14 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
- Full Text
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3. Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates.
- Author
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Desloges I, Taylor JA, Leclerc JM, Brannon JR, Portt A, Spencer JD, Dewar K, Marczynski GT, Manges A, Gruenheid S, Le Moual H, and Thomassin JL
- Subjects
- Antimicrobial Cationic Peptides metabolism, Bacterial Outer Membrane Proteins chemistry, Bacterial Outer Membrane Proteins genetics, Escherichia coli Infections microbiology, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Humans, Hydrolysis, Peptide Hydrolases chemistry, Peptide Hydrolases genetics, Polymerase Chain Reaction, Substrate Specificity, Urinary Tract Infections microbiology, Uropathogenic Escherichia coli isolation & purification, Virulence Factors analysis, Virulence Factors chemistry, Virulence Factors genetics, Whole Genome Sequencing, Bacterial Outer Membrane Proteins analysis, Escherichia coli Proteins analysis, Peptide Hydrolases analysis, Uropathogenic Escherichia coli enzymology
- Abstract
Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI-causing strains, including ompT. As OmpT homologs cleave and inactivate AMPs, we hypothesized that UPEC strains from patients with symptomatic UTIs have high OmpT protease activity. Therefore, we measured OmpT activity in 58 clinical E. coli isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harbored an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence of ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)
- Published
- 2019
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4. Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity.
- Author
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Brannon JR, Thomassin JL, Gruenheid S, and Le Moual H
- Subjects
- Amino Acid Sequence, Antimicrobial Cationic Peptides chemistry, Bacterial Outer Membrane Proteins genetics, Citrobacter rodentium genetics, Citrobacter rodentium metabolism, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins genetics, Gene Expression Regulation, Bacterial physiology, Gene Expression Regulation, Enzymologic, Molecular Sequence Data, Peptide Hydrolases genetics, Protein Conformation, Serine Endopeptidases genetics, Antimicrobial Cationic Peptides metabolism, Bacterial Outer Membrane Proteins metabolism, Citrobacter rodentium enzymology, Escherichia coli enzymology, Escherichia coli Proteins metabolism, Peptide Hydrolases metabolism, Serine Endopeptidases metabolism
- Abstract
Unlabelled: Bacterial proteases contribute to virulence by cleaving host or bacterial proteins to promote survival and dissemination. Omptins are a family of proteases embedded in the outer membrane of Gram-negative bacteria that cleave various substrates, including host antimicrobial peptides, with a preference for cleaving at dibasic motifs. OmpT, the enterohemorrhagic Escherichia coli (EHEC) omptin, cleaves and inactivates the human cathelicidin LL-37. Similarly, the omptin CroP, found in the murine pathogen Citrobacter rodentium, which is used as a surrogate model to study human-restricted EHEC, cleaves the murine cathelicidin-related antimicrobial peptide (CRAMP). Here, we compared the abilities of OmpT and CroP to cleave LL-37 and CRAMP. EHEC OmpT degraded LL-37 and CRAMP at similar rates. In contrast, C. rodentium CroP cleaved CRAMP more rapidly than LL-37. The different cleavage rates of LL-37 and CRAMP were independent of the bacterial background and substrate sequence specificity, as OmpT and CroP have the same preference for cleaving at dibasic sites. Importantly, LL-37 was α-helical and CRAMP was unstructured under our experimental conditions. By altering the α-helicity of LL-37 and CRAMP, we found that decreasing LL-37 α-helicity increased its rate of cleavage by CroP. Conversely, increasing CRAMP α-helicity decreased its cleavage rate. This structural basis for CroP substrate specificity highlights differences between the closely related omptins of C. rodentium and E. coli. In agreement with previous studies, this difference in CroP and OmpT substrate specificity suggests that omptins evolved in response to the substrates present in their host microenvironments., Importance: Omptins are recognized as key virulence factors for various Gram-negative pathogens. Their localization to the outer membrane, their active site facing the extracellular environment, and their unique catalytic mechanism make them attractive targets for novel therapeutic strategies. Gaining insights into similarities and variations between the different omptin active sites and subsequent substrate specificities will be critical to develop inhibitors that can target multiple omptins. Here, we describe subtle differences between the substrate specificities of two closely related omptins, CroP and OmpT. This is the first reported example of substrate conformation acting as a structural determinant for omptin activity between OmpT-like proteases., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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5. ER egress of invariant chain isoform p35 requires direct binding to MHCII molecules and is inhibited by the NleA virulence factor of enterohaemorrhagic Escherichia coli.
- Author
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Cloutier M, Gauthier C, Fortin JS, Genève L, Kim K, Gruenheid S, Kim J, and Thibodeau J
- Subjects
- Antigens, Differentiation, B-Lymphocyte genetics, Enterohemorrhagic Escherichia coli pathogenicity, Escherichia coli Proteins genetics, HEK293 Cells, HLA Antigens metabolism, Histocompatibility Antigens Class II genetics, Humans, Protein Binding, Protein Isoforms genetics, Protein Isoforms metabolism, Protein Multimerization, Protein Sorting Signals genetics, Protein Transport drug effects, Protein Transport genetics, Virulence Factors genetics, Antigens, Differentiation, B-Lymphocyte metabolism, Endoplasmic Reticulum metabolism, Enterohemorrhagic Escherichia coli immunology, Escherichia coli Proteins metabolism, Histocompatibility Antigens Class II metabolism, Virulence Factors metabolism
- Abstract
Four invariant chain (Ii) isoforms assist the folding and trafficking of human MHC class II (MHCIIs). The main isoforms, Iip33 and Iip35, assemble in the ER into homo- and/or hetero-trimers. The sequential binding of up to three MHCII αβ heterodimers to Ii trimers results in the formation of pentamers, heptamers and nonamers. MHCIIs are required to overcome the p35-encoded di-arginine (RxR) ER retention motif and to allow anterograde trafficking of the complex. Here, we show that inactivation of the RxR motif requires a direct cis interaction between p35 and the MHCII, precluding ER egress of some unsaturated Ii trimers. Interestingly, as opposed to MHCII/p33 complexes, those including p35 remained in the ER when co-expressed with the NleA protein of enterohaemorrhagic Escherichia coli. Taken together, our results demonstrate that p35 influences distinctively MHCII/Ii assembly and trafficking., (Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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6. Enterohemorrhagic Escherichia coli OmpT regulates outer membrane vesicle biogenesis.
- Author
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Premjani V, Tilley D, Gruenheid S, Le Moual H, and Samis JA
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- Animals, Bacterial Outer Membrane Proteins genetics, Biological Transport, Enterohemorrhagic Escherichia coli pathogenicity, Escherichia coli Infections prevention & control, Escherichia coli Proteins genetics, Gene Deletion, Mice, Microscopy, Electron, Transmission, Peptide Hydrolases genetics, Bacterial Outer Membrane Proteins metabolism, Cell Membrane chemistry, Enterohemorrhagic Escherichia coli genetics, Escherichia coli Proteins metabolism, Gene Expression Regulation, Bacterial, Peptide Hydrolases metabolism
- Abstract
Enterohemorrhagic Escherichia coli (EHEC) infection from food or water often results in severe diarrheal disease and is a leading cause of death globally. Outer membrane vesicles (OMVs) secreted from E. coli induce lethality in mice. The omptin outer membrane protease OmpT from E. coli inactivates antimicrobial peptides and may enhance colonization of the uroepithelium, but its precise function remains unclear. Given OmpT is an outer membrane protease, we hypothesized it may have a role in OMV biogenesis. To further characterize the effect of OmpT on OMV production, a genetic approach using wild type, an ompT deletion mutant and an ompT overexpressing construct in EHEC were employed. ompT gene deletion markedly decreased OMV production and stainable lipid but increased vesicle diameter. Conversely, ompT overexpression profoundly increased OMV biogenesis but decreased stainable lipid, protein content, and vesicle diameter. Alterations in EHEC ompT gene expression have an impact on the biogenesis, composition, and size of OMVs. Changes in ompT gene expression may dynamically alter OMV formation, composition, and diameter in response to different host environments and contribute to cell-free intercellular communication to enhance bacterial growth and survival., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)
- Published
- 2014
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7. 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
- Full Text
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8. 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
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9. Role of uropathogenic Escherichia coli OmpT in the resistance against human cathelicidin LL-37.
- Author
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Brannon JR, Thomassin JL, Desloges I, Gruenheid S, and Le Moual H
- Subjects
- Anti-Bacterial Agents pharmacology, Antimicrobial Cationic Peptides pharmacology, Bacterial Outer Membrane Proteins genetics, Drug Resistance, Bacterial, Escherichia coli Proteins genetics, Humans, Peptide Hydrolases genetics, Uropathogenic Escherichia coli drug effects, Uropathogenic Escherichia coli genetics, Cathelicidins, Anti-Bacterial Agents metabolism, Antimicrobial Cationic Peptides metabolism, Bacterial Outer Membrane Proteins metabolism, Escherichia coli Infections microbiology, Escherichia coli Proteins metabolism, Peptide Hydrolases metabolism, Uropathogenic Escherichia coli enzymology
- Abstract
Uropathogenic Escherichia coli (UPEC) strains are among the most prevalent causative agents of urinary tract infections. To establish infection, UPEC must overcome the bactericidal action of host antimicrobial peptides. Previously, the enterohaemorrhagic E. coli outer membrane protease, OmpT, was shown to degrade and inactivate the human antimicrobial peptide LL-37. This study aims to investigate the involvement of UPEC OmpT in LL-37 degradation. An ompT deletion mutant was generated in the prototypical UPEC strain CFT073. Western blot analysis showed that the OmpT protein level is moderate in CFT073. In agreement, OmpT was shown to partially cleave LL-37. However, no difference in the minimum inhibitory concentration of LL-37 was observed between CFT073 and the ompT mutant. Plasmid complementation of ompT, which led to increased OmpT levels, resulted in complete cleavage of LL-37 and a fourfold increase in the minimum inhibitory concentration. The analysis of other UPEC isolates showed similar OmpT activity levels as CFT073. Although UPEC OmpT can cleave LL-37, we conclude that the low level of OmpT limits its contribution to LL-37 resistance. Collectively, these data suggest that UPEC OmpT is likely accompanied by other LL-37 resistance mechanisms., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)
- Published
- 2013
- Full Text
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10. 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.
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- 2010
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11. Characterization of the NleF effector protein from attaching and effacing bacterial pathogens.
- Author
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Echtenkamp F, Deng W, Wickham ME, Vazquez A, Puente JL, Thanabalasuriar A, Gruenheid S, Finlay BB, and Hardwidge PR
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, Cytoplasm chemistry, Enterobacteriaceae Infections microbiology, Epithelial Cells chemistry, Epithelial Cells microbiology, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Female, Gastrointestinal Tract microbiology, HeLa Cells, Humans, Mice, Mice, Inbred C3H, Mice, Inbred C57BL, Swine, Virulence, Virulence Factors chemistry, Virulence Factors genetics, Citrobacter rodentium genetics, Citrobacter rodentium pathogenicity, Escherichia coli O157 genetics, Escherichia coli O157 pathogenicity, Escherichia coli Proteins metabolism, Virulence Factors metabolism
- Abstract
Enterohemorrhagic Escherichia coli (EHEC) is a water- and food-borne pathogen that causes hemorrhagic colitis. EHEC uses a type III secretion system (T3SS) to translocate effector proteins that subvert host cell function. T3SS-substrates encoded outside of the locus of enterocyte effacement are important to E. coli pathogenesis. We discovered an EHEC secreted protein, NleF, encoded by z6020 in O-island 71 of E. coli EDL933 that we hypothesized to be a T3SS substrate. Experiments are presented that probe the function of NleF and its role in virulence. Immunoblotting of secreted and translocated proteins suggest that NleF is secreted by the T3SS and is translocated into host cells in vitro where it localizes to the host cytoplasm. Infection of HeLa cells with E. coli possessing or lacking nleF and transient expression of NleF-GFP via transfection did not reveal a significant role for NleF in several assays of bacterial adherence, host cytoskeletal remodeling, or host protein secretion. However, competitive coinfection of mice with Citrobacter rodentium strains possessing or lacking nleF suggested a contribution of NleF to bacterial colonization. Challenge of gnotobiotic piglets also revealed a role for NleF in colonization of the piglet colon and rectoanal junction.
- Published
- 2008
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12. 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
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13. Regulation of type III secretion hierarchy of translocators and effectors in attaching and effacing bacterial pathogens.
- Author
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Deng W, Li Y, Hardwidge PR, Frey EA, Pfuetzner RA, Lee S, Gruenheid S, Strynakda NC, Puente JL, and Finlay BB
- Subjects
- Bacterial Outer Membrane Proteins physiology, Calcium pharmacology, Citrobacter rodentium pathogenicity, Escherichia coli pathogenicity, Humans, Citrobacter rodentium metabolism, Escherichia coli metabolism, Escherichia coli Proteins physiology
- Abstract
Human enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and the mouse pathogen Citrobacter rodentium (CR) belong to the family of attaching and effacing (A/E) bacterial pathogens. They possess the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system. These pathogens secrete a number of proteins into culture media, including type III effector proteins and translocators that are required for the translocation of effectors into host cells. Preliminary evidence indicated that the LEE-encoded SepL and Rorf6/SepD may form a molecular switch that controls the secretion of translocators and effectors in CR. Here, we show that SepL and SepD indeed perform this function in A/E pathogens such as EHEC and EPEC. Their sepL and sepD mutants do not secrete translocators but exhibit enhanced secretion of effectors. We demonstrate that SepL and SepD interact with each other and that both SepL and SepD are localized to the bacterial membranes. Furthermore, we demonstrate that culture media influence the type III secretion profile of EHEC, EPEC, and CR and that low-calcium concentrations inhibit secretion of translocators but promote the secretion of effectors, similar to effects on type III secretion by mutations in sepL and sepD. However, the secretion profile of the sepD and sepL mutants is not affected by these culture conditions. Collectively, our results suggest that SepL and SepD not only are necessary for efficient translocator secretion in A/E pathogens but also control a switch from translocator to effector secretion by sensing certain environmental signals such as low calcium.
- Published
- 2005
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14. Identification and characterization of NleA, a non-LEE-encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7.
- Author
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Gruenheid S, Sekirov I, Thomas NA, Deng W, O'Donnell P, Goode D, Li Y, Frey EA, Brown NF, Metalnikov P, Pawson T, Ashman K, and Finlay BB
- Subjects
- Amino Acid Sequence, Animals, Citrobacter genetics, Citrobacter metabolism, Citrobacter pathogenicity, Colon cytology, Colon microbiology, Colon pathology, Enterobacteriaceae Infections microbiology, Escherichia coli O157 genetics, Escherichia coli O157 pathogenicity, Escherichia coli Proteins chemistry, Genome, Bacterial, Golgi Apparatus metabolism, HeLa Cells, Humans, Mice, Mice, Inbred Strains, Molecular Sequence Data, Open Reading Frames, Protein Transport, Proteome analysis, Sequence Alignment, Spleen cytology, Spleen microbiology, Spleen pathology, Virulence Factors chemistry, Escherichia coli O157 metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Genomic Islands, Virulence Factors genetics, Virulence Factors metabolism
- Abstract
Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 uses a specialized protein translocation apparatus, the type III secretion system (TTSS), to deliver bacterial effector proteins into host cells. These effectors interfere with host cytoskeletal pathways and signalling cascades to facilitate bacterial survival and replication and promote disease. The genes encoding the TTSS and all known type III secreted effectors in EHEC are localized in a single pathogenicity island on the bacterial chromosome known as the locus for enterocyte effacement (LEE). In this study, we performed a proteomic analysis of proteins secreted by the LEE-encoded TTSS of EHEC. In addition to known LEE-encoded type III secreted proteins, such as EspA, EspB and Tir, a novel protein, NleA (non-LEE-encoded effector A), was identified. NleA is encoded in a prophage-associated pathogenicity island within the EHEC genome, distinct from the LEE. The LEE-encoded TTSS directs translocation of NleA into host cells, where it localizes to the Golgi apparatus. In a panel of strains examined by Southern blot and database analyses, nleA was found to be present in all other LEE-containing pathogens examined, including enteropathogenic E. coli and Citrobacter rodentium, and was absent from non-pathogenic strains of E. coli and non-LEE-containing pathogens. NleA was determined to play a key role in virulence of C. rodentium in a mouse infection model.
- Published
- 2004
- Full Text
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