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    EspFU, an Enterohemorrhagic E. Coli Secreted Effector, Hijacks Mammalian Actin Assembly Proteins by Molecular Mimicry and Repetition: A Dissertation

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    Authors
    Lai, YuShuan (Cindy)
    Faculty Advisor
    John M. Leong, MD, PhD
    Academic Program
    MD/PhD
    UMass Chan Affiliations
    Molecular Genetics and Microbiology
    Document Type
    Doctoral Dissertation
    Publication Date
    2014-04-25
    Keywords
    Dissertations, UMMS
    Actin Cytoskeleton
    Actins
    Calpain
    Cytoskeletal Proteins
    Enterohemorrhagic Escherichia coli
    Escherichia coli Infections
    Escherichia coli Proteins
    Microvilli
    Actin Cytoskeleton
    Actins
    Calpain
    Cytoskeletal Proteins
    Enterohemorrhagic Escherichia coli
    Escherichia coli Infections
    Escherichia coli Proteins
    Microvilli
    Bacterial Infections and Mycoses
    Bacteriology
    Cellular and Molecular Physiology
    Microbial Physiology
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    Abstract
    Enterohemorrhagic E. coli (EHEC) is a major cause of food borne diarrheal illness worldwide. While disease symptoms are usually self-resolving and limited to severe gastroenteritis with bloody diarrhea, EHEC infection can lead to a life threatening complication known as Hemolytic Uremic Syndrome (HUS), which strikes children disproportionately and is the leading cause of kidney failure in children. Upon infection of gut epithelia, EHEC produces characteristic lesions called actin pedestals. These striking formations involve dramatic rearrangement of host cytoskeletal proteins. EHEC hijacks mammalian signaling pathways to cause destruction of microvilli and rebuilds the actin cytoskeleton underneath sites of bacterial attachment. Here, we present a brief study on a host factor, Calpain, involved in microvilli effacement, and an in depth investigation on a bacterial factor, EspFU, required for actin pedestal formation in intestinal cell models. Calpain is activated by both EHEC and the related pathogen, enteropathogenic E. coli (EPEC), during infection and facilitates microvilli disassembly by cleavage of a key membrane-cytoskeleton anchoring substrate, Ezrin. Actin pedestal formation is facilitated by the injection of two bacterial effectors, Tir and EspFU, into host cells, which work in concert to manipulate the host actin nucleators N-WASP and Arp2/3. EspFU hijacks key host signaling proteins N-WASP and IRTKS by mimetic displacement and has evolved to outcompete mammalian host ligands. Multiple repeats of key functional domains of EspFU are essential for actin pedestal activity through proper localization and competition against the an abundant host factor Eps8 for binding to IRTKS.
    DOI
    10.13028/M2HW2H
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/32074
    Notes

    Copyright by (Cindy) YuShuan Lai 2014 All Rights Reserved

    Rights
    Copyright is held by the author, with all rights reserved.
    ae974a485f413a2113503eed53cd6c53
    10.13028/M2HW2H
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      Attaching-and-Effacing Pathogens Exploit Junction Regulatory Activities of N-WASP and SNX9 to Disrupt the Intestinal Barrier

      Garber, John J.; Mallick, Emily M.; Scanlon, Karen M.; Turner, Jerrold R.; Donnenberg, Michael S.; Leong, John M.; Snapper, Scott B. (2017-12-15)
      Background and Aims: Neural Wiskott-Aldrich Syndrome protein (N-WASP) is a key regulator of the actin cytoskeleton in epithelial tissues and is poised to mediate cytoskeletal-dependent aspects of apical junction complex (AJC) homeostasis. Attaching-and-effacing (AE) pathogens disrupt this homeostasis through translocation of the effector molecule early secreted antigenic target-6 (ESX)-1 secretion-associated protein F (EspF). Although the mechanisms underlying AJC disruption by EspF are unknown, EspF contains putative binding sites for N-WASP and the endocytic regulator sorting nexin 9 (SNX9). We hypothesized that N-WASP regulates AJC integrity and AE pathogens use EspF to induce junction disassembly through an N-WASP- and SNX9-dependent pathway. Methods: We analyzed mice with intestine-specific N-WASP deletion and generated cell lines with N-WASP and SNX9 depletion for dynamic functional assays. We generated EPEC and Citrobacter rodentium strains complemented with EspF bearing point mutations abolishing N-WASP and SNX9 binding to investigate the requirement for these interactions. Results: Mice lacking N-WASP in the intestinal epithelium showed spontaneously increased permeability, abnormal AJC morphology, and mislocalization of occludin. N-WASP depletion in epithelial cell lines led to impaired assembly and disassembly of tight junctions in response to changes in extracellular calcium. Cells lacking N-WASP or SNX9 supported actin pedestals and type III secretion, but were resistant to EPEC-induced AJC disassembly and loss of transepithelial resistance. We found that during in vivo infection with AE pathogens, EspF must bind both N-WASP and SNX9 to disrupt AJCs and induce intestinal barrier dysfunction. Conclusions: Overall, these studies show that N-WASP critically regulates AJC homeostasis, and the AE pathogen effector EspF specifically exploits both N-WASP and SNX9 to disrupt intestinal barrier integrity during infection.
    • Thumbnail

      EspFU is a translocated EHEC effector that interacts with Tir and N-WASP and promotes Nck-independent actin assembly

      Campellone, Kenneth Geno; Robbins, Douglas; Leong, John M. (2004-08-07)
      Several microbial pathogens including enteropathogenic E. coli (EPEC) exploit mammalian tyrosine-kinase signaling cascades to recruit Nck adaptor proteins and activate N-WASP-Arp2/3-mediated actin assembly. To promote localized actin "pedestal formation," EPEC translocates the bacterial effector protein Tir into the plasma membrane, where it is tyrosine-phosphorylated and binds Nck. Enterohemorrhagic E. coli (EHEC) also generates Tir-dependent pedestals, but in the absence of phosphotyrosines and Nck recruitment. To identify additional EHEC effectors that stimulate phosphotyrosine-independent actin assembly, we systematically generated EHEC mutants containing specific deletions in putative pathogenicity-islands. Among 0.33 Mb of deleted sequences, only one ORF was critical for pedestal formation. It lies within prophage-U, and encodes a protein similar to the known effector EspF. This proline-rich protein, EspFU, is the only EHEC effector of actin assembly absent from EPEC. Whereas EHEC Tir cannot efficiently recruit N-WASP or trigger actin polymerization, EspFU associates with Tir, binds N-WASP, and potently stimulates Nck-independent actin assembly.
    • Thumbnail

      Increased adherence and actin pedestal formation by dam-deficient enterohaemorrhagic Escherichia coli O157:H7

      Campellone, Kenneth Geno; Roe, Andrew J.; Lobner-Olesen, Anders; Murphy, Kenan C.; Magoun, Loranne; Brady, Michael John; Donohue-Rolfe, Arthur; Tzipori, Saul; Gally, David L.; Leong, John M.; et al. (2007-02-17)
      Enterohaemorrhagic Escherichia coli (EHEC) are highly infectious pathogens capable of causing severe diarrhoeal illnesses. As a critical step during their colonization, EHEC adhere intimately to intestinal epithelial cells and generate F-actin 'pedestal' structures that elevate them above surrounding cell surfaces. Intimate adhesion and pedestal formation result from delivery of the EHEC type III secretion system (TTSS) effector proteins Tir and EspF(U) into the host cell and expression of the bacterial outer membrane adhesin, intimin. To investigate a role for DNA methylation during the regulation of adhesion and pedestal formation in EHEC, we deleted the dam (DNA adenine methyltransferase) gene from EHEC O157:H7 and demonstrate that this mutation results in increased interactions with cultured host cells. EHECDeltadam exhibits dramatically elevated levels of adherence and pedestal formation when compared with wild-type EHEC, and expresses significantly higher protein levels of intimin, Tir and EspF(U). Analyses of GFP fusions, Northern blotting, reverse transcription polymerase chain reaction, and microarray experiments indicate that the abundance of Tir in the dam mutant is not due to increased transcription levels, raising the possibility that Dam methylation can indirectly control protein expression by a post-transcriptional mechanism. In contrast to other dam-deficient pathogens, EHECDeltadam is capable of robust intestinal colonization of experimentally infected animals.
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