Rho-Modifying Cytotoxic Necrotizing Factor of E. coli

Summary

Principal Investigator: ALISON DAVIS O'BRIEN
Abstract: Cytotoxic necrotizing factor type 1 (CNF1) is a member of a family of bacterial toxins that deamidate single glutamine residues in RhoA, Rac, and Cdc42 and thereby constitutively activate these small GTPases. These deamidation events trigger a myriad of effects on the target cells such as actin cytoskeleton rearrangements, cell cycle abnormalities, and alterations in signaling pathways. CNF1 and a membrane-lytic toxin, hemolysin (Hly), are often co- expressed by Escherichia coli strains that cause urinary tract infections (UTIs), i.e., cystitis or pyelonephritis, or acute prostatitis. In fact, the cnf1 locus and a hly operon are co-transcribed and co-regulated from a prototypic uropathogenic E. coli (UPEC) strain during culture in vitro. Of particular relevance to this proposal, CNF1/Hly-producing UPEC isolates are more frequently isolated from humans with blood and high levels of certain pro-inflammatory cytokines in their urine than are toxin-negative UPEC. The latter observation is consistent with our prior findings that CNF1+ UPEC strains elicit a more intense inflammatory response than do isogenic CNF1- mutants in a mouse model of ascending UTI and in a rat model of acute prostatitis and that the CNF1-positive UPEC strain CP9 survives better than does its cnf1 isogenic mutant in human and mouse polymorphonuclear leukocytes (PMNs). We also reported that Hly provokes loss of surface uroepithelial cells in culture and a 3-D organoid model, and we recently found that Hly damages the uroepithelium and evokes hemorrhage in the bladders of mice 24 hours after intraurethral inoculation with CP9. We therefore theorize that CNF1 and Hly enhance the pathogenicity of UPEC strains by: I.) promoting uroepithelial cell shedding and tissue hemorrhage (Hly);ii.) evoking a large influx of potentially tissue-damaging PMNs (CNF1 and Hly) while simultaneously protecting the bacterium from phagocyte-mediated killing (CNF1), and;iii.) eliciting submucosal edema (CNF1). The specific aims designed to test this hypothesis are to: 1.) delineate the impact of CNF1 and Hly alone and together on the levels of blood, PMNs, and selected pro-inflammatory cytokines in the urine of mice during the first 24 hours after intraurethral infection with CP9 and its cnf1 and hlyA1 single and double mutants and to more broadly compare the host response to these isogenic strains through microarray transcriptional analyses of infected bladders;2.) monitor expression kinetics of cnf1 and the contiguous hly operon by real time RT-PCR in the urine and/or bladders of CP9-challenged mice to ask whether cnf1 and the linked hly operon are co-transcribed in vivo;3.) determine whether CNF1 actually modifies small GTPases in vivo by measuring the extent of activation of Rho, Rac and Cdc42 in uroepithelial cells from bladders of mice infected with CP9 or its CNF mutant;and, 4.) attempt to reduce the extent of inflammation and damage evoked by CP9 through parenteral and/or mucosal immunization of mice with a CNF1/Hly toxoid cocktail.
Funding Period: 1995-09-30 - 2015-04-30
more information: NIH RePORT

Top Publications

  1. pmc Novel three-dimensional organoid model for evaluation of the interaction of uropathogenic Escherichia coli with terminally differentiated human urothelial cells
    Yarery C Smith
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Rm B4052, 4301 Jones Bridge Rd, Bethesda, MD 20814 4799, USA
    Infect Immun 74:750-7. 2006
  2. ncbi A single amino acid substitution in the enzymatic domain of cytotoxic necrotizing factor type 1 of Escherichia coli alters the tissue culture phenotype to that of the dermonecrotic toxin of Bordetella spp
    Beth A McNichol
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA
    Mol Microbiol 60:939-50. 2006
  3. pmc Cytotoxic necrotizing factor type 1 delivered by outer membrane vesicles of uropathogenic Escherichia coli attenuates polymorphonuclear leukocyte antimicrobial activity and chemotaxis
    Jon M Davis
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, B4052, 4301 Jones Bridge Road, Bethesda, Maryland 20814 4799, USA
    Infect Immun 74:4401-8. 2006
  4. pmc Two domains of cytotoxic necrotizing factor type 1 bind the cellular receptor, laminin receptor precursor protein
    Beth A McNichol
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 4799, USA
    Infect Immun 75:5095-104. 2007
  5. pmc Hemolysin of uropathogenic Escherichia coli evokes extensive shedding of the uroepithelium and hemorrhage in bladder tissue within the first 24 hours after intraurethral inoculation of mice
    Yarery C Smith
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 4799, USA
    Infect Immun 76:2978-90. 2008
  6. pmc Cytotoxic necrotizing factor type 1-neutralizing monoclonal antibody NG8 recognizes three amino acids in a C-terminal region of the toxin and reduces toxin binding to HEp-2 cells
    Kerian K Grande
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 4799, USA
    Infect Immun 77:170-9. 2009
  7. pmc Cytotoxic necrotizing factor 1 and hemolysin from uropathogenic Escherichia coli elicit different host responses in the murine bladder
    Tamako A Garcia
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
    Infect Immun 81:99-109. 2013

Detail Information

Publications7

  1. pmc Novel three-dimensional organoid model for evaluation of the interaction of uropathogenic Escherichia coli with terminally differentiated human urothelial cells
    Yarery C Smith
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Rm B4052, 4301 Jones Bridge Rd, Bethesda, MD 20814 4799, USA
    Infect Immun 74:750-7. 2006
    ..We propose these uro-organoids as models that simulate the interactions between UPEC and terminally differentiated human urothelium...
  2. ncbi A single amino acid substitution in the enzymatic domain of cytotoxic necrotizing factor type 1 of Escherichia coli alters the tissue culture phenotype to that of the dermonecrotic toxin of Bordetella spp
    Beth A McNichol
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA
    Mol Microbiol 60:939-50. 2006
    ..Nevertheless, the substitution of threonine for Lys(1310) in the DNT-based mutant, while affecting transglutamination efficiency of the toxin, did not abrogate that enzymatic activity...
  3. pmc Cytotoxic necrotizing factor type 1 delivered by outer membrane vesicles of uropathogenic Escherichia coli attenuates polymorphonuclear leukocyte antimicrobial activity and chemotaxis
    Jon M Davis
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, B4052, 4301 Jones Bridge Road, Bethesda, Maryland 20814 4799, USA
    Infect Immun 74:4401-8. 2006
    ..We conclude that OMVs provide a means for delivery of CNF1 from a UPEC strain to PMNs and thus negatively affect the efficacy of the acute inflammatory response to these organisms...
  4. pmc Two domains of cytotoxic necrotizing factor type 1 bind the cellular receptor, laminin receptor precursor protein
    Beth A McNichol
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 4799, USA
    Infect Immun 75:5095-104. 2007
    ..The data further indicate that CNF1 can bind to an additional receptor(s) on HEp-2 cells and that LRP can also serve as a cellular receptor for CNF2...
  5. pmc Hemolysin of uropathogenic Escherichia coli evokes extensive shedding of the uroepithelium and hemorrhage in bladder tissue within the first 24 hours after intraurethral inoculation of mice
    Yarery C Smith
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 4799, USA
    Infect Immun 76:2978-90. 2008
    ..From these data, we speculate that Hly and CNF1 may be largely responsible for the signs and symptoms of cystitis in humans infected with toxigenic UPEC...
  6. pmc Cytotoxic necrotizing factor type 1-neutralizing monoclonal antibody NG8 recognizes three amino acids in a C-terminal region of the toxin and reduces toxin binding to HEp-2 cells
    Kerian K Grande
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 4799, USA
    Infect Immun 77:170-9. 2009
    ..Taken together, these results pinpoint three amino acids in CNF1 that are key amino acids for recognition by neutralizing MAb NG8 and further help define a region in CNF1 that is critical for full toxin binding to HEp-2 cells...
  7. pmc Cytotoxic necrotizing factor 1 and hemolysin from uropathogenic Escherichia coli elicit different host responses in the murine bladder
    Tamako A Garcia
    Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
    Infect Immun 81:99-109. 2013
    ..Our data demonstrate substantial roles for CNF1 and HlyA1 in initiation of a strong proinflammatory response to UPEC in the bladder...

Research Grants30

  1. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert S Sandler; Fiscal Year: 2013
    ..Through all of its activities, the Center improves communication, promotes collaboration, develops careers and generally enriches the intellectual climate for digestive disease research. ..
  2. AMPK Endothelial Cell Dysfunction and the Metabolic Syndrome (PROGRAM PROJECT)
    Neil B Ruderman; Fiscal Year: 2013
    ..The proposed studies should both yield novel insights into the biological bases for the premature atherosclerosis and impaired angiogenesis associated with this entity and suggest new therapeutic targets for their prevention...
  3. Vesicle Trafficking and Bacteria Invasion
    Yehia Daaka; Fiscal Year: 2013
    ..We identified NOS/NO and dynamin2 as regulators of E. coli invasion into bladder cells. The targeting of eNOS and dynamin2 may provide a window of opportunity to interfere with currently untreatable recurrent UTIs. ..
  4. RESOLUTION OF CLINICAL LUNG INJURY
    Michael A Matthay; Fiscal Year: 2013
    ..abstract_text> ..
  5. Oklahoma Center for Respiratory and Infectious Diseases
    Lin Liu; Fiscal Year: 2013
    ..The completion of the goals of the present COBRE will have a major impact on research programs on respiratory infectious diseases in the State of Oklahoma. ..
  6. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013
    ..pseudomallei host pathogen response during both the septicemic as well as the intracellular phases of the disease. ..
  7. New England Regional Center of Excellence in Biodefense and Emerging Infectious D
    Dennis L Kasper; Fiscal Year: 2013
    ..NERCE will also continue its Developmental Projects program and Career Development in Biodefense program in an effort to initiate new research efforts and to attract new investigators to this field. ..
  8. Pathophysiology of Alveolar Epithelial Lung Injury
    Jacob I Sznajder; Fiscal Year: 2013
    ..The insights gained from the data generated from these studies will provide novel molecular targets for the development of new therapeutic strategies to treat patients with lung injury. ..