10 results on '"Gruenheid, Samantha"'
Search Results
2. The bacterial virulence factor NleA undergoes host-mediated O-linked glycosylation.
- Author
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Burns L, Giannakopoulou N, Zhu L, Xu YZ, Khan RH, Bekal S, Schurr E, Schmeing TM, and Gruenheid S
- Subjects
- Humans, Animals, Mice, Virulence Factors metabolism, HeLa Cells, Glycosylation, Mice, Inbred C57BL, Escherichia coli Proteins metabolism, Enteropathogenic Escherichia coli
- Abstract
Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are gastrointestinal pathogens responsible for severe diarrheal illness. EHEC and EPEC form "attaching and effacing" lesions during colonization and, upon adherence, inject proteins directly into host intestinal cells via the type III secretion system (T3SS). Injected bacterial proteins have a variety of functions but generally alter host cell biology to favor survival and/or replication of the pathogen. Non-LEE-encoded effector A (NleA) is a T3SS-injected effector of EHEC, EPEC, and the related mouse pathogen Citrobacter rodentium. Studies in mouse models indicate that NleA has an important role in bacterial virulence. However, the mechanism by which NleA contributes to disease remains unknown. We have determined that the following translocation into host cells, a serine and threonine-rich region of NleA is modified by host-mediated mucin-type O-linked glycosylation. Surprisingly, this region was not present in several clinical EHEC isolates. When expressed in C. rodentium, a non-modifiable variant of NleA was indistinguishable from wildtype NleA in an acute mortality model but conferred a modest increase in persistence over the course of infection in mixed infections in C57BL/6J mice. This is the first known example of a bacterial effector being modified by host-mediated O-linked glycosylation. Our data also suggests that this modification may confer a selective disadvantage to the bacteria during in vivo infection., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)
- Published
- 2023
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3. Both group 4 capsule and lipopolysaccharide O-antigen contribute to enteropathogenic Escherichia coli resistance to human α-defensin 5.
- Author
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Thomassin JL, Lee MJ, Brannon JR, Sheppard DC, Gruenheid S, and Le Moual H
- Subjects
- Bacterial Capsules metabolism, Enteropathogenic Escherichia coli metabolism, Escherichia coli Proteins metabolism, Gene Expression Regulation, Bacterial, Humans, O Antigens metabolism, Polysaccharides, Bacterial pharmacology, Sequence Deletion, Anti-Infective Agents pharmacology, Bacterial Capsules genetics, Drug Resistance, Bacterial genetics, Enteropathogenic Escherichia coli drug effects, Enteropathogenic Escherichia coli genetics, Escherichia coli Proteins genetics, O Antigens genetics, alpha-Defensins pharmacology
- Abstract
Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that colonize the small intestine and colon, respectively. To cause disease, these pathogens must overcome the action of different host antimicrobial peptides (AMPs) secreted into these distinct niches. We have shown previously that EHEC expresses high levels of the OmpT protease to inactivate the human cathelicidin LL-37, an AMP present in the colon. In this study, we investigate the mechanisms used by EPEC to resist human α-defensin 5 (HD-5), the most abundant AMP in the small intestine. Quantitative PCR was used to measure transcript levels of various EPEC surface structures. High transcript levels of gfcA, a gene required for group 4 capsule (G4C) production, were observed in EPEC, but not in EHEC. The unencapsulated EPEC ∆gfcA and EHEC wild-type strains were more susceptible to HD-5 than EPEC wild-type. Since the G4C is composed of the same sugar repeats as the lipopolysaccharide O-antigen, an -antigen ligase (waaL) deletion mutant was generated in EPEC to assess its role in HD-5 resistance. The ∆waaL EPEC strain was more susceptible to HD-5 than both the wild-type and ∆gfcA strains. The ∆gfcA∆waaL EPEC strain was not significantly more susceptible to HD-5 than the ∆waaL strain, suggesting that the absence of -antigen influences G4C formation. To determine whether the G4C and -antigen interact with HD-5, total polysaccharide was purified from wild-type EPEC and added to the ∆gfcA∆waaL strain in the presence of HD-5. The addition of exogenous polysaccharide protected the susceptible strain against HD-5 killing in a dose-dependent manner, suggesting that HD-5 binds to the polysaccharides present on the surface of EPEC. Altogether, these findings indicate that EPEC relies on both the G4C and the -antigen to resist the bactericidal activity of HD-5.
- Published
- 2013
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4. The inhibition of COPII trafficking is important for intestinal epithelial tight junction disruption during enteropathogenic Escherichia coli and Citrobacter rodentium infection.
- Author
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Thanabalasuriar A, Kim J, and Gruenheid S
- Subjects
- Animals, Diarrhea pathology, Disease Models, Animal, Epithelial Cells microbiology, Host-Pathogen Interactions, Mice, Mice, Inbred C3H, COP-Coated Vesicles metabolism, Citrobacter rodentium physiology, Diarrhea microbiology, Enteropathogenic Escherichia coli physiology, Epithelial Cells physiology, Escherichia coli Proteins metabolism, Tight Junctions, Virulence Factors metabolism
- Abstract
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are bacterial pathogens that cause severe illnesses in humans. Citrobacter rodentium is a related mouse pathogen that serves as a small animal model for EPEC and EHEC infections. EPEC, EHEC and C. rodentium translocate bacterial virulence proteins directly into host intestinal cells via a type III secretion system (T3SS). Non-LEE-encoded effector A (NleA) is a T3SS effector that is common to EPEC, EHEC and C. rodentium. NleA interacts with and inhibits the mammalian COPII complex, impairing cellular secretion; this interaction is required for bacterial virulence. Although diarrhea is a hallmark of EPEC, EHEC and C. rodentium infections, the underlying mechanisms are not well characterized. One of the essential functions of the intestine is to maintain a barrier between the lumen and submucosa. Tight junctions seal the space between adjacent epithelial cells creating this barrier. Consequently, it is thought that the disruption of intestinal epithelial tight junctions by EPEC, EHEC, and C. rodentium could result in a loss of barrier function. In this study, we demonstrate that NleA mediated COPII inhibition is required for EPEC- and C. rodentium-mediated disruption of tight junction proteins and increases in fecal water content., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
5. Identification of potentially diarrheagenic atypical enteropathogenic Escherichia coli strains present in Canadian food animals at slaughter and in retail meats.
- Author
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Comery R, Thanabalasuriar A, Garneau P, Portt A, Boerlin P, Reid-Smith RJ, Harel J, Manges AR, and Gruenheid S
- Subjects
- Abattoirs standards, Animals, Blotting, Western veterinary, Canada epidemiology, Cattle, Cattle Diseases microbiology, Cluster Analysis, Drug Resistance, Microbial genetics, Escherichia coli Infections epidemiology, Food Supply standards, Microscopy, Fluorescence, Polymerase Chain Reaction veterinary, Poultry Diseases microbiology, Swine, Swine Diseases microbiology, Cattle Diseases epidemiology, Chickens, Enteropathogenic Escherichia coli genetics, Escherichia coli Infections veterinary, Meat microbiology, Poultry Diseases epidemiology, Swine Diseases epidemiology
- Abstract
This study identified and characterized enteropathogenic Escherichia coli (EPEC) in the Canadian food supply. Eighteen of 450 E. coli isolates from food animal sources were identified as atypical EPEC (aEPEC). Several of the aEPEC isolates identified in this study possessed multiple virulence genes, exhibited adherence and attaching and effacing (A/E) lesion formation, disrupted tight junctions, and were coclassified with the extraintestinal pathogenic E. coli (ExPEC) and enterotoxigenic E. coli (ETEC) pathotypes.
- Published
- 2013
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6. Enterohemorrhagic and enteropathogenic Escherichia coli evolved different strategies to resist antimicrobial peptides.
- Author
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Thomassin JL, Brannon JR, Kaiser J, Gruenheid S, and Le Moual H
- Subjects
- Antimicrobial Cationic Peptides immunology, Antimicrobial Cationic Peptides metabolism, Bacterial Outer Membrane Proteins metabolism, Escherichia coli Infections immunology, Escherichia coli Infections microbiology, Escherichia coli Proteins metabolism, Gene Expression, Humans, Peptide Hydrolases metabolism, Promoter Regions, Genetic, Proteolysis, Cathelicidins, Antimicrobial Cationic Peptides pharmacology, Drug Resistance, Microbial, Enterohemorrhagic Escherichia coli drug effects, Enterohemorrhagic Escherichia coli pathogenicity, Enteropathogenic Escherichia coli drug effects, Enteropathogenic Escherichia coli pathogenicity, Immune Evasion
- Abstract
Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are enteric human pathogens that colonize the large and small intestines, respectively. To establish infection EHEC and EPEC must overcome innate host defenses, such as antimicrobial peptides (AMPs) produced by the intestinal epithelium. Gram-negative pathogens have evolved different mechanisms to resist AMPs, including outer-membrane proteases that degrade AMPs. We showed that the protease OmpT degrades the human AMP LL-37 more rapidly in EHEC than in EPEC. Promoter-swap experiments showed that this is due to differences in the promoters of the two genes, leading to greater ompT expression and subsequently greater levels of OmpT in EHEC. Here, we propose that the different ompT expression in EHEC and EPEC reflects the varying levels of LL-37 throughout the human intestinal tract. These data suggest that EHEC and EPEC adapted to their specific niches by developing distinct AMP-specific resistance mechanisms.
- Published
- 2012
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7. OmpT outer membrane proteases of enterohemorrhagic and enteropathogenic Escherichia coli contribute differently to the degradation of human LL-37.
- Author
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Thomassin JL, Brannon JR, Gibbs BF, Gruenheid S, and Le Moual H
- Subjects
- Antimicrobial Cationic Peptides pharmacology, Drug Resistance, Bacterial, Fluorescence Resonance Energy Transfer, Gene Deletion, Gene Expression Regulation, Bacterial physiology, Gene Expression Regulation, Enzymologic physiology, Humans, Microbial Sensitivity Tests, Promoter Regions, Genetic, Serine Endopeptidases genetics, Cathelicidins, Antimicrobial Cationic Peptides metabolism, Enterohemorrhagic Escherichia coli enzymology, Enteropathogenic Escherichia coli enzymology, Serine Endopeptidases metabolism
- Abstract
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are food-borne pathogens that cause serious diarrheal diseases. To colonize the human intestine, these pathogens must overcome innate immune defenses such as antimicrobial peptides (AMPs). Bacterial pathogens have evolved various mechanisms to resist killing by AMPs, including proteolytic degradation of AMPs. To examine the ability of the EHEC and EPEC OmpT outer membrane (OM) proteases to degrade α-helical AMPs, ompT deletion mutants were generated. Determination of MICs of various AMPs revealed that both mutant strains are more susceptible than their wild-type counterparts to α-helical AMPs, although to different extents. Time course assays monitoring the degradation of LL-37 and C18G showed that EHEC cells degraded both AMPs faster than EPEC cells in an OmpT-dependent manner. Mass spectrometry analyses of proteolytic fragments showed that EHEC OmpT cleaves LL-37 at dibasic sites. The superior protection provided by EHEC OmpT compared to EPEC OmpT against α-helical AMPs was due to higher expression of the ompT gene and, in turn, higher levels of the OmpT protein in EHEC. Fusion of the EPEC ompT promoter to the EHEC ompT open reading frame resulted in decreased OmpT expression, indicating that transcriptional regulation of ompT is different in EHEC and EPEC. We hypothesize that the different contributions of EHEC and EPEC OmpT to the degradation and inactivation of LL-37 may be due to their adaptation to their respective niches within the host, the colon and small intestine, respectively, where the environmental cues and abundance of AMPs are different.
- Published
- 2012
- Full Text
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8. Perturbation of host cell cytoskeleton by cranberry proanthocyanidins and their effect on enteric infections.
- Author
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Harmidy K, Tufenkji N, and Gruenheid S
- Subjects
- Bacterial Adhesion, Bacterial Proteins metabolism, Cell Survival drug effects, Epithelial Cells microbiology, Escherichia coli Infections drug therapy, Escherichia coli Infections microbiology, HeLa Cells, Humans, Salmonella Infections drug therapy, Salmonella Infections microbiology, Signal Transduction, Actins metabolism, Cytoskeleton drug effects, Enteropathogenic Escherichia coli drug effects, Epithelial Cells drug effects, Proanthocyanidins pharmacology, Salmonella typhimurium drug effects, Vaccinium macrocarpon chemistry
- Abstract
Cranberry-derived compounds, including a fraction known as proanthocyanidins (PACs) exhibit anti-microbial, anti-infective, and anti-adhesive properties against a number of disease-causing organisms. In this study, the effect of cranberry proanthocyanidins (CPACs) on the infection of epithelial cells by two enteric bacterial pathogens, enteropathogenic Escherichia coli (EPEC) and Salmonella Typhimurium was investigated. Immunofluorescence data showed that actin pedestal formation, required for infection by enteropathogenic Escherichia coli (EPEC), was disrupted in the presence of CPACs. In addition, invasion of HeLa cells by Salmonella Typhimurium was significantly reduced, as verified by gentamicin protection assay and immunofluorescence. CPACs had no effect on bacterial growth, nor any detectable effect on the production of bacterial effector proteins of the type III secretion system. Furthermore, CPACs did not affect the viability of host cells. Interestingly, we found that CPACs had a potent and dose-dependent effect on the host cell cytoskeleton that was evident even in uninfected cells. CPACs inhibited the phagocytosis of inert particles by a macrophage cell line, providing further evidence that actin-mediated host cell functions are disrupted in the presence of cranberry CPACs. Thus, although CPAC treatment inhibited Salmonella invasion and EPEC pedestal formation, our results suggest that this is likely primarily because of the perturbation of the host cell cytoskeleton by CPACs rather than an effect on bacterial virulence itself. These findings have significant implications for the interpretation of experiments on the effects of CPACs on bacteria-host cell interactions.
- Published
- 2011
- Full Text
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9. The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic Escherichia coli.
- Author
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Thanabalasuriar A, Koutsouris A, Weflen A, Mimee M, Hecht G, and Gruenheid S
- Subjects
- Brefeldin A pharmacology, Caco-2 Cells, Carrier Proteins genetics, Carrier Proteins metabolism, Carrier Proteins physiology, Cell Line, Tumor, Endoplasmic Reticulum drug effects, Endoplasmic Reticulum metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Genetic Complementation Test, Golgi Apparatus drug effects, Golgi Apparatus metabolism, HeLa Cells, Humans, Intracellular Signaling Peptides and Proteins, Microscopy, Fluorescence, Two-Hybrid System Techniques, Virulence genetics, Virulence Factors genetics, Virulence Factors metabolism, Enteropathogenic Escherichia coli pathogenicity, Enteropathogenic Escherichia coli physiology, Escherichia coli Proteins physiology, Intestines microbiology, Tight Junctions metabolism, Virulence physiology, Virulence Factors physiology
- Abstract
Enteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen that adheres to epithelial cells of the small intestine and uses a type III secretion system to inject effector proteins into host cells. EPEC infection leads to disruption of host intestinal tight junctions that are important for maintaining intestinal barrier function. This disruption is dependent on the bacterial type III secretion system, as well as the translocated effectors EspF and Map. Here we show that a third type III translocated bacterial effector protein, NleA, is also involved in tight junction disruption during EPEC infection. Using the drug Brefeldin A, we demonstrate that the effect of NleA on tight junction integrity is related to its inhibition of host cell protein trafficking through COPII-dependent pathways. These results suggest that NleA's striking effect on virulence is mediated, at least in part, via its role in disruption of intestinal barrier function.
- Published
- 2010
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10. The bacterial virulence factor NleA inhibits cellular protein secretion by disrupting mammalian COPII function.
- Author
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Kim J, Thanabalasuriar A, Chaworth-Musters T, Fromme JC, Frey EA, Lario PI, Metalnikov P, Rizg K, Thomas NA, Lee SF, Hartland EL, Hardwidge PR, Pawson T, Strynadka NC, Finlay BB, Schekman R, and Gruenheid S
- Subjects
- Animals, CHO Cells, Cricetinae, Cricetulus, Enteropathogenic Escherichia coli pathogenicity, HeLa Cells, Humans, COP-Coated Vesicles metabolism, Enteropathogenic Escherichia coli physiology, Escherichia coli Proteins physiology, Proteins metabolism, Vesicular Transport Proteins metabolism, Virulence Factors physiology
- Abstract
Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) maintain an extracellular lifestyle and use a type III secretion system to translocate effector proteins into the host cytosol. These effectors manipulate host pathways to favor bacterial replication and survival. NleA is an EHEC/EPEC- and related species-specific translocated effector protein that is essential for bacterial virulence. However, the mechanism by which NleA impacts virulence remains undetermined. Here we demonstrate that NleA compromises the Sec23/24 complex, a component of the mammalian COPII protein coat that shapes intracellular protein transport vesicles, by directly binding Sec24. Expression of an NleA-GFP fusion protein reduces the efficiency of cellular secretion by 50%, and secretion is inhibited in EPEC-infected cells. Direct biochemical experiments show that NleA inhibits COPII-dependent protein export from the endoplasmic reticulum. Collectively, these findings indicate that disruption of COPII function in host cells contributes to the virulence of EPEC and EHEC.
- Published
- 2007
- Full Text
- View/download PDF
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