Advertisement

Enteropathogenic Escherichia coli EspH-Mediated Rho GTPase Inhibition Results in Desmosomal Perturbations

  • Jennifer Lising Roxas
    Affiliations
    School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, Arizona
    Search for articles by this author
  • Ross Calvin Monasky
    Affiliations
    School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, Arizona
    Search for articles by this author
  • Bryan Angelo P. Roxas
    Affiliations
    School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, Arizona
    Search for articles by this author
  • Al B. Agellon
    Affiliations
    School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, Arizona

    BIO5 Institute for Collaborative Research, University of Arizona, Tucson, Arizona
    Search for articles by this author
  • Asad Mansoor
    Affiliations
    School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, Arizona
    Search for articles by this author
  • James B. Kaper
    Affiliations
    University of Maryland School of Medicine, Baltimore, Maryland
    Search for articles by this author
  • Author Footnotes
    § Authors share co-senior authorship.
    Gayatri Vedantam
    Footnotes
    § Authors share co-senior authorship.
    Affiliations
    School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, Arizona

    BIO5 Institute for Collaborative Research, University of Arizona, Tucson, Arizona

    Department of Immunobiology, University of Arizona, Tucson, Arizona

    Southern Arizona VA Healthcare System, Tucson, Arizona
    Search for articles by this author
  • Author Footnotes
    § Authors share co-senior authorship.
    V.K. Viswanathan
    Correspondence
    Correspondence Address correspondence to: V. K. Viswanathan, PhD, School of Animal and Comparative Biomedical Sciences, 1006 E. Lowell, Building 106, Room 231, University of Arizona, Tucson, Arizona 85721. fax: (520) 621-6366.
    Footnotes
    § Authors share co-senior authorship.
    Affiliations
    School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, Arizona

    BIO5 Institute for Collaborative Research, University of Arizona, Tucson, Arizona

    Department of Immunobiology, University of Arizona, Tucson, Arizona
    Search for articles by this author
  • Author Footnotes
    § Authors share co-senior authorship.
Open AccessPublished:April 27, 2018DOI:https://doi.org/10.1016/j.jcmgh.2018.04.007

      Background & Aims

      The diarrheagenic pathogen, enteropathogenic Escherichia coli (EPEC), uses a type III secretion system to deliver effector molecules into intestinal epithelial cells (IECs). While exploring the basis for the lateral membrane separation of EPEC-infected IECs, we observed infection-induced loss of the desmosomal cadherin desmoglein-2 (DSG2). We sought to identify the molecule(s) involved in, and delineate the mechanisms and consequences of, EPEC-induced DSG2 loss.

      Methods

      DSG2 abundance and localization was monitored via immunoblotting and immunofluorescence, respectively. Junctional perturbations were visualized by electron microscopy, and cell–cell adhesion was assessed using dispase assays. EspH alanine-scan mutants as well as pharmacologic agents were used to evaluate impacts on desmosomal alterations. EPEC-mediated DSG2 loss, and its impact on bacterial colonization in vivo, was assessed using a murine model.

      Results

      The secreted virulence protein EspH mediates EPEC-induced DSG2 degradation, and contributes to desmosomal perturbation, loss of cell junction integrity, and barrier disruption in infected IECs. EspH sequesters Rho guanine nucleotide exchange factors and inhibits Rho guanosine triphosphatase signaling; EspH mutants impaired for Rho guanine nucleotide exchange factor interaction failed to inhibit RhoA or deplete DSG2. Cytotoxic necrotizing factor 1, which locks Rho guanosine triphosphatase in the active state, jasplakinolide, a molecule that promotes actin polymerization, and the lysosomal inhibitor bafilomycin A, respectively, rescued infected cells from EPEC-induced DSG2 loss. Wild-type EPEC, but not an espH-deficient strain, colonizes mouse intestines robustly, widens paracellular junctions, and induces DSG2 re-localization in vivo.

      Conclusions

      Our studies define the mechanism and consequences of EPEC-induced desmosomal alterations in IECs. These perturbations contribute to the colonization and virulence of EPEC, and likely related pathogens.

      Graphical abstract

      Keywords

      Abbreviations used in this paper:

      A/E (attaching and effacing), BSA (bovine serum albumin), CM (calcium and magnesium), DMEM (Dulbecco's modified Eagle medium), DSC (desmocollin), DSG (desmoglein), EPEC (enteropathogenic Escherichia coli), GEF (guanine nucleotide exchange factors), GTPase (guanosine triphosphatase), IF (intermediate filament), IEC (intestinal epithelial cell), PBS (phosphate-buffered saline), T3SS (type 3 secretion system), TER (transepithelial electrical resistance), TJ (tight junction), WT (wild-type)
      See editorial on page 225.
      EspH, a protein secreted by the diarrheagenic pathogen enteropathogenic Escherichia coli into intestinal epithelial cells, sequesters Rho guanine nucleotide exchange factors and inhibits Rho guanosine triphosphatases. These EspH effects cause desmosomal perturbations and loss of epithelial barrier integrity, and contribute to bacterial persistence in the intestine.
      The intestinal epithelial monolayer facilitates absorption of nutrients and fluids, and, simultaneously, serves as a barrier against the entry of harmful intraluminal components such as bacteria and toxins into the underlying tissue.
      • Groschwitz K.R.
      • Hogan S.P.
      Intestinal barrier function: molecular regulation and disease pathogenesis.
      The paracellular space between adjacent intestinal epithelial cells (IECs) is bridged by protein complexes including tight junctions (TJs), adherens junctions, gap junctions, and desmosomal junctions.
      • Giepmans B.N.
      • van Ijzendoorn S.C.
      Epithelial cell-cell junctions and plasma membrane domains.
      The apical-most TJs act as fences that maintain polarity by separating apical and basolateral membrane components of the epithelium, and form the major paracellular barrier.
      • Anderson J.M.
      • Van Itallie C.M.
      Tight junctions and the molecular basis for regulation of paracellular permeability.
      Adherens junctions, located immediately below TJs, comprise transmembrane cadherins and cytoplasmically attached α- and β-catenins that connect to cytoskeletal actin filaments.
      • Niessen C.M.
      Tight junctions/adherens junctions: basic structure and function.
      Desmosomal junctions, comprising membrane-anchored desmoglein (DSG) and desmocollin (DSC), form spot-welds that confer stability and strength to cell junctions. Four DSG (DSG1–4) and 3 DSC (DSC1–3) isoforms are expressed in human beings in a tissue-specific manner.
      • Nekrasova O.
      • Green K.J.
      Desmosome assembly and dynamics.
      Only DSG2 and DSC2 are expressed in normal human intestinal epithelium.
      • Holthöfer B.
      • Windoffer R.
      • Troyanovsky S.
      • Leube R.E.
      Structure and function of desmosomes.
      The N-termini of DSG and DSC are located in the intercellular space, and form homotypic and heterotypic interactions with desmosomal cadherins of adjacent cells.
      • Shabih-e-Hassnain S.
      • Trinnaman B.
      • Martin S.
      • Major S.
      • Hutchinson J.
      • Magee A.I.
      Molecular interactions between desmosomal cadherins.
      The plaque proteins plakoglobin, plakophilin, and desmoplakin connect the C-termini of DSG and DSC to intermediate filaments (IFs).
      • Capaldo C.T.
      • Farkas A.E.
      • Nusrat A.
      Epithelial adhesive junctions.
      Many disease states are marked by relocalization of junctional proteins, increased paracellular permeability, loss of cell–cell adhesion, and, in extreme cases, the gross exfoliation of cells.
      • Barreau F.
      • Hugot J.
      Intestinal barrier dysfunction triggered by invasive bacteria.
      Enteropathogenic Escherichia coli (EPEC) is a leading cause of juvenile diarrheal disease mortality.
      • Lanata C.F.
      • Fischer-Walker C.L.
      • Olascoaga A.C.
      • Torres C.X.
      • Aryee M.J.
      • Black R.E.
      Child Health Epidemiology Reference Group of the World Health Organization and UNICEF. Global causes of diarrheal disease mortality in children <5 years of age: a systematic review.
      EPEC belongs to the attaching and effacing (A/E) family of pathogens that intimately attach to epithelial cells and efface brush-border microvilli.
      • Nataro J.P.
      • Kaper J.B.
      Diarrheagenic Escherichia coli.
      EPEC elaborates a type III secretion system (T3SS) that translocates effector proteins into host cells and thereby modulates host cell signaling. Notably, effector protein-dependent barrier perturbation is thought to contribute to EPEC-induced diarrhea.
      • Berkes J.
      • Viswanathan V.K.
      • Savkovic S.D.
      • Hecht G.
      Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation.
      The EPEC effectors EspF, EspG1/G2, Map, and NleA disrupt TJs and compromise epithelial barrier function.
      • McNamara B.P.
      • Koutsouris A.
      • O'Connell C.B.
      • Nougayrede J.P.
      • Donnenberg M.S.
      • Hecht G.
      Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function.
      • Garber J.J.
      • Mallick E.M.
      • Scanlon K.M.
      • Turner J.R.
      • Donnenberg M.S.
      • Leong J.M.
      • Snapper S.B.
      Attaching-and-effacing pathogens exploit junction regulatory activities of N-WASP and SNX9 to disrupt the intestinal barrier.
      • Tomson F.L.
      • Viswanathan V.K.
      • Kanack K.J.
      • Kanteti R.P.
      • Straub K.V.
      • Menet M.
      • Kaper J.B.
      • Hecht G.
      EPEC EspG disrupts microtubules and in conjunction with Orf3 enhances perturbation of tight junction barrier.
      • Dean P.
      • Kenny B.
      Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein.
      • Thanabalasuriar A.
      • Koutsouris A.
      • Weflen A.
      • Mimee M.
      • Hecht G.
      • Gruenheid S.
      The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic Escherichia coli.
      EPEC outer-membrane proteins disrupt adherens junctions by inducing the dissociation of cadherin/β-catenin complexes.
      • Malladi V.
      • Shankar B.
      • Williams P.H.
      • Balakrishnan A.
      Enteropathogenic Escherichia coli outer membrane proteins induce changes in cadherin junctions of Caco-2 cells through activation of PKCalpha.
      Early electron microscopy studies on small intestinal biopsy samples from EPEC-infected children showed a separation of the lateral intercellular junctions of epithelial cells.
      • Rothbaum R.J.
      • Partin J.C.
      • Saalfield K.
      • McAdams A.J.
      An ultrastructural study of enteropathogenic Escherichia coli infection in human infants.
      We observed similar widening of paracellular junctions in EPEC-infected IECs, specifically in regions below the TJs. To test the hypothesis that the loss of desmosomes contributed to these changes, we evaluated desmosomal protein abundance in infected IECs. EPEC infection induced a marked decrease in DSG2, the only IEC desmoglein isoform. The aim of this study, therefore, was to investigate the mechanisms and impact of EPEC-induced DSG2 down-regulation in vitro and in vivo. Our studies show a novel role for the effector protein EspH, and for Rho guanosine triphosphatase (GTPase) signaling, in modulating epithelial monolayer integrity, barrier function, and bacterial colonization.

      Materials and Methods

      Cell Lines

      The human IEC line C2BBe, a brush-border–expressing Caco-2 subclone,
      • Peterson M.D.
      • Mooseker M.S.
      Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco-2.
      was used and cultured as reported previously.
      • Roxas J.L.
      • Ryan K.
      • Vedantam G.
      • Viswanathan V.K.
      Enteropathogenic Escherichia coli dynamically regulates EGFR signaling in intestinal epithelial cells.

      Bacterial Strains and Generation of Mutants

      The EPEC O127:H6 strain E2348/69 and its isogenic derivatives, and the commensal E coli strain HS4, were used (Table 1). E2348/69 espH was disrupted via homologous recombination.
      • Elliott S.J.
      • Krejany E.O.
      • Mellies J.L.
      • Robins-Browne R.M.
      • Sasakawa C.
      • Kaper J.B.
      EspG, a novel type III system-secreted protein from enteropathogenic Escherichia coli with similarities to VirA of Shigella flexneri.
      A 202-bp espH internal fragment (base pairs 181–382) was cloned into the suicide vector pJP5603,
      • Penfold R.J.
      • Pemberton J.M.
      An improved suicide vector for construction of chromosomal insertion mutations in bacteria.
      and the resulting construct, pSE864, was introduced into E2348/69 via conjugation. Transconjugants were confirmed for espH disruption by polymerase chain reaction and Southern blot, and 1 representative espH mutant, SE874, was used. For complementation studies, full-length espH was amplified and cloned into pTrcHis2-TOPO TA (Thermo Fisher Scientific, Waltham, MA) to generate pJLR1, and transformed into SE874 to generate SE874/C. espH also was cloned in-frame with the 6XHis tag sequence of pTrcHis2-TOPO TA (pJLR2). Alanine scans have been empirically found to be a particularly effective method for assessing the functional importance of residues within a protein.
      • Bromberg Y.
      • Rost B.
      Comprehensive in silico mutagenesis highlights functionally important residues in proteins.
      To identify EspH residues involved in RhoA inhibition, a panel of 29 espH alanine-scan mutants were engineered by sequentially replacing 5–amino acid stretches of EspH (excluding the N-terminal 25 amino acids containing the secretion signal) with alanine residues. To facilitate cloning of these mutants, an XhoI restriction site was introduced into pJLR2 via a 703G→A silent substitution to generate pJLR4. Dispase assay screens (described later) identified EspH mutants M14 and M16, corresponding to alterations of amino acids 88–92 and 98–102, respectively, as candidates for further study. To verify that M14 and M16 retained wild-type levels of EspH secretion, culture supernatants of wild-type (WT) EPEC and mutant strains grown in serum-free Dulbecco's modified Eagle medium (DMEM) were immunoblotted as described later.
      Table 1Bacterial Strains, Plasmids, and Primers
      StrainGenotype/descriptionReference
      HS4Nonpathogenic E coli
      • Myhal M.L.
      • Laux D.C.
      • Cohen P.S.
      Relative colonizing abilities of human fecal and K 12 strains of Escherichia coli in the large intestines of streptomycin-treated mice.
      EPECE coli O127:H6 strain E2348/69, NalR ; parent strain for all mutants/complements below
      • Taylor J.
      Infectious infantile enteritis, yesterday and today.
      • Levine M.M.
      • Nataro J.P.
      • Karch H.
      • Baldini M.M.
      • Kaper J.B.
      • Black R.E.
      • Clements M.L.
      • O'Brien A.D.
      The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor.
      ΔespANonpolar insertion in espA; NalR, KanR
      • Kenny B.
      • Lai L.C.
      • Finlay B.B.
      • Donnenberg M.S.
      EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells.
      SE874Insertional disruption of espH; KanR, NalRThis study
      SE874/CSE874 complemented with pJLR1This study
      Plasmids
      pJP5603Suicide vector
      • Penfold R.J.
      • Pemberton J.M.
      An improved suicide vector for construction of chromosomal insertion mutations in bacteria.
      pSE864pJP5603 with 202-bp insert corresponding to espH bp181-382; used for generating SE874This study
      SE874 complementation plasmids: vector: pTrcHis2TOPO-TA
      pJLR1espH amplified with primers EspH1 and EspH2 Expresses WT EspHThis study
      pJLR2espH amplified with primers EspH1 and EspH3

      Expresses WT EspH with C-terminal 6xHis tag
      This study
      pJLR4espH with silent mutation at (703G>A) to introduce XhoI restriction site

      Expresses WT EspH with C-terminal 6xHis tag
      This study
      M14Alanine-scan mutant

      Expresses EspH 88AAAAA92, His-tagged
      This study
      M16Alanine-scan mutant

      Expresses EspH 98AAAAA102, His-tagged
      This study
      Primers
      EspH15’-ATGTCGTCATCATTATCAG-3’This study
      EspH25’-TTAAACTGTCACACCTGAT-3’This study
      EspH35’-AACTGTCACACCTGAT-3’This study

      Growth of Bacteria and Infection of IECs

      C2BBe culture medium was changed to serum-free DMEM 16 hours before infection. For infections, overnight bacterial cultures (in Luria–Bertani broth with appropriate antibiotics for plasmid selection) were subcultured at 1:20 dilution in serum-free DMEM and grown to midlogarithmic phase (OD600nm, 0.4) at 37°C. Bacteria were added to the apical surface of IECs corresponding to an initial multiplicity of infection of 100, and infections were allowed to proceed for 3 hours unless otherwise indicated. For studies involving Rho activation or actin polymerization, IECs were pretreated with 300 ng/mL cytotoxic necrotizing factor 1–based Rho activator CN03 (Cytoskeleton, Inc, Denver, CO) or 1 μmol/L jasplakinolide (Thermo Fisher Scientific), for 1 hour before infection. For inhibitor studies, IECs were pretreated with 50 μmol/L pan-caspase inhibitor z-DEVD-fmk, 52 nmol/L calpain inhibitor I, 10 μmol/L proteasomal inhibitor MG132, or 250 nmol/L lysosomal inhibitor bafilomycin A1 (Sigma-Aldrich, St. Louis, MO) for 2 hours before infection, and inhibitor levels were maintained through the course of infection.

      Protein Extraction

      Mock-treated and infected monolayers were washed with ice-cold, sterile phosphate-buffered saline (PBS) with calcium and magnesium (PBS-CM), scraped, harvested via centrifugation, resuspended in urea buffer (7 mol/L urea, 2 mol/L thiourea, 100 mmol/L 1,4-dithiothreitol, and 4% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate), and lysed via sonication or by silica bead-beating (Bead Ruptor; Omni International, NW Kennesaw, GA).

      Immunoblot Analyses

      Protein extracts (50–100 μg) were separated by 7% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to 0.2-μm nitrocellulose membranes, which were blocked with 5% nonfat milk in Tris-buffered saline containing Tween 20 and incubated with anti-DSG2, anti-DSC2, or anti-actin antisera (Abcam, Cambridge, MA; Sigma-Aldrich) following product recommendations. Horseradish-peroxidase–conjugated goat anti-rabbit and anti-mouse antisera (Sigma-Aldrich) were used as secondary antisera. Membranes were developed with SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific).

      Co-immunoprecipitation Studies

      C2BBe cells were grown to 60% confluency and transfected with pEnter expression plasmid harboring C-terminal Flag-tagged p115-Rho guanine nucleotide exchange factor (GEF) (Vigene, Rockville, MD). Transfection was facilitated using jetPRIME transfection reagent (Polyplus Transfection, Illkirch, France) following the manufacturer’s protocol. Two days after transfection, C2BBe cells were infected with EPEC strains at a multiplicity of infection of 100 for 3 hours. Samples were washed with ice-cold PBS, scraped, and harvested via centrifugation. Proteins were extracted in immunoprecipitation lysis buffer containing 50 mmol/L TrisCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 1 × Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Immunoprecipitation was performed on 5-mg protein extracts using Surebeads Protein A Magnetic beads (Bio-Rad Laboratories, Hercules, CA) bound with anti-Flag M2 antibody (Sigma-Aldrich). The presence of Flag-tagged p115-RhoGEF and His-tagged EspH in the immunoprecipitated samples was determined via Western blot analysis using p115-RhoGEF antibody (Abcam) and anti-6xHis-tag antibody (Cell Signaling, Danvers, MA). To assess EspH secretion, 30-mL cultures of EPEC strains inoculated in serum-free DMEM and normalized to midlogarithmic phase (OD600nm, 0.4) were grown for 3 hours at 37°C. Cultures were centrifuged at 400g for 10 minutes. Bacterial supernatants were filter-sterilized and concentrated using Amicon 3-kilodalton cut-off filters (MilliporeSigma, Burlington, MA). EspH secretion was assessed via immunoblot analysis using anti-6xHis-tag antibody.

      Dispase Assay

      Cell adhesion strength was assessed using a modified dispase assay.
      • Jiang K.
      • Rankin C.R.
      • Nava P.
      • Sumagin R.
      • Kamekura R.
      • Stowell S.R.
      • Feng M.
      • Parkos C.A.
      • Nusrat A.
      Galectin-3 regulates desmoglein-2 and intestinal epithelial intercellular adhesion.
      Briefly, mock-treated and infected IEC monolayers were washed with Hank’s balanced salt solution with CM, and incubated with dispase (20 mg/mL in Hank’s balanced salt solution with CM) at 37°C and 5% CO2 for 30 minutes. Detached monolayers were agitated mechanically for 5 minutes, and monolayer disruption was scored qualitatively (none, partial, or full).

      RhoA Activity Assays

      The low RhoA activity signal under standard infection conditions impeded detection of further EPEC/EspH-mediated inhibition. To overcome this, baseline RhoA activity was boosted by pretreating serum-starved, confluent IECs with 5 μmol/L lysophosphatidic acid for 1 hour before infection. RhoA activity in cell extracts were measured using a RhoA G-LISA Activation Assay Kit (Cytoskeleton, Inc).

      Transepithelial Electrical Resistance Measurements

      Polarized IECs grown on 0.33-cm2 collagen-coated Corning Transwells (Thermo Fisher Scientific) were apically infected with EPEC strains (WT, SE874, and SE874/C) cultured in DMEM as described earlier. Measurements were made every hour for 6 hours after infection using an epithelial volt-Ohm voltmeter (World Precision Instruments, Sarasota, FL), and transepithelial electrical resistance (TER) was calculated by applying Ohm’s Law.

      Mouse Model of EPEC Infection

      All procedures were approved by the University of Arizona Institutional Animal Care and Use Committee. Six-week-old C57BL/6J male mice (Jackson Laboratory, Bar Harbor, ME) were infected as reported previously.
      • Royan S.V.
      • Jones R.M.
      • Koutsouris A.
      • Roxas J.L.
      • Falzari K.
      • Weflen A.W.
      • Kim A.
      • Bellmeyer A.
      • Turner J.R.
      • Neish A.S.
      • Rhee K.J.
      • Viswanathan V.K.
      • Hecht G.A.
      Enteropathogenic E. coli non-LEE encoded effectors NleH1 and NleH2 attenuate NF-kappaB activation.
      Briefly, streptomycin-treated mice (5 g/L streptomycin in drinking water ad libitum for 24 hours, followed by sterile water for 24 hours), were gavaged with 150 μL sterile PBS (control, uninfected), or 2 × 108 bacteria suspended in 150 μL sterile PBS, and monitored for up to 4 days after infection.

      Attachment Assays

      To assess EPEC attachment to the intestinal mucosa, WT- or SE874-infected C57BL/6J mice were euthanized on days 3 and 4 after infection (4 animals per condition). Segments of the cecum and proximal colon were isolated, cleaned, weighed, homogenized, serially diluted in PBS, and bacteria were enumerated on MacConkey agar containing 100 μg/mL nalidixic acid.

      Fecal Shedding of EPEC

      Mouse stool samples were collected daily for 4 days after infection, and weighed, homogenized in PBS, serially diluted, and bacteria were enumerated on MacConkey agar containing 100 μg/mL nalidixic acid. No colonies were recovered from the stool of uninfected animals, consistent with media selectivity for EPEC.

      Immunofluorescence Microscopy

      Cultured monolayers were grown on poly-L-lysine–coated coverslips. For mouse intestinal tissues, samples were frozen in OCT embedding medium (Tissue-Tek; Sakura Finetek, Torrance, CA) and stored at -80°C. Cultured monolayers and OCT-mounted tissue samples cut at 4-micron thickness were fixed in a 1:1 mixture of methanol and acetone for 20 minutes at -20°C, air-dried, rehydrated in PBS for 5 minutes, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in PBS for 15 minutes, and blocked with 5% IgG-free bovine serum albumin (BSA) in PBS for 1 hour. Primary antibodies used for cultured cells were 1:40 dilution of mouse anti-DSG2 and 1:1000 dilution of rabbit anti–lysosomal-associated membrane protein 1 (Abcam); mouse tissues were probed with 1:40 dilution of rabbit anti-DSG2 (Sigma Aldrich). Samples were incubated with primary antisera overnight at 4°C, and then washed 3 times with 1% IgG-free BSA in PBS. Secondary antibodies (Alexa 488–conjugated goat anti-mouse IgG, Alexa Fluor 555–conjugated anti-rabbit IgG, or Alexa 488 goat anti-rabbit IgG antisera; Thermo Fisher Scientific) were added at 8 μg/mL in 5% IgG-free BSA for 1 hour. Samples were washed with PBS, stained with 4,6-diamidino-2-phenylindole, and mounted in ProLong Diamond Antifade reagent (Thermo Fisher Scientific). Images were captured on a DeltaVision Elite Deconvolution Microscope (GE Healthcare, Pittsburgh, PA) using an Olympus 60×/1.42 objective with 1.59× auxiliary magnification or an Olympus 100×/1.4 objective (immersion oil refractive index, n = 1.516) (Sigma-Aldrich).

      Transmission Electron Microscopy

      Polarized IECs grown on 0.33-cm2 collagen-coated Transwells (Thermo Fisher Scientific) were apically infected with EPEC strains cultured in DMEM. IECs and tissue samples were fixed in Karnovsky’s Fixative (Electron Microscopy Sciences, Hatfield, PA) overnight at 4°C, neutralized with 125 mmol/L glycine in PBS, postfixed in 1% osmium tetroxide, and sequentially dehydrated with 15%, 30%, 50%, 70%, 90%, and 100% ethanol. Samples then were infiltrated with Spurr’s Resin (Electron Microscopy Sciences). Ultrathin sections were contrasted with 2% uranyl acetate, followed by Reynold’s lead citrate, and visualized with an FEI Tecnai Spirit transmission electron microscope (FEI, Hillsboro, OR).

      Statistical Analyses

      Significant changes for all pairwise sample comparisons in experiments involving quantitation were assessed by analysis of variance with Bonferroni post hoc test.
      All authors had access to the study data and have reviewed and approved the final manuscript.

      Results

      EspH Mediates Desmoglein-2 Loss in EPEC-Infected Epithelial Cells

      To explore the molecular basis for the cell–cell separation previously seen in EPEC-infected patient tissues,
      • Rothbaum R.J.
      • Partin J.C.
      • Saalfield K.
      • McAdams A.J.
      An ultrastructural study of enteropathogenic Escherichia coli infection in human infants.
      infected IEC extracts were immunoblotted for the transmembrane, spot-weld protein DSG2, the only desmoglein isoform present in the intestine. We observed substantial DSG2 loss at 3 hours after EPEC infection, but not in mock-treated cells (Figure 1A). Immunofluorescence of mock-treated cells, and cells infected with WT EPEC for up to 1 hour, showed the typical junctional localization of DSG2 noted previously.
      • Schlegel N.
      • Meir M.
      • Heupel W.M.
      • Holthofer B.
      • Leube R.E.
      • Waschke J.
      Desmoglein 2-mediated adhesion is required for intestinal epithelial barrier integrity.
      At 2 hours after infection, there was evidence of DSG2 displacement from the cell junctions, and internalization of this protein into the cytosol (Figure 1B); by 3 hours, DSG2 was substantially depleted from infected cells, mirroring the immunoblot results.
      Figure thumbnail gr1
      Figure 1EPEC induces desmoglein-2 loss. C2BBe monolayers were mock-treated, or infected with EPEC at a multiplicity of infection of 100 for 0.5, 1, 2, and 3 hours. (A) Whole-cell extracts of mock-treated or EPEC-infected C2BBe were immunoblotted for DSG2 and actin loading control. Scatter plot shows the densitometry analysis of the abundance of DSG2/actin of each sample compared with mock 0.5 h (3 independent experiments). The median values are indicated by horizontal lines. P values for the DSG2/actin ratio of EPEC 3 hours vs mock 3 hours were computed using analysis of variance with the Bonferroni post hoc test. (B) Immunofluorescence staining of DSG2 (magenta) in mock-treated or EPEC-infected C2BBe imaged with the DeltaVision Elite Deconvolution Microscope equipped with an Olympus 100×/1.40 oil objective and using immersion oil (n = 1.516). 4,6-Diamidino-2-phenylindole (blue) was used to stain DNA. Scale bar: 10 μm. Blots and images are representative of 3 independent experiments. For immunofluorescence staining, at least 6 fields per sample were captured in each experiment.
      In contrast to WT EPEC, an isogenic T3SS-deficient strain (ΔespA) failed to deplete DSG2 (Figure 2A), suggesting a role for 1 or more secreted effectors in the process. To identify the effector proteins involved, we assessed DSG2 abundance in IECs infected with isogenic EPEC mutants lacking specific effector molecules. EPEC Δmap, Δtir, ΔespG, ΔespF, and ΔespZ caused DSG2 loss similar to the WT strain, ruling out a role for the corresponding effectors in DSG2 down-regulation (not shown). A strain lacking EspH (SE874), however, failed to deplete DSG2, and this defect was reversed by espH complementation (SE874/C) (Figure 2B). The major heterotypic partner of DSG2, DSC2, also was depleted in EPEC-infected IECs in an EspH-dependent manner (Figure 2C). Although DSC2 is the primary isoform expressed in the normal intestine, other desmocollin isoforms have been observed in disease states.
      • Khan K.
      • Hardy R.
      • Haq A.
      • Ogunbiyi O.
      • Morton D.
      • Chidgey M.
      Desmocollin switching in colorectal cancer.
      Therefore, all further studies focus on DSG2, with DSC2 included as a validation control where appropriate. In contrast to WT-infected cells, junctional DSG2 staining was maintained in mock-treated and SE874-infected cells (Figure 2D); complementation (SE874/C) restored the ability of the espH mutant to displace DSG2 from the junctions and cause its depletion.
      Figure thumbnail gr2
      Figure 2EPEC EspH induces desmoglein-2 redistribution and loss. (A–C) Whole-cell extracts of C2BBe mock-treated, or infected with EPEC, (A) the T3SS mutant ΔespA, or (B and C) espH mutant SE874 and corresponding complement SE874/C were immunoblotted for (A and B) DSG2 or (C) DSC2, and (A–C) actin loading control. Blots shown are representative of 4 independent experiments. Scatter plot shows densitometry analysis of the abundance of DSG2 or DSC2 relative to actin, respectively, for each sample compared with mock. Horizontal lines represent median values. *P = .0001 for the DSG2/actin ratio of specific sample comparisons calculated using analysis of variance with the Bonferroni post hoc test. (D) Immunofluorescence staining of DSG2 (magenta) in mock-, EPEC-, SE874-, and SE874/C-infected C2BBe cells imaged using the DeltaVision Elite Deconvolution Microscope equipped with an Olympus 100×/1.40 oil objective and immersion oil (n = 1.516). 4,6-Diamidino-2-phenylindole (blue) was used to stain DNA. Scale bar: 6 μm. Images are representative of at least 3 independent experiments. All immunofluorescence images are representative of >6 fields per sample captured for each independent experiment.
      Previous studies have shown the perturbation of TJs in EPEC-infected intestinal epithelial cells, notably the displacement of occludin and zonula occludens 1 from the TJ regions.
      • McNamara B.P.
      • Koutsouris A.
      • O'Connell C.B.
      • Nougayrede J.P.
      • Donnenberg M.S.
      • Hecht G.
      Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function.
      • Garber J.J.
      • Mallick E.M.
      • Scanlon K.M.
      • Turner J.R.
      • Donnenberg M.S.
      • Leong J.M.
      • Snapper S.B.
      Attaching-and-effacing pathogens exploit junction regulatory activities of N-WASP and SNX9 to disrupt the intestinal barrier.
      • Tomson F.L.
      • Viswanathan V.K.
      • Kanack K.J.
      • Kanteti R.P.
      • Straub K.V.
      • Menet M.
      • Kaper J.B.
      • Hecht G.
      EPEC EspG disrupts microtubules and in conjunction with Orf3 enhances perturbation of tight junction barrier.
      • Dean P.
      • Kenny B.
      Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein.
      • Thanabalasuriar A.
      • Koutsouris A.
      • Weflen A.
      • Mimee M.
      • Hecht G.
      • Gruenheid S.
      The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic Escherichia coli.
      In some images of intestinal epithelia from biopsy specimens of EPEC-infected children, however, TJs appeared intact in regions where basolateral unzipping was evident. We therefore used immunofluorescence to assess the temporal relationship between TJ and desmosomal disruptions in EPEC-infected epithelial cells. At 3 hours after infection, occludin staining was restricted primarily to the junctions of infected epithelial cells compared with mock-treated cells (Figure 3A). In contrast, DSG2 is substantially internalized and lost by this time point, suggesting that desmosomal alterations precede TJ disruption.
      Figure thumbnail gr3
      Figure 3Desmoglein-2 perturbation precedes tight junction disruption in EPEC-infected cells. (A and B) C2BBe cells were infected with EPEC at a multiplicity of infection of 100 for 3 hours. (A) Cells were stained for desmoglein-2 (magenta), tight junction protein occludin (green), and 4,6-diamidino-2-phenylindole (DAPI; blue). All images were imaged using the DeltaVision Elite Deconvolution Microscope equipped with an Olympus 60×/1.42 oil objective and 1.59 auxiliary magnification and using immersion oil (n = 1.516). Scale bar: 6 μm. (B) EPEC EspH perturbs desmosomes. Transmission electron micrographs of mock-treated (left-most panel) or infected IECs showing desmosomes (magenta arrows) and areas of unzipping and separation of lateral membranes (blue arrows). Green arrowheads point to TJs. Scale bar: 500 nm. All images are representative of at >6 fields captured per sample from 3 independent experiments.
      Because EPEC depletes DSG2 and DSC2, we assessed desmosomal integrity in infected cells via transmission electron microscopy. Densely staining desmosomes tracked along the lateral walls of mock-treated IECs, as well as cells infected with SE874 (Figure 3B). In contrast, WT EPEC- or SE874/C-infected cells had reduced desmosomal staining, areas of unzipping, and junctional separation. Notably, despite separation of the lateral membranes, TJs appeared intact, further indicating that desmosomal perturbations likely precede EPEC-induced TJ disruption. Collectively, these data suggest that EspH induces DSG2 displacement and loss from cell junctions, leading to desmosomal perturbations and loss of cell–cell contacts.

      EPEC EspH Compromises Cell–Cell Adhesion and Epithelial Barrier Function

      Because desmosomes are critical scaffolds between the lateral membranes of epithelial cells, we assessed the impact of EPEC-induced DSG2 loss on cell junction strength. Dispase-treated mock-infected and commensal E coli–infected cells, as well as ΔespA-infected and SE874-infected cells, were displaced from the plastic substratum, but remained as intact sheets (Figure 4A). In contrast, and consistent with EspH-dependent DSG2 loss, EPEC-infected and SE874/C-infected monolayers disintegrated into small fragments.
      Figure thumbnail gr4
      Figure 4EspH weakens junctional integrity and disrupts barrier function of infected host epithelial monolayers. (A) Images showing C2BBe monolayer integrity after mock treatment, or infection with commensal E coli HS4, EPEC WT, ΔespA, SE874, and SE874/C, respectively, followed by dispase treatment and agitation. Images are representative of 3 independent experiments. (B) TER assays to measure barrier integrity in mock-, EPEC-, SE874-, or SE874/C-infected cells. Cells were infected at an initial multiplicity of infection of 100. *P = .0001 for EPEC or SE874/C compared with SE874 for time points indicated. Data are representative of 3 independent TER assays with at least 3 sample replicates per condition.
      To determine if DSG2 loss impacted barrier function, we monitored infection-mediated TER changes in polarized C2BBe monolayers. WT EPEC induced a progressive decrease in TER relative to mock-treated epithelial cells (Figure 4B). Lack of EspH (SE874) markedly impaired the ability to decrease TER, while complementation (SE874/C) restored the phenotype. Collectively, this suggests that EspH-induced DSG2 loss and desmosomal perturbations compromise epithelial monolayer integrity, leading to loss of epithelial barrier function.

      EspH-Dependent DSG2 Redistribution and Loss Correlates With RhoA Inhibition

      EspH (∼168 amino acids) is unique to A/E pathogens, and does not show significant sequence similarity to other proteins. EspH interaction with Rho GEFs Dbl homology-plecstrin homology domains inhibits Rho GTPase activation, and perturbs the actin cytoskeletion.
      • Dong N.
      • Liu L.
      • Shao F.
      A bacterial effector targets host DH-PH domain RhoGEFs and antagonizes macrophage phagocytosis.
      However, the specific EspH residues involved in these processes remain undefined. To identify EspH domains involved in Rho GTPase inhibition, and in DSG2 relocalization, we generated a panel of alanine-scan mutants. The parent plasmid (encoding WT EspH) and mutant derivatives were used to complement the espH-deficient SE874 strain. Transfected C2BBe cells expressing p115-RhoGEF were infected with this panel of strains, and the extracts were assessed for EspH-p115 RhoGEF interactions. Co-immunoprecipitation studies confirmed interaction of EspH with p115-RhoGEF (Figure 5A); in contrast, 2 alanine-scan mutants, M14 and M16, with replacements of amino acids 88–92 and 98–102, respectively, failed to pull down p115-RhoGEF. The lesions in M14 and M16 did not compromise EspH secretion via the T3SS (Figure 5A).
      Figure thumbnail gr5
      Figure 5EspH inhibition of RhoA correlates with DSG2 loss and weakening of cell–cell interactions in EPEC-infected monolayers. (A) Transfected C2BBe cells expressing Flag-tagged p115-RhoGEF were infected with EPEC, SE874, or SE874 complemented with pJLR4 (WT, His-tagged) or alanine scan mutants M14 (88AAAAA92) or M16 (98AAAAA102). Flag-tagged p115-RhoGEF were immunoprecipitated from whole-cell extracts using anti-Flag antibody. Immunoprecipitated samples were blotted for p115-RhoGEF and His-tagged EspH. Blots shown are representative of 3 independent experiments. Densitometry analysis shows abundance of co-immunoprecipitated His-tagged EspH relative to p115-RhoGEF. Horizontal line represents median. Corresponding bacterial supernatants were concentrated and blotted for His-tagged EspH. Image is of a single gel with irrelevant lanes removed. P values for complemented strains, relative to SE874, are .0001 (pJLR4), .222 (M14), and 1.000 (M16), respectively. (B) RhoA activity in lysophosphatidic acid–treated C2BBe cells infected with EPEC, SE874, SE874/C, or SE874 complemented with pJLR4 (his-tagged EspH), M14, or M16 (alanine-scan mutants). Images are representative of 2 independent RhoA activity assays with 3 sample replicates per condition. P values are .0192 (mock vs EPEC) and .9721 (mock vs SE874); for the complemented strains, relative to SE874, P values are .0145 (pJLR1), .0364 (pJLR4), .0303 (M14), and .1797 (M16), respectively. (C) Extracts of mock-treated C2BBe cells, or C2BBe infected with the same strains as in panel B immunoblotted for DSG2 and DSC2 (upper two panels), and actin (loading control, lower panel). Scatter plot shows densitometry analysis of the abundance of DSG2/actin or DSC2/actin for each sample compared with mock. Horizontal line represents median. For DSG2/actin abundance ratios, P = .0001 for mock vs EPEC and mock vs SE874. P for the complemented strains, relative to SE874, are .0001 (pJLR1), .0002 (pJLR4), .4506 (M14), and .7078 (M16), respectively. For DSC2/actin abundance ratios, P = .0001 for mock vs EPEC and mock vs SE874. P values for the complemented strains, relative to SE874, are .0001 (pJLR1), .0001 (pJLR4), .9030 (M14), and .3527 (M16), respectively. (D) Dispase assays using the same strains described in panel B, and visualization of cell junctional integrity after mechanical agitation. All images for immunoblot and dispase assay are representative of 3 independent experiments. All infections were performed at an initial multiplicity of infection of 100 for 3 hours. COIP, co-immunoprecipitation.
      The corresponding mutants were assessed for their ability to inhibit Rho GTPase activty. WT EPEC, but not SE874, inhibited RhoA activity in IECs (Figure 5B). Complementation of SE874 (SE874/C [=SE874/pJLR1] or SE874/pJLR4 [His-tagged EspH]) restored the ability to inhibit RhoA. In contrast, the RhoGEF interaction-defective mutants M14 and M16 failed to complement SE874 for RhoA inhibition (Figure 5B). Unlike WT espH-complemented SE874, M14- and M16-complemented SE874 also failed to induce the loss of DSG2, or its major heterotypic partner DSC2 (Figure 5C). Finally, M14 and M16 failed to complement SE874 for monolayer fragmentation, as assessed in the dispase assay (Figure 5D). Host-cell adherence of all strains was comparable with WT EPEC (not shown). These data support a model whereby EPEC EspH-mediated Rho GTPase inhibition promotes internalization and loss of the desmosomal proteins DSG2 and DSC2, causes the separation of the lateral membranes of adjacent cells, and compromises cell–cell adhesion and barrier function.

      Mechanism of EspH-Mediated DSG2 Loss in Infected Cells

      EspH competitively binds to RhoGEFs and inhibits Rho GTPase activation.
      • Dong N.
      • Liu L.
      • Shao F.
      A bacterial effector targets host DH-PH domain RhoGEFs and antagonizes macrophage phagocytosis.
      Although Rho GTPases were implicated in desmosomal protein anchoring to IFs in keratinocytes,
      • Waschke J.
      • Spindler V.
      • Bruggeman P.
      • Zillikens D.
      • Schmidt G.
      • Drenckhahn D.
      Inhibition of Rho A activity causes pemphigus skin blistering.
      their contributions to desmosomal integrity in IECs has not been investigated. We therefore explored if Rho activator II CN03 could rescue RhoA activation and restore DSG2 levels downstream of the EspH-RhoGEF block. CN03 is based on the catalytic domain of cytotoxic necrotizing factor 1, which irreversibly deamidates Gln63 of RhoA and locks it in the active confirmation.
      • Schmidt G.
      • Sehr P.
      • Wilm M.
      • Selzer J.
      Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1.
      • Lemichez E.
      • Flatau G.
      • Bruzzone M.
      • Boquet P.
      • Gauthier M.
      Molecular localization of the Escherichia coli cytotoxic necrotizing factor CNF1 cell-binding and catalytic domains.
      Although EPEC infection depleted DSG2 in infected cells, CN03 treatment restored DSG2 abundance (Figure 6A) and its localization (Figure 6B). CN03 alone did not have discernable impacts on DSG2 abundance or localization in mock-treated cells (Figure 6A and B). This further supports the contention that EspH induces DSG2 loss by inhibiting Rho GTPases.
      Figure thumbnail gr6
      Figure 6Rho GTPase activation, as well as actin stabilization, rescues EPEC-infected cells from DSG2 loss. (A and B) C2BBe cells were pretreated with H2O vehicle control (A, lanes 1 and 2; B, upper row), or 300 ng/mL CN03 (A, lanes 3 and 4; B, bottom row) for 1 hour before, and continuing through, EPEC infection. Total proteins were extracted and analyzed for the abundance of (A) DSG2, or infected cells fixed and immunostained for (B) DSG2 (magenta) and 4,6-diamidino-2-phenylindole (DAPI; blue). (A) Scatter plot shows densitometry analysis of the abundance of DSG2/actin of EPEC-infected sample compared with corresponding mock. Horizontal line represents median. Immunoblots are representative of 4 independent experiments. P = .0001 (mock + vehicle vs EPEC + vehicle), .6657 (mock + CN03 vs EPEC + CN03), and .0001 (EPEC + vehicle vs EPEC + CN03). (C) C2BBe cells were pretreated with dimethyl sulfoxide vehicle control or 1 μmol/L jasplakinolide for 1 hour before, and continuing through, infection (mock, WT EPEC, SE874, or SE874/C), and fixed and immunostained for DSG2 (magenta), cytokeratin 8/18 [green], and DAPI [blue]. Infections were performed at an initial multiplicity of infection of 100 for 3 hours. Arrows mark IF retraction from cell membranes. All immunofluorescence images were captured using the DeltaVision Elite Deconvolution Microscope equipped with an Olympus 60×/1.42 oil objective and using immersion oil (n = 1.516), and are representative of >6 fields per sample from 3 independent experiments. Scale bar: 11 μm.
      To our knowledge, there is no known pathway linking Rho GTPases to DSG2 stabilization at cell junctions. Because Rho GTPase activation can stimulate actin polymerization, we considered a role for cytoskeletal modulation in EPEC-induced DSG2 down-regulation. The actin cytoskeleton plays a role in maintaining keratin IFs, and facilitates IF anchorage to desmosomes via the cytolinker desmoplakin.
      • Godsel L.M.
      • Hsieh S.N.
      • Amargo E.V.
      • Bass A.E.
      • Pascoe-McGillicuddy L.T.
      • Huen A.C.
      • Thorne M.E.
      • Gaudry C.A.
      • Park J.K.
      • Myung K.
      • Goldman R.D.
      • Chew T.L.
      • Green K.J.
      Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin.
      • Kouklis P.D.
      • Hutton E.
      • Fuchs E.
      Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.
      Keratin IF precursors nucleate at desmosomes, and then translocate continuously toward the nucleus until they integrate end-on into the peripheral IF network.
      • Windoffer R.
      • Beil M.
      • Magin T.M.
      • Leube R.E.
      Cytoskeleton in motion: the dynamics of keratin intermediate filaments in epithelia.
      Importantly, this inward-directed transport of keratin IF precursors depends on actin retrograde flow.
      • Kolsch A.
      • Windoffer R.
      • Leube R.E.
      Actin-dependent dynamics of keratin filament precursors.
      We therefore hypothesized that EspH-mediated Rho inactivation and consequent actin destabilization impairs the normal actin-dependent transport of keratin IFs, ultimately disrupting DSG2 and desmosome -anchoring at the junctions. To test this hypothesis, we infected IECs with EPEC strains and visualized IFs (via cytokeratin 8/18 staining) and DSG2. Consistent with our hypothesis, keratin staining was disrupted, or absent, in IECs infected with WT EPEC and SE874/C (Figure 6C); in several cells, the staining was suggestive of IF collapse away from the cell periphery, and toward the nucleus. Keratin staining remained intact, and comparable with mock-treated cells, in SE874-infected IECs (Figure 6C). This suggests that EspH impedes the inward-directed transport of new and maturing keratin IFs. Consistent with a role for cytoskeletal perturbation in this process, the actin-stabilizing drug jasplakinolide preserved IF integrity and DSG2 membrane localization in EPEC- and SE874/C-infected cells (Figure 6C). In the absence of infection, jasplakinolide did not impact DSG2 localization or abundance. Together, these data suggest that EspH-dependent Rho GTPase inactivation leads to actin depolymerization; this, in turn, perturbs IF stability and, ultimately, leads to destabilization of desmosomes and DSG2 down-regulation.

      DSG2 Is Internalized and Degraded in the Lysosomes of EPEC-Infected Cells

      Destabilized desmosomes can split into half-desmosomes and be internalized; in keratinocytes, desmogleins are degraded via the lysosomal or proteasomal pathways.
      • McHarg S.
      • Hopkins G.
      • Lim L.
      • Garrod D.
      Down-regulation of desmosomes in cultured cells: the roles of PKC, microtubules and lysosomal/proteasomal degradation.
      To assess DSG2 fate in EPEC-infected IECs, we determined the effects of the lysosomal inhibitor bafilomycin A1 and the proteasomal inhibitor MG132. Bafilomycin A1, but not the vehicle dimethyl sulfoxide, prevented EPEC-induced DSG2 loss (Figure 7A). MG132 had no apparent impact on infection-induced DSG2 displacement from the cell junctions to the cytosol (Figure 7B), but partially protected against DSG2 loss (Figure 7A). In contrast, lysosomal inhibition protected against EPEC-induced DSG2 loss (Figure 7A); moreover, DSG2 was internalized, and co-localized with lysosomal-associated membrane protein 1 in bafilomycin-treated, EPEC-infected IECs (Figure 7B). Taken together, these results suggest that EPEC EspH promotes junctional instability of DSG2, leading to its internalization and subsequent degradation primarily within lysosomes.
      Figure thumbnail gr7
      Figure 7DSG2 is degraded in the lysosomes of EPEC-infected cells. C2BBe cells were pretreated with 250 nmol/L lysosomal inhibitor bafilomycin A1, 10 μmol/L proteasomal inhibitor MG132, or dimethyl sulfoxide vehicle control for 2 hours before infection with EPEC at a multiplicity of infection of 100 for 3 hours. Inhibitor concentrations were maintained throughout the course of infection. (A) Total protein extracts immunoblotted for DSG2 (or actin; loading control). Scatter plot shows densitometry analysis of the abundance of DSG2/actin of EPEC-infected sample compared with corresponding mock. Horizontal line represents median. Immunoblots are representative of 3 independent experiments. P = .0001 for EPEC + MG132 and EPEC + bafilomycin A1 compared with EPEC + vehicle, respectively. (B) Mock-treated and infected cells were fixed and immunostained for DSG2 (magenta), lysosomal-associated membrane protein 1 (LAMP1; green), and 4,6-diamidino-2-phenylindole (blue). Arrows point to co-localized DSG2 and LAMP1. All images were captured using the DeltaVision Elite Deconvolution Microscope equipped with an Olympus 100×/1.40 oil objective and using immersion oil (n = 1.516), and are representative of >6 fields per sample from 3 independent experiments.
      We explored 2 alternate pathways for direct DSG2 cleavage in EPEC-infected cells. EspH activates caspase-3, a cysteine–aspartic acid protease involved in programmed cell death.
      • Wong A.R.
      • Clements A.
      • Raymond B.
      • Crepin V.F.
      • Frankel G.
      The interplay between the Escherichia coli Rho guanine nucleotide exchange factor effectors and the mammalian RhoGEF inhibitor EspH.
      EPEC also activates calcium-dependent nonlysosomal enzymes (calpains), although EspH has not been implicated in this pathway.
      • Serapio-Palacios A.
      • Navarro-Garcia F.
      EspC, an autotransporter protein secreted by enteropathogenic Escherichia coli, causes apoptosis and necrosis through caspase and calpain activation, including direct procaspase-3 cleavage.
      Because DSG2 is a known target of both caspases and calpains,
      • Nava P.
      • Laukoetter M.G.
      • Hopkins A.M.
      • Laur O.
      • Gerner-Smidt K.
      • Green K.J.
      • Parkos C.A.
      • Nusrat A.
      Desmoglein-2: a novel regulator of apoptosis in the intestinal epithelium.
      we assessed the effects of calpain inhibitor I and the pan-caspase inhibitor z-DEVD-fmk, respectively, on EPEC-induced DSG2 loss. Neither inhibitor impacted infection-induced DSG2 loss, indicating that caspases and calpains are not involved in this process (data not shown).

      EspH Contributes to EPEC-Induced DSG2 Relocalization, and to Intestinal Colonization, in a Mouse Model of Infection

      Finally, we assessed EPEC-induced DSG2 down-regulation and desmosomal perturbation in vivo using a mouse colonization model.
      • Royan S.V.
      • Jones R.M.
      • Koutsouris A.
      • Roxas J.L.
      • Falzari K.
      • Weflen A.W.
      • Kim A.
      • Bellmeyer A.
      • Turner J.R.
      • Neish A.S.
      • Rhee K.J.
      • Viswanathan V.K.
      • Hecht G.A.
      Enteropathogenic E. coli non-LEE encoded effectors NleH1 and NleH2 attenuate NF-kappaB activation.
      • Roxas J.L.
      • Koutsouris A.
      • Bellmeyer A.
      • Tesfay S.
      • Royan S.
      • Falzari K.
      • Harris A.
      • Cheng H.
      • Rhee K.J.
      • Hecht G.
      Enterohemorrhagic E. coli alters murine intestinal epithelial tight junction protein expression and barrier function in a Shiga toxin independent manner.
      WT EPEC robustly colonized the mouse cecum and colon on days 3 and 4 after infection (Figure 8A). Although SE874 colonization of the cecum and colon were comparable with WT EPEC on day 3 after infection, its levels decreased at both sites by day 4 after infection (Figure 8A), suggesting a role for EspH in EPEC persistence in the mouse intestine. Both strains were shed at comparable levels on days 1–4 after infection (data not shown).
      Figure thumbnail gr8
      Figure 8EspH contributes to EPEC persistence in the mouse gut, DSG2 redistribution, and disruption of mouse intestinal epithelial cell junctions. C57BL/6J mice were mock-treated or infected with EPEC or SE874. (A) Colonization of the cecum and colon on days 3 and 4 after infection (N = 4 per sample group) were evaluated by plating on selective media. Horizontal line represents median. P values were computed using analysis of variance with the Bonferroni post hoc test. Colon tissues of mock-treated or day 3 postinfected mice (N = 4 per sample group) were used for (B) immunofluorescence staining and (C) transmission electron microscopy experiments. (A) Blue boxes correspond to representative tissue samples of infected animals shown in panels B and C. (B) Colon tissues of mock-treated, EPEC-infected or SE874-infected mice were stained for DSG2 (magenta) and 4,6-diamidino-2-phenylindole (blue). Arrows point to punctate cytoplasmic DSG2 staining. Images were captured using the DeltaVision Elite Deconvolution Microscope equipped with an Olympus 40×/1.35 oil objective and using immersion oil (n = 1.516). Scale bar: 17 μm. (C) Transmission electron micrographs of colon tissues of mock-treated (left panel), EPEC-infected (middle panel), or SE874-infected (right panel) showing tight junctions (magenta arrowheads) and areas of unzipping and separation of lateral membranes (blue arrows). Scale bar: 500 nm. All immunofluorescence and transmission electron microscopy images are representative of at least 6 fields captured per sample from the 3 sample groups (n = 4 per sample group). CFU, colony-forming unit.
      To investigate infection-induced DSG2 alterations in vivo, colonic tissues isolated on day 3 after infection, when WT EPEC and SE874 colonized at comparable levels, were subjected to immunofluorescence staining. In uninfected and SE874-infected mice, DSG2 was distributed uniformly along the colonic epithelial cell junctions (Figure 8B). In colon tissues of EPEC-infected mice, however, DSG2 was relocalized intracellularly into distinct puncta, consistent with our in vitro observations. Electron micrographs images of infected tissues showed separation of the lateral membranes of colonic epithelial cells in WT-infected mice, but not mock-treated or SE874-infected animals (Figure 8C). Notably, as previously seen in infant colon biopsy specimens,
      • Rothbaum R.J.
      • Partin J.C.
      • Saalfield K.
      • McAdams A.J.
      An ultrastructural study of enteropathogenic Escherichia coli infection in human infants.
      and in vitro (Figure 4B), basolateral unzipping was evident in EPEC-infected mice even in areas where the TJs appeared unperturbed. Collectively, these data suggest that EspH mediates EPEC-induced DSG2 relocalization from intestinal epithelial junctions, its subsequent loss in the cytosol, and the consequent perturbation of cell–cell contacts. These changes likely contribute to EPEC colonization of the mouse intestine.

      Discussion

      As its primary virulence strategy, EPEC secretes effector proteins into IECs and alters their physiology.
      • Berkes J.
      • Viswanathan V.K.
      • Savkovic S.D.
      • Hecht G.
      Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation.
      We show that the RhoGEF-sequestering effector EspH inhibits RhoA activity and, correspondingly, induces the internalization and degradation of DSG2 and its major heterotypic partner, DSC2. This results in the loss of desmosomal contacts between adjacent cells, weakened cell–cell adhesion, and disruption of epithelial barrier function. The loss of desmosomal contacts could be a precursor to the cell rounding phenotype observed in EspH-expressing cells,
      • Dong N.
      • Liu L.
      • Shao F.
      A bacterial effector targets host DH-PH domain RhoGEFs and antagonizes macrophage phagocytosis.
      • Tu X.
      • Nisan I.
      • Yona C.
      • Hanski E.
      • Rosenshine I.
      EspH, a new cytoskeleton-modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli.
      and the epithelial erosion previously seen in infected animal tissues.
      • Al-Mamun A.
      • Mily A.
      • Sarker P.
      • Tiash S.
      • Navarro A.
      • Akter M.
      • Talukder K.A.
      • Islam M.F.
      • Agerberth B.
      • Gudmundsson G.H.
      • Cravioto A.
      • Raqib R.
      Treatment with phenylbutyrate in a pre-clinical trial reduces diarrhea due to enteropathogenic Escherichia coli: link to cathelicidin induction.
      Importantly, the mechanism outlined for EspH-dependent DSG2 depletion and desmosomal perturbation may provide a molecular basis for the basolateral junction widening observed in intestinal biopsy specimens from EPEC-infected infants.
      • Rothbaum R.J.
      • Partin J.C.
      • Saalfield K.
      • McAdams A.J.
      An ultrastructural study of enteropathogenic Escherichia coli infection in human infants.
      Rho GTPases were implicated in the maintenance of desmosomal integrity of keratinocytes and myocardial cells,
      • Waschke J.
      • Spindler V.
      • Bruggeman P.
      • Zillikens D.
      • Schmidt G.
      • Drenckhahn D.
      Inhibition of Rho A activity causes pemphigus skin blistering.
      • Gliem M.
      • Heupel W.M.
      • Spindler V.
      • Harms G.S.
      • Waschke J.
      Actin reorganization contributes to loss of cell adhesion in pemphigus vulgaris.
      • Spindler V.
      • Meir M.
      • Vigh B.
      • Flemming S.
      • Hutz K.
      • Germer C.T.
      • Waschke J.
      • Schlegel N.
      Loss of desmoglein 2 contributes to the pathogenesis of Crohn's disease.
      • Ellawindy A.
      • Satoh K.
      • Sunamura S.
      • Kikuchi N.
      • Suzuki K.
      • Minami T.
      • Ikeda S.
      • Tanaka S.
      • Shimizu T.
      • Enkhjargal B.
      • Miyata S.
      • Taguchi Y.
      • Handoh T.
      • Kobayashi K.
      • Kobayashi K.
      • Nakayama K.
      • Miura M.
      • Shimokawa H.
      Rho-kinase inhibition during early cardiac development causes arrhythmogenic right ventricular cardiomyopathy in mice.
      but their impact on desmosomes of the very different simple columnar intestinal epithelia is unknown. On the other hand, the effects of Rho GTPase signaling on intestinal epithelial TJs and adherens junctions have been characterized in some detail.
      • Citi S.
      • Guerrera D.
      • Spadaro D.
      • Shah J.
      Epithelial junctions and Rho family GTPases: the zonular signalosome.
      Our studies provide 2 lines of evidence for a key role for Rho GTPase signaling in maintaining IEC desmosomal junctions: EspH mutants that failed to inhibit RhoA also were unable to perturb desmosomes, and CN03-mediated Rho GTPase activation rescued EPEC-induced DSG2 loss.
      EspH-induced Rho GTPase inhibition leads to actin cytoskeleton disruption
      • Dong N.
      • Liu L.
      • Shao F.
      A bacterial effector targets host DH-PH domain RhoGEFs and antagonizes macrophage phagocytosis.
      ; our studies also implicate a role for EspH in perturbing the IF network. Our observations are consistent with a model whereby EspH-mediated Rho GTPase inactivation impedes the actin-dependent, inward-directed transport of cytokeratins from the periphery to the cell center, leading to IF retraction
      • Windoffer R.
      • Beil M.
      • Magin T.M.
      • Leube R.E.
      Cytoskeleton in motion: the dynamics of keratin intermediate filaments in epithelia.
      • Kolsch A.
      • Windoffer R.
      • Leube R.E.
      Actin-dependent dynamics of keratin filament precursors.
      ; this, possibly in conjunction with displacement of the IF-desmosome linker, desmoplakin, induces DSG2 internalization into, and degradation in, lysosomes.
      • Godsel L.M.
      • Hsieh S.N.
      • Amargo E.V.
      • Bass A.E.
      • Pascoe-McGillicuddy L.T.
      • Huen A.C.
      • Thorne M.E.
      • Gaudry C.A.
      • Park J.K.
      • Myung K.
      • Goldman R.D.
      • Chew T.L.
      • Green K.J.
      Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin.
      • Kouklis P.D.
      • Hutton E.
      • Fuchs E.
      Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.
      We previously described cytokeratin alterations in EPEC-infected cells partially mediated by EspF.
      • Viswanathan V.K.
      • Lukic S.
      • Koutsouris A.
      • Miao R.
      • Muza M.M.
      • Hecht G.
      Cytokeratin 18 interacts with the enteropathogenic Escherichia coli secreted protein F (EspF) and is redistributed after infection.
      EspF therefore also may contribute to desmosomal perturbations. However, given the profound deficiency of cytokeratin and DSG2 perturbations in cells infected with SE874 (which lacks EspH, but expresses EspF), EspF may have only an ancillary role in this process.
      Desmosomal perturbations have been reported in the context of other intestinal infections, notably with Entamoeba histolytica, Giardia duodenalis, and Campylobacter jejuni.
      • Hernández-Nava E.
      • Cuellar P.
      • Nava P.
      • Chávez-Munguía B.
      • Schnoor M.
      • Orozco E.
      • Betanzos A.
      Adherens junctions and desmosomes are damaged by Entamoeba histolytica: participation of EhCPADH complex and EhCP112 protease.
      • Maia-Brigagão C.
      • Morgado-Díaz J.
      • De Souza W.
      Giardia disrupts the arrangement of tight, adherens and desmosomal junction proteins of intestinal cells.
      • Wine E.
      • Chan V.L.
      • Sherman P.M.
      Campylobacter jejuni mediated disruption of polarized epithelial monolayers is cell-type specific, time dependent, and correlates with bacterial invasion.
      E histolytica EhCP112 protease cleaves several desmosomal proteins, including DSG2, but the underlying mechanisms for C jejuni– and G duodenalis–mediated desmosomal perturbation are unknown. The current study is a mechanistic characterization of Rho-dependent intestinal epithelial desmosomal perturbation. By extension, other enteric pathogens that harbor Rho GTPase-inactivating virulence proteins, such as Clostridium difficile (toxins TcdA and TcdB),
      • Aktories K.
      Rho-modifying bacterial protein toxins.
      Vibrio cholerae (MARTX toxin),
      • Ahrens S.
      • Geissler B.
      • Satchell K.J.
      Identification of a His-Asp-Cys catalytic triad essential for function of the Rho inactivation domain (RID) of Vibrio cholerae MARTX toxin.
      and Yersinia (YopE, YpkA, and YopT)
      • Prehna G.
      • Ivanov M.I.
      • Bliska J.B.
      • Stebbins C.E.
      Yersinia virulence depends on mimicry of host Rho-family nucleotide dissociation inhibitors.
      may also promote desmosome destabilization. Beyond a possible role in the etiology of infectious diseases, a robust decrease in DSG2 levels was observed in the mucosa of patients with Crohn’s disease.
      • Spindler V.
      • Meir M.
      • Vigh B.
      • Flemming S.
      • Hutz K.
      • Germer C.T.
      • Waschke J.
      • Schlegel N.
      Loss of desmoglein 2 contributes to the pathogenesis of Crohn's disease.
      It is noteworthy that there was an increased abundance of specific pathogenic E coli strains in a subset of Crohn’s disease patients; many of these strains disrupt epithelial barrier function, but their impacts on desmosomal junctions have not been explored.
      • Shawki A.
      • McCole D.F.
      Mechanisms of intestinal epithelial barrier dysfunction by adherent-invasive Escherichia coli.
      Our work now adds EspH to the list of EPEC effector proteins that perturb epithelial paracellular permeability.
      • Singh A.P.
      • Aijaz S.
      Enteropathogenic E. coli: breaking the intestinal tight junction barrier.
      The impact of EspH on epithelial barrier function is novel and noteworthy because it suggests a role for desmosomes beyond that of inert scaffolding. Consistent with this hypothesis, small interfering RNA–mediated DSG2 down-regulation in IECs alters epithelial barrier function.
      • Spindler V.
      • Meir M.
      • Vigh B.
      • Flemming S.
      • Hutz K.
      • Germer C.T.
      • Waschke J.
      • Schlegel N.
      Loss of desmoglein 2 contributes to the pathogenesis of Crohn's disease.
      Interestingly, a tandem peptide-mediated stabilization of junctional DSG2 linkages in Caco-2 cells ameliorated TNF-α–induced barrier disruption.
      • Spindler V.
      • Meir M.
      • Vigh B.
      • Flemming S.
      • Hutz K.
      • Germer C.T.
      • Waschke J.
      • Schlegel N.
      Loss of desmoglein 2 contributes to the pathogenesis of Crohn's disease.
      Electron micrographs images of EPEC-infected epithelial cells suggest that DSG2 depletion and separation of lateral cell surfaces precede complete dissociation of TJs. Our data, however, do not rule out the possibility of a direct impact of EspH-mediated Rho inhibition on TJs, independent of, and in addition to, the desmosomal alterations.
      In contrast to our observations, Guttman et al
      • Guttman J.A.
      • Kazemi P.
      • Lin A.E.
      • Vogl A.W.
      • Finlay B.B.
      Desmosomes are unaltered during infections by attaching and effacing pathogens.
      noted that desmoglein and desmocollin remain unchanged during EPEC infection in vitro. The differences in experimental conditions between the 2 studies is a likely explanation for this discrepancy. Whereas Guttman et al
      • Guttman J.A.
      • Kazemi P.
      • Lin A.E.
      • Vogl A.W.
      • Finlay B.B.
      Desmosomes are unaltered during infections by attaching and effacing pathogens.
      used subconfluent Caco-2 cells, our studies used confluent C2BBe cells that reportedly have maximal cell-junctional DSG2 distribution and stable desmosomes.
      Interestingly, Guttman et al
      • Guttman J.A.
      • Kazemi P.
      • Lin A.E.
      • Vogl A.W.
      • Finlay B.B.
      Desmosomes are unaltered during infections by attaching and effacing pathogens.
      also did not detect desmoglein redistribution in colonic tissues of mice infected with Citrobacter rodentium. This may well represent a true distinction between EPEC and C rodentium. Unlike the prototype EPEC strain used in this study, C rodentium secretes EspM2 and EspT, which mimic GEF activity for RhoA and Rac1, respectively. These effectors were shown to mitigate some impacts of EspH-mediated Rho GTPase suppression.
      • Dong N.
      • Liu L.
      • Shao F.
      A bacterial effector targets host DH-PH domain RhoGEFs and antagonizes macrophage phagocytosis.
      • Bulgin R.R.
      • Arbeloa A.
      • Chung J.C.
      • Frankel G.
      EspT triggers formation of lamellipodia and membrane ruffles through activation of Rac-1 and Cdc42.
      • Arbeloa A.
      • Blanco M.
      • Moreira F.C.
      • Bulgin R.
      • Lopez C.
      • Dahbi G.
      • Blanco J.E.
      • Mora A.
      • Alonso M.P.
      • Mamani R.C.
      • Gomes T.A.
      • Blanco J.
      • Frankel G.
      Distribution of espM and espT among enteropathogenic and enterohaemorrhagic Escherichia coli.
      • Arbeloa A.
      • Garnett J.
      • Lillington J.
      • Bulgin R.R.
      • Berger C.N.
      • Lea S.M.
      • Matthews S.
      • Frankel G.
      EspM2 is a RhoA guanine nucleotide exchange factor.
      Consistent with this, C rodentium mutants lacking espH were not impaired for virulence in mouse infection studies, although they fared poorer in competition experiments with WT C rodentium.
      • Mundy R.
      • MacDonald T.T.
      • Dougan G.
      • Frankel G.
      • Wiles S.
      Citrobacter rodentium of mice and man.
      In contrast, EPEC mutants lacking espH initially colonized the mouse cecum and colon at levels comparable with the parent WT strain (3 days after infection) but were recovered at lower levels on day 4 after infection. EspH-induced barrier perturbations may enhance nutrient flux into the intestine, which, in turn, may facilitate EPEC expansion; alternatively, the concomitant activation of inflammatory responses may deplete commensal organisms and provide EPEC a competitive edge. In a previous study, enterohemorrhagic E coli mutants lacking espH also were shown to be impaired for virulence in a rabbit model of infection.
      • Ritchie J.M.
      • Waldor M.K.
      The locus of enterocyte effacement-encoded effector proteins all promote enterohemorrhagic Escherichia coli pathogenicity in infant rabbits.
      A comprehensive understanding of the role of EspH, as well as Rho GTPase inhibition and desmosomal changes in A/E pathogen virulence, therefore, requires due consideration of the specific model systems, and an appreciation for the cooperative action of effector molecules. In summary, we identified a novel role for EPEC EspH in perturbing intestinal epithelial desmosomal junctions, which likely contributes to disease. Our delineation of the underlying pathway of desmosomal disruption, involving Rho GTPases and the actin cytoskeleton, has broader implications for both normal intestinal function and for various pathologic states.

      Acknowledgments

      The authors thank Professor Mrinalini Rao for critically reading the manuscript and suggesting substantial improvements. The authors also thank Anthony Joseph Santiago for assisting with the animal experiments. The authors gratefully acknowledge the University of Arizona Microscopy Alliance for providing access to their FEI Tecnai Spirit 120kV Transmission Electron Microscope and DeltaVision Elite Deconvolution Microscope, and William Day, Patricia Jansma, and Allison Buchanan for technical support.

      References

        • Groschwitz K.R.
        • Hogan S.P.
        Intestinal barrier function: molecular regulation and disease pathogenesis.
        J Allergy Clin Immunol. 2009; 124: 3-20
        • Giepmans B.N.
        • van Ijzendoorn S.C.
        Epithelial cell-cell junctions and plasma membrane domains.
        Biochim Biophys Acta. 2009; 1788: 820-831
        • Anderson J.M.
        • Van Itallie C.M.
        Tight junctions and the molecular basis for regulation of paracellular permeability.
        Am J Physiol. 1995; 269: G467-G475
        • Niessen C.M.
        Tight junctions/adherens junctions: basic structure and function.
        J Invest Dermatol. 2007; 127: 2525-2532
        • Nekrasova O.
        • Green K.J.
        Desmosome assembly and dynamics.
        Trends Cell Biol. 2013; 23: 537-546
        • Holthöfer B.
        • Windoffer R.
        • Troyanovsky S.
        • Leube R.E.
        Structure and function of desmosomes.
        Int Rev Cytol. 2007; 264: 65-163
        • Shabih-e-Hassnain S.
        • Trinnaman B.
        • Martin S.
        • Major S.
        • Hutchinson J.
        • Magee A.I.
        Molecular interactions between desmosomal cadherins.
        Biochem J. 2002; 362: 317-327
        • Capaldo C.T.
        • Farkas A.E.
        • Nusrat A.
        Epithelial adhesive junctions.
        F1000Prime Rep. 2014; 6: 1
        • Barreau F.
        • Hugot J.
        Intestinal barrier dysfunction triggered by invasive bacteria.
        Curr Opin Microbiol. 2014; 17: 91-98
        • Lanata C.F.
        • Fischer-Walker C.L.
        • Olascoaga A.C.
        • Torres C.X.
        • Aryee M.J.
        • Black R.E.
        Child Health Epidemiology Reference Group of the World Health Organization and UNICEF. Global causes of diarrheal disease mortality in children <5 years of age: a systematic review.
        PLoS One. 2013; 8: e72788
        • Nataro J.P.
        • Kaper J.B.
        Diarrheagenic Escherichia coli.
        Clin Microbiol Rev. 1998; 11: 142-201
        • Berkes J.
        • Viswanathan V.K.
        • Savkovic S.D.
        • Hecht G.
        Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation.
        Gut. 2003; 52: 439-451
        • McNamara B.P.
        • Koutsouris A.
        • O'Connell C.B.
        • Nougayrede J.P.
        • Donnenberg M.S.
        • Hecht G.
        Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function.
        J Clin Invest. 2001; 107: 621-629
        • Garber J.J.
        • Mallick E.M.
        • Scanlon K.M.
        • Turner J.R.
        • Donnenberg M.S.
        • Leong J.M.
        • Snapper S.B.
        Attaching-and-effacing pathogens exploit junction regulatory activities of N-WASP and SNX9 to disrupt the intestinal barrier.
        Cell Mol Gastroenterol Hepatol. 2017; 5: 273-288
        • Tomson F.L.
        • Viswanathan V.K.
        • Kanack K.J.
        • Kanteti R.P.
        • Straub K.V.
        • Menet M.
        • Kaper J.B.
        • Hecht G.
        EPEC EspG disrupts microtubules and in conjunction with Orf3 enhances perturbation of tight junction barrier.
        Mol Microbiol. 2005; 2: 447-464
        • Dean P.
        • Kenny B.
        Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein.
        Mol Microbiol. 2004; 54: 665-675
        • Thanabalasuriar A.
        • Koutsouris A.
        • Weflen A.
        • Mimee M.
        • Hecht G.
        • Gruenheid S.
        The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic Escherichia coli.
        Cell Microbiol. 2010; 12: 31-41
        • Malladi V.
        • Shankar B.
        • Williams P.H.
        • Balakrishnan A.
        Enteropathogenic Escherichia coli outer membrane proteins induce changes in cadherin junctions of Caco-2 cells through activation of PKCalpha.
        Microbes Infect. 2004; 6: 38-50
        • Rothbaum R.J.
        • Partin J.C.
        • Saalfield K.
        • McAdams A.J.
        An ultrastructural study of enteropathogenic Escherichia coli infection in human infants.
        Ultrastruct Pathol. 1983; 4: 291-304
        • Peterson M.D.
        • Mooseker M.S.
        Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco-2.
        J Cell Sci. 1992; 102: 581-600
        • Roxas J.L.
        • Ryan K.
        • Vedantam G.
        • Viswanathan V.K.
        Enteropathogenic Escherichia coli dynamically regulates EGFR signaling in intestinal epithelial cells.
        Am J Physiol Gastrointest Liver Physiol. 2014; 307: G374-G380
        • Elliott S.J.
        • Krejany E.O.
        • Mellies J.L.
        • Robins-Browne R.M.
        • Sasakawa C.
        • Kaper J.B.
        EspG, a novel type III system-secreted protein from enteropathogenic Escherichia coli with similarities to VirA of Shigella flexneri.
        Infect Immun. 2001; 69: 4027-4033
        • Penfold R.J.
        • Pemberton J.M.
        An improved suicide vector for construction of chromosomal insertion mutations in bacteria.
        Gene. 1992; 118: 145-146
        • Bromberg Y.
        • Rost B.
        Comprehensive in silico mutagenesis highlights functionally important residues in proteins.
        Bioinformatics. 2008; 24: i207-i212
        • Jiang K.
        • Rankin C.R.
        • Nava P.
        • Sumagin R.
        • Kamekura R.
        • Stowell S.R.
        • Feng M.
        • Parkos C.A.
        • Nusrat A.
        Galectin-3 regulates desmoglein-2 and intestinal epithelial intercellular adhesion.
        J Biol Chem. 2014; 289: 10510-10517
        • Royan S.V.
        • Jones R.M.
        • Koutsouris A.
        • Roxas J.L.
        • Falzari K.
        • Weflen A.W.
        • Kim A.
        • Bellmeyer A.
        • Turner J.R.
        • Neish A.S.
        • Rhee K.J.
        • Viswanathan V.K.
        • Hecht G.A.
        Enteropathogenic E. coli non-LEE encoded effectors NleH1 and NleH2 attenuate NF-kappaB activation.
        Mol Microbiol. 2010; 78: 1232-1245
        • Schlegel N.
        • Meir M.
        • Heupel W.M.
        • Holthofer B.
        • Leube R.E.
        • Waschke J.
        Desmoglein 2-mediated adhesion is required for intestinal epithelial barrier integrity.
        Am J Physiol Gastrointest Liver Physiol. 2010; 298: G774-G783
        • Khan K.
        • Hardy R.
        • Haq A.
        • Ogunbiyi O.
        • Morton D.
        • Chidgey M.
        Desmocollin switching in colorectal cancer.
        Br J Cancer. 2006; 95: 1367-1370
        • Dong N.
        • Liu L.
        • Shao F.
        A bacterial effector targets host DH-PH domain RhoGEFs and antagonizes macrophage phagocytosis.
        EMBO J. 2010; 29: 1363-1376
        • Waschke J.
        • Spindler V.
        • Bruggeman P.
        • Zillikens D.
        • Schmidt G.
        • Drenckhahn D.
        Inhibition of Rho A activity causes pemphigus skin blistering.
        J Cell Biol. 2006; 175: 721-727
        • Schmidt G.
        • Sehr P.
        • Wilm M.
        • Selzer J.
        Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1.
        Nature. 1997; 387: 725
        • Lemichez E.
        • Flatau G.
        • Bruzzone M.
        • Boquet P.
        • Gauthier M.
        Molecular localization of the Escherichia coli cytotoxic necrotizing factor CNF1 cell-binding and catalytic domains.
        Mol Microbiol. 1997; 24: 1061-1070
        • Godsel L.M.
        • Hsieh S.N.
        • Amargo E.V.
        • Bass A.E.
        • Pascoe-McGillicuddy L.T.
        • Huen A.C.
        • Thorne M.E.
        • Gaudry C.A.
        • Park J.K.
        • Myung K.
        • Goldman R.D.
        • Chew T.L.
        • Green K.J.
        Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin.
        J Cell Biol. 2005; 171: 1045-1059
        • Kouklis P.D.
        • Hutton E.
        • Fuchs E.
        Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.
        J Cell Biol. 1994; 127: 1049-1060
        • Windoffer R.
        • Beil M.
        • Magin T.M.
        • Leube R.E.
        Cytoskeleton in motion: the dynamics of keratin intermediate filaments in epithelia.
        J Cell Biol. 2011; 194: 669-678
        • Kolsch A.
        • Windoffer R.
        • Leube R.E.
        Actin-dependent dynamics of keratin filament precursors.
        Cell Motil Cytoskeleton. 2009; 66: 976-985
        • McHarg S.
        • Hopkins G.
        • Lim L.
        • Garrod D.
        Down-regulation of desmosomes in cultured cells: the roles of PKC, microtubules and lysosomal/proteasomal degradation.
        PLoS One. 2014; 9: e108570
        • Wong A.R.
        • Clements A.
        • Raymond B.
        • Crepin V.F.
        • Frankel G.
        The interplay between the Escherichia coli Rho guanine nucleotide exchange factor effectors and the mammalian RhoGEF inhibitor EspH.
        MBio. 2012; 3: 1
        • Serapio-Palacios A.
        • Navarro-Garcia F.
        EspC, an autotransporter protein secreted by enteropathogenic Escherichia coli, causes apoptosis and necrosis through caspase and calpain activation, including direct procaspase-3 cleavage.
        MBio. 2016; 7: 3
        • Nava P.
        • Laukoetter M.G.
        • Hopkins A.M.
        • Laur O.
        • Gerner-Smidt K.
        • Green K.J.
        • Parkos C.A.
        • Nusrat A.
        Desmoglein-2: a novel regulator of apoptosis in the intestinal epithelium.
        Mol Biol Cell. 2007; 18: 4565-4578
        • Roxas J.L.
        • Koutsouris A.
        • Bellmeyer A.
        • Tesfay S.
        • Royan S.
        • Falzari K.
        • Harris A.
        • Cheng H.
        • Rhee K.J.
        • Hecht G.
        Enterohemorrhagic E. coli alters murine intestinal epithelial tight junction protein expression and barrier function in a Shiga toxin independent manner.
        Lab Invest. 2010; 90: 1152-1168
        • Tu X.
        • Nisan I.
        • Yona C.
        • Hanski E.
        • Rosenshine I.
        EspH, a new cytoskeleton-modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli.
        Mol Microbiol. 2003; 47: 595-606
        • Al-Mamun A.
        • Mily A.
        • Sarker P.
        • Tiash S.
        • Navarro A.
        • Akter M.
        • Talukder K.A.
        • Islam M.F.
        • Agerberth B.
        • Gudmundsson G.H.
        • Cravioto A.
        • Raqib R.
        Treatment with phenylbutyrate in a pre-clinical trial reduces diarrhea due to enteropathogenic Escherichia coli: link to cathelicidin induction.
        Microbes Infect. 2013; 15: 939-950
        • Gliem M.
        • Heupel W.M.
        • Spindler V.
        • Harms G.S.
        • Waschke J.
        Actin reorganization contributes to loss of cell adhesion in pemphigus vulgaris.
        Am J Physiol Cell Physiol. 2010; 299: C606-C613
        • Spindler V.
        • Meir M.
        • Vigh B.
        • Flemming S.
        • Hutz K.
        • Germer C.T.
        • Waschke J.
        • Schlegel N.
        Loss of desmoglein 2 contributes to the pathogenesis of Crohn's disease.
        Inflamm Bowel Dis. 2015; 21: 2349-2359
        • Ellawindy A.
        • Satoh K.
        • Sunamura S.
        • Kikuchi N.
        • Suzuki K.
        • Minami T.
        • Ikeda S.
        • Tanaka S.
        • Shimizu T.
        • Enkhjargal B.
        • Miyata S.
        • Taguchi Y.
        • Handoh T.
        • Kobayashi K.
        • Kobayashi K.
        • Nakayama K.
        • Miura M.
        • Shimokawa H.
        Rho-kinase inhibition during early cardiac development causes arrhythmogenic right ventricular cardiomyopathy in mice.
        Arterioscler Thromb Vasc Biol. 2015; 35: 2172-2184
        • Citi S.
        • Guerrera D.
        • Spadaro D.
        • Shah J.
        Epithelial junctions and Rho family GTPases: the zonular signalosome.
        Small GTPases. 2014; 5: e973760
        • Viswanathan V.K.
        • Lukic S.
        • Koutsouris A.
        • Miao R.
        • Muza M.M.
        • Hecht G.
        Cytokeratin 18 interacts with the enteropathogenic Escherichia coli secreted protein F (EspF) and is redistributed after infection.
        Cell Microbiol. 2004; 6: 987-997
        • Hernández-Nava E.
        • Cuellar P.
        • Nava P.
        • Chávez-Munguía B.
        • Schnoor M.
        • Orozco E.
        • Betanzos A.
        Adherens junctions and desmosomes are damaged by Entamoeba histolytica: participation of EhCPADH complex and EhCP112 protease.
        Cell Microbiol. 2017; 19: e12761
        • Maia-Brigagão C.
        • Morgado-Díaz J.
        • De Souza W.
        Giardia disrupts the arrangement of tight, adherens and desmosomal junction proteins of intestinal cells.
        Parasitol Int. 2012; 61: 280-287
        • Wine E.
        • Chan V.L.
        • Sherman P.M.
        Campylobacter jejuni mediated disruption of polarized epithelial monolayers is cell-type specific, time dependent, and correlates with bacterial invasion.
        Pediatr Res. 2008; 64: 599-604
        • Aktories K.
        Rho-modifying bacterial protein toxins.
        Pathog Dis. 2015; 73: ftv091
        • Ahrens S.
        • Geissler B.
        • Satchell K.J.
        Identification of a His-Asp-Cys catalytic triad essential for function of the Rho inactivation domain (RID) of Vibrio cholerae MARTX toxin.
        J Biol Chem. 2013; 288: 1397-1408
        • Prehna G.
        • Ivanov M.I.
        • Bliska J.B.
        • Stebbins C.E.
        Yersinia virulence depends on mimicry of host Rho-family nucleotide dissociation inhibitors.
        Cell. 2006; 126: 869-880
        • Shawki A.
        • McCole D.F.
        Mechanisms of intestinal epithelial barrier dysfunction by adherent-invasive Escherichia coli.
        Cell Mol Gastroenterol Hepatol. 2017; 3: 41-50
        • Singh A.P.
        • Aijaz S.
        Enteropathogenic E. coli: breaking the intestinal tight junction barrier.
        F1000Res. 2015; 4: 231
        • Guttman J.A.
        • Kazemi P.
        • Lin A.E.
        • Vogl A.W.
        • Finlay B.B.
        Desmosomes are unaltered during infections by attaching and effacing pathogens.
        Anat Rec (Hoboken). 2007; 290: 199-205
        • Bulgin R.R.
        • Arbeloa A.
        • Chung J.C.
        • Frankel G.
        EspT triggers formation of lamellipodia and membrane ruffles through activation of Rac-1 and Cdc42.
        Cell Microbiol. 2009; 11: 217-229
        • Arbeloa A.
        • Blanco M.
        • Moreira F.C.
        • Bulgin R.
        • Lopez C.
        • Dahbi G.
        • Blanco J.E.
        • Mora A.
        • Alonso M.P.
        • Mamani R.C.
        • Gomes T.A.
        • Blanco J.
        • Frankel G.
        Distribution of espM and espT among enteropathogenic and enterohaemorrhagic Escherichia coli.
        J Med Microbiol. 2009; 58: 988-995
        • Arbeloa A.
        • Garnett J.
        • Lillington J.
        • Bulgin R.R.
        • Berger C.N.
        • Lea S.M.
        • Matthews S.
        • Frankel G.
        EspM2 is a RhoA guanine nucleotide exchange factor.
        Cell Microbiol. 2010; 12: 654-664
        • Mundy R.
        • MacDonald T.T.
        • Dougan G.
        • Frankel G.
        • Wiles S.
        Citrobacter rodentium of mice and man.
        Cell Microbiol. 2005; 7: 1697-1706
        • Ritchie J.M.
        • Waldor M.K.
        The locus of enterocyte effacement-encoded effector proteins all promote enterohemorrhagic Escherichia coli pathogenicity in infant rabbits.
        Infect Immun. 2005; 73: 1466-1474
        • Myhal M.L.
        • Laux D.C.
        • Cohen P.S.
        Relative colonizing abilities of human fecal and K 12 strains of Escherichia coli in the large intestines of streptomycin-treated mice.
        Eur J Clin Microbiol. 1982; 1: 186-192
        • Taylor J.
        Infectious infantile enteritis, yesterday and today.
        Proc R Soc Med. 1970; 63: 1297-1301
        • Levine M.M.
        • Nataro J.P.
        • Karch H.
        • Baldini M.M.
        • Kaper J.B.
        • Black R.E.
        • Clements M.L.
        • O'Brien A.D.
        The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor.
        J Infect Dis. 1985; 152: 550-559
        • Kenny B.
        • Lai L.C.
        • Finlay B.B.
        • Donnenberg M.S.
        EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells.
        Mol Microbiol. 1996; 20: 313-323