Figure 2).Figure 2lifA in C. rodentium. C. rodentium lifA is 9627 bp in size, encoding for a putative 364-kDa protein with three distinct motifs: glucosyltransferase (1.6 kb, DXD), protease motif (4.5 to 4.8 kb, C, H, D), and aminotransferase II (5.8 kb, TMGKALSASA). We generated three mutations in C. rodentium lifA: CrGlM21 at 1.6 kb, EID3 at 3.5 kb, and CrPrM5 at 4.5 kb.View Large Image Figure ViewerDownload Hi-res image Download (PPT)C. rodentium Lymphostatin Mutants Retain Basic Pathogenicity FeaturesTo determine whether mutations in C. rodentium lymphostatin influence basic bacterial functions, we first examined growth curves of mutant strains CrGlM21 and CrPrM5 in comparison with those of C. rodentium wild type for up to 15 hours. Proliferation of C. rodentium wild type, CrGlM21, and CrPrM5 strains were indistinguishable during lag (0 to 2 hours), log (2 to 9 hours), and plateau phase (9 to 13 hours) as determined by OD600(Figure 3A) and by plating of serial dilutions on LB agar plates on analysis the following day.Figure 3Mutations in lymphostatin do not interfere with basic pathogenicity features. A: Bacterial proliferation of C. rodentium wild type (open diamonds), CrGlM21 (closed squares), and CrPrM5 (closed triangles) strains was indistinguishable during lag (0 to 2 hours), log (2 to 9 hours), and plateau phase (9 to 13 hours) as determined by OD600 and plating of serial dilutions on LB agar (data not shown). B: Mutations of lymphostatin glucosyltransferase (CrGlM21) and protease motif (CrPrM5) did not interfere with formation of A/E lesions in 3T3 fibroblast cultures (white arrows) in comparison with C. rodentium wild type (CrWT) and EPEC strain E2348/69.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Lymphostatin has also been characterized as an adhesion factor for EHEC serotype O111:H−.25Nicholls L Grant TH Robins-Browne RM Identification of a novel genetic locus that is required for in vitro adhesion of a clinical isolate of enterohaemorrhagic Escherichia coli to epithelial cells.Mol Microbiol. 2000; 35: 275-288Crossref PubMed Scopus (209) Google Scholar Mutation of O111:H− lymphostatin lead to an almost sevenfold reduction in bacterial adhesion to CHO cells in vitro. We therefore examined whether mutation of C. rodentium lymphostatin impairs its ability to adhere to intestinal epithelial cells. No significant difference was observed in bacterial-epithelial adhesion after infection of the polarized model intestinal epithelial cell line, Caco-2, with C. rodentium wild type and mutant strains (data not shown). Mean CFU/1 × 106 Caco-2 epithelial cells from three independent experiments were 1.9 × 106 for C. rodentium wild type, 1.42 × 106 for CrGlM21, and 1.47 × 106 for CrPrM5 (P > 0.05).Next, we determined whether mutation of lymphostatin glucosyltransferase or protease motifs impairs the ability of C. rodentium to form attaching and effacing (A/E) lesions. For these experiments, we used 3T3 fibroblasts, which have been classically used for such studies, and bacterial strain E2348/69 was used as a positive control. C. rodentium wild type and both lymphostatin mutants were able to form A/E lesions similar to E2348/69 (Figure 3B). Taken together, the above results support that the basic yet critical pathogenic properties of C. rodentium are not altered by inactivating either the lymphostatin glucosyltransferase or protease motifs.Effects of C. rodentium and Lymphostatin Mutants on Epithelial Barrier FunctionBecause C. rodentium demonstrated the ability to compromise the intestinal epithelial barrier and disseminate in mice, we analyzed the effects of C. rodentium wild type, CrGlM21, and CrPrM5 on intestinal epithelial barrier properties. TEER of monolayers was measured for up to 15 hours after infection. Infection with all three C. rodentium strains, ultimately resulted in a significant and sustained decrease in TEER (Figure 4A). However, the decrease in TEER observed after infection with CrGlM21 was on average 20% reduced after 6 hours compared with infections with C. rodentium wild type and CrPrM5.Figure 4Lymphostatin regulates intestinal epithelial barrier function. A: Caco-2 cell monolayers were infected at MOI 10:1 with C. rodentium strains for 15 hours. All three strains decreased TEER throughout time, however, infection with CrGlM21 resulted in some attenuation of the decline in TEER, which became significant after 6 hours of infection (*P < 0.05). B: Caco-2 cell monolayers were infected at MOI 10:1 with C. rodentium wild type, CrGlM21, or CrPrM5 for 15 hours. After 5 hours of infection, monolayers were apically loaded with FD-3. Samples were taken from the lower chamber 60 minutes later, and fluorescence intensity determined. Data are expressed as mean with SEM from three individual experiments. In comparison with C. rodentium wild type (CrWT) and CrPrM5, dextran flux was reduced in CrGlM21-infected monolayers (*P < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Subsequently, we assessed paracellular permeability of FITC-dextran across the epithelial monolayers after infection with C. rodentium wild type or the lymphostatin mutant strains. Paracellular flux of FITC-dextran increased in response to C. rodentium wild type and both mutants. However, the degree of dextran flux was attenuated after CrGlM21 infection compared with C. rodentium wild type and CrPrM5, consistent with the TEER results (Figure 4B, P < 0.05). Because mutation of the lymphostatin glucosyltransferase motif attenuated the decrease in TEER and increased FITC-dextran flux in response to C. rodentium infection, these data implicate a role of C. rodentium lymphostatin in compromising the intestinal epithelial barrier.Lymphostatin Plays a Role in the Disruption of the AJC in VitroGiven that infection with C. rodentium wild type and lymphostatin mutants compromised epithelial barrier function, we n
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