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Gad Frankel - One of the best experts on this subject based on the ideXlab platform.

  • Enteropathogenic Escherichia Coli stimulates effector driven rapid caspase 4 activation in human macrophages
    Cell Reports, 2019
    Co-Authors: Philippa J Goddard, Julia Sanchezgarrido, Sabrina L Slater, Mohini Kalyan, David Ruanogallego, Olivier Marches, Luis Angel Fernandez, Gad Frankel, Avinash R. Shenoy
    Abstract:

    Summary Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen Enteropathogenic Escherichia Coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. Coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.

  • Enteropathogenic Escherichia Coli stimulates effector driven rapid caspase 4 activation in human macrophages
    Cell Reports, 2019
    Co-Authors: Philippa J Goddard, Julia Sanchezgarrido, Sabrina L Slater, Mohini Kalyan, David Ruanogallego, Olivier Marches, Luis Angel Fernandez, Gad Frankel, Avinash R. Shenoy
    Abstract:

    Summary Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen Enteropathogenic Escherichia Coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. Coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.

  • hierarchical protein targeting and secretion is controlled by an affinity switch in the type iii secretion system of Enteropathogenic Escherichia Coli
    The EMBO Journal, 2017
    Co-Authors: Athina G Portaliou, Gad Frankel, Konstantinos C Tsolis, Maria S Loos, Vassileia Balabanidou, Josep Rayo, Alexandra Tsirigotaki, Valerie F Crepin, Charalampos G Kalodimos, Spyridoula Karamanou
    Abstract:

    Type III secretion (T3S), a protein export pathway common to Gram-negative pathogens, comprises a trans-envelope syringe, the injectisome, with a cytoplasm-facing translocase channel. Exported substrates are chaperone-delivered to the translocase, EscV in Enteropathogenic Escherichia Coli, and cross it in strict hierarchical manner, for example, first "translocators", then "effectors". We dissected T3S substrate targeting and hierarchical switching by reconstituting them in vitro using inverted inner membrane vesicles. EscV recruits and conformationally activates the tightly membrane-associated pseudo-effector SepL and its chaperone SepD. This renders SepL a high-affinity receptor for translocator/chaperone pairs, recognizing specific chaperone signals. In a second, SepD-coupled step, translocators docked on SepL become secreted. During translocator secretion, SepL/SepD suppress effector/chaperone binding to EscV and prevent premature effector secretion. Disengagement of the SepL/SepD switch directs EscV to dedicated effector export. These findings advance molecular understanding of T3S and reveal a novel mechanism for hierarchical trafficking regulation in protein secretion channels.

  • a type iii effector protease nlec from Enteropathogenic Escherichia Coli targets nf κb for degradation
    Molecular Microbiology, 2011
    Co-Authors: Jaclyn S Pearson, Olivier Marches, Gad Frankel, Patrice Riedmaier, Elizabeth L Hartland
    Abstract:

    Summary Many bacterial pathogens utilize a type III secretion system (T3SS) to inject virulence effector proteins into host cells during infection. Previously, we found that Enteropathogenic Escherichia Coli (EPEC) uses the type III effector, NleE, to block the inflammatory response by inhibiting IkB degradation and nuclear translocation of the p65 subunit of NF-kB. Here we screened further effectors with unknown function for their capacity to prevent p65 nuclear translocation. We observed that ectopic expression of GFP-NleC in HeLa cells led to the degradation of p65. Delivery of NleC by the T3SS of EPEC also induced degradation of p65 in infected cells as well as other NF-kB compo- nents, c-Rel and p50. Recombinant His 6 -NleC induced p65 and p50 cleavage in HeLa cell lysates and muta- tion of a consensus zinc metalloprotease motif, HEIIH, abrogated NleC proteolytic activity. NleC inhibited IL-8 production during prolonged EPEC infection of HeLa cells in a protease activity-dependent manner. A double nleE/nleC mutant was further impaired for its ability to inhibit IL-8 secretion than either a single nleE or a single nleC mutant. We conclude that NleC is a type III effector protease that degrades NF-kB thereby contributing the arsenal of bacterial effectors that inhibit innate immune activation.

  • Comparison of colonization dynamics and pathology of mice infected with Enteropathogenic Escherichia Coli, enterohaemorrhagic E. Coli and Citrobacter rodentium.
    FEMS Microbiology Letters, 2006
    Co-Authors: Rosanna Mundy, Francis Girard, Anthony J. Fitzgerald, Gad Frankel
    Abstract:

    Enteropathogenic Escherichia Coli (EPEC), enterohaemorrhagic E. Coli (EHEC) and Citrobacter rodentium (CR) colonize the gastrointestinal tract epithelium via attaching and effacing lesions. While humans are believed to be the only living reservoir of typical EPEC and EHEC to have border host specificity, CR is a restricted mouse pathogen. Recently, conflicting conclusions were reported concerning the utility of a murine model to study mechanisms of EPEC and EHEC colonization and infection. We therefore aimed to compare colonization dynamics of EPEC, EHEC and CR, together with a commensal E. Coli (Nissle) as a control, in the murine. We show that all strains are equally shed in stools over the first 48 h post inoculation. However, while the CR population then rapidly expanded the EPEC, EHEC and Nissle populations quickly declined to a level just above detection. We conclude that following oral inoculation EPEC and EHEC develop a commensal, rather than pathogenic, interaction within the mouse host.

Brett B Finlay - One of the best experts on this subject based on the ideXlab platform.

  • in vitro and in vivo model systems for studying Enteropathogenic Escherichia Coli infections
    Cold Spring Harbor Perspectives in Medicine, 2013
    Co-Authors: Robyn J Law, Ilan Rosenshine, Lihi Gurarie, Brett B Finlay
    Abstract:

    Enteropathogenic Escherichia Coli (EPEC) and enterohemorrhagic E. Coli (EHEC) belong to a group of bacteria known as attaching and effacing (A/E) pathogens that cause disease by adhering to the lumenal surfaces of their host's intestinal epithelium. EPEC and EHEC are major causes of infectious diarrhea that result in significant childhood morbidity and mortality worldwide. Recent advances in in vitro and in vivo modeling of these pathogens have contributed to our knowledge of how EPEC and EHEC attach to host cells and subvert host-cell signaling pathways to promote infection and cause disease. A more detailed understanding of how these pathogenic microbes infect their hosts and how the host responds to infection could ultimately lead to new therapeutic strategies to help control these significant enteric pathogens.

  • role for cd2ap and other endocytosis associated proteins in Enteropathogenic Escherichia Coli pedestal formation
    Infection and Immunity, 2010
    Co-Authors: Julian A. Guttman, Esteban Veiga, Pascale Cossart, Brett B Finlay
    Abstract:

    Enteropathogenic Escherichia Coli (EPEC) strains are extracellular pathogens that generate actin-rich structures (pedestals) beneath the adherent bacteria as part of their virulence strategy. Pedestals are hallmarks of EPEC infections, and their efficient formation in vitro routinely requires phosphorylation of the EPEC effector protein Tir at tyrosine 474 (Y474). This phosphorylation results in the recruitment and direct attachment of the host adaptor protein Nck to Tir at Y474, which is utilized for actin nucleation through a downstream N-WASP-Arp2/3-based mechanism. Recently, the endocytic protein clathrin was demonstrated to be involved in EPEC pedestal formation. Here we examine the organization of clathrin in pedestals and report that CD2AP, an endocytosis-associated and cortactin-binding protein, is a novel and important component of EPEC pedestal formation that also utilizes Y474 phosphorylation of EPEC Tir. We also demonstrate the successive recruitment of Nck and then clathrin prior to actin polymerization at pedestals during the Nck-dependent pathway of pedestal formation. This study further demonstrates that endocytic proteins are key components of EPEC pedestals and suggests a novel endocytosis subversion strategy employed by these extracellular bacteria.

  • transcriptional inhibitor of virulence factors in Enteropathogenic Escherichia Coli
    Antimicrobial Agents and Chemotherapy, 2005
    Co-Authors: Annick Gauthier, Marilyn Robertson, Michael J Lowden, Antonio J Ibarra, Jose L Puente, Brett B Finlay
    Abstract:

    The type III secretion system (TTSS) is a key virulence mechanism of many important gram-negative bacterial pathogens. The TTSS is conserved among different bacterial pathogens, and mutations and deletions to the system significantly decrease virulence, making the TTSS an important potential therapeutic target. We have developed a high-throughput assay to search for inhibitors of the TTSS. We screened a commercial library of 20,000 small molecules for their ability to inhibit type III secretion by Enteropathogenic Escherichia Coli (EPEC). After discarding compounds that had no effect on secretion, inhibited bacterial growth, and/or caused degradation of EPEC-secreted proteins, the search was focused on a class of compounds that, while not direct inhibitors of type III secretion, inhibit expression of TTSS-related genes and other genes involved in virulence. This class of compounds does not affect bacterial viability or motility, indicating that it is not significantly affecting the expression of essential genes and is specific to virulence-associated genes. Transcriptional fusion assays confirmed that virulence-associated promoters were more sensitive to inhibition by this class of compounds. Overall, we have identified a class of compounds that can be used as a tool to probe the mechanism(s) that regulates virulence gene expression in EPEC.

  • exploitation of host cells by Enteropathogenic Escherichia Coli
    Proceedings of the National Academy of Sciences of the United States of America, 2000
    Co-Authors: Bruce A Vallance, Brett B Finlay
    Abstract:

    Microbial pathogens have evolved many ingenious ways to infect their hosts and cause disease, including the subversion and exploitation of target host cells. One such subversive microbe is Enteropathogenic Escherichia Coli (EPEC). A major cause of infantile diarrhea in developing countries, EPEC poses a significant health threat to children worldwide. Central to EPEC-mediated disease is its colonization of the intestinal epithelium. After initial adherence, EPEC causes the localized effacement of microvilli and intimately attaches to the host cell surface, forming characteristic attaching and effacing (A/E) lesions. Considered the prototype for a family of A/E lesion-causing bacteria, recent in vitro studies of EPEC have revolutionized our understanding of how these pathogens infect their hosts and cause disease. Intimate attachment requires the type III-mediated secretion of bacterial proteins, several of which are translocated directly into the infected cell, including the bacteria's own receptor (Tir). Binding to this membrane-bound, pathogen-derived protein permits EPEC to intimately attach to mammalian cells. The translocated EPEC proteins also activate signaling pathways within the underlying cell, causing the reorganization of the host actin cytoskeleton and the formation of pedestal-like structures beneath the adherent bacteria. This review explores what is known about EPEC's subversion of mammalian cell functions and how this knowledge has provided novel insights into bacterial pathogenesis and microbe-host interactions. Future studies of A/E pathogens in animal models should provide further insights into how EPEC exploits not only epithelial cells but other host cells, including those of the immune system, to cause diarrheal disease.

  • crystal structure of Enteropathogenic Escherichia Coli intimin receptor complex
    Nature, 2000
    Co-Authors: Yu Luo, Brett B Finlay, Elizabeth A Frey, Richard A Pfuetzner, A L Creagh, D G Knoechel, Charles A Haynes, Natalie C J Strynadka
    Abstract:

    Intimin and its translocated intimin receptor (Tir) are bacterial proteins that mediate adhesion between mammalian cells and attaching and effacing (A/E) pathogens. Enteropathogenic Escherichia Coli (EPEC) causes significant paediatric morbidity and mortality world-wide. A related A/E pathogen, enterohaemorrhagic E. Coli (EHEC; O157:H7) is one of the most important food-borne pathogens in North America, Europe and Japan. A unique and essential feature of A/E bacterial pathogens is the formation of actin-rich pedestals beneath the intimately adherent bacteria and localized destruction of the intestinal brush border. The bacterial outer membrane adhesin, intimin, is necessary for the production of the A/E lesion and diarrhoea. The A/E bacteria translocate their own receptor for intimin, Tir, into the membrane of mammalian cells using the type III secretion system. The translocated Tir triggers additional host signalling events and actin nucleation, which are essential for lesion formation. Here we describe the the crystal structures of an EPEC intimin carboxy-terminal fragment alone and in complex with the EPEC Tir intimin-binding domain, giving insight into the molecular mechanisms of adhesion of A/E pathogens.

Michael S Donnenberg - One of the best experts on this subject based on the ideXlab platform.

  • the bundlin pilin protein of Enteropathogenic Escherichia Coli is an n acetyllactosamine specific lectin
    Cellular Microbiology, 2007
    Co-Authors: Romney M Hyland, Jiangxiao Sun, Thomas P Griener, George L Mulvey, John S Klassen, Michael S Donnenberg, Glen D Armstrong
    Abstract:

    Synthetic N-acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit Enteropathogenic Escherichia Coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAc-specific adhesin appears to be associated with the bundle-forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp-2 cells and binds to HEp-2 cells. Recombinant bundlin also binds, with millimolar association constants (K(assoc)), to synthetic LacNAc-Benzene and LacNAc-O(CH(2))(8)CONH(2) glycosides as assessed in the gas phase by nanoelectrospray ionization mass spectrometry. Furthermore, LacNAc-BSA inhibits LA only of EPEC strains that express alpha bundlin alleles, suggesting putative locations for the LacNAc-binding pocket in the alpha bundlin monomer. Collectively, these results suggest that alpha bundlin possesses lectin-like properties that are responsible for LacNAc-specific initial adherence of alpha bundlin-expressing EPEC strains to host intestinal epithelial cells.

  • Enteropathogenic Escherichia Coli espf is targeted to mitochondria and is required to initiate the mitochondrial death pathway
    Cellular Microbiology, 2004
    Co-Authors: Jeanphilippe Nougayrede, Michael S Donnenberg
    Abstract:

    Enteropathogenic Escherichia Coli (EPEC) is a causative agent of infant diarrhoea in developing countries. The EspF protein is the product of the espF gene found on the locus of enterocyte effacement, the key pathogenicity island carried by EPEC and enterohemorrhagic E. Coli. EspF is injected from adherent EPEC into host cells via a type III secretion system and was previously shown to induce apoptotic cell death and to be required for disruption of host intestinal barrier function. In this work, we show by immunofluorescence and fractionation studies that EspF is targeted to host mitochondria. The N-terminal region of EspF serves as a mitochondrial import signal and, when expressed within cells, can target hybrid green fluorescent protein to mitochondria. Assessment of mitochondrial membrane potential in infected epithelial cells indicated that EspF plays a role in the mitochondrial membrane permeabilization induced by EPEC infection. Furthermore, EspF was associated with the release of cytochrome c from mitochondria into the cytoplasm and with caspase-9 and caspase-3 cleavage. These findings indicate a role for EspF in initiating the mitochondrial death pathway.

  • adhesion of Enteropathogenic Escherichia Coli to host cells
    Cellular Microbiology, 2003
    Co-Authors: Jeanphilippe Nougayrede, Paula J Fernandes, Michael S Donnenberg
    Abstract:

    Enteropathogenic Escherichia Coli (EPEC) adhere to the intestinal mucosa and to tissue culture cells in a distinctive fashion, destroying microvilli, altering the cytoskeleton and attaching intimately to the host cell membrane in a manner termed the attaching and effacing effect. Typical EPEC strains also form three-dimensional microcolonies in a pattern termed localized adherence. Attaching and effacing, and in particular intimate attachment requires an outer membrane adhesin called intimin, which binds to the translocated intimin receptor, Tir. Tir is produced by the bacteria and delivered to the host cell via a type III secretion system. In addition to this well-established adhesin-receptor pair, numerous other adhesin interactions between EPEC and host cells have been described including those between intimin and cellular receptors and those involving a bundle-forming pilus and flagella and unknown receptors. Much additional work is needed before a full understanding of EPEC adhesion to host cells comes to light.

  • translocated espf protein from Enteropathogenic Escherichia Coli disrupts host intestinal barrier function
    Journal of Clinical Investigation, 2001
    Co-Authors: Barry P Mcnamara, Athanasia Koutsouris, Michael S Donnenberg, Jeanphilippe Nougayrede, Colin B Oconnell, Gail Hecht
    Abstract:

    The mechanisms by which Enteropathogenic Escherichia Coli (EPEC), an important cause of diarrhea among infants in developing countries, induce symptoms are not defined. EPEC have a type III secretion system required for characteristic attaching and effacing changes that modify the cytoskeleton and apical surface of host cells. Infection of polarized intestinal epithelial cell monolayers by EPEC leads to a loss of transepithelial electrical resistance, which also requires the type III secretion system. We demonstrate here that EspF, a protein that is secreted by EPEC via the type III secretion system, is not required for quantitatively and qualitatively typical attaching and effacing lesion formation in intestinal epithelial cells. However, EspF is required in a dose-dependent fashion for the loss of transepithelial electrical resistance, for increased monolayer permeability, and for redistribution of the tight junction-associated protein occludin. Furthermore, the analysis of EPEC strains expressing EspF-adenylate cyclase fusion proteins indicates that EspF is translocated via the type III secretion system to the cytoplasm of host cells, a result confirmed by immunofluorescence microscopy. These studies suggest a novel role for EspF as an effector protein that disrupts intestinal barrier function without involvement in attaching and effacing lesion formation.

  • the espb protein of Enteropathogenic Escherichia Coli is targeted to the cytoplasm of infected hela cells
    Infection and Immunity, 1998
    Co-Authors: Kathleen A Taylor, Colin B Oconnell, Paul W Luther, Michael S Donnenberg
    Abstract:

    The EspB protein of Enteropathogenic Escherichia Coli (EPEC) is exported via a type III secretion apparatus. EspB is critical for signaling the host cell and for the development of the attaching and effacing lesion characteristic of EPEC infection. We used cellular fractionation and confocal laser scanning microscopy to determine the cellular location of EspB during infection of HeLa cells. Both methods indicated that EspB is targeted to the cytoplasm of infected cells. Using mutants, we found that EspB targeting to the host cell cytoplasm requires the type III secretion apparatus and the secreted proteins EspA and EspD, but not intimin. These results provide insights into the function of the type III secretion apparatus of EPEC and the functions of the Esp proteins.

Avinash R. Shenoy - One of the best experts on this subject based on the ideXlab platform.

  • Enteropathogenic Escherichia Coli stimulates effector driven rapid caspase 4 activation in human macrophages
    Cell Reports, 2019
    Co-Authors: Philippa J Goddard, Julia Sanchezgarrido, Sabrina L Slater, Mohini Kalyan, David Ruanogallego, Olivier Marches, Luis Angel Fernandez, Gad Frankel, Avinash R. Shenoy
    Abstract:

    Summary Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen Enteropathogenic Escherichia Coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. Coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.

  • Enteropathogenic Escherichia Coli stimulates effector driven rapid caspase 4 activation in human macrophages
    Cell Reports, 2019
    Co-Authors: Philippa J Goddard, Julia Sanchezgarrido, Sabrina L Slater, Mohini Kalyan, David Ruanogallego, Olivier Marches, Luis Angel Fernandez, Gad Frankel, Avinash R. Shenoy
    Abstract:

    Summary Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen Enteropathogenic Escherichia Coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. Coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.

James B Kaper - One of the best experts on this subject based on the ideXlab platform.

  • Enteropathogenic Escherichia Coli EspH-Mediated Rho GTPase Inhibition Results in Desmosomal PerturbationsSummary
    'Elsevier BV', 2018
    Co-Authors: Jennifer Lising Roxas, James B Kaper, Ross Calvin Monasky, Bryan Angelo P. Roxas, Al B. Agellon, Asad Mansoor, Gayatri Vedantam, V.k. Viswanathan
    Abstract:

    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. Keywords: EPEC, Desmoglein, DSG2, Host–Pathogen Interactio

  • flagellin of Enteropathogenic Escherichia Coli stimulates interleukin 8 production in t84 cells
    Infection and Immunity, 2003
    Co-Authors: Xin Zhou, Jorge A Giron, Alfredo G Torres, Adam J Crawford, Erasmo Negrete, Stefanie N Vogel, James B Kaper
    Abstract:

    The type III secretion system (TTSS) of Enteropathogenic Escherichia Coli (EPEC) has been associated with the ability of these bacteria to induce secretion of proinflammatory cytokines, including interleukin-8 (IL-8), in cultured epithelial cells. However, the identity of the effector molecule directly involved in this event is unknown. In this study, we determined that the native flagellar filament and its flagellin monomer are activators of IL-8 release in T84 epithelial cells. Supernatants of wild-type EPEC strain E2348/69 and its isogenic mutants deficient in TTSS (escN) and in production of intimin (eae), grown in Luria-Bertani broth, elicited similar amounts of IL-8 secretion by T84 cells. In contrast, supernatants of EPEC fliC mutants and of B171, a nonflagellated EPEC strain, were defective in inducing IL-8 release, a phenotype that was largely restored by complementation of the fliC gene in the mutant lacking flagella. Purified flagella from E. Coli K-12, EPEC serotypes H6 and H34, and enterohemorrhagic E. Coli serotype H7 all induced IL-8 release in T84 cells. Induction of IL-8 by purified flagella or His-tagged FliC from EPEC strain E2348/69 was dose dependent and was blocked by a polyclonal anti-H6 antibody. Finally, the mitogen-activated protein kinases (Erk1 and -2 and Jnk) were phosphorylated in flagellin-treated T84 cells, and inhibition of the p38 and Erk pathways significantly decreased the IL-8 response induced by EPEC flagellin. Our data clearly indicate that FliC of EPEC is sufficient to induce IL-8 release in T84 cells and that activation of the Erk and p38 pathways is required for IL-8 induction.

  • the flagella of Enteropathogenic Escherichia Coli mediate adherence to epithelial cells
    Molecular Microbiology, 2002
    Co-Authors: Jorge A Giron, Alfredo G Torres, Enrique Freer, James B Kaper
    Abstract:

    Enteropathogenic Escherichia Coli (EPEC) utilizes a type III protein secretion system to target effector molecules into the host cell leading to effacement of the intestinal mucosa. This secretion apparatus shares many structural features of the flagellar type III export system involved in flagella assembly and motility. We report here that fliC insertional mutants constructed in two wild-type EPEC strains were markedly impaired in adherence and microcolony formation on cultured cells. An E. Coli K-12 strain harbouring the EPEC H6 fliC gene on a plasmid showed discrete adhering clusters on HeLa cells, albeit to less extent than the wild-type EPEC strain. Flagella purified from EPEC bound to cultured epithelial cells and antiflagella antibodies blocked adherence of several EPEC serotypes. We determined that eukaryotic cells in culture stimulate expression of flagella by motile and non-motile EPEC. Isogenic strains mutated in perA (a transcriptional activator), bfpA (a type IV pilin), luxS (a quorum-sensing autoinducer gene) and in the type III secretion genes were reduced for motility in Dulbecco's modified Eagle medium (DMEM) motility agar and produced none or few flagella when associated with epithelial cells. Growth of these mutants in preconditioned tissue culture medium restored motility and their ability to produce flagella, suggesting the influence of a signal provided by mammalian cells that triggers flagella production. This study shows for the first time that the flagella of EPEC are directly involved in the adherence of these bacteria and supports the existence of a molecular relationship between the two existing type III secretion pathways of EPEC, the EPEC adherence factor (EAF) plasmid-encoded regulator, quorum sensing and epithelial cells.

  • identification of cest a chaperone for the type iii secretion of tir in Enteropathogenic Escherichia Coli
    Molecular Microbiology, 2002
    Co-Authors: Simon J Elliott, Gad Frankel, Miranda Batchelor, Jay L Mellies, Stuart Knutton, Steven W Hutcheson, Maria S Dubois, Leslie A Wainwright, James B Kaper
    Abstract:

    The locus of enterocyte effacement of Enteropathogenic Escherichia Coli encodes a type III secretion system, an outer membrane protein adhesin (intimin, the product of eae ) and Tir, a translocated protein that becomes a host cell receptor for intimin. Many type III secreted proteins require chaperones, which function to stabilize proteins, prevent inappropriate protein–protein interactions and aid in secretion. An open reading frame located between tir and eae, previously named orfU, was predicted to encode a protein with partial similarity to the Yersinia SycH chaperone. We examined the potential of the orfU gene product to serve as a chaperone for Tir. The orfU gene encoded a 15 kDa cytoplasmic protein that specifically interacted with Tir as demonstrated by the yeast two-hybrid assay, column binding and coimmunoprecipitation experiments. An orfU mutant was defective in attaching–effacing lesion formation and Tir secretion, but was unaffected in expression of other virulence factors. OrfU appeared to stabilize Tir levels in the cytoplasm, but was not absolutely necessary for secretion of Tir. Based upon the physical similarities, phenotypic characteristics and the demonstrated interaction with Tir, orfU is redesignated as cesT for the chaperone for E. Coli secretion of T ir.

  • espc pathogenicity island of Enteropathogenic Escherichia Coli encodes an enterotoxin
    Infection and Immunity, 2001
    Co-Authors: Jay L Mellies, Fernando Navarrogarcia, Iruka N Okeke, Julie Frederickson, James P Nataro, James B Kaper
    Abstract:

    At least five proteins are secreted extracellularly by Enteropathogenic Escherichia Coli (EPEC), a leading cause of infant diarrhea in developing countries. However only one, EspC, is known to be secreted independently of the type III secretion apparatus encoded by genes located within the 35.6-kb locus of enterocyte effacement pathogenicity island. EspC is a member of the autotransporter family of proteins, and the secreted portion of the molecule is 110 kDa. Here we determine that the espC gene is located within a second EPEC pathogenicity island at 60 min on the chromosome of E. Coli. We also show that EspC is an enterotoxin, indicated by rises in short-circuit current and potential difference in rat jejunal tissue mounted in Ussing chambers. In addition, preincubation with antiserum against the homologous Pet enterotoxin of enteroaggregative E. Coli eliminated EspC enterotoxin activity. Like the EAF plasmid, the espC pathogenicity island was found only in a subset of EPEC, suggesting that EspC may play a role as an accessory virulence factor in some but not all EPEC strains.