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1
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Badilla Lobo A, Soutourina O, Peltier J. The current riboswitch landscape in Clostridioides difficile. MICROBIOLOGY (READING, ENGLAND) 2024; 170:001508. [PMID: 39405103 PMCID: PMC11477304 DOI: 10.1099/mic.0.001508] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/15/2024] [Accepted: 09/26/2024] [Indexed: 10/19/2024]
Abstract
Riboswitches are 5' RNA regulatory elements that are capable of binding to various ligands, such as small metabolites, ions and tRNAs, leading to conformational changes and affecting gene transcription or translation. They are widespread in bacteria and frequently control genes that are essential for the survival or virulence of major pathogens. As a result, they represent promising targets for the development of new antimicrobial treatments. Clostridioides difficile, a leading cause of antibiotic-associated nosocomial diarrhoea in adults, possesses numerous riboswitches in its genome. Accumulating knowledge of riboswitch-based regulatory mechanisms provides insights into the potential therapeutic targets for treating C. difficile infections. This review offers an in-depth examination of the current state of knowledge regarding riboswitch-mediated regulation in C. difficile, highlighting their importance in bacterial adaptability and pathogenicity. Particular attention is given to the ligand specificity and function of known riboswitches in this bacterium. The review also discusses the recent progress that has been made in the development of riboswitch-targeting compounds as potential treatments for C. difficile infections. Future research directions are proposed, emphasizing the need for detailed structural and functional analyses of riboswitches to fully harness their regulatory capabilities for developing new antimicrobial strategies.
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Affiliation(s)
- Adriana Badilla Lobo
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Olga Soutourina
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Johann Peltier
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
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2
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Nhu NTQ, Lin H, Pigli Y, Sia JK, Kuhn P, Snitkin ES, Young V, Kamboj M, Pamer EG, Rice PA, Shen A, Dong Q. Flagellar switch inverted repeat impacts flagellar invertibility and varies Clostridioides difficile RT027/MLST1 virulence. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.06.22.546185. [PMID: 39386689 PMCID: PMC11463649 DOI: 10.1101/2023.06.22.546185] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 10/12/2024]
Abstract
Clostridioides difficile RT027 strains cause infections that vary in severity from asymptomatic to lethal, but the molecular basis for this variability is poorly understood. Through comparative analyses of RT027 clinical isolates, we determined that isolates that exhibit greater variability in their flagellar gene expression exhibit greater virulence in vivo. C. difficile flagellar genes are phase-variably expressed due to the site-specific inversion of the flgB 5'UTR region, which reversibly generates ON vs. OFF orientations for the flagellar switch. We found that longer inverted repeat (IR) sequences in this switch region correlate with greater disease severity, with RT027 strains carrying 6A/6T IR sequences exhibiting greater phenotypic heterogeneity in flagellar gene expression (60%-75% ON) and causing more severe disease than those with shorter IRs (> 99% ON or OFF). Taken together, our results reveal that phenotypic heterogeneity in flagellar gene expression may contribute to the variable disease severity observed in C. difficile patients.
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Affiliation(s)
- Nguyen T. Q. Nhu
- Department of Medicine, University of Chicago, Chicago, Illinois, USA
- Duchossois Family Institute, University of Chicago, Chicago, Illinois, USA
| | - Huaiying Lin
- Duchossois Family Institute, University of Chicago, Chicago, Illinois, USA
| | - Ying Pigli
- Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA
| | - Jonathan K. Sia
- Immunology Program, Memorial Sloan Kettering Cancer Center, New York City, New York, USA
| | - Pola Kuhn
- Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts, USA
| | - Evan S. Snitkin
- Division of Infectious Diseases, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA
| | - Vincent Young
- Division of Infectious Diseases, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA
- Department of Microbiology & Immunology, University of Michigan, Ann Arbor, MI, USA
| | - Mini Kamboj
- Infection Control, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA
| | - Eric G. Pamer
- Department of Medicine, University of Chicago, Chicago, Illinois, USA
- Duchossois Family Institute, University of Chicago, Chicago, Illinois, USA
| | - Phoebe A. Rice
- Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA
| | - Aimee Shen
- Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts, USA
| | - Qiwen Dong
- Department of Medicine, University of Chicago, Chicago, Illinois, USA
- Duchossois Family Institute, University of Chicago, Chicago, Illinois, USA
- Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts, USA
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3
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Ouyang Z, Zhao M, Li J, Zhang Y, Zhao J. Cyclic diguanylate differentially regulates the expression of virulence factors and pathogenesis-related phenotypes in Clostridioides difficile. Microbiol Res 2024; 286:127811. [PMID: 38909416 DOI: 10.1016/j.micres.2024.127811] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2024] [Revised: 06/03/2024] [Accepted: 06/12/2024] [Indexed: 06/25/2024]
Abstract
Clostridioides difficile infection (CDI) caused by toxigenic C. difficile is the leading cause of antimicrobial and healthcare-associated diarrhea. The pathogenicity of C. difficile relies on the synergistic effect of multiple virulence factors, including spores, flagella, type IV pili (T4P), toxins, and biofilm. Spores enable survival and transmission of C. difficile, while adhesion factors such as flagella and T4P allow C. difficile to colonize and persist in the host intestine. Subsequently, C. difficile produces the toxins TcdA and TcdB, causing pseudomembranous colitis and other C. difficile-associated diseases; adhesion factors bind to the extracellular matrix to form biofilm, allowing C. difficile to evade drug and immune system attack and cause recurrent infection. Cyclic diguanylate (c-di-GMP) is a near-ubiquitous second messenger that extensively regulates morphology, the expression of virulence factors, and multiple physiological processes in C. difficile. In this review, we summarize current knowledge of how c-di-GMP differentially regulates the expression of virulence factors and pathogenesis-related phenotypes in C. difficile. We highlight that C. difficile spore formation and expression of toxin and flagella genes are inhibited at high intracellular levels of c-di-GMP, while T4P biosynthesis, cell aggregation, and biofilm formation are induced. Recent studies have enhanced our understanding of the c-di-GMP signaling networks in C. difficile and provided insights for the development of c-di-GMP-dependent strategies against CDI.
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Affiliation(s)
- Zirou Ouyang
- Hebei Provincial Center for Clinical Laboratories, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China
| | - Min Zhao
- Hebei Provincial Center for Clinical Laboratories, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China
| | - Jiayiren Li
- Hebei Provincial Center for Clinical Laboratories, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China
| | - Yulian Zhang
- Hebei Provincial Center for Clinical Laboratories, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China
| | - Jianhong Zhao
- Hebei Provincial Center for Clinical Laboratories, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China.
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Cassona CP, Ramalhete S, Amara K, Candela T, Kansau I, Denève-Larrazet C, Janoir-Jouveshomme C, Mota LJ, Dupuy B, Serrano M, Henriques AO. Spores of Clostridioides difficile are toxin delivery vehicles. Commun Biol 2024; 7:839. [PMID: 38987278 PMCID: PMC11237016 DOI: 10.1038/s42003-024-06521-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2023] [Accepted: 06/28/2024] [Indexed: 07/12/2024] Open
Abstract
Clostridioides difficile causes a wide range of intestinal diseases through the action of two main cytotoxins, TcdA and TcdB. Ingested spores germinate in the intestine establishing a population of cells that produce toxins and spores. The pathogenicity locus, PaLoc, comprises several genes, including those coding for TcdA/B, for the holin-like TcdE protein, and for TcdR, an auto-regulatory RNA polymerase sigma factor essential for tcdA/B and tcdE expression. Here we show that tcdR, tcdA, tcdB and tcdE are expressed in a fraction of the sporulating cells, in either the whole sporangium or in the forespore. The whole sporangium pattern is due to protracted expression initiated in vegetative cells by σD, which primes the TcdR auto-regulatory loop. In contrast, the forespore-specific regulatory proteins σG and SpoVT control TcdR production and tcdA/tcdB and tcdE expression in this cell. We detected TcdA at the spore surface, and we show that wild type and ΔtcdA or ΔtcdB spores but not ΔtcdR or ΔtcdA/ΔtcdB spores are cytopathic against HT29 and Vero cells, indicating that spores may serve as toxin-delivery vehicles. Since the addition of TcdA and TcdB enhance binding of spores to epithelial cells, this effect may occur independently of toxin production by vegetative cells.
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Affiliation(s)
- Carolina P Cassona
- Instituto de Tecnologia Química e Biológica, NOVA University Lisbon, Oeiras, Portugal
| | - Sara Ramalhete
- Instituto de Tecnologia Química e Biológica, NOVA University Lisbon, Oeiras, Portugal
| | - Khira Amara
- Instituto de Tecnologia Química e Biológica, NOVA University Lisbon, Oeiras, Portugal
| | - Thomas Candela
- Micalis Institute, Université Paris-Saclay, INRAE, AgroParisTech, Jouy-en-Josas, France
| | - Imad Kansau
- Micalis Institute, Université Paris-Saclay, INRAE, AgroParisTech, Jouy-en-Josas, France
| | | | | | - Luís Jaime Mota
- Associate Laboratory i4HB, Institute for Health and Bioeconomy, NOVA School of Science and Technology, NOVA University Lisbon, Caparica, Portugal
- UCIBIO, Applied Molecular Biosciences Unit, Department of Life Sciences, NOVA School of Science and Technology, NOVA University Lisbon, Caparica, Portugal
| | - Bruno Dupuy
- Institut Pasteur, Université Paris-Cité, UMR-CNRS 6047, Laboratoire Pathogenèse des Bactéries Anaérobies, F-75015, Paris, France
| | - Mónica Serrano
- Instituto de Tecnologia Química e Biológica, NOVA University Lisbon, Oeiras, Portugal
| | - Adriano O Henriques
- Instituto de Tecnologia Química e Biológica, NOVA University Lisbon, Oeiras, Portugal.
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DiCandia MA, Edwards AN, Alcaraz YB, Monteiro MP, Lee CD, Vargas Cuebas G, Bagchi P, McBride SM. A conserved switch controls virulence, sporulation, and motility in C. difficile. PLoS Pathog 2024; 20:e1012224. [PMID: 38739653 PMCID: PMC11115286 DOI: 10.1371/journal.ppat.1012224] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2023] [Revised: 05/23/2024] [Accepted: 04/25/2024] [Indexed: 05/16/2024] Open
Abstract
Spore formation is required for environmental survival and transmission of the human enteropathogenic Clostridioides difficile. In all bacterial spore formers, sporulation is regulated through activation of the master response regulator, Spo0A. However, the factors and mechanisms that directly regulate C. difficile Spo0A activity are not defined. In the well-studied Bacillus species, Spo0A is directly inactivated by Spo0E, a small phosphatase. To understand Spo0E function in C. difficile, we created a null mutation of the spo0E ortholog and assessed sporulation and physiology. The spo0E mutant produced significantly more spores, demonstrating Spo0E represses C. difficile sporulation. Unexpectedly, the spo0E mutant also exhibited increased motility and toxin production, and enhanced virulence in animal infections. We uncovered that Spo0E interacts with both Spo0A and the toxin and motility regulator, RstA. Direct interactions between Spo0A, Spo0E, and RstA constitute a previously unknown molecular switch that coordinates sporulation with motility and toxin production. Reinvestigation of Spo0E function in B. subtilis revealed that Spo0E induced motility, demonstrating Spo0E regulation of motility and sporulation among divergent species. Further, 3D structural analyses of Spo0E revealed specific and exclusive interactions between Spo0E and binding partners in C. difficile and B. subtilis that provide insight into the conservation of this regulatory mechanism among different species.
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Affiliation(s)
- Michael A. DiCandia
- Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, Georgia, United States of America
| | - Adrianne N. Edwards
- Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, Georgia, United States of America
| | - Ysabella B. Alcaraz
- Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, Georgia, United States of America
| | - Marcos P. Monteiro
- Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, Georgia, United States of America
| | - Cheyenne D. Lee
- Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, Georgia, United States of America
| | - Germán Vargas Cuebas
- Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, Georgia, United States of America
| | - Pritha Bagchi
- Emory Integrated Proteomics Core, Emory University, Atlanta, Georgia, United States of America
| | - Shonna M. McBride
- Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, Georgia, United States of America
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6
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Warren Norris MAH, Plaskon DM, Tamayo R. Phase Variation of Flagella and Toxins in Clostridioides difficile is Mediated by Selective Rho-dependent Termination. J Mol Biol 2024; 436:168456. [PMID: 38278436 PMCID: PMC10942720 DOI: 10.1016/j.jmb.2024.168456] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2023] [Revised: 01/16/2024] [Accepted: 01/20/2024] [Indexed: 01/28/2024]
Abstract
Clostridioides difficile is an intestinal pathogen that exhibits phase variation of flagella and toxins through inversion of the flagellar (flg) switch controlling flagellar and toxin gene expression. The transcription termination factor Rho preferentially inhibits swimming motility of bacteria with the 'flg-OFF' switch sequence. How C. difficile Rho mediates this selectivity was unknown. C. difficile Rho contains an N-terminal insertion domain (NID) which is found in a subset of Rho orthologues and confers diverse functions. Here we determined how Rho distinguishes between flg-ON and -OFF mRNAs and the roles of the NID and other domains of C. difficile Rho. Using in vitro ATPase assays, we determined that Rho specifically binds a region containing the left inverted repeat of the flg switch, but only of flg-OFF mRNA, indicating that differential termination is mediated by selective Rho binding. Using a suite of in vivo and in vitro assays in C. difficile, we determined that the NID is essential for Rho termination of flg-OFF mRNA, likely by influencing the ability to form stable hexamers, and the RNA binding domain is critical for flg-OFF specific termination. This work gives insight into the novel mechanism by which Rho interacts with flg mRNA to mediate phase variation of flagella and toxins in C. difficile and broadens our understanding of Rho-mediated termination in an organism with an AT-rich genome.
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Affiliation(s)
- Mercedes A H Warren Norris
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA
| | - Dylan M Plaskon
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA
| | - Rita Tamayo
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
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7
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Gong JJ, Huang IH, Su MSW, Xie SX, Liu WY, Huang CR, Hung YP, Wu SR, Tsai PJ, Ko WC, Chen JW. Phage transcriptional regulator X (PtrX)-mediated augmentation of toxin production and virulence in Clostridioides difficile strain R20291. Microbiol Res 2024; 280:127576. [PMID: 38183754 DOI: 10.1016/j.micres.2023.127576] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2023] [Revised: 11/24/2023] [Accepted: 12/13/2023] [Indexed: 01/08/2024]
Abstract
Clostridioides difficile is a Gram-positive, anaerobic, and spore-forming bacterial member of the human gut microbiome. The primary virulence factors of C. difficile are toxin A and toxin B. These toxins damage the cell cytoskeleton and cause various diseases, from diarrhea to severe pseudomembranous colitis. Evidence suggests that bacteriophages can regulate the expression of the pathogenicity locus (PaLoc) genes of C. difficile. We previously demonstrated that the genome of the C. difficile RT027 strain NCKUH-21 contains a prophage-like DNA sequence, which was found to be markedly similar to that of the φCD38-2 phage. In the present study, we investigated the mechanisms underlying the φNCKUH-21-mediated regulation of the pathogenicity and the PaLoc genes expression in the lysogenized C. difficile strain R20291. The carriage of φNCKUH-21 in R20291 cells substantially enhanced toxin production, bacterial motility, biofilm formation, and spore germination in vitro. Subsequent mouse studies revealed that the lysogenized R20291 strain caused a more severe infection than the wild-type strain. We screened three φNCKUH-21 genes encoding DNA-binding proteins to check their effects on PaLoc genes expression. The overexpression of NCKUH-21_03890, annotated as a transcriptional regulator (phage transcriptional regulator X, PtrX), considerably enhanced toxin production, biofilm formation, and bacterial motility of R20291. Transcriptome analysis further confirmed that the overexpression of ptrX led to the upregulation of the expression of toxin genes, flagellar genes, and csrA. In the ptrX-overexpressing R20291 strain, PtrX influenced the expression of flagellar genes and the sigma factor gene sigD, possibly through an increased flagellar phase ON configuration ratio.
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Affiliation(s)
- Jun-Jia Gong
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - I-Hsiu Huang
- Department of Biochemistry and Microbiology, Oklahoma State University Center for Health Sciences, Tulsa, OK 74107, USA
| | - Marcia Shu-Wei Su
- Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Si-Xuan Xie
- Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Wei-Yong Liu
- Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Cheng-Rung Huang
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Yuan-Pin Hung
- Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Department of Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Shang-Rung Wu
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Institute of Oral Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Pei-Jane Tsai
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwan; Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan; Center for Clinical Medicine Research, National Cheng Kung University Hospital, Tainan, Taiwan
| | - Wen-Chien Ko
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Department of Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Jenn-Wei Chen
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
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Zeng J, Fang S, Guo J, Dong M, Tian G, Tao L. Fight or flee, a vital choice for Clostridioides difficile. MLIFE 2024; 3:14-20. [PMID: 38827507 PMCID: PMC11139204 DOI: 10.1002/mlf2.12102] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/10/2023] [Revised: 09/17/2023] [Accepted: 10/08/2023] [Indexed: 06/04/2024]
Abstract
Clostridioides difficile is a leading cause of healthcare-associated infections, causing billions of economic losses every year. Its symptoms range from mild diarrhea to life-threatening damage to the colon. Transmission and recurrence of C. difficile infection (CDI) are mediated by the metabolically dormant spores, while the virulence of C. difficile is mainly due to the two large clostridial toxins, TcdA and TcdB. Producing toxins or forming spores are two different strategies for C. difficile to cope with harsh environmental conditions. It is of great significance to understand the molecular mechanisms for C. difficile to skew to either of the cellular processes. Here, we summarize the current understanding of the regulation and connections between toxin production and sporulation in C. difficile and further discuss the potential solutions for yet-to-be-answered questions.
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Affiliation(s)
- Ji Zeng
- School of Biomedical and Pharmaceutical SciencesGuangdong University of TechnologyGuangzhouChina
| | - Shuying Fang
- School of Biomedical and Pharmaceutical SciencesGuangdong University of TechnologyGuangzhouChina
| | - Jinquan Guo
- Department of Brest SurgeryPanyu Central HospitalGuangzhouChina
| | - Min Dong
- Department of MicrobiologyHarvard Medical SchoolBostonMassachusettsUSA
- Department of Urology, Boston Children's HospitalHarvard Medical SchoolBostonMassachusettsUSA
| | - Guo‐Bao Tian
- Department of MicrobiologyZhongshan School of Medicine, Sun Yat‐sen UniversityGuangzhouChina
- Advanced Medical Technology Center, The First Affiliated Hospital, Zhongshan School of MedicineSun Yat‐sen UniversityGuangzhouChina
- Key Laboratory of Tropical Diseases Control (Sun Yat‐sen University), Ministry of EducationGuangzhouChina
- School of MedicineXizang Minzu UniversityXianyangChina
| | - Liang Tao
- Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and BiomedicineWestlake UniversityHangzhouChina
- Research Center for Industries of the Future, School of Life SciencesWestlake UniversityHangzhouChina
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Ghosh S, Erickson D, Chua MJ, Collins J, Jala VR. The microbial metabolite urolithin A reduces Clostridioides difficile toxin expression and toxin-induced epithelial damage. mSystems 2024; 9:e0125523. [PMID: 38193707 PMCID: PMC10878087 DOI: 10.1128/msystems.01255-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2023] [Accepted: 12/04/2023] [Indexed: 01/10/2024] Open
Abstract
Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacterium responsible for antibiotic-associated pseudomembranous colitis. Clostridioides difficile infection (CDI) symptoms can range from diarrhea to life-threatening colon damage. Toxins produced by C. difficile (TcdA and TcdB) cause intestinal epithelial injury and lead to severe gut barrier dysfunction, stem cell damage, and impaired regeneration of the gut epithelium. Current treatment options for intestinal repair are limited. In this study, we demonstrate that treatment with the microbial metabolite urolithin A (UroA) attenuates CDI-induced adverse effects on the colon epithelium in a preclinical model of CDI-induced colitis. Moreover, our analysis suggests that UroA treatment protects against C. difficile-induced inflammation, disruption of gut barrier integrity, and intestinal tight junction proteins in the colon of CDI mice. Importantly, UroA treatment significantly reduced the expression and release of toxins from C. difficile without inducing bacterial cell death. These results indicate the direct regulatory effects of UroA on bacterial gene regulation. Overall, our findings reveal a novel aspect of UroA activity, as it appears to act at both the bacterial and host levels to protect against CDI-induced colitis pathogenesis. This research sheds light on a promising avenue for the development of novel treatments for C. difficile infection.IMPORTANCETherapy for Clostridioides difficile infections includes the use of antibiotics, immunosuppressors, and fecal microbiota transplantation. However, these treatments have several drawbacks, including the loss of colonization resistance, the promotion of autoimmune disorders, and the potential for unknown pathogens in donor samples. To date, the potential benefits of microbial metabolites in CDI-induced colitis have not been fully investigated. Here, we report for the first time that the microbial metabolite urolithin A has the potential to block toxin production from C. difficile and enhance gut barrier function to mitigate CDI-induced colitis.
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Affiliation(s)
- Sweta Ghosh
- Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky, USA
- UofL-Brown Cancer Center, Louisville, Kentucky, USA
| | - Daniel Erickson
- Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky, USA
| | - Michelle J. Chua
- Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky, USA
| | - James Collins
- Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky, USA
- Center for Predictive Medicine, University of Louisville, Louisville, Kentucky, USA
- Center for Microbiomics, Inflammation and Pathogenicity, University of Louisville, Louisville, Kentucky, USA
| | - Venkatakrishna Rao Jala
- Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky, USA
- UofL-Brown Cancer Center, Louisville, Kentucky, USA
- Center for Microbiomics, Inflammation and Pathogenicity, University of Louisville, Louisville, Kentucky, USA
- Center for Integrative Environmental Health Sciences, University of Louisville, Louisville, Kentucky, USA
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10
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Marvaud JC, Bouttier S, Saunier J, Kansau I. Clostridioides difficile Flagella. Int J Mol Sci 2024; 25:2202. [PMID: 38396876 PMCID: PMC10889297 DOI: 10.3390/ijms25042202] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2024] [Revised: 02/04/2024] [Accepted: 02/09/2024] [Indexed: 02/25/2024] Open
Abstract
Clostridioides difficile is an important pathogen for humans with a lead in nosocomial infection, but it is also more and more common in communities. Our knowledge of the pathology has historically been focused on the toxins produced by the bacteria that remain its major virulence factors. But the dysbiosis of the intestinal microbiota creating the conditions for the colonization appears to be fundamental for our understanding of the disease. Colonization implies several steps for the bacteria that do or do not use their capacity of motility with the synthesis of flagella. In this review, we focus on the current understanding of different topics on the C. difficile flagellum, ranging from its genetic organization to the vaccinal interest in it.
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Affiliation(s)
- Jean-Christophe Marvaud
- Institut MICALIS, INRAE, AgroParisTech, Equipe Bactéries Pathogènes et Santé, Faculté de Pharmacie, Université Paris-Saclay, 91400 Orsay, France (I.K.)
| | - Sylvie Bouttier
- Institut MICALIS, INRAE, AgroParisTech, Equipe Bactéries Pathogènes et Santé, Faculté de Pharmacie, Université Paris-Saclay, 91400 Orsay, France (I.K.)
| | - Johanna Saunier
- Matériaux et Santé, Faculté de pharmacie, Université Paris Saclay, 91400 Orsay, France
| | - Imad Kansau
- Institut MICALIS, INRAE, AgroParisTech, Equipe Bactéries Pathogènes et Santé, Faculté de Pharmacie, Université Paris-Saclay, 91400 Orsay, France (I.K.)
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Wetzel D, Rizvi A, Edwards AN, McBride SM. The predicted acetoin dehydrogenase pathway represses sporulation of Clostridioides difficile. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.07.28.551048. [PMID: 37546766 PMCID: PMC10402147 DOI: 10.1101/2023.07.28.551048] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/08/2023]
Abstract
Clostridioides difficile is a major gastrointestinal pathogen that is transmitted as a dormant spore. As an intestinal pathogen, C. difficile must contend with variable environmental conditions, including fluctuations in pH and nutrient availability. Nutrition and pH both influence growth and spore formation, but how pH and nutrition jointly influence sporulation are not known. In this study, we investigated the dual impact of pH and pH-dependent metabolism on C. difficile sporulation. Specifically, we examined the impacts of pH and the metabolite acetoin on C. difficile growth and sporulation. We found that expression of the predicted acetoin dehydrogenase operon, acoRABCL , was pH-dependent and regulated by acetoin. Regulation of the C. difficile aco locus is distinct from other characterized systems and appears to involve a co-transcribed DeoR-family regulator rather than the sigma 54 -dependent activator. In addition, an acoA null mutant produced significantly more spores and initiated sporulation earlier than the parent strain. However, unlike other Firmicutes, growth and culture density of C. difficile was not increased by acetoin availability or disruption of the aco pathway. Together, these results indicate that acetoin, pH, and the aco pathway play important roles in nutritional repression of sporulation in C. difficile , but acetoin metabolism does not support cell growth as a stationary phase energy source. IMPORTANCE Clostridioides difficile, or C. diff , is an anaerobic bacterium that lives within the gut of many mammals and causes infectious diarrhea. C. difficile is able to survive outside of the gut and transmit to new hosts by forming dormant spores. It is known that the pH of the intestine and the nutrients available both affect the growth and sporulation of C. diffiicile, but the specific conditions that result in sporulation in the host are not clear. In this study, we investigated how pH and the metabolite acetoin affect the ability of C. difficile to grow, proliferate, and form spores. We found that a mutant lacking the predicted acetoin metabolism pathway form more spores, but their growth is not impacted. These results show that C. difficile uses acetoin differently than many other species and that acetoin has an important role as an environmental metabolite that influences spore formation.
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Ghosh S, Erickson D, Chua MJ, Collins J, Jala VR. The microbial metabolite Urolithin A reduces C. difficile toxin expression and repairs toxin-induced epithelial damage. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.07.24.550342. [PMID: 37546803 PMCID: PMC10402075 DOI: 10.1101/2023.07.24.550342] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/08/2023]
Abstract
Clostridioides difficile is a gram-positive, anaerobic, spore-forming bacterium that is responsible for antibiotic-associated pseudomembranous colitis. Clostridioides difficile infection (CDI) symptoms can range from diarrhea to life-threatening colon damage. Toxins produced by C. difficile (TcdA and TcdB) cause intestinal epithelial injury and lead to severe gut barrier dysfunction, stem cell damage, and impaired regeneration of the gut epithelium. Current treatment options for intestinal repair are limited. In this study, we demonstrate that treatment with the microbial metabolite urolithin A (UroA) attenuates CDI-induced adverse effects on the colon epithelium in a preclinical model of CDI-induced colitis. Moreover, our analysis suggests that UroA treatment protects against C. difficile-induced inflammation, disruption of gut barrier integrity, and intestinal tight junction proteins in the colon of CDI mice. Importantly, UroA treatment significantly reduced the expression and release of toxins from C. difficile, without inducing bacterial cell death. These results indicate the direct regulatory effects of UroA on bacterial gene regulation. Overall, our findings reveal a novel aspect of UroA activities, as it appears to act at both the bacterial and host levels to protect against CDI-induced colitis pathogenesis. This research sheds light on a promising avenue for the development of novel treatments for C. difficile infection.
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Affiliation(s)
- Sweta Ghosh
- Department of Microbiology & Immunology, University of Louisville, Louisville, KY, USA
- UofL-Brown Cancer Center, Louisville, KY, USA
| | - Daniel Erickson
- Department of Microbiology & Immunology, University of Louisville, Louisville, KY, USA
| | - Michelle J Chua
- Department of Microbiology & Immunology, University of Louisville, Louisville, KY, USA
| | - James Collins
- Department of Microbiology & Immunology, University of Louisville, Louisville, KY, USA
- Center for Predictive Medicine, University of Louisville, Louisville, KY, USA
- Center for Microbiomics, Inflammation and Pathogenicity, University of Louisville, Louisville, KY, USA
| | - Venkatakrishna Rao Jala
- Department of Microbiology & Immunology, University of Louisville, Louisville, KY, USA
- UofL-Brown Cancer Center, Louisville, KY, USA
- Center for Microbiomics, Inflammation and Pathogenicity, University of Louisville, Louisville, KY, USA
- Center for Integrative Environmental Health Sciences, University of Louisville, Louisville, KY, USA
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13
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Dupuy B. Regulation of Clostridial Toxin Gene Expression: A Pasteurian Tradition. Toxins (Basel) 2023; 15:413. [PMID: 37505682 PMCID: PMC10467148 DOI: 10.3390/toxins15070413] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2023] [Revised: 06/13/2023] [Accepted: 06/19/2023] [Indexed: 07/29/2023] Open
Abstract
The alarming symptoms attributed to several potent clostridial toxins enabled the early identification of the causative agent of tetanus, botulism, and gas gangrene diseases, which belongs to the most famous species of pathogenic clostridia. Although Clostridioides difficile was identified early in the 20th century as producing important toxins, it was identified only 40 years later as the causative agent of important nosocomial diseases upon the advent of antibiotic therapies in hospital settings. Today, C. difficile is a leading public health issue, as it is the major cause of antibiotic-associated diarrhea in adults. In particular, severe symptoms within the spectrum of C. difficile infections are directly related to the levels of toxins produced in the host. This highlights the importance of understanding the regulation of toxin synthesis in the pathogenicity process of C. difficile, whose regulatory factors in response to the gut environment were first identified at the Institut Pasteur. Subsequently, the work of other groups in the field contributed to further deciphering the complex mechanisms controlling toxin production triggered by the intestinal dysbiosis states during infection. This review summarizes the Pasteurian contribution to clostridial toxin regulation studies.
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Affiliation(s)
- Bruno Dupuy
- Institut Pasteur, Université Paris-Cité, UMR-CNRS 6047, Laboratoire Pathogenèse des Bactéries Anaérobies, F-75015 Paris, France
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14
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Bocchetti M, Ferraro MG, Melisi F, Grisolia P, Scrima M, Cossu AM, Yau TO. Overview of current detection methods and microRNA potential in Clostridioides difficile infection screening. World J Gastroenterol 2023; 29:3385-3399. [PMID: 37389232 PMCID: PMC10303512 DOI: 10.3748/wjg.v29.i22.3385] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/25/2023] [Revised: 03/23/2023] [Accepted: 05/04/2023] [Indexed: 06/06/2023] Open
Abstract
Clostridioides difficile (formerly called Clostridium difficile, C. difficile) infection (CDI) is listed as an urgent threat on the 2019 antibiotic resistance threats report in the United States by the Centers for Disease Control and Prevention. Early detection and appropriate disease management appear to be essential. Meanwhile, although the majority of cases are hospital-acquired CDI, community-acquired CDI cases are also on the rise, and this vulnerability is not limited to immunocompromised patients. Gastrointestinal treatments and/or gastrointestinal tract surgeries may be required for patients diagnosed with digestive diseases. Such treatments could suppress or interfere with the patient's immune system and disrupt gut flora homeostasis, creating a suitable microecosystem for C. difficile overgrowth. Currently, stool-based non-invasive screening is the first-line approach to CDI diagnosis, but the accuracy is varied due to different clinical microbiology detection methods; therefore, improving reliability is clearly required. In this review, we briefly summarised the life cycle and toxicity of C. difficile, and we examined existing diagnostic approaches with an emphasis on novel biomarkers such as microRNAs. These biomarkers can be easily detected through non-invasive liquid biopsy and can yield crucial information about ongoing pathological phenomena, particularly in CDI.
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Affiliation(s)
- Marco Bocchetti
- Department of Precision Medicine, University of Campania “Luigi Vanvitelli,” Naples 80138, Italy
- Department of Molecular Oncology, Precision Medicine Laboratory and COVID19 Laboratory, Biogem Scarl, Ariano Irpino 83031, Italy
| | - Maria Grazia Ferraro
- School of Infection and Immunity, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom
- Department of Pharmacy, School of Medicine and Surgery, University of Naples “Federico II,” Naples 80131, Italy
| | - Federica Melisi
- Department of Precision Medicine, University of Campania “Luigi Vanvitelli,” Naples 80138, Italy
- Department of Molecular Oncology, Precision Medicine Laboratory and COVID19 Laboratory, Biogem Scarl, Ariano Irpino 83031, Italy
| | - Piera Grisolia
- Department of Precision Medicine, University of Campania “Luigi Vanvitelli,” Naples 80138, Italy
- Department of Molecular Oncology, Precision Medicine Laboratory and COVID19 Laboratory, Biogem Scarl, Ariano Irpino 83031, Italy
| | - Marianna Scrima
- Department of Molecular Oncology, Precision Medicine Laboratory and COVID19 Laboratory, Biogem Scarl, Ariano Irpino 83031, Italy
| | - Alessia Maria Cossu
- Department of Precision Medicine, University of Campania “Luigi Vanvitelli,” Naples 80138, Italy
- Department of Molecular Oncology, Precision Medicine Laboratory and COVID19 Laboratory, Biogem Scarl, Ariano Irpino 83031, Italy
| | - Tung On Yau
- School of Science and Technology, Nottingham Trent University, Nottingham NG11 8NS, United Kingdom
- Department of Rural Land Use, Scotland’s Rural College, Aberdeen AB21 9YA, Scotland, United Kingdom
- Department of Health Science, University of the People, Pasadena, CA 9110112, United States
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15
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Chandra H, Sorg JA, Hassett DJ, Sun X. Regulatory transcription factors of Clostridioides difficile pathogenesis with a focus on toxin regulation. Crit Rev Microbiol 2023; 49:334-349. [PMID: 35389761 PMCID: PMC11209739 DOI: 10.1080/1040841x.2022.2054307] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2021] [Revised: 02/26/2022] [Accepted: 03/14/2022] [Indexed: 11/03/2022]
Abstract
Clostridioides difficile (CD), a nosocomial gut pathogen, produces two major exotoxins, TcdA and TcdB, which disrupt the gut epithelial barrier and induce inflammatory/immune responses, leading to symptoms ranging from mild diarrhoea to pseudomembranous colitis and potentially to death. The expression of toxins is regulated by various transcription factors (TFs) which are induced in response to CD physiological life stages, nutritional availability, and host environment. This review summarises our current understanding on the regulation of toxin expression by TFs that interconnect with pathways of flagellar synthesis, quorum sensing, motility, biofilm formation, sporulation, and phase variation. The pleiotropic roles of some key TFs suggest that toxin production is tightly linked to other cellular processes of the CD physiology.
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Affiliation(s)
- Harish Chandra
- Department of Environmental Microbiology, School of Environmental and Earth Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow, India
- Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA
| | - Joseph A. Sorg
- Department of Biology, Texas A&M University, College Station, TX, USA
| | - Daniel J Hassett
- Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA
| | - Xingmin Sun
- Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
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16
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Zhang X, Li J, Chen C, Liu YJ, Cui Q, Hong W, Chen Z, Feng Y, Cui G. Molecular Basis of TcdR-Dependent Promoter Activity for Toxin Production by Clostridioides difficile Studied by a Heterologous Reporter System. Toxins (Basel) 2023; 15:toxins15050306. [PMID: 37235341 DOI: 10.3390/toxins15050306] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2023] [Revised: 04/17/2023] [Accepted: 04/19/2023] [Indexed: 05/28/2023] Open
Abstract
The alternative σ factor TcdR controls the synthesis of two major enterotoxins: TcdA and TcdB in Clostridioides difficile. Four potential TcdR-dependent promoters in the pathogenicity locus of C. difficile showed different activities. In this study, we constructed a heterologous system in Bacillus subtilis to investigate the molecular basis of TcdR-dependent promoter activity. The promoters of the two major enterotoxins showed strong TcdR-dependent activity, while the two putative TcdR-dependent promoters in the upstream region of the tcdR gene did not show detectable activity, suggesting that the autoregulation of TcdR may need other unknown factors involved. Mutation analysis indicated that the divergent -10 region is the key determinant for different activities of the TcdR-dependent promoters. Analysis of the TcdR model predicted by AlphaFold2 suggested that TcdR should be classified into group 4, i.e., extracytoplasmic function, σ70 factors. The results of this study provide the molecular basis of the TcdR-dependent promoter recognition for toxin production. This study also suggests the feasibility of the heterologous system in analyzing σ factor functions and possibly in drug development targeting these factors.
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Affiliation(s)
- Xinyue Zhang
- Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang 550025, China
- CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Shandong Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China
- Joint Laboratory of Helicobacter Pylori and Intestinal Microecology of Affiliated Hospital of Guizhou Medical University, Guiyang 550025, China
- Shandong Energy Institute, Qingdao 266101, China
- Qingdao New Energy Shandong Laboratory, Qingdao 266101, China
| | - Jie Li
- CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Shandong Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China
- Shandong Energy Institute, Qingdao 266101, China
- Qingdao New Energy Shandong Laboratory, Qingdao 266101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Chao Chen
- CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Shandong Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China
- Shandong Energy Institute, Qingdao 266101, China
- Qingdao New Energy Shandong Laboratory, Qingdao 266101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Ya-Jun Liu
- CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Shandong Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China
- Shandong Energy Institute, Qingdao 266101, China
- Qingdao New Energy Shandong Laboratory, Qingdao 266101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Qiu Cui
- CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Shandong Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China
- Shandong Energy Institute, Qingdao 266101, China
- Qingdao New Energy Shandong Laboratory, Qingdao 266101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Wei Hong
- Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang 550025, China
| | - Zhenghong Chen
- Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang 550025, China
- Joint Laboratory of Helicobacter Pylori and Intestinal Microecology of Affiliated Hospital of Guizhou Medical University, Guiyang 550025, China
| | - Yingang Feng
- Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang 550025, China
- CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Shandong Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China
- Shandong Energy Institute, Qingdao 266101, China
- Qingdao New Energy Shandong Laboratory, Qingdao 266101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Guzhen Cui
- Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang 550025, China
- Joint Laboratory of Helicobacter Pylori and Intestinal Microecology of Affiliated Hospital of Guizhou Medical University, Guiyang 550025, China
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DiCandia MA, Edwards AN, Lee CD, Monteiro MP, Cuebas GNV, Bagchi P, McBride SM. A Conserved Switch Controls Virulence, Sporulation, and Motility in C. difficile. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.03.28.534590. [PMID: 37034656 PMCID: PMC10081167 DOI: 10.1101/2023.03.28.534590] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/19/2023]
Abstract
Spore formation is required for environmental survival and transmission of the human enteropathogenic Clostridioides difficile . In all bacterial spore formers, sporulation is regulated through activation of the master response regulator, Spo0A. However, the factors and mechanisms that directly regulate C. difficile Spo0A activity are not defined. In the well-studied Bacillus species, Spo0A is directly inactivated by Spo0E, a small phosphatase. To understand Spo0E function in C. difficile , we created a null mutation of the spo0E ortholog and assessed sporulation and physiology. The spo0E mutant produced significantly more spores, demonstrating Spo0E represses C. difficile sporulation. Unexpectedly, the spo0E mutant also exhibited increased motility and toxin production, and enhanced virulence in animal infections. We uncovered that Spo0E interacts with both Spo0A and the toxin and motility regulator, RstA. Direct interactions between Spo0A, Spo0E, and RstA constitute a previously unknown molecular switch that coordinates sporulation with motility and toxin production. Reinvestigation of Spo0E function in B. subtilis revealed that Spo0E induced motility, demonstrating Spo0E regulation of motility and sporulation among divergent species. Further, we found that Spo0E orthologs are widespread among prokaryotes, suggesting that Spo0E performs conserved regulatory functions in diverse bacteria.
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18
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Cheng JKJ, Unnikrishnan M. Clostridioides difficile infection: traversing host-pathogen interactions in the gut. MICROBIOLOGY (READING, ENGLAND) 2023; 169. [PMID: 36848200 DOI: 10.1099/mic.0.001306] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/01/2023]
Abstract
C. difficile is the primary cause for nosocomial infective diarrhoea. For a successful infection, C. difficile must navigate between resident gut bacteria and the harsh host environment. The perturbation of the intestinal microbiota by broad-spectrum antibiotics alters the composition and the geography of the gut microbiota, deterring colonization resistance, and enabling C. difficile to colonize. This review will discuss how C. difficile interacts with and exploits the microbiota and the host epithelium to infect and persist. We provide an overview of C. difficile virulence factors and their interactions with the gut to aid adhesion, cause epithelial damage and mediate persistence. Finally, we document the host responses to C. difficile, describing the immune cells and host pathways that are associated and triggered during C. difficile infection.
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Affiliation(s)
- Jeffrey K J Cheng
- Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, CV4 7AL, UK
| | - Meera Unnikrishnan
- Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, CV4 7AL, UK
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19
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Reyes Ruiz LM, King KA, Agosto-Burgos C, Gamez IS, Gadda NC, Garrett EM, Tamayo R. Coordinated modulation of multiple processes through phase variation of a c-di-GMP phosphodiesterase in Clostridioides difficile. PLoS Pathog 2022; 18:e1010677. [PMID: 35789350 PMCID: PMC9286219 DOI: 10.1371/journal.ppat.1010677] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Revised: 07/15/2022] [Accepted: 06/15/2022] [Indexed: 11/19/2022] Open
Abstract
The opportunistic nosocomial pathogen Clostridioides difficile exhibits phenotypic heterogeneity through phase variation, a stochastic, reversible process that modulates expression. In C. difficile, multiple sequences in the genome undergo inversion through site-specific recombination. Two such loci lie upstream of pdcB and pdcC, which encode phosphodiesterases (PDEs) that degrade the signaling molecule c-di-GMP. Numerous phenotypes are influenced by c-di-GMP in C. difficile including cell and colony morphology, motility, colonization, and virulence. In this study, we aimed to assess whether PdcB phase varies, identify the mechanism of regulation, and determine the effects on intracellular c-di-GMP levels and regulated phenotypes. We found that expression of pdcB is heterogeneous and the orientation of the invertible sequence, or ‘pdcB switch’, determines expression. The pdcB switch contains a promoter that when properly oriented promotes pdcB expression. Expression is augmented by an additional promoter upstream of the pdcB switch. Mutation of nucleotides at the site of recombination resulted in phase-locked strains with significant differences in pdcB expression. Characterization of these mutants showed that the pdcB locked-ON mutant has reduced intracellular c-di-GMP compared to the locked-OFF mutant, consistent with increased and decreased PdcB activity, respectively. These alterations in c-di-GMP had concomitant effects on multiple known c-di-GMP regulated processes, indicating that phase variation of PdcB allows C. difficile to coordinately diversify multiple phenotypes in the population to enhance survival. Phase variation is a mechanism by which many bacteria introduce phenotypic heterogeneity into a population as a bet-hedging strategy to help ensure survival under detrimental conditions. In C. difficile, the intracellular signaling molecule c-di-GMP regulates production of flagella, toxins, adhesins, and other factors that impact virulence. C. difficile encodes multiple c-di-GMP synthases and hydrolases that modulate intracellular c-di-GMPs and control these processes. Here, we show that production of a c-di-GMP hydrolytic enzyme, PdcB, undergoes phase variation in C. difficile. We generated phase-locked mutants unable to phase vary and found that PdcB affects global intracellular c-di-GMP levels, swimming and surface motility, and biofilm formation. These findings suggest that phase variation of PdcB enables C. difficile to coordinately regulate the production multiple factors by generating heterogeneity in intracellular c-di-GMP levels among bacteria in the population.
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Affiliation(s)
- Leila M. Reyes Ruiz
- Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America
| | - Kathleen A. King
- Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America
| | - Christian Agosto-Burgos
- Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America
| | - Isabella S. Gamez
- Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America
| | - Nicole C. Gadda
- Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America
| | - Elizabeth M. Garrett
- Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America
| | - Rita Tamayo
- Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America
- * E-mail:
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20
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Saund K, Pirani A, Lacy DB, Hanna PC, Snitkin E. Strain Variation in Clostridioides difficile Cytotoxicity Associated with Genomic Variation at Both Pathogenic and Nonpathogenic Loci. mSphere 2022; 7:e0017422. [PMID: 35766503 PMCID: PMC9241522 DOI: 10.1128/msphere.00174-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Accepted: 04/13/2022] [Indexed: 11/20/2022] Open
Abstract
Clinical disease from Clostridioides difficile infection can be mediated by two toxins and their neighboring regulatory genes located within the five-gene pathogenicity locus (PaLoc). We provide several lines of evidence that the cytotoxicity of C. difficile may be modulated by genomic variants outside the PaLoc. We used a phylogenetic tree-based approach to demonstrate discordance between cytotoxicity and PaLoc evolutionary history, an elastic net method to show the insufficiency of PaLoc variants alone to model cytotoxicity, and a convergence-based bacterial genome-wide association study (GWAS) to identify correlations between non-PaLoc loci and changes in cytotoxicity. Combined, these data support a model of C. difficile disease wherein cytotoxicity may be strongly affected by many non-PaLoc loci. Additionally, we characterize multiple other in vitro phenotypes relevant to human infections, including germination and sporulation. These phenotypes vary greatly in their clonality, variability, convergence, and concordance with genomic variation. Finally, we highlight the intersection of loci identified by the GWAS for different phenotypes and clinical severity. This strategy to identify overlapping loci can facilitate the identification of genetic variation linking phenotypic variation to clinical outcomes. IMPORTANCE Clostridioides difficile has two major disease-mediating toxins, A and B, encoded within the pathogenicity locus (PaLoc). In this study, we demonstrate via multiple approaches that genomic variants outside the PaLoc are associated with changes in cytotoxicity. These genomic variants may provide new avenues of exploration in the hunt for novel disease-modifying interventions. Additionally, we provide insight into the evolution of several additional phenotypes also critical for clinical infection, such as sporulation, germination, and growth rate. These in vitro phenotypes display a range of responses to evolutionary pressures and, as such, vary in their appropriateness for certain bacterial genome-wide association study approaches. We used a convergence-based association method to identify the genomic variants most correlated with both changes in these phenotypes and disease severity. These overlapping loci may be important for both bacterial function and human clinical disease.
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Affiliation(s)
- Katie Saund
- Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA
| | - Ali Pirani
- Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA
- Department of Internal Medicine, Division of Infectious Diseases, University of Michigan, Ann Arbor, Michigan, USA
| | - D. Borden Lacy
- Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
- The Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
| | - Philip C. Hanna
- Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA
| | - Evan Snitkin
- Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA
- Department of Internal Medicine, Division of Infectious Diseases, University of Michigan, Ann Arbor, Michigan, USA
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21
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Clark AE, Adamson CC, Carothers KE, Roxas BAP, Viswanathan VK, Vedantam G. The Alternative Sigma Factor SigL Influences Clostridioides difficile Toxin Production, Sporulation, and Cell Surface Properties. Front Microbiol 2022; 13:871152. [PMID: 35633701 PMCID: PMC9130780 DOI: 10.3389/fmicb.2022.871152] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Accepted: 04/06/2022] [Indexed: 12/18/2022] Open
Abstract
The alternative sigma factor SigL (Sigma-54) facilitates bacterial adaptation to the extracellular environment by modulating the expression of defined gene subsets. A homolog of the gene encoding SigL is conserved in the diarrheagenic pathogen Clostridioides difficile. To explore the contribution of SigL to C. difficile biology, we generated sigL-disruption mutants (sigL::erm) in strains belonging to two phylogenetically distinct lineages-the human-relevant Ribotype 027 (strain BI-1) and the veterinary-relevant Ribotype 078 (strain CDC1). Comparative proteomics analyses of mutants and isogenic parental strains revealed lineage-specific SigL regulons. Concomitantly, loss of SigL resulted in pleiotropic and distinct phenotypic alterations in the two strains. Sporulation kinetics, biofilm formation, and cell surface-associated phenotypes were altered in CDC1 sigL::erm relative to the isogenic parent strain but remained unchanged in BI-1 sigL::erm. In contrast, secreted toxin levels were significantly elevated only in the BI-1 sigL::erm mutant relative to its isogenic parent. We also engineered SigL overexpressing strains and observed enhanced biofilm formation in the CDC1 background, and reduced spore titers as well as dampened sporulation kinetics in both strains. Thus, we contend that SigL is a key, pleiotropic regulator that dynamically influences C. difficile's virulence factor landscape, and thereby, its interactions with host tissues and co-resident microbes.
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Affiliation(s)
- Andrew E. Clark
- School of Animal and Comparative Biomedical Sciences, Tucson, AZ, United States
| | - Chelsea C. Adamson
- School of Animal and Comparative Biomedical Sciences, Tucson, AZ, United States
| | | | | | - V. K. Viswanathan
- School of Animal and Comparative Biomedical Sciences, Tucson, AZ, United States
- Department of Immunobiology, University of Arizona, Tucson, AZ, United States
- BIO5 Institute for Collaborative Research, University of Arizona, Tucson, AZ, United States
| | - Gayatri Vedantam
- School of Animal and Comparative Biomedical Sciences, Tucson, AZ, United States
- Department of Immunobiology, University of Arizona, Tucson, AZ, United States
- BIO5 Institute for Collaborative Research, University of Arizona, Tucson, AZ, United States
- Southern Arizona VA Healthcare System, Tucson, AZ, United States
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22
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Metzendorf NG, Lange LM, Lainer N, Schlüter R, Dittmann S, Paul LS, Troitzsch D, Sievers S. Destination and Specific Impact of Different Bile Acids in the Intestinal Pathogen Clostridioides difficile. Front Microbiol 2022; 13:814692. [PMID: 35401433 PMCID: PMC8989276 DOI: 10.3389/fmicb.2022.814692] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2021] [Accepted: 02/09/2022] [Indexed: 01/05/2023] Open
Abstract
The anaerobic bacterium Clostridioides difficile represents one of the most problematic pathogens, especially in hospitals. Dysbiosis has been proven to largely reduce colonization resistance against this intestinal pathogen. The beneficial effect of the microbiota is closely associated with the metabolic activity of intestinal microbes such as the ability to transform primary bile acids into secondary ones. However, the basis and the molecular action of bile acids (BAs) on the pathogen are not well understood. We stressed the pathogen with the four most abundant human bile acids: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and lithocholic acid (LCA). Thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM), and electron microscopy (EM) were employed to track the enrichment and destination of bile acids in the bacterial cell. TLC not only revealed a strong accumulation of LCA in C. difficile, but also indicated changes in the composition of membrane lipids in BA-treated cells. Furthermore, morphological changes induced by BAs were determined, most pronounced in the virtually complete loss of flagella in LCA-stressed cells and a flagella reduction after DCA and CDCA challenge. Quantification of both, protein and RNA of the main flagella component FliC proved the decrease in flagella to originate from a change in gene expression on transcriptional level. Notably, the loss of flagella provoked by LCA did not reduce adhesion ability of C. difficile to Caco-2 cells. Most remarkably, extracellular toxin A levels in the presence of BAs showed a similar pattern as flagella expression. That is, CA did not affect toxin expression, whereas lower secretion of toxin A was determined in cells stressed with LCA, DCA or CDCA. In summary, the various BAs were shown to differentially modify virulence determinants, such as flagella expression, host cell adhesion and toxin synthesis. Our results indicate differences of BAs in cellular localization and impact on membrane composition, which could be a reason of their diverse effects. This study is a starting point in the elucidation of the molecular mechanisms underlying the differences in BA action, which in turn can be vital regarding the outcome of a C. difficile infection.
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Affiliation(s)
| | - Lena Melanie Lange
- Department of Microbial Physiology and Molecular Biology, Institute of Microbiology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
| | - Nina Lainer
- Department of Microbial Physiology and Molecular Biology, Institute of Microbiology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
| | - Rabea Schlüter
- Imaging Center of the Department of Biology, University of Greifswald, Greifswald, Germany
| | - Silvia Dittmann
- Department of Microbial Physiology and Molecular Biology, Institute of Microbiology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
| | - Lena-Sophie Paul
- Department of Microbial Physiology and Molecular Biology, Institute of Microbiology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
| | - Daniel Troitzsch
- Department of Microbial Physiology and Molecular Biology, Institute of Microbiology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
| | - Susanne Sievers
- Department of Microbial Physiology and Molecular Biology, Institute of Microbiology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
- *Correspondence: Susanne Sievers,
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23
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Shivaperumal N, Knight DR, Imwattana K, Androga GO, Chang BJ, Riley TV. Esculin hydrolysis negative and TcdA‐only producing strains of
Clostridium (Clostridiodes) difficile
from the environment in Western Australia. J Appl Microbiol 2022; 133:1183-1196. [PMID: 35184359 PMCID: PMC9544920 DOI: 10.1111/jam.15500] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2021] [Revised: 02/10/2022] [Accepted: 02/15/2022] [Indexed: 12/03/2022]
Abstract
Background and Aims Clostridium (Clostridiodes) difficile clade 3 ribotype (RT) 023 strains that fail to produce black colonies on bioMérieux ChromID agar have been reported, as well as variant strains of C. difficile that produce only toxin A. We have recently isolated strains of C. difficile from the environment in Western Australia (WA) with similar characteristics. The objective of this study was to characterize these strains. It was hypothesized that a putative β‐glucosidase gene was lacking in these strains of C. difficile, including RT 023, leading to white colonies. Methods and Results A total of 17 environmental isolates of C. difficile from garden soil and compost, and gardening shoe soles in Perth, WA, failed to produce black colonies on ChromID agar. MALDI‐TOF MS analysis confirmed these strains as C. difficile. Four strains contained only a tcdA gene (A+B−CDT−) by PCR and were a novel RT (QX 597). All isolates were susceptible to all antimicrobials tested except one with low‐level resistance to clindamycin (MIC = 8 mg/L). The four tcdA‐positive strains were motile. All isolates contained neither bgl locus but only bgl K or a putative β‐glucosidase gene by PCR. Whole‐genome sequencing showed the 17 strains belonged to novel multi‐locus sequence types 632, 848, 849, 850, 851, 852 and 853, part of the evolutionarily divergent clade C‐III. Four isolates carried a full‐length tcdA but not tcdB nor binary toxin genes. Conclusions ChromID C. difficile agar is used for the specific detection of C. difficile in the samples. To date, all strains except RT 023 strains from clinical samples hydrolyse esculin. This is the first report to provide insights into the identification of esculin hydrolysis negative and TcdA‐only producing (A+B−CDT−) strains of C. difficile from environmental samples. Significance and Impact of the Study White colonies of C. difficile from environmental samples could be overlooked when using ChromID C. difficile agar, leading to false‐negative results, however, whether these strains are truly pathogenic remains to be proven.
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Affiliation(s)
| | - Daniel R. Knight
- Biosecurity and One Health Research Centre, Harry Butler Institute Murdoch University Murdoch Western Australia Australia
| | | | - Grace O. Androga
- School of Biomedical Sciences Nedlands Western Australia Australia
- Current address: HIV, Inflammation and Microbiome Group Burnet Institute Melbourne Victoria Australia
| | - Barbara J. Chang
- School of Biomedical Sciences Nedlands Western Australia Australia
| | - Thomas V. Riley
- School of Biomedical Sciences Nedlands Western Australia Australia
- Biosecurity and One Health Research Centre, Harry Butler Institute Murdoch University Murdoch Western Australia Australia
- PathWest Laboratory Medicine, Department of Microbiology, Queen Elizabeth II Medical Centre Nedlands Western Australia Australia
- School of Medical and Health Sciences Edith Cowan University Joondalup Western Australia Australia
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24
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Regulation of Clostridioides difficile toxin production. Curr Opin Microbiol 2022; 65:95-100. [PMID: 34781095 PMCID: PMC8792210 DOI: 10.1016/j.mib.2021.10.018] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2021] [Revised: 10/19/2021] [Accepted: 10/25/2021] [Indexed: 02/03/2023]
Abstract
Clostridioides difficile produces toxins TcdA and TcdB during infection. Since the severity of the illness is directly correlated with the level of toxins produced, researchers have long been interested in the regulation mechanisms of toxin production. The advent of new genetics and mutagenesis technologies in C. difficile has allowed a slew of new investigations in the last decade, which considerably improved our understanding of this crucial regulatory network. The current body of work shows that the toxin regulatory network overlaps with the regulatory networks of sporulation, motility, and key metabolic pathways. This implies that toxin production is a complicated process initiated by bacteria in response to numerous host factors during infection. We summarize the existing knowledge about the toxin gene regulatory network here.
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25
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Ahmed UKB, Ballard JD. Autoinducing peptide-based quorum signaling systems in Clostridioides difficile. Curr Opin Microbiol 2022; 65:81-86. [PMID: 34773906 PMCID: PMC8792308 DOI: 10.1016/j.mib.2021.10.017] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2021] [Revised: 10/20/2021] [Accepted: 10/25/2021] [Indexed: 02/03/2023]
Abstract
The autoinducing peptide-based Agr system in Clostridioides difficile is involved in virulence factor expression, motility, and sporulation. This review highlights several of the recent discoveries regarding C. difficile Agr. Typical Agr systems rely on the combined activities of four proteins involved in peptide expression, peptide processing, peptide sensing, and transcriptional regulation. As emphasized in this review, at least two C. difficile Agr systems (Agr1 and Agr3) lack the set of proteins associated with this regulatory network. In line with this, recent finding indicate Agr1 can function in ways that may not depend on accumulation of extracellular peptide. Also, described are the similarities and differences in Agr systems within the pathogenic Clostridia.
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26
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Trzilova D, Warren MAH, Gadda NC, Williams CL, Tamayo R. Flagellum and toxin phase variation impacts intestinal colonization and disease development in a mouse model of Clostridioides difficile infection. Gut Microbes 2022; 14:2038854. [PMID: 35192433 PMCID: PMC8890394 DOI: 10.1080/19490976.2022.2038854] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Accepted: 01/14/2022] [Indexed: 02/04/2023] Open
Abstract
Clostridioides difficile is a major nosocomial pathogen that can cause severe, toxin-mediated diarrhea and pseudomembranous colitis. Recent work has shown that C. difficile exhibits heterogeneity in swimming motility and toxin production in vitro through phase variation by site-specific DNA recombination. The recombinase RecV reversibly inverts the flagellar switch sequence upstream of the flgB operon, leading to the ON/OFF expression of flagellum and toxin genes. How this phenomenon impacts C. difficile virulence in vivo remains unknown. We identified mutations in the right inverted repeat that reduced or prevented flagellar switch inversion by RecV. We introduced these mutations into C. difficile R20291 to create strains with the flagellar switch "locked" in either the ON or OFF orientation. These mutants exhibited a loss of flagellum and toxin phase variation during growth in vitro, yielding precisely modified mutants suitable for assessing virulence in vivo. In a hamster model of acute C. difficile infection, the phase-locked ON mutant caused greater toxin accumulation than the phase-locked OFF mutant but did not differ significantly in the ability to cause acute disease symptoms. In contrast, in a mouse model, preventing flagellum and toxin phase variation affected the ability of C. difficile to colonize the intestinal tract and to elicit weight loss, which is attributable to differences in toxin production during infection. These results show that the ability of C. difficile to phase vary flagella and toxins influences colonization and disease development and suggest that the phenotypic variants generated by flagellar switch inversion have distinct capacities for causing disease.
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Affiliation(s)
- Dominika Trzilova
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA
| | - Mercedes A. H. Warren
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA
| | - Nicole C. Gadda
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA
| | - Caitlin L. Williams
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA
| | - Rita Tamayo
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA
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27
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Edwards AN, Willams CL, Pareek N, McBride SM, Tamayo R. c-di-GMP Inhibits Early Sporulation in Clostridioides difficile. mSphere 2021; 6:e0091921. [PMID: 34878288 PMCID: PMC8653836 DOI: 10.1128/msphere.00919-21] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Accepted: 11/19/2021] [Indexed: 12/02/2022] Open
Abstract
The formation of dormant spores is essential for the anaerobic pathogen Clostridioides difficile to survive outside the host gastrointestinal tract. The regulatory pathways and environmental signals that initiate C. difficile spore formation within the host are not well understood. One second-messenger signaling molecule, cyclic diguanylate (c-di-GMP), modulates several physiological processes important for C. difficile pathogenesis and colonization, but the impact of c-di-GMP on sporulation is unknown. In this study, we investigated the contribution of c-di-GMP to C. difficile sporulation. The overexpression of a gene encoding a diguanylate cyclase, dccA, decreased the sporulation frequency and early sporulation gene transcription in both the epidemic R20291 and historical 630Δerm strains. The expression of a dccA allele encoding a catalytically inactive DccA that is unable to synthesize c-di-GMP no longer inhibited sporulation, indicating that the accumulation of intracellular c-di-GMP reduces C. difficile sporulation. A null mutation in dccA slightly increased sporulation in R20291 and slightly decreased sporulation in 630Δerm, suggesting that DccA contributes to the intracellular pool of c-di-GMP in a strain-dependent manner. However, these data were highly variable, underscoring the complex regulation involved in modulating intracellular c-di-GMP concentrations. Finally, the overexpression of dccA in known sporulation mutants revealed that c-di-GMP is likely signaling through an unidentified regulatory pathway to control early sporulation events in C. difficile. c-di-GMP-dependent regulation of C. difficile sporulation may represent an unexplored avenue of potential environmental and intracellular signaling that contributes to the complex regulation of sporulation initiation. IMPORTANCE Many bacterial organisms utilize the small signaling molecule cyclic diguanylate (c-di-GMP) to regulate important physiological processes, including motility, toxin production, biofilm formation, and colonization. c-di-GMP inhibits motility and toxin production and promotes biofilm formation and colonization in the anaerobic, gastrointestinal pathogen Clostridioides difficile. However, the impact of c-di-GMP on C. difficile spore formation, a critical step in this pathogen's life cycle, is unknown. Here, we demonstrate that c-di-GMP negatively impacts sporulation in two clinically relevant C. difficile strains, the epidemic strain R20291 and the historical strain 630Δerm. The pathway through which c-di-GMP controls sporulation was investigated, and our results suggest that c-di-GMP is likely signaling through an unidentified regulatory pathway to control C. difficile sporulation. This work implicates c-di-GMP metabolism as a mechanism to integrate environmental and intracellular cues through c-di-GMP levels to influence C. difficile sporulation.
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Affiliation(s)
- Adrianne N. Edwards
- Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, Georgia, USA
| | - Caitlin L. Willams
- Department of Microbiology and Immunology, University of North Carolina—Chapel Hill, Chapel Hill, North Carolina, USA
| | - Nivedita Pareek
- Department of Microbiology and Immunology, University of North Carolina—Chapel Hill, Chapel Hill, North Carolina, USA
| | - Shonna M. McBride
- Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, Georgia, USA
| | - Rita Tamayo
- Department of Microbiology and Immunology, University of North Carolina—Chapel Hill, Chapel Hill, North Carolina, USA
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28
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Purcell EB. Second messenger signaling in Clostridioides difficile. Curr Opin Microbiol 2021; 65:138-144. [PMID: 34864551 DOI: 10.1016/j.mib.2021.11.006] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2021] [Revised: 11/09/2021] [Accepted: 11/10/2021] [Indexed: 12/18/2022]
Abstract
Small, diffusible second messenger molecules transmit information about extracellular conditions to intracellular machinery in order to influence transcription, translation, and metabolism. The enteropathogenic bacterium Clostridioides difficile coordinates its response to a dynamic and hostile environment via nucleotide second messengers. While riboswitch-mediated cyclic diguanylate regulation has been extensively characterized in C. difficile, signaling by cyclic diadenylate and by guanosine alarmones has only recently been confirmed in this organism. This review summarizes the current knowledge of how nucleotide second messenger signaling regulates physiological processes in C. difficile.
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Affiliation(s)
- Erin B Purcell
- Old Dominion University, Department of Chemistry and Biochemistry, 4501 Elkhorn Ave, Norfolk, VA 23529, United States.
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29
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Arrieta-Ortiz ML, Immanuel SRC, Turkarslan S, Wu WJ, Girinathan BP, Worley JN, DiBenedetto N, Soutourina O, Peltier J, Dupuy B, Bry L, Baliga NS. Predictive regulatory and metabolic network models for systems analysis of Clostridioides difficile. Cell Host Microbe 2021; 29:1709-1723.e5. [PMID: 34637780 PMCID: PMC8595754 DOI: 10.1016/j.chom.2021.09.008] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2021] [Revised: 07/29/2021] [Accepted: 09/16/2021] [Indexed: 12/15/2022]
Abstract
We present predictive models for comprehensive systems analysis of Clostridioides difficile, the etiology of pseudomembranous colitis. By leveraging 151 published transcriptomes, we generated an EGRIN model that organizes 90% of C. difficile genes into a transcriptional regulatory network of 297 co-regulated modules, implicating genes in sporulation, carbohydrate transport, and metabolism. By advancing a metabolic model through addition and curation of metabolic reactions including nutrient uptake, we discovered 14 amino acids, diverse carbohydrates, and 10 metabolic genes as essential for C. difficile growth in the intestinal environment. Finally, we developed a PRIME model to uncover how EGRIN-inferred combinatorial gene regulation by transcription factors, such as CcpA and CodY, modulates essential metabolic processes to enable C. difficile growth relative to commensal colonization. The C. difficile interactive web portal provides access to these model resources to support collaborative systems-level studies of context-specific virulence mechanisms in C. difficile.
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Affiliation(s)
| | | | | | - Wei-Ju Wu
- Institute for Systems Biology, Seattle, WA 98109, USA
| | - Brintha P Girinathan
- Massachusetts Host-Microbiome Center, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
| | - Jay N Worley
- Massachusetts Host-Microbiome Center, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
| | - Nicholas DiBenedetto
- Massachusetts Host-Microbiome Center, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
| | - Olga Soutourina
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-yvette 91198, France
| | - Johann Peltier
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-yvette 91198, France
| | - Bruno Dupuy
- Laboratoire Pathogenèse des Bactéries anaérobies, Institut Pasteur, Université de Paris, UMR CNRS 2001, Paris 75015, France
| | - Lynn Bry
- Massachusetts Host-Microbiome Center, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
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30
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Dhungel BA, Govind R. Phase-variable expression of pdcB, a phosphodiesterase, influences sporulation in Clostridioides difficile. Mol Microbiol 2021; 116:1347-1360. [PMID: 34606654 DOI: 10.1111/mmi.14828] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2021] [Revised: 09/30/2021] [Accepted: 10/01/2021] [Indexed: 02/07/2023]
Abstract
Clostridioides difficile is the causative agent of antibiotic-associated diarrhea and is the leading cause of nosocomial infection in developed countries. An increasing number of C. difficile infections are attributed to epidemic strains that produce more toxins and spores. C. difficile spores are the major factor for the transmission and persistence of the organism. Previous studies have identified global regulators that influence sporulation in C. difficile. This study discovers that PdcB, a phosphodiesterase, enhances sporulation in C. difficile strain UK1. Through genetic and biochemical assays, we show that phase-variable expression of pdcB results in hypo- and hyper-sporulation phenotypes. In the "ON" orientation, the identified promotor is in the right orientation to drive the expression of pdcB. Production of the PdcB phosphodiesterase reduces the intracellular cyclic-di-GMP (c-di-GMP) concentration, resulting in a hyper-sporulation phenotype. Loss of PdcB due to the pdcB promoter being in the OFF orientation or mutation of pdcB results in increased c-di-GMP levels and a hypo-sporulation phenotype. Additionally, we demonstrate that CodY binds to the upstream region of pdcB. DNA inversion reorients the CodY binding site so that in the OFF orientation, CodY binds a site that is upstream of the pdcB promoter and can further repress gene expression.
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Affiliation(s)
| | - Revathi Govind
- Division of Biology, Kansas State University, Manhattan, Kansas, USA
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31
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An RNA-centric global view of Clostridioides difficile reveals broad activity of Hfq in a clinically important gram-positive bacterium. Proc Natl Acad Sci U S A 2021; 118:2103579118. [PMID: 34131082 PMCID: PMC8237595 DOI: 10.1073/pnas.2103579118] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The gram-positive human pathogen Clostridioides difficile has emerged as the leading cause of antibiotic-associated diarrhea. However, little is known about the bacterium's transcriptome architecture and mechanisms of posttranscriptional control. Here, we have applied transcription start site and termination mapping to generate a single-nucleotide-resolution RNA map of C. difficile 5' and 3' untranslated regions, operon structures, and noncoding regulators, including 42 sRNAs. Our results indicate functionality of many conserved riboswitches and predict cis-regulatory RNA elements upstream of multidrug resistance (MDR)-type ATP-binding cassette (ABC) transporters and transcriptional regulators. Despite growing evidence for a role of Hfq in RNA-based gene regulation in C. difficile, the functions of Hfq-based posttranscriptional regulatory networks in gram-positive pathogens remain controversial. Using Hfq immunoprecipitation followed by sequencing of bound RNA species (RIP-seq), we identify a large cohort of transcripts bound by Hfq and show that absence of Hfq affects transcript stabilities and steady-state levels. We demonstrate sRNA expression during intestinal colonization by C. difficile and identify infection-related signals impacting its expression. As a proof of concept, we show that the utilization of the abundant intestinal metabolite ethanolamine is regulated by the Hfq-dependent sRNA CDIF630nc_085. Overall, our study lays the foundation for understanding clostridial riboregulation with implications for the infection process and provides evidence for a global role of Hfq in posttranscriptional regulation in a gram-positive bacterium.
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32
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Brauer M, Lassek C, Hinze C, Hoyer J, Becher D, Jahn D, Sievers S, Riedel K. What's a Biofilm?-How the Choice of the Biofilm Model Impacts the Protein Inventory of Clostridioides difficile. Front Microbiol 2021; 12:682111. [PMID: 34177868 PMCID: PMC8225356 DOI: 10.3389/fmicb.2021.682111] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Accepted: 05/12/2021] [Indexed: 12/18/2022] Open
Abstract
The anaerobic pathogen Clostridioides difficile is perfectly equipped to survive and persist inside the mammalian intestine. When facing unfavorable conditions C. difficile is able to form highly resistant endospores. Likewise, biofilms are currently discussed as form of persistence. Here a comprehensive proteomics approach was applied to investigate the molecular processes of C. difficile strain 630Δerm underlying biofilm formation. The comparison of the proteome from two different forms of biofilm-like growth, namely aggregate biofilms and colonies on agar plates, revealed major differences in the formation of cell surface proteins, as well as enzymes of its energy and stress metabolism. For instance, while the obtained data suggest that aggregate biofilm cells express both flagella, type IV pili and enzymes required for biosynthesis of cell-surface polysaccharides, the S-layer protein SlpA and most cell wall proteins (CWPs) encoded adjacent to SlpA were detected in significantly lower amounts in aggregate biofilm cells than in colony biofilms. Moreover, the obtained data suggested that aggregate biofilm cells are rather actively growing cells while colony biofilm cells most likely severely suffer from a lack of reductive equivalents what requires induction of the Wood-Ljungdahl pathway and C. difficile’s V-type ATPase to maintain cell homeostasis. In agreement with this, aggregate biofilm cells, in contrast to colony biofilm cells, neither induced toxin nor spore production. Finally, the data revealed that the sigma factor SigL/RpoN and its dependent regulators are noticeably induced in aggregate biofilms suggesting an important role of SigL/RpoN in aggregate biofilm formation.
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Affiliation(s)
- Madita Brauer
- Department for Microbial Physiology and Molecular Biology, Institute of Microbiology, University of Greifswald, Greifswald, Germany
| | - Christian Lassek
- Department for Microbial Physiology and Molecular Biology, Institute of Microbiology, University of Greifswald, Greifswald, Germany
| | - Christian Hinze
- Department for Microbial Physiology and Molecular Biology, Institute of Microbiology, University of Greifswald, Greifswald, Germany
| | - Juliane Hoyer
- Department for Microbial Proteomics, Institute of Microbiology, University of Greifswald, Greifswald, Germany
| | - Dörte Becher
- Department for Microbial Proteomics, Institute of Microbiology, University of Greifswald, Greifswald, Germany
| | - Dieter Jahn
- Braunschweig Integrated Centre of Systems Biology (BRICS), Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Susanne Sievers
- Department for Microbial Physiology and Molecular Biology, Institute of Microbiology, University of Greifswald, Greifswald, Germany
| | - Katharina Riedel
- Department for Microbial Physiology and Molecular Biology, Institute of Microbiology, University of Greifswald, Greifswald, Germany
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Garrett EM, Mehra A, Sekulovic O, Tamayo R. Multiple Regulatory Mechanisms Control the Production of CmrRST, an Atypical Signal Transduction System in Clostridioides difficile. mBio 2021; 13:e0296921. [PMID: 35164558 PMCID: PMC8844915 DOI: 10.1128/mbio.02969-21] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Accepted: 01/20/2022] [Indexed: 12/16/2022] Open
Abstract
Clostridioides difficile, an intestinal pathogen and leading cause of nosocomial infection, exhibits extensive phenotypic heterogeneity through phase variation. The signal transduction system CmrRST, which encodes two response regulators (CmrR and CmrT) and a sensor kinase (CmrS), impacts C. difficile cell and colony morphology, surface and swimming motility, biofilm formation, and virulence in an animal model. CmrRST is subject to phase variation through site-specific recombination and reversible inversion of the "cmr switch," and expression of cmrRST is also regulated by cyclic diguanylate (c-di-GMP) through a riboswitch. The goal of this study was to determine how the cmr switch and c-di-GMP work together to regulate cmrRST expression. We generated "phase-locked" strains by mutating key residues in the right inverted repeat flanking the cmr switch. Phenotypic characterization of these phase-locked cmr-ON and -OFF strains demonstrates that they cannot switch between rough and smooth colony morphologies, respectively, or other CmrRST-associated phenotypes. Manipulation of c-di-GMP levels in these mutants showed that c-di-GMP promotes cmrRST expression and associated phenotypes independently of cmr switch orientation. We identified multiple promoters controlling cmrRST transcription, including one within the ON orientation of the cmr switch and another that is positively autoregulated by CmrR. Overall, this work reveals a complex regulatory network that governs cmrRST expression and a unique intersection of phase variation and c-di-GMP signaling. These findings suggest that multiple environmental signals impact the production of this signaling transduction system. IMPORTANCE Clostridioides difficile is a leading cause of hospital-acquired intestinal infections in the United States. The CmrRST signal transduction system controls numerous physiological traits and processes in C. difficile, including cell and colony morphology, motility, biofilm formation, and virulence. Here, we define the complex, multilevel regulation of cmrRST expression, including stochastic control through phase variation, modulation by the second messenger c-di-GMP, and positive autoregulation by CmrR. The results of this study suggest that multiple, distinct environmental stimuli and selective pressures must be integrated to appropriately control cmrRST expression.
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Affiliation(s)
- Elizabeth M. Garrett
- Department of Microbiology and Immunology, University of North Carolina Chapel Hill, Chapel Hill, North Carolina, USA
| | - Anchal Mehra
- Department of Microbiology and Immunology, University of North Carolina Chapel Hill, Chapel Hill, North Carolina, USA
| | - Ognjen Sekulovic
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA
| | - Rita Tamayo
- Department of Microbiology and Immunology, University of North Carolina Chapel Hill, Chapel Hill, North Carolina, USA
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Combined and Distinct Roles of Agr Proteins in Clostridioides difficile 630 Sporulation, Motility, and Toxin Production. mBio 2020; 11:mBio.03190-20. [PMID: 33443122 PMCID: PMC8534292 DOI: 10.1128/mbio.03190-20] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The Clostridioides difficile accessory gene regulator 1 (agr1) locus consists of two genes, agrB1 and agrD1, that presumably constitute an autoinducing peptide (AIP) system. Typically, AIP systems function through the AgrB-mediated processing of AgrD to generate a processed form of the AIP that provides a concentration-dependent extracellular signal. Here, we show that the C. difficile 630 Agr1 system has multiple functions, not all of which depend on AgrB1. CRISPR-Cas9n deletion of agrB1, agrD1, or the entire locus resulted in changes in transcription of sporulation-related factors and an overall loss in spore formation. Sporulation was recovered in the mutants by providing supernatant from stationary-phase cultures of the parental strain. In contrast, C. difficile motility was reduced only when both AgrB1 and AgrD1 were disrupted. Finally, in the absence of AgrB1, the AgrD1 peptide accumulated within the cytoplasm and this correlated with increased expression of tcdR (15-fold), as well as tcdA (20-fold) and tcdB (5-fold), which encode the two major C. difficile toxins. The combined deletion of agrB1/agrD1 or deletion of only agrD1 did not significantly alter expression of tcdR or tcdB but did show a minor effect on tcdA expression. Overall, these data indicate that the Agr1-based system in C. difficile 630 carries out multiple functions, some of which are associated with prototypical AIP signaling and others of which involve previously undescribed mechanisms of action.IMPORTANCE C. difficile is a spore-forming, toxigenic, anaerobic bacterium that causes severe gastrointestinal illness. Understanding the ways in which C. difficile senses growth conditions to regulate toxin expression and sporulation is essential to advancing our understanding of this pathogen. The Agr1 system in C. difficile has been thought to function by generating an extracellular autoinducing peptide that accumulates and exogenously activates two-component signaling. The absence of the peptide or protease should, in theory, result in similar phenotypes. However, in contrast to longstanding assumptions about Agr, we found that mutants of individual agr1 genes exhibit distinct phenotypes in C. difficile These findings suggest that the Agr1 system may have other regulatory mechanisms independent of the typical Agr quorum sensing system. These data not only challenge models for Agr's mechanism of action in C. difficile but also may expand our conceptions of how this system works in other Gram-positive pathogens.
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Homologous Recombination in Clostridioides difficile Mediates Diversification of Cell Surface Features and Transport Systems. mSphere 2020; 5:5/6/e00799-20. [PMID: 33208516 PMCID: PMC7677006 DOI: 10.1128/msphere.00799-20] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Infections with C. difficile result in up to half a million illnesses and tens of thousands of deaths annually in the United States. The severity of C. difficile illness is dependent on both host and bacterial factors. Illness caused by the pathogen Clostridioides difficile is widespread and can range in severity from mild diarrhea to sepsis and death. Strains of C. difficile isolated from human infections exhibit great genetic diversity, leading to the hypothesis that the genetic background of the infecting strain at least partially determines a patient’s clinical course. However, although certain strains of C. difficile have been suggested to be associated with increased severity, strain typing alone has proved insufficient to explain infection severity. The limited explanatory power of strain typing has been hypothesized to be due to genetic variation within strain types, as well as genetic elements shared between strain types. Homologous recombination is an evolutionary mechanism that can result in large genetic differences between two otherwise clonal isolates, and also lead to convergent genotypes in distantly related strains. More than 400 C. difficile genomes were analyzed here to assess the effect of homologous recombination within and between C. difficile clades. Almost three-quarters of single nucleotide variants in the C. difficile phylogeny are predicted to be due to homologous recombination events. Furthermore, recombination events were enriched in genes previously reported to be important to virulence and host-pathogen interactions, such as flagella, cell wall proteins, and sugar transport and metabolism. Thus, by exploring the landscape of homologous recombination in C. difficile, we identified genetic loci whose elevated rates of recombination mediated diversification, making them strong candidates for being mediators of host-pathogen interaction in diverse strains of C. difficile. IMPORTANCE Infections with C. difficile result in up to half a million illnesses and tens of thousands of deaths annually in the United States. The severity of C. difficile illness is dependent on both host and bacterial factors. Studying the evolutionary history of C. difficile pathogens is important for understanding the variation in pathogenicity of these bacteria. This study examines the extent and targets of homologous recombination, a mechanism by which distant strains of bacteria can share genetic material, in hundreds of C. difficile strains and identifies hot spots of realized recombination events. The results of this analysis reveal the importance of homologous recombination in the diversification of genetic loci in C. difficile that are significant in its pathogenicity and host interactions, such as flagellar construction, cell wall proteins, and sugar transport and metabolism.
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Abstract
Clostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with toxin production, and spores are responsible for the transmission and persistence of the organism. Previously, we characterized sin locus regulators SinR and SinR' (we renamed it SinI), where SinR is the regulator of toxin production and sporulation. The SinI regulator acts as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, controls SinR by regulating the expression of its antagonist, sinI However, the role of Spo0A in the expression of sinR and sinI in C. difficile had not yet been reported. In this study, we tested spo0A mutants in three different C. difficile strains, R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. Quantitative reverse transcription-PCR (qRT-PCR) analysis of its expression further supported these data. By carrying out genetic and biochemical assays, we show that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates the sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficile IMPORTANCE Clostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, the sin locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls sin locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.
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Trzilova D, Tamayo R. Site-Specific Recombination - How Simple DNA Inversions Produce Complex Phenotypic Heterogeneity in Bacterial Populations. Trends Genet 2020; 37:59-72. [PMID: 33008627 DOI: 10.1016/j.tig.2020.09.004] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2020] [Revised: 08/26/2020] [Accepted: 09/01/2020] [Indexed: 02/07/2023]
Abstract
Many bacterial species generate phenotypically heterogeneous subpopulations as a strategy for ensuring the survival of the population as a whole - an environmental stress that eradicates one subpopulation may leave other phenotypic groups unharmed, allowing the lineage to continue. Phase variation, a process that functions as an ON/OFF switch for gene expression, is one way that bacteria achieve phenotypic heterogeneity. Phase variation occurs stochastically and reversibly, and in the presence of a selective pressure the advantageous phenotype(s) predominates in the population. Phase variation can occur through multiple genetic and epigenetic mechanisms. This review focuses on conservative site-specific recombination that generates reversible DNA inversions as a genetic mechanism mediating phase variation. Recent studies have sparked a renewed interest in phase variation mediated through DNA inversion, revealing a high level of complexity beyond simple ON/OFF switching, including unusual modes of gene regulation, and highlighting an underappreciation of the use of these mechanisms by bacteria.
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Affiliation(s)
- Dominika Trzilova
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA
| | - Rita Tamayo
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
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Redefining the Clostridioides difficile σ B Regulon: σ B Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide. mSphere 2020; 5:5/5/e00728-20. [PMID: 32938698 PMCID: PMC7494833 DOI: 10.1128/msphere.00728-20] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile, σB has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allows for the refinement of the previously proposed σB regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σB was demonstrated through in vitro runoff transcription of specific target genes (cd0350, cd3614, cd3605, and cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to σB-dependent gene activation.IMPORTANCE Sigma B is the alternative sigma factor governing stress response in many Gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together, our data suggest that σB is a key player in dealing with toxic radicals.
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Clostridioides difficile para-Cresol Production Is Induced by the Precursor para-Hydroxyphenylacetate. J Bacteriol 2020; 202:JB.00282-20. [PMID: 32631945 PMCID: PMC7925072 DOI: 10.1128/jb.00282-20] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2020] [Accepted: 06/24/2020] [Indexed: 12/12/2022] Open
Abstract
Clostridioides difficile is an etiological agent for antibiotic-associated diarrheal disease. C. difficile produces a phenolic compound, para-cresol, which selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. C. difficile decarboxylates para-hydroxyphenylacetate (p-HPA) to produce p-cresol by the action of the HpdBCA decarboxylase encoded by the hpdBCA operon. Here, we investigate regulation of the hpdBCA operon and directly compare three independent reporter systems; SNAP-tag, glucuronidase gusA, and alkaline phosphatase phoZ reporters to detect basal and inducible expression. We show that expression of hpdBCA is upregulated in response to elevated p-HPA. In silico analysis identified three putative promoters upstream of hpdBCA operon-P1, P2, and Pσ54; only the P1 promoter was responsible for both basal and p-HPA-inducible expression of hpdBCA We demonstrated that turnover of tyrosine, a precursor for p-HPA, is insufficient to induce expression of the hpdBCA operon above basal levels because it is inefficiently converted to p-HPA in minimal media. We show that induction of the hpdBCA operon in response to p-HPA occurs in a dose-dependent manner. We also identified an inverted palindromic repeat (AAAAAG-N13-CTTTTT) upstream of the hpdBCA start codon (ATG) that is essential for inducing transcription of the hpdBCA operon in response to p-HPA, which drives the production of p-cresol. This provides insights into the regulatory control of p-cresol production, which affords a competitive advantage for C. difficile over other intestinal bacteria, promoting dysbiosis.IMPORTANCE Clostridioides difficile infection results from antibiotic-associated dysbiosis. para-Cresol, a phenolic compound produced by C. difficile, selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. Here, we demonstrate that expression of the hpdBCA operon, encoding the HpdBCA decarboxylase which converts p-HPA to p-cresol, is upregulated in response to elevated exogenous p-HPA, with induction occurring between >0.1 and ≤0.25 mg/ml. We determined a single promoter and an inverted palindromic repeat responsible for basal and p-HPA-inducible hpdBCA expression. We identified turnover of tyrosine, a p-HPA precursor, does not induce hpdBCA expression above basal level, indicating that exogenous p-HPA was required for p-cresol production. Identifying regulatory controls of p-cresol production will provide novel therapeutic targets to prevent p-cresol production, reducing C. difficile's competitive advantage.
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Soutourina O, Dubois T, Monot M, Shelyakin PV, Saujet L, Boudry P, Gelfand MS, Dupuy B, Martin-Verstraete I. Genome-Wide Transcription Start Site Mapping and Promoter Assignments to a Sigma Factor in the Human Enteropathogen Clostridioides difficile. Front Microbiol 2020; 11:1939. [PMID: 32903654 PMCID: PMC7438776 DOI: 10.3389/fmicb.2020.01939] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2020] [Accepted: 07/23/2020] [Indexed: 12/12/2022] Open
Abstract
The emerging human enteropathogen Clostridioides difficile is the main cause of diarrhea associated with antibiotherapy. Regulatory pathways underlying the adaptive responses remain understudied and the global view of C. difficile promoter structure is still missing. In the genome of C. difficile 630, 22 genes encoding sigma factors are present suggesting a complex pattern of transcription in this bacterium. We present here the first transcriptional map of the C. difficile genome resulting from the identification of transcriptional start sites (TSS), promoter motifs and operon structures. By 5′-end RNA-seq approach, we mapped more than 1000 TSS upstream of genes. In addition to these primary TSS, this analysis revealed complex structure of transcriptional units such as alternative and internal promoters, potential RNA processing events and 5′ untranslated regions. By following an in silico iterative strategy that used as an input previously published consensus sequences and transcriptomic analysis, we identified candidate promoters upstream of most of protein-coding and non-coding RNAs genes. This strategy also led to refine consensus sequences of promoters recognized by major sigma factors of C. difficile. Detailed analysis focuses on the transcription in the pathogenicity locus and regulatory genes, as well as regulons of transition phase and sporulation sigma factors as important components of C. difficile regulatory network governing toxin gene expression and spore formation. Among the still uncharacterized regulons of the major sigma factors of C. difficile, we defined the SigL regulon by combining transcriptome and in silico analyses. We showed that the SigL regulon is largely involved in amino-acid degradation, a metabolism crucial for C. difficile gut colonization. Finally, we combined our TSS mapping, in silico identification of promoters and RNA-seq data to improve gene annotation and to suggest operon organization in C. difficile. These data will considerably improve our knowledge of global regulatory circuits controlling gene expression in C. difficile and will serve as a useful rich resource for scientific community both for the detailed analysis of specific genes and systems biology studies.
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Affiliation(s)
- Olga Soutourina
- Laboratoire Pathogenèses des Bactéries Anaérobies, Institut Pasteur, UMR CNRS 2001, Université de Paris, Paris, France.,Institut Universitaire de France, Paris, France.,Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Thomas Dubois
- Laboratoire Pathogenèses des Bactéries Anaérobies, Institut Pasteur, UMR CNRS 2001, Université de Paris, Paris, France
| | - Marc Monot
- Laboratoire Pathogenèses des Bactéries Anaérobies, Institut Pasteur, UMR CNRS 2001, Université de Paris, Paris, France
| | | | - Laure Saujet
- Laboratoire Pathogenèses des Bactéries Anaérobies, Institut Pasteur, UMR CNRS 2001, Université de Paris, Paris, France
| | - Pierre Boudry
- Laboratoire Pathogenèses des Bactéries Anaérobies, Institut Pasteur, UMR CNRS 2001, Université de Paris, Paris, France
| | - Mikhail S Gelfand
- Institute for Information Transmission Problems, Moscow, Russia.,Skolkovo Institute of Science and Technology, Moscow, Russia
| | - Bruno Dupuy
- Laboratoire Pathogenèses des Bactéries Anaérobies, Institut Pasteur, UMR CNRS 2001, Université de Paris, Paris, France
| | - Isabelle Martin-Verstraete
- Laboratoire Pathogenèses des Bactéries Anaérobies, Institut Pasteur, UMR CNRS 2001, Université de Paris, Paris, France.,Institut Universitaire de France, Paris, France
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Trzilova D, Anjuwon-Foster BR, Torres Rivera D, Tamayo R. Rho factor mediates flagellum and toxin phase variation and impacts virulence in Clostridioides difficile. PLoS Pathog 2020; 16:e1008708. [PMID: 32785266 PMCID: PMC7446863 DOI: 10.1371/journal.ppat.1008708] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2020] [Revised: 08/24/2020] [Accepted: 06/16/2020] [Indexed: 12/17/2022] Open
Abstract
The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the "flagellar switch" upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5' leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile.
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Affiliation(s)
- Dominika Trzilova
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, United States of America
| | - Brandon R. Anjuwon-Foster
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, United States of America
| | - Dariana Torres Rivera
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, United States of America
| | - Rita Tamayo
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, United States of America
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Affiliation(s)
- Leila M. Reyes Ruiz
- Department of Microbiology and Immunology, University of North Carolina Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Caitlin L. Williams
- Department of Microbiology and Immunology, University of North Carolina Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Rita Tamayo
- Department of Microbiology and Immunology, University of North Carolina Chapel Hill, Chapel Hill, North Carolina, United States of America
- * E-mail:
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The Impact of pH on Clostridioides difficile Sporulation and Physiology. Appl Environ Microbiol 2020; 86:AEM.02706-19. [PMID: 31811041 DOI: 10.1128/aem.02706-19] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2019] [Accepted: 12/03/2019] [Indexed: 12/18/2022] Open
Abstract
Clostridioides difficile is a pathogenic bacterium that infects the human colon to cause diarrheal disease. Growth of the bacterium is known to be dependent on certain bile acids, oxygen levels, and nutrient availability in the intestine, but how the environmental pH can influence C. difficile is mostly unknown. Previous studies indicated that C. difficile modulates the intestinal pH, and prospective cohort studies have found a strong association between a more alkaline fecal pH and C. difficile infection. Based on these data, we hypothesized that C. difficile physiology can be affected by various pH conditions. In this study, we investigated the impact of a range of pH conditions on C. difficile to assess potential effects on growth, sporulation, motility, and toxin production in the strains 630Δerm and R20291. We observed pH-dependent differences in sporulation rate, spore morphology, and viability. Sporulation frequency was lowest under acidic conditions, and differences in cell morphology were apparent at low pH. In alkaline environments, C. difficile sporulation was greater for strain 630Δerm, whereas R20291 produced relatively high levels of spores in a broad range of pH conditions. Rapid changes in pH during exponential growth impacted sporulation similarly among the strains. Furthermore, we observed an increase in C. difficile motility with increases in pH, and strain-dependent differences in toxin production under acidic conditions. The data demonstrate that pH is an important parameter that affects C. difficile physiology and may reveal relevant insights into the growth and dissemination of this pathogen.IMPORTANCE Clostridioides difficile is an anaerobic bacterium that causes gastrointestinal disease. C. difficile forms dormant spores which can survive harsh environmental conditions, allowing their spread to new hosts. In this study, we determine how intestinally relevant pH conditions impact C. difficile physiology in the two divergent strains, 630Δerm and R20291. Our data demonstrate that low pH conditions reduce C. difficile growth, sporulation, and motility. However, toxin production and spore morphology were differentially impacted in the two strains at low pH. In addition, we observed that alkaline environments reduce C. difficile growth, but increase cell motility. When pH was adjusted rapidly during growth, we observed similar impacts on both strains. This study provides new insights into the phenotypic diversity of C. difficile grown under diverse pH conditions present in the intestinal tract, and demonstrates similarities and differences in the pH responses of different C. difficile isolates.
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Strain-Dependent RstA Regulation of Clostridioides difficile Toxin Production and Sporulation. J Bacteriol 2020; 202:JB.00586-19. [PMID: 31659010 DOI: 10.1128/jb.00586-19] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2019] [Accepted: 10/21/2019] [Indexed: 02/04/2023] Open
Abstract
The anaerobic spore former Clostridioides difficile causes significant diarrheal disease in humans and other mammals. Infection begins with the ingestion of dormant spores, which subsequently germinate within the host gastrointestinal tract. There, the vegetative cells proliferate and secrete two exotoxins, TcdA and TcdB, which cause disease symptoms. Although spore formation and toxin production are critical for C. difficile pathogenesis, the regulatory links between these two physiological processes are not well understood and are strain dependent. Previously, we identified a conserved C. difficile regulator, RstA, that promotes sporulation initiation through an unknown mechanism and directly and indirectly represses toxin and motility gene transcription in the historical isolate 630Δerm To test whether perceived strain-dependent differences in toxin production and sporulation are mediated by RstA, we created an rstA mutant in the epidemic ribotype 027 strain R20291. RstA affected sporulation and toxin gene expression similarly but more robustly in R20291 than in 630Δerm In contrast, no effect on motility gene expression was observed in R20291. Reporter assays measuring transcriptional regulation of tcdR, the sigma factor gene essential for toxin gene expression, identified sequence-dependent effects influencing repression by RstA and CodY, a global nutritional sensor, in four diverse C. difficile strains. Finally, sequence- and strain-dependent differences were evident in RstA negative autoregulation of rstA transcription. Altogether, our data suggest that strain-dependent differences in RstA regulation contribute to the sporulation and toxin phenotypes observed in R20291. Our data establish RstA as an important regulator of C. difficile virulence traits.IMPORTANCE Two critical traits of Clostridioides difficile pathogenesis are toxin production, which causes disease symptoms, and spore formation, which permits survival outside the gastrointestinal tract. The multifunctional regulator RstA promotes sporulation and prevents toxin production in the historical strain 630Δerm Here, we show that RstA exhibits stronger effects on these phenotypes in an epidemic isolate, R20291, and additional strain-specific effects on toxin and rstA expression are evident. Our data demonstrate that sequence-specific differences within the promoter for the toxin regulator TcdR contribute to the regulation of toxin production by RstA and CodY. These sequence differences account for some of the variability in toxin production among isolates and may allow strains to differentially control toxin production in response to a variety of signals.
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Shen A, Edwards AN, Sarker MR, Paredes-Sabja D. Sporulation and Germination in Clostridial Pathogens. Microbiol Spectr 2019; 7:10.1128/microbiolspec.GPP3-0017-2018. [PMID: 31858953 PMCID: PMC6927485 DOI: 10.1128/microbiolspec.gpp3-0017-2018] [Citation(s) in RCA: 66] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2018] [Indexed: 12/14/2022] Open
Abstract
As obligate anaerobes, clostridial pathogens depend on their metabolically dormant, oxygen-tolerant spore form to transmit disease. However, the molecular mechanisms by which those spores germinate to initiate infection and then form new spores to transmit infection remain poorly understood. While sporulation and germination have been well characterized in Bacillus subtilis and Bacillus anthracis, striking differences in the regulation of these processes have been observed between the bacilli and the clostridia, with even some conserved proteins exhibiting differences in their requirements and functions. Here, we review our current understanding of how clostridial pathogens, specifically Clostridium perfringens, Clostridium botulinum, and Clostridioides difficile, induce sporulation in response to environmental cues, assemble resistant spores, and germinate metabolically dormant spores in response to environmental cues. We also discuss the direct relationship between toxin production and spore formation in these pathogens.
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Affiliation(s)
- Aimee Shen
- Department of Molecular Biology and Microbiology, Tufts University Medical School, Boston, MA
| | - Adrianne N Edwards
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA
| | - Mahfuzur R Sarker
- Department of Microbiology, College of Science, Oregon State University, Corvallis, OR
- Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR
| | - Daniel Paredes-Sabja
- Department of Gut Microbiota and Clostridia Research Group, Departamento de Ciencias Biolo gicas, Facultad de Ciencias Biologicas, Universidad Andres Bello, Santiago, Chile
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Chen KY, Rathod J, Chiu YC, Chen JW, Tsai PJ, Huang IH. The Transcriptional Regulator Lrp Contributes to Toxin Expression, Sporulation, and Swimming Motility in Clostridium difficile. Front Cell Infect Microbiol 2019; 9:356. [PMID: 31681632 PMCID: PMC6811523 DOI: 10.3389/fcimb.2019.00356] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2019] [Accepted: 10/02/2019] [Indexed: 12/17/2022] Open
Abstract
Clostridium difficile is a Gram-positive, spore-forming bacterium, and major cause of nosocomial diarrhea. Related studies have identified numerous factors that influence virulence traits such as the production of the two primary toxins, toxin A (TcdA) and toxin B (TcdB), as well as sporulation, motility, and biofilm formation. However, multiple putative transcriptional regulators are reportedly encoded in the genome, and additional factors are likely involved in virulence regulation. Although the leucine-responsive regulatory protein (Lrp) has been studied extensively in Gram-negative bacteria, little is known about its function in Gram-positive bacteria, although homologs have been identified in the genome. This study revealed that disruption of the lone lrp homolog in C. difficile decelerated growth under nutrient-limiting conditions, increased TcdA and TcdB production. Lrp was also found to negatively regulate sporulation while positively regulate swimming motility in strain R20291, but not in strain 630. The C. difficile Lrp appeared to function through transcriptional repression or activation. In addition, the lrp mutant was relatively virulent in a mouse model of infection. The results of this study collectively demonstrated that Lrp has broad regulatory function in C. difficile toxin expression, sporulation, motility, and pathogenesis.
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Affiliation(s)
- Kuan-Yu Chen
- Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Jagat Rathod
- Department of Earth Sciences, National Cheng Kung University, Tainan, Taiwan
| | - Yi-Ching Chiu
- Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Jenn-Wei Chen
- Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.,Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan
| | - Pei-Jane Tsai
- Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan.,Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - I-Hsiu Huang
- Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.,Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan
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Garrett EM, Sekulovic O, Wetzel D, Jones JB, Edwards AN, Vargas-Cuebas G, McBride SM, Tamayo R. Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence. PLoS Biol 2019; 17:e3000379. [PMID: 31658249 PMCID: PMC6837544 DOI: 10.1371/journal.pbio.3000379] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2019] [Revised: 11/07/2019] [Accepted: 10/15/2019] [Indexed: 12/20/2022] Open
Abstract
Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. Many C. difficile strains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show that C. difficile reversibly differentiates into rough and smooth colony morphologies and that bacteria derived from the isolates display discrete motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein colony morphology regulators RST (CmrRST), which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of flagella and type IV pili, suggesting a novel mechanism of cell migration in C. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration on surfaces. In a hamster model of acute C. difficile disease, the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. Our findings indicate that C. difficile employs phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.
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Affiliation(s)
- Elizabeth M. Garrett
- Department of Microbiology and Immunology, University of North Carolina Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Ognjen Sekulovic
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America
| | - Daniela Wetzel
- Department of Microbiology and Immunology, Emory University, Rollins Research Center, Atlanta, Georgia, United States of America
| | - Joshua B. Jones
- Department of Microbiology and Immunology, Emory University, Rollins Research Center, Atlanta, Georgia, United States of America
| | - Adrianne N. Edwards
- Department of Microbiology and Immunology, Emory University, Rollins Research Center, Atlanta, Georgia, United States of America
| | - Germán Vargas-Cuebas
- Department of Microbiology and Immunology, Emory University, Rollins Research Center, Atlanta, Georgia, United States of America
| | - Shonna M. McBride
- Department of Microbiology and Immunology, Emory University, Rollins Research Center, Atlanta, Georgia, United States of America
| | - Rita Tamayo
- Department of Microbiology and Immunology, University of North Carolina Chapel Hill, Chapel Hill, North Carolina, United States of America
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Ciftci Y, Girinathan BP, Dhungel BA, Hasan MK, Govind R. Clostridioides difficile SinR' regulates toxin, sporulation and motility through protein-protein interaction with SinR. Anaerobe 2019; 59:1-7. [PMID: 31077800 PMCID: PMC6785386 DOI: 10.1016/j.anaerobe.2019.05.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2018] [Revised: 04/10/2019] [Accepted: 05/07/2019] [Indexed: 12/18/2022]
Abstract
Clostridioides difficile is a Gram-positive, anaerobic bacterium. It is known that C. difficile is one of the major causes of antibiotic associated diarrhea. The enhanced antibiotic resistance observed in C. difficile is the result of highly resistant spores produced by the bacterium. In Bacillus subtilis, the sin operon is involved in sporulation inhibition. Two proteins coded within this operon, SinR and SinI, have an antagonistic relationship; SinR acts as an inhibitor to sporulation whereas SinI represses the activity of SinR, thus allowing the bacterium to sporulate. In a previous study, we examined the sin locus in C. difficile and named the two genes associated with this operon sinR and sinR', analogous to sinR and sinI in B. subtilis, respectively. We have shown that SinR and SinR' have pleiotropic roles in pathogenesis pathways and interact antagonistically with each other. Unlike B. subtilis SinI, SinR' in C. difficile carries two domains: the HTH domain and the Multimerization Domain (MD). In this study, we first performed a GST Pull-down experiment to determine the domain within SinR' that interacts with SinR. Second, the effect of these two domains on three phenotypes; sporulation, motility, and toxin production was examined. The findings of this study confirmed the prediction that the Multimerization Domain (MD) of SinR' is responsible for the interaction between SinR and SinR'. It was also discovered that SinR' regulates sporulation, toxin production and motility primarily by inhibiting SinR activity through the Multimerization Domain (MD).
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Affiliation(s)
- Yusuf Ciftci
- Division of Biology, Kansas State University, Manhattan, KS, USA
| | - Brintha Parasumanna Girinathan
- Division of Biology, Kansas State University, Manhattan, KS, USA; Department of Pathology, Harvard Medical School, Boston, MA, USA
| | | | - Md Kamrul Hasan
- Division of Biology, Kansas State University, Manhattan, KS, USA
| | - Revathi Govind
- Division of Biology, Kansas State University, Manhattan, KS, USA.
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49
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Zhu D, Bullock J, He Y, Sun X. Cwp22, a novel peptidoglycan cross-linking enzyme, plays pleiotropic roles in Clostridioides difficile. Environ Microbiol 2019; 21:3076-3090. [PMID: 31173438 DOI: 10.1111/1462-2920.14706] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2018] [Revised: 06/01/2019] [Accepted: 06/05/2019] [Indexed: 12/23/2022]
Abstract
Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe pathogen, and can induce nosocomial antibiotic-associated intestinal disease. While production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, adhesion and colonization of C. difficile in the host gut are prerequisites for disease onset. Previous cell wall proteins (CWPs) were identified that were implicated in C. difficile adhesion and colonization. In this study, we predicted and characterized Cwp22 (CDR20291_2601) from C. difficile R20291 to be involved in bacterial adhesion based on the Vaxign reverse vaccinology tool. The ClosTron-generated cwp22 mutant showed decreased TcdA and TcdB production during early growth, and increased cell permeability and autolysis. Importantly, the cwp22 mutation impaired cellular adherence in vitro and decreased cytotoxicity and fitness over the parent strain in a mouse infection model. Furthermore, lactate dehydrogenase cytotoxicity assay, live-dead cell staining and transmission electron microscopy confirmed the decreased cell viability of the cwp22 mutant. Thus, Cwp22 is involved in cell wall integrity and cell viability, which could affect most phenotypes of R20291. Our data suggest that Cwp22 is an attractive target for C. difficile infection therapeutics and prophylactics.
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Affiliation(s)
- Duolong Zhu
- Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
| | - Jessica Bullock
- Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
| | - Yongqun He
- Department of Microbiology and Immunology, and Center for Computational Medicine and Bioinformatics, Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Xingmin Sun
- Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
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50
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Bouillaut L, Dubois T, Francis MB, Daou N, Monot M, Sorg JA, Sonenshein AL, Dupuy B. Role of the global regulator Rex in control of NAD + -regeneration in Clostridioides (Clostridium) difficile. Mol Microbiol 2019; 111:1671-1688. [PMID: 30882947 DOI: 10.1111/mmi.14245] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/13/2019] [Indexed: 12/31/2022]
Abstract
For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D-proline reductase (PR), a Stickland reaction that regenerates NAD+ from NADH. Many Clostridium spp. use Stickland metabolism (co-fermentation of pairs of amino acids) to generate ATP and NAD+ . Synthesis of PR is activated by PrdR, a proline-responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD+ -generating pathways, such as the glycine reductase and succinate-acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox-sensing regulator that responds to the NAD+ /NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD+ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR-regulated Stickland metabolism in the virulence of C. difficile.
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Affiliation(s)
- Laurent Bouillaut
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, 02111
| | - Thomas Dubois
- Pathogenesis of Bacterial Anaerobes Laboratory, Institut Pasteur, 75724, Paris, France.,Université Paris Diderot, Sorbonne Paris Cité, Paris, France
| | - Michael B Francis
- Department of Biology, Texas A&M University, College Station, TX, 77843
| | - Nadine Daou
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, 02111
| | - Marc Monot
- Pathogenesis of Bacterial Anaerobes Laboratory, Institut Pasteur, 75724, Paris, France.,Université Paris Diderot, Sorbonne Paris Cité, Paris, France
| | - Joseph A Sorg
- Department of Biology, Texas A&M University, College Station, TX, 77843
| | - Abraham L Sonenshein
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, 02111
| | - Bruno Dupuy
- Pathogenesis of Bacterial Anaerobes Laboratory, Institut Pasteur, 75724, Paris, France.,Université Paris Diderot, Sorbonne Paris Cité, Paris, France
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