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Yang J, Qian Y, Kim C, Birhanu BT, Cal Y Mayor-Luna C, Ding D, Yu X, Schroeder VA, Mobashery S, Chang M. Targeting SleC and CspB in the Inhibition of Spore Germination in Clostridioides difficile. J Med Chem 2025; 68:9357-9370. [PMID: 40286328 DOI: 10.1021/acs.jmedchem.4c03090] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/29/2025]
Abstract
Clostridioides difficile, a Gram-positive, spore-forming anaerobic bacterium, is a major healthcare threat. Its spores colonize the gut following dysbiosis caused by broad-spectrum antibiotics, remaining dormant until host's bile acid triggers germination into vegetative cells that produce toxins, leading to diarrhea, colitis, and potentially death. Current antibiotics to treat C. difficile infection target vegetative cells but not spore germination, a pivotal step in infection development. This study unveils 1,2,4-oxadiazoles as a novel class of spore germination inhibitors and delineates the structure-activity relationship. Screening of 120 oxadiazoles revealed compound 110 (IC50 = 14 ± 1 μM or 6.3 ± 0.4 μg/mL). Compound 110 targets mature SleC (Kd = 12 ± 1.0 μM) and CspB (Kd = 8.0 ± 1.0 μM) on spores, inhibiting their enzymatic activities, thus preventing spore germination. To our knowledge, compound 110 is the first reported spore germination inhibitor targeting SleC/CspB, offering a promising avenue for C. difficile therapies.
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Affiliation(s)
- Jingdong Yang
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
| | - Yuanyuan Qian
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
| | - Choon Kim
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
| | - Biruk T Birhanu
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
| | - Carlos Cal Y Mayor-Luna
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
| | - Derong Ding
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
| | - Xiaotan Yu
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
| | - Valerie A Schroeder
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
| | - Shahriar Mobashery
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
| | - Mayland Chang
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
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2
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Yang H, Kuang Y, Wang L, Ma X, Gálvez JAV, Lu J, Dai Y, Liu S, Yao J, Chen X, Cao Y. Pterostilbene attenuates intestinal barrier damage and secondary liver oxidative stress in a murine model of Clostridium difficile infection by regulating the gut microbiota. Food Funct 2025; 16:3325-3343. [PMID: 40190207 DOI: 10.1039/d4fo06413e] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/07/2025]
Abstract
Clostridium difficile infection (CDI) is a significant infectious disease with limited treatment options. Pterostilbene, an active compound found in blueberries, is known for its antioxidant and anti-inflammatory properties. This study investigated the effects of pterostilbene on intestinal barrier damage and secondary liver oxidative stress induced by CDI in mice. Pathological changes in the colon and liver, the levels of anti-inflammatory cytokines and antioxidants, and the expression of related genes were evaluated. Additionally, 16S rRNA sequencing and targeted metabolomics analyses of the gut microbiota and bile acids were conducted. Pterostilbene reduced the abundance of harmful bacteria such as Enterococcus, while increasing beneficial bacteria like Lactobacillus, thereby reshaping the gut microbiota and bile acid profile and reducing the accumulation of T-βMCA. This process activated intestinal FXR signaling, which alleviated colonic inflammation and reduced intestinal permeability. The reduction in intestinal permeability prevented the translocation of bacteria and bacterial toxins into the liver via the portal vein, thereby reducing liver inflammation and oxidative stress. Pterostilbene presented a promising strategy for maintaining intestinal health through the regulation of dysbiosis and bile acid disturbances caused by CDI. When integrated into the food system, pterostilbene has the potential to improve intestinal health, mitigate the risk of CDI associated with contaminated agricultural products, and enhance public health and food safety. Additionally, we identified that regulating the intestinal bile acid profile and the FXR receptor could serve as potential therapeutic targets for CDI, thereby facilitating the development of novel treatment options and dietary strategies.
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Affiliation(s)
- Hao Yang
- College of Animal Science and Technology, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi, 712100, China.
| | - Yanling Kuang
- College of Animal Science and Technology, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi, 712100, China.
| | - Lamei Wang
- College of Animal Science and Technology, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi, 712100, China.
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215, USA.
| | - Xinru Ma
- College of Animal Science and Technology, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi, 712100, China.
| | - Javier A Villafuerte Gálvez
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215, USA.
| | - Jing Lu
- College of Animal Science and Technology, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi, 712100, China.
| | - Yanfei Dai
- College of Animal Science and Technology, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi, 712100, China.
| | - Shimin Liu
- Institute of Agriculture, The University of Western Australia, Perth, Australia
| | - Junhu Yao
- College of Animal Science and Technology, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi, 712100, China.
| | - Xinhua Chen
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215, USA.
| | - Yangchun Cao
- College of Animal Science and Technology, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi, 712100, China.
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215, USA.
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Huang X, Johnson AE, Brehm JN, Do TVT, Auchtung TA, McCullough HC, Lerma AI, Haidacher SJ, Hoch KM, Horvath TD, Sorg JA, Haag AM, Auchtung JM. Clostridioides difficile colonization is not mediated by bile salts and utilizes Stickland fermentation of proline in an in vitro model. mSphere 2025; 10:e0104924. [PMID: 39817755 PMCID: PMC11852769 DOI: 10.1128/msphere.01049-24] [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: 12/10/2024] [Accepted: 12/18/2024] [Indexed: 01/18/2025] Open
Abstract
Treatment with antibiotics is a major risk factor for Clostridioides difficile infection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limit C. difficile colonization include the conversion of conjugated primary bile salts into secondary bile salts toxic to C. difficile growth and competition between the microbiota and C. difficile for limiting nutrients. Using a continuous flow model that simulates the nutrient conditions of the distal colon, we investigated how treatment with 6 clinically used antibiotics influenced susceptibility to C. difficile infection in 12 different microbial communities cultivated from healthy individuals. Antibiotic treatment reduced microbial richness; disruption varied by antibiotic class and microbiota composition, but did not correlate with C. difficile susceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate. However, changes in bile salt did not correlate with increased C. difficile susceptibility. Furthermore, bile salts were not required to inhibit C. difficile colonization. We tested whether amino acid fermentation contributed to the persistence of C. difficile in antibiotic-treated communities. C. difficile mutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (prdB-) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. The inability to ferment glycine or leucine as electron acceptors, however, was not sufficient to limit colonization in any communities. The data provide further support for the importance of bile salt-independent mechanisms in regulating the colonization of C. difficile.IMPORTANCEClostridioides difficile is one of the leading causes of hospital-acquired infections and antibiotic-associated diarrhea. Several potential mechanisms through which the microbiota can limit C. difficile infection have been identified and are potential targets for new therapeutics. However, it is unclear which mechanisms of C. difficile inhibition represent the best targets for the development of new therapeutics. These studies demonstrate that in a complex in vitro model of C. difficile infection, colonization resistance is independent of microbial bile salt metabolism. Instead, the ability of C. difficile to colonize is dependent upon its ability to metabolize proline, although proline-dependent colonization is context dependent and is not observed in all disrupted communities. Altogether, these studies support the need for further work to understand how bile-independent mechanisms regulate C. difficile colonization.
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Affiliation(s)
- Xiaoyun Huang
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, Nebraska, USA
| | - April E. Johnson
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, Nebraska, USA
| | - Joshua N. Brehm
- Department of Biology, Texas A&M University, College Station, Texas, USA
| | - Thi Van Thanh Do
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, Nebraska, USA
| | - Thomas A. Auchtung
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, Nebraska, USA
| | - Hugh C. McCullough
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, Nebraska, USA
| | - Armando I. Lerma
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, Nebraska, USA
| | - Sigmund J. Haidacher
- Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA
- Department of Pathology, Texas Children’s Microbiome Center, Texas Children’s Hospital, Houston, Texas, USA
| | - Kathleen M. Hoch
- Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA
- Department of Pathology, Texas Children’s Microbiome Center, Texas Children’s Hospital, Houston, Texas, USA
| | - Thomas D. Horvath
- Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA
- Department of Pathology, Texas Children’s Microbiome Center, Texas Children’s Hospital, Houston, Texas, USA
- Department of Pharmacy Practice & Translational Research, University of Houston, Houston, Texas, USA
| | - Joseph A. Sorg
- Department of Biology, Texas A&M University, College Station, Texas, USA
| | - Anthony M. Haag
- Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA
- Department of Pathology, Texas Children’s Microbiome Center, Texas Children’s Hospital, Houston, Texas, USA
| | - Jennifer M. Auchtung
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, Nebraska, USA
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Vinay G, Seppen J, Setlow P, Brul S. Bile acids as germinants for Clostridioides difficile spores, evidence of adaptation to the gut? FEMS Microbiol Rev 2025; 49:fuaf005. [PMID: 39924167 PMCID: PMC11878537 DOI: 10.1093/femsre/fuaf005] [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: 03/30/2024] [Revised: 12/16/2024] [Accepted: 02/07/2025] [Indexed: 02/11/2025] Open
Abstract
Bacterial spores formed upon metabolic stress have minimal metabolic activity and can remain dormant for years. Nevertheless, they can sense the environment and germinate quickly upon exposure to various germinants. Germinated spores can then outgrow into vegetative cells. Germination of spores of some anaerobes, especially Clostridioides difficile, is triggered by cholic acid and taurocholic acid. Elevated levels of these bile acids are thought to correlate with a perturbed gut microbiome, which cannot efficiently convert primary bile acids into secondary bile acids. That bile acids are germination-triggers suggests these bacteria have a life cycle taking place partially in the mammalian digestive tract where bile acids are plentiful; notably bile acids can be made by all vertebrates. Thus, spores survive in the environment until taken up by a host where they encounter an environment suitable for germination and then proliferate in the largely anaerobic large intestine; some ultimately sporulate there, regenerating environmentally resistant spores in the C. difficile life cycle. This review summarizes current literature on the effects of bile acids and their metabolites on spore germination in the gut and evidence that adaptation to bile acids as germinants is a consequence of a life cycle both inside and outside the digestive tract.
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Affiliation(s)
- Gianni Vinay
- Tytgat Institute for Liver and Intestinal Research, Amsterdam University Medical Centers, Location AMC, University of Amsterdam, Amsterdam 1105 AZ, The Netherlands
| | - Jurgen Seppen
- Tytgat Institute for Liver and Intestinal Research, Amsterdam University Medical Centers, Location AMC, University of Amsterdam, Amsterdam 1105 AZ, The Netherlands
| | - Peter Setlow
- Department of Molecular Biology and Biophysics, UConn Health, Farmington, CT 06030-3305, United States
| | - Stanley Brul
- Department of Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam 1098 XH, The Netherlands
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5
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Daniel SL, Ridlon JM. Clostridium scindens: history and current outlook for a keystone species in the mammalian gut involved in bile acid and steroid metabolism. FEMS Microbiol Rev 2025; 49:fuaf016. [PMID: 40307670 PMCID: PMC12065433 DOI: 10.1093/femsre/fuaf016] [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: 08/12/2024] [Revised: 04/10/2025] [Accepted: 04/29/2025] [Indexed: 05/02/2025] Open
Abstract
Clostridium scindens is a keystone bacterial species in the mammalian gut that, while low in abundance, has a significant impact on bile acid and steroid metabolism. Numerous studies indicate that the two most studied strains of C. scindens (i.e. ATCC 35704 and VPI 12708) are important for a myriad of physiological processes in the host. We focus on both historical and current microbiological and molecular biology work on the Hylemon-Björkhem pathway and the steroid-17,20-desmolase pathway that were first discovered in C. scindens. Our most recent analysis now calls into question whether strains currently defined as C. scindens represent two separate taxonomic groups. Future directions include developing genetic tools to further explore the physiological role of bile acid and steroid metabolism by strains of C. scindens and the causal role of these pathways in host physiology and disease.
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Affiliation(s)
- Steven L Daniel
- Department of Biological Sciences, Eastern Illinois University, Charleston, IL 61920, United States
- Department of Animal Sciences, University of Illinois Urbana-Champaign, Urbana, IL 61801, United States
| | - Jason M Ridlon
- Department of Animal Sciences, University of Illinois Urbana-Champaign, Urbana, IL 61801, United States
- Carl R. Woese Institute for Genomic Biology, Urbana, IL 61801, United States
- Division of Nutritional Sciences, University of Illinois Urbana-Champaign, Urbana, IL 61801, United States
- Cancer Center at Illinois, University of Illinois Urbana-Champaign, Urbana, IL 61801, United States
- Center for Advanced Study, University of Illinois Urbana-Champaign, Urbana, IL 61801, United States
- Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, United States
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6
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Beebe MA, Paredes-Sabja D, Kociolek LK, Rodríguez C, Sorg JA. Phenotypic analysis of various Clostridioides difficile ribotypes reveals consistency among core processes. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.10.632434. [PMID: 39829883 PMCID: PMC11741275 DOI: 10.1101/2025.01.10.632434] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/22/2025]
Abstract
Clostridioides difficile infections (CDI) cause almost 300,000 hospitalizations per year of which ~15-30% are the result of recurring infections. The prevalence and persistence of CDI in hospital settings has resulted in an extensive collection of C. difficile clinical isolates and their classification, typically by ribotype. While much of the current literature focuses on one or two prominent ribotypes (e.g., RT027), recent years have seen several other ribotypes dominate the clinical landscape (e.g., RT106 and RT078). Some ribotypes are associated with severe disease and / or increased recurrence rates, but why are certain ribotypes more prominent or harmful than others remains unknown. Because C. difficile has a large, open pan-genome, this observed relationship between ribotype and clinical outcome could be a result of the genetic diversity of C. difficile. Thus, we hypothesize that core biological processes of C. difficile are conserved across ribotypes / clades. We tested this hypothesis by observing the growth kinetics, sporulation, germination, bile acid sensitivity, bile salt hydrolase activity, and surface motility of fifteen strains belonging to various ribotypes spanning each known C. difficile clade. In viewing these phenotypes across each strain, we see that core phenotypes (growth, germination, sporulation, and resistance to bile salt toxicity) are remarkably consistent across clades / ribotypes. This suggests that variations observed in the clinical setting may be due to unidentified factors in the accessory genome or due to unknown host-factors. Importance C. difficile infections impact thousands of individuals every year many of whom experience recurring infections. Clinical studies have reported an unexplained correlation between some clades / ribotypes of C. difficile and disease severity / recurrence. Here, we demonstrate that C. difficile strains across the major clades / ribotypes are consistent in their core phenotypes. This suggests that such phenotypes are not responsible for variations in disease severity / recurrence and are ideal targets for the development of therapeutics meant to treat C. difficile related infections.
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Affiliation(s)
- Merilyn A. Beebe
- Department of Biology, Texas A&M University, College Station, TX 77845
| | | | - Larry K. Kociolek
- Division of Pediatric Infectious Diseases, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL 60611
| | - César Rodríguez
- Facultad de Microbiología & Centro de Investigación en Enfermedades Tropicales, Universidad de Costa Rica, San José, 11501-2060, Costa Rica
| | - Joseph A. Sorg
- Department of Biology, Texas A&M University, College Station, TX 77845
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Vernon JJ. Modulation of the Human Microbiome: Probiotics, Prebiotics, and Microbial Transplants. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2025; 1472:277-294. [PMID: 40111698 DOI: 10.1007/978-3-031-79146-8_17] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/22/2025]
Abstract
The balance between health and disease is intrinsically linked to the interactions between microbial communities and the host. This complex environment of antagonism and synergy involves both prokaryotic and eukaryotic cells, whose collaborative metabolic pathways and immunomodulatory elements influence system homeostasis. As with the gut and other niches, the oral microbiome has the capacity to affect distal host sites. The ability to manipulate this environment holds the potential to impact local and systemic disease.With the increasing threat of antimicrobial resistance, novel approaches to reduce the burden of disease are essential. The use of probiotics and prebiotics is one such strategy. Probiotics introduce non-pathogenic bacteria into the environment to compete with pathogens for nutrients and attachment sites, or to produce metabolites that counteract disease aetiologies. Prebiotic compounds enhance the growth of health-associated organisms, offering additional benefits, whilst a conjunctive approach with probiotics potentially holds even greater promise. Though widely studied in the gastrointestinal context, their potential for treating oral diseases, such as dental caries and periodontitis, is less understood. Additionally, the use of microbial transplantations has demonstrated efficacy in other areas, reducing systemic inflammation and recolonising with commensal bacteria. Here we evaluate their use in the oral context and their modulatory impact on overall health.In this chapter, we discuss how pro- and prebiotic strategies seek to modulate both the oral and gut environments to promote oral health and prevent disease. We assess novel approaches for utilising health-associated microorganisms to combat oral disorders, either administered locally in the mouth or imparting influence through immune modulation via the oral-gut axis. By examining available clinical trial data, we aim to further understand the intricacies involved in this discipline. Furthermore, we consider the challenges facing the research community, including optimal candidate organism/compound selection and colonisation retention, as well as considerations for future research.
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Affiliation(s)
- Jon J Vernon
- Division of Oral Biology, School of Dentistry, University of Leeds, Leeds, UK.
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8
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Huang X, Johnson AE, Brehm JN, Thanh Do TV, Auchtung TA, McCullough HC, Lerma AI, Haidacher SJ, Hoch KM, Horvath TD, Sorg JA, Haag AM, Auchtung JM. Clostridioides difficile colonization is not mediated by bile salts and utilizes Stickland fermentation of proline in an in vitro model. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.07.17.603937. [PMID: 39071387 PMCID: PMC11275744 DOI: 10.1101/2024.07.17.603937] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/30/2024]
Abstract
Treatment with antibiotics is a major risk factor for Clostridioides difficile infection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limit C. difficile colonization include conversion of conjugated primary bile salts into secondary bile salts toxic to C. difficile growth, and competition between the microbiota and C. difficile for limiting nutrients. Using a continuous flow model that simulates the nutrient conditions of the distal colon, we investigated how treatment with six clinically-used antibiotics influenced susceptibility to C. difficile infection in 12 different microbial communities cultivated from healthy individuals. Antibiotic treatment reduced microbial richness; disruption varied by antibiotic class and microbiota composition, but did not correlate with C. difficile susceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate. However, changes in bile salt did not correlate with increased C. difficile susceptibility. Further, bile salts were not required to inhibit C. difficile colonization. We tested whether amino acid fermentation contributed to persistence of C. difficile in antibiotic-treated communities. C. difficile mutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (prdB-) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. Inability to ferment glycine or leucine as electron acceptors, however, was not sufficient to limit colonization in any communities. This data provides further support for the importance of bile salt-independent mechanisms in regulating colonization of C. difficile.
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Affiliation(s)
- Xiaoyun Huang
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, NE 68588 USA
| | - April E. Johnson
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, NE 68588 USA
| | - Joshua N. Brehm
- Department of Biology, Texas A&M University, College Station, TX USA
| | - Thi Van Thanh Do
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, NE 68588 USA
| | - Thomas A. Auchtung
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, NE 68588 USA
| | - Hugh C. McCullough
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, NE 68588 USA
| | - Armando I. Lerma
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, NE 68588 USA
| | - Sigmund J. Haidacher
- Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX USA
- Texas Children’s Microbiome Center, Department of Pathology, Texas Children’s Hospital, Houston, TX USA
| | - Kathleen M. Hoch
- Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX USA
- Texas Children’s Microbiome Center, Department of Pathology, Texas Children’s Hospital, Houston, TX USA
| | - Thomas D. Horvath
- Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX USA
- Texas Children’s Microbiome Center, Department of Pathology, Texas Children’s Hospital, Houston, TX USA
- Department of Pharmacy Practice & Translational Research, University of Houston, Houston, TX USA
| | - Joseph A. Sorg
- Department of Biology, Texas A&M University, College Station, TX USA
| | - Anthony M. Haag
- Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX USA
- Texas Children’s Microbiome Center, Department of Pathology, Texas Children’s Hospital, Houston, TX USA
| | - Jennifer M. Auchtung
- Department of Food Science and Technology and Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, NE 68588 USA
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9
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Claushuis B, de Ru AH, van Veelen PA, Hensbergen PJ, Corver J. Characterization of the Clostridioides difficile 630Δerm putative Pro-Pro endopeptidase CD1597. Access Microbiol 2024; 6:000855.v3. [PMID: 39381498 PMCID: PMC11460543 DOI: 10.1099/acmi.0.000855.v3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2024] [Accepted: 09/03/2024] [Indexed: 10/10/2024] Open
Abstract
Clostridioides difficile is the leading cause of antibiotic-associated infections worldwide. Within the host, C. difficile can transition from a sessile to a motile state by secreting PPEP-1, which releases the cells from the intestinal epithelium by cleaving adhesion proteins. PPEP-1 belongs to the group of Pro-Pro endopeptidases (PPEPs), which are characterized by their unique ability to cleave proline-proline bonds. Interestingly, another putative member of this group, CD1597, is present in C. difficile. Although it possesses a domain similar to other PPEPs, CD1597 displays several distinct features that suggest a markedly different role for this protein. We investigated the proteolytic activity of CD1597 by testing various potential substrates. In addition, we investigated the effect of the absence of CD1597 by generating an insertional mutant of the cd1597 gene. Using the cd1597 mutant, we sought to identify phenotypic changes through a series of in vitro experiments and quantitative proteomic analyses. Furthermore, we aimed to study the localization of this protein using a fluorogenic fusion protein. Despite its similarities to PPEP-1, CD1597 did not show proteolytic activity. In addition, the absence of CD1597 caused an increase in various sporulation proteins during the stationary phase, yet we did not observe any alterations in the sporulation frequency of the cd1597 mutant. Furthermore, a promoter activity assay indicated a very low expression level of cd1597 in vegetative cells, which was independent of the culture medium and growth stage. The low expression was corroborated by our comprehensive proteomic analysis of the whole cell cultures, which failed to identify CD1597. However, an analysis of purified C. difficile spores identified CD1597 as part of the spore proteome. Hence, we predict that the protein is involved in sporulation, although we were unable to define a precise role for CD1597 in C. difficile.
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Affiliation(s)
- Bart Claushuis
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands
| | - Arnoud H. de Ru
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands
| | - Peter A. van Veelen
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands
| | - Paul J. Hensbergen
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands
| | - Jeroen Corver
- Leiden University Center for Infectious Diseases, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands
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10
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Osborne MS, Brehm JN, Olivença C, Cochran AM, Serrano M, Henriques AO, Sorg JA. The Impact of YabG Mutations on Clostridioides difficile Spore Germination and Processing of Spore Substrates. Mol Microbiol 2024; 122:534-548. [PMID: 39258427 PMCID: PMC12016784 DOI: 10.1111/mmi.15316] [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: 05/24/2024] [Revised: 08/27/2024] [Accepted: 08/30/2024] [Indexed: 09/12/2024]
Abstract
YabG is a sporulation-specific protease that is conserved among sporulating bacteria. Clostridioides difficile YabG processes the cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co-germinant receptor, mutations in yabG were found which altered the co-germinants required to initiate spore germination. To understand how these mutations in the yabG locus contribute to C. difficile spore germination, we introduced these mutations into an isogenic background. Spores derived from C. difficile yabGC207A (a catalytically inactive allele), C. difficile yabGA46D, C. difficile yabGG37E, and C. difficile yabGP153L strains germinated in response to taurocholic acid alone. Recombinantly expressed and purified preproSleC incubated with E. coli lysate expressing wild type YabG resulted in the removal of the presequence from preproSleC. Interestingly, only YabGA46D showed any activity toward purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in yabG expression under the mutant promoters were analyzed using a SNAP-tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.
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Affiliation(s)
- Morgan S. Osborne
- Department of Biology, Texas A&M University, College Station, TX 77845, USA
| | - Joshua N. Brehm
- Department of Biology, Texas A&M University, College Station, TX 77845, USA
| | - Carmen Olivença
- Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Alicia M. Cochran
- Department of Biology, Texas A&M University, College Station, TX 77845, USA
| | - Mónica Serrano
- Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Adriano O. Henriques
- Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Joseph A. Sorg
- Department of Biology, Texas A&M University, College Station, TX 77845, USA
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11
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Martinez E, Berg N, Rodriguez C, Daube G, Taminiau B. Influence of microbiota on the growth and gene expression of Clostridioides difficile in an in vitro coculture model. Microbiologyopen 2024; 13:e70001. [PMID: 39404502 PMCID: PMC11633334 DOI: 10.1002/mbo3.70001] [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: 04/02/2024] [Revised: 09/02/2024] [Accepted: 09/09/2024] [Indexed: 12/13/2024] Open
Abstract
Clostridioides difficile is an anaerobic, spore-forming, Gram-positive pathogenic bacterium. This study aimed to analyze the effect of two samples of healthy fecal microbiota on C. difficile gene expression and growth using an in vitro coculture model. The inner compartment was cocultured with spores of the C. difficile polymerase chain reaction (PCR)-ribotype 078, while the outer compartment contained fecal samples from donors to mimic the microbiota (FD1 and FD2). A fecal-free plate served as a control (CT). RNA-Seq and quantitative PCR confirmation were performed on the inner compartment sample. Similarities in gene expression were observed in the presence of the microbiota. After 12 h, the expression of genes associated with germination, sporulation, toxin production, and growth was downregulated in the presence of the microbiota. At 24 h, in an iron-deficient environment, C. difficile activated several genes to counteract iron deficiency. The expression of genes associated with germination and sporulation was upregulated at 24 h compared with 12 h in the presence of microbiota from donor 1 (FD1). This study confirmed previous findings that C. difficile can use ethanolamine as a primary nutrient source. To further investigate this interaction, future studies will use a simplified coculture model with an artificial bacterial consortium instead of fecal samples.
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Affiliation(s)
- Elisa Martinez
- Department of Food Sciences, Food MicrobiologyFundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of LiegeLiegeBelgium
| | - Noémie Berg
- Department of Food Sciences, Food MicrobiologyFundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of LiegeLiegeBelgium
| | - Cristina Rodriguez
- Instituto de Investigación Biomédica de Málaga‐IBIMAMálagaSpain
- Unidadde Gestión Clínica de Aparato DigestivoHospital Universitario Virgen de laVictoriaMálagaSpain
| | - Georges Daube
- Department of Food Sciences, Food MicrobiologyFundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of LiegeLiegeBelgium
| | - Bernard Taminiau
- Department of Food Sciences, Food MicrobiologyFundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of LiegeLiegeBelgium
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12
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González A, Fullaondo A, Odriozola I, Odriozola A. Microbiota and other detrimental metabolites in colorectal cancer. ADVANCES IN GENETICS 2024; 112:309-365. [PMID: 39396839 DOI: 10.1016/bs.adgen.2024.08.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/15/2024]
Abstract
Increasing scientific evidence demonstrates that gut microbiota plays an essential role in the onset and development of Colorectal cancer (CRC). However, the mechanisms by which these microorganisms contribute to cancer development are complex and far from completely clarified. Specifically, the impact of gut microbiota-derived metabolites on CRC is undeniable, exerting both protective and detrimental effects. This paper examines the effects and mechanisms by which important bacterial metabolites exert detrimental effects associated with increased risk of CRC. Metabolites considered include heterocyclic amines and polycyclic aromatic hydrocarbons, heme iron, secondary bile acids, ethanol, and aromatic amines. It is necessary to delve deeper into the mechanisms of action of these metabolites in CRC and identify the microbiota members involved in their production. Furthermore, since diet is the main factor capable of modifying the intestinal microbiota, conducting studies that include detailed descriptions of dietary interventions is crucial. All this knowledge is essential for developing precision nutrition strategies to optimise a protective intestinal microbiota against CRC.
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Affiliation(s)
- Adriana González
- Hologenomics Research Group, Department of Genetics, Physical Anthropology, and Animal Physiology, University of the Basque Country, Spain
| | - Asier Fullaondo
- Hologenomics Research Group, Department of Genetics, Physical Anthropology, and Animal Physiology, University of the Basque Country, Spain
| | - Iñaki Odriozola
- Health Department of Basque Government, Donostia-San Sebastián, Spain
| | - Adrian Odriozola
- Hologenomics Research Group, Department of Genetics, Physical Anthropology, and Animal Physiology, University of the Basque Country, Spain.
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13
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Daniel SL, Ridlon JM. Clostridium scindens : an endocrine keystone species in the mammalian gut. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.08.23.609444. [PMID: 39229245 PMCID: PMC11370556 DOI: 10.1101/2024.08.23.609444] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/05/2024]
Abstract
Clostridium scindens is a keystone human gut microbial taxonomic group that, while low in abundance, has a disproportionate effect on bile acid and steroid metabolism in the mammalian gut. Numerous studies indicate that the two most studied strains of C. scindens (i.e., ATCC 35704 and VPI 12708) are important for a myriad of physiological processes in the host. We focus on both historical and current microbiological and molecular biology work on the Hylemon-Björkhem pathway and the steroid-17,20-desmolase pathway that were first discovered in C. scindens. Our most recent analysis now calls into question whether strains currently defined as C. scindens represent two separate taxonomic groups. Future directions include developing genetic tools to further explore the physiological role bile acid and steroid metabolism by strains of C. scindens , and the causal role of these pathways in host physiology and disease.
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14
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Sum R, Lim SJM, Sundaresan A, Samanta S, Swaminathan M, Low W, Ayyappan M, Lim TW, Choo MD, Huang GJ, Cheong I. Clostridium septicum manifests a bile salt germinant response mediated by Clostridioides difficile csp gene orthologs. Commun Biol 2024; 7:947. [PMID: 39103440 PMCID: PMC11300598 DOI: 10.1038/s42003-024-06617-4] [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: 05/09/2023] [Accepted: 07/23/2024] [Indexed: 08/07/2024] Open
Abstract
Clostridium septicum infections are highly predictive of certain malignancies in human patients. To initiate infections, C. septicum spores must first germinate and regain vegetative growth. Yet, what triggers the germination of C. septicum spores is still unknown. Here, we observe that C. septicum germinates in response to specific bile salts. Putative bile salt recognition genes are identified in C. septicum based on their similarity in sequence and organization to bile salt-responsive csp genes in Clostridioides difficile. Inactivating two of these csp orthologs (cspC-82 and cspC-1718) results in mutant spores that no longer germinate in the presence of their respective cognate bile salts. Additionally, inactivating the putative cspBA or sleC genes in C. septicum abrogates the germination response to all bile salt germinants, suggesting that both act at a convergent point downstream of cspC-82 and cspC-1718. Molecular dynamics simulations show that both CspC-82 and CspC-1718 bear a strong structural congruence with C. difficile's CspC. The existence of functional bile salt germination sensors in C. septicum may be relevant to the association between infection and malignancy.
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Affiliation(s)
- Rongji Sum
- Temasek Life Sciences Laboratory, Singapore, Singapore
- Department of Biological Sciences, National University of Singapore, Singapore, Singapore
| | - Sylvester Jian Ming Lim
- Temasek Life Sciences Laboratory, Singapore, Singapore
- Department of Biological Sciences, National University of Singapore, Singapore, Singapore
| | - Ajitha Sundaresan
- Temasek Life Sciences Laboratory, Singapore, Singapore
- Department of Biological Sciences, National University of Singapore, Singapore, Singapore
| | | | | | - Wayne Low
- Temasek Life Sciences Laboratory, Singapore, Singapore
| | - Madhumitha Ayyappan
- Temasek Life Sciences Laboratory, Singapore, Singapore
- NUS High School of Mathematics and Sciences, Singapore, Singapore
| | - Ting Wei Lim
- Temasek Life Sciences Laboratory, Singapore, Singapore
- NUS High School of Mathematics and Sciences, Singapore, Singapore
| | - Marvin Dragon Choo
- Temasek Life Sciences Laboratory, Singapore, Singapore
- NUS High School of Mathematics and Sciences, Singapore, Singapore
| | | | - Ian Cheong
- Temasek Life Sciences Laboratory, Singapore, Singapore.
- Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
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15
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Nerber HN, Baloh M, Brehm JN, Sorg JA. The small acid-soluble proteins of Clostridioides difficile regulate sporulation in a SpoIVB2-dependent manner. PLoS Pathog 2024; 20:e1012507. [PMID: 39213448 PMCID: PMC11392383 DOI: 10.1371/journal.ppat.1012507] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2024] [Revised: 09/12/2024] [Accepted: 08/14/2024] [Indexed: 09/04/2024] Open
Abstract
Clostridioides difficile is a pathogen whose transmission relies on the formation of dormant endospores. Spores are highly resilient forms of bacteria that resist environmental and chemical insults. In recent work, we found that C. difficile SspA and SspB, two small acid-soluble proteins (SASPs), protect spores from UV damage and, interestingly, are necessary for the formation of mature spores. Here, we build upon this finding and show that C. difficile sspA and sspB are required for the formation of the spore cortex layer. Moreover, using an EMS mutagenesis selection strategy, we identified mutations that suppressed the defect in sporulation of C. difficile SASP mutants. Many of these strains contained mutations in CDR20291_0714 (spoIVB2) revealing a connection between the SpoIVB2 protease and the SASPs in the sporulation pathway. This work builds upon the hypothesis that the small acid-soluble proteins can regulate gene expression.
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Affiliation(s)
- Hailee N Nerber
- Department of Biology, Texas A&M University, College Station, Texas, United States of America
| | - Marko Baloh
- Department of Biology, Texas A&M University, College Station, Texas, United States of America
| | - Joshua N Brehm
- Department of Biology, Texas A&M University, College Station, Texas, United States of America
| | - Joseph A Sorg
- Department of Biology, Texas A&M University, College Station, Texas, United States of America
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16
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Boyle BL, Khanna S. Fecal microbiota live - jslm (Rebyota™/RBL) for management of recurrent Clostridioides difficile infection. Future Microbiol 2024; 19:1243-1251. [PMID: 38989699 PMCID: PMC11633411 DOI: 10.1080/17460913.2024.2364583] [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: 08/16/2023] [Accepted: 06/03/2024] [Indexed: 07/12/2024] Open
Abstract
There is an unmet need for effective treatments of Clostridioides difficile infection, an emerging health crisis in the United States. The management of C. difficile infection should include treatment of active infection and a strategy to prevent recurrence. Current gold standard therapy includes oral antibiotics which predispose patients to gut dysbiosis and increase the risk of recurrent infection. Addressing dysbiosis via fecal microbiota transplantation is an active and promising area of research, but studies have lacked standardization which makes outcome and safety data difficult to interpret. Rebyota™, formerly known as RBX2660, is a live biotherapeutic product designed using a standardized protocol and manufacturing process that has been shown to be effective for preventing recurrent C. difficile infection.
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Affiliation(s)
| | - Sahil Khanna
- Division of Gastroenterology & Hepatology, Mayo Clinic, Rochester, MN55905, USA
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17
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Camargo A, Bohorquez L, López DP, Ferrebuz-Cardozo A, Castellanos-Rozo J, Díaz-Ovalle J, Rada M, Camargo M, Ramírez JD, Muñoz M. Clostridium perfringens in central Colombia: frequency, toxin genes, and risk factors. Gut Pathog 2024; 16:32. [PMID: 38965598 PMCID: PMC11225238 DOI: 10.1186/s13099-024-00629-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/29/2024] [Accepted: 06/28/2024] [Indexed: 07/06/2024] Open
Abstract
Clostridium perfringens is an opportunistic bacterium that causes intestinal diseases in both humans and animals. This study aimed to assess the frequency of C. perfringens and the presence of toxin-encoding genes in fecal samples from individuals with or without gastrointestinal symptoms in the Department of Boyacá, Colombia. Additionally, risk factors associated with carriage and disease development were analyzed. A total of 114 stool samples were analyzed using a molecular test based on specific polymerase chain reaction (PCR) targeting 16S-rRNA and alpha toxin (cpa) genes. For individuals with a positive result for the PCR test, stool samples were cultured on Tryptose Sulfite Cycloserine (TSC) agar. Two to five colonies forming units were selected based on phenotypic characteristics, resulting in 56 bacterial isolates. These isolates were then analyzed for toxin-coding genes associated with gastrointestinal diseases. In addition, sociodemographic and clinical data from 77 individuals were also analyzed. The overall frequency of C. perfringens was 19.3% (n = 22/114). The detection frequency in 77 individuals with clinical data was 16.6% (n = 5/30) among symptomatic individuals and 21.2% (n = 10/47) among asymptomatic individuals. All 56 isolates obtained carried the cpa gene, while cpb2 was present in 10.7% (n = 6/56); cpe and cpb genes were not detected. Notably, diabetes and autoimmune diseases are significantly associated with an increased risk of C. perfringens detection (adjusted OR 8.41: 95% CI 1.32-35.89). This study highlights an elevated frequency of C. perfringens and the presence of the cpb2 gene in asymptomatic individuals compared with their symptomatic counterparts. These findings offer insights into the distribution and virulence factors of C. perfringens at a micro-geographical level. This information supports the need for developing tailored prevention strategies based on local characteristics to promote active surveillance programs based on molecular epidemiology.
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Affiliation(s)
- Anny Camargo
- Centro de Investigaciones en Microbiología y Biotecnología-UR (CIMBIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia
- Universidad de Boyacá, Tunja, Colombia
| | - Laura Bohorquez
- Centro de Investigaciones en Microbiología y Biotecnología-UR (CIMBIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia
| | | | | | | | | | | | - Milena Camargo
- Centro de Investigaciones en Microbiología y Biotecnología-UR (CIMBIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia
- Centro de Tecnología en Salud (CETESA), Innovaseq SAS, Funza, Cundinamarca, Colombia
| | - Juan David Ramírez
- Centro de Investigaciones en Microbiología y Biotecnología-UR (CIMBIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia
- Molecular Microbiology Laboratory, Department of Pathology, Molecular and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Marina Muñoz
- Centro de Investigaciones en Microbiología y Biotecnología-UR (CIMBIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia.
- Instituto de Biotecnología-UN (IBUN), Universidad Nacional de Colombia, Bogotá, Colombia.
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18
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Nerber HN, Baloh M, Brehm JN, Sorg JA. The small acid-soluble proteins of Clostridioides difficile regulate sporulation in a SpoIVB2-dependent manner. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.05.17.541253. [PMID: 37292792 PMCID: PMC10245694 DOI: 10.1101/2023.05.17.541253] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/10/2023]
Abstract
Clostridioides difficile is a pathogen whose transmission relies on the formation of dormant endospores. Spores are highly resilient forms of bacteria that resist environmental and chemical insults. In recent work, we found that C. difficile SspA and SspB, two small acid-soluble proteins (SASPs), protect spores from UV damage and, interestingly, are necessary for the formation of mature spores. Here, we build upon this finding and show that C. difficile sspA and sspB are required for the formation of the spore cortex layer. Moreover, using an EMS mutagenesis selection strategy, we identified mutations that suppressed the defect in sporulation of C. difficile SASP mutants. Many of these strains contained mutations in CDR20291_0714 (spoIVB2) revealing a connection between the SpoIVB2 protease and the SASPs in the sporulation pathway. This work builds upon the hypothesis that the small acid-soluble proteins can regulate gene expression.
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Affiliation(s)
- Hailee N. Nerber
- Department of Biology, Texas A&M University, College Station, TX 77845
| | - Marko Baloh
- Department of Biology, Texas A&M University, College Station, TX 77845
| | - Joshua N. Brehm
- Department of Biology, Texas A&M University, College Station, TX 77845
| | - Joseph A. Sorg
- Department of Biology, Texas A&M University, College Station, TX 77845
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19
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Okhuysen PC, Ramesh MS, Louie T, Kiknadze N, Torre-Cisneros J, de Oliveira CM, Van Steenkiste C, Stychneuskaya A, Garey KW, Garcia-Diaz J, Li J, Duperchy E, Chang BY, Sukbuntherng J, Montoya JG, Styles L, Clow F, James D, Dubberke ER, Wilcox M. A Randomized, Double-Blind, Phase 3 Safety and Efficacy Study of Ridinilazole Versus Vancomycin for Treatment of Clostridioides difficile Infection: Clinical Outcomes With Microbiome and Metabolome Correlates of Response. Clin Infect Dis 2024; 78:1462-1472. [PMID: 38305378 PMCID: PMC11175683 DOI: 10.1093/cid/ciad792] [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: 10/02/2023] [Indexed: 02/03/2024] Open
Abstract
BACKGROUND Exposure to antibiotics predisposes to dysbiosis and Clostridioides difficile infection (CDI) that can be severe, recurrent (rCDI), and life-threatening. Nonselective drugs that treat CDI and perpetuate dysbiosis are associated with rCDI, in part due to loss of microbiome-derived secondary bile acid (SBA) production. Ridinilazole is a highly selective drug designed to treat CDI and prevent rCDI. METHODS In this phase 3 superiority trial, adults with CDI, confirmed with a stool toxin test, were randomized to receive 10 days of ridinilazole (200 mg twice daily) or vancomycin (125 mg 4 times daily). The primary endpoint was sustained clinical response (SCR), defined as clinical response and no rCDI through 30 days after end of treatment. Secondary endpoints included rCDI and change in relative abundance of SBAs. RESULTS Ridinilazole and vancomycin achieved an SCR rate of 73% versus 70.7%, respectively, a treatment difference of 2.2% (95% CI: -4.2%, 8.6%). Ridinilazole resulted in a 53% reduction in recurrence compared with vancomycin (8.1% vs 17.3%; 95% CI: -14.1%, -4.5%; P = .0002). Subgroup analyses revealed consistent ridinilazole benefit for reduction in rCDI across subgroups. Ridinilazole preserved microbiota diversity, increased SBAs, and did not increase the resistome. Conversely, vancomycin worsened CDI-associated dysbiosis, decreased SBAs, increased Proteobacteria abundance (∼3.5-fold), and increased the resistome. CONCLUSIONS Although ridinilazole did not meet superiority in SCR, ridinilazole greatly reduced rCDI and preserved microbiome diversity and SBAs compared with vancomycin. These findings suggest that treatment of CDI with ridinilazole results in an earlier recovery of gut microbiome health. Clinical Trials Registration.Ri-CoDIFy 1 and 2: NCT03595553 and NCT03595566.
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Affiliation(s)
- Pablo C Okhuysen
- Department of Infectious Diseases, Infection Control, and Employee Heatlh, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | | | - Thomas Louie
- Foothills Medical Center and University of Calgary, Calgary, Canada
| | | | - Julian Torre-Cisneros
- Reina Sofia University Hospital-IMIBIC, University of Córdoba, CIBERINFEC, Cordoba, Spain
| | | | | | | | - Kevin W Garey
- University of Houston College of Pharmacy, Houston, Texas, USA
| | | | - Jianling Li
- Summit Therapeutics, Menlo Park, California, USA
| | | | | | | | - Jose G Montoya
- Summit Therapeutics, Menlo Park, California, USA
- Dr. Jack S. Remington Laboratory for Specialty Diagnostics, Palo Alto Medical Foundation, Palo Alto, California, USA
| | - Lori Styles
- Summit Therapeutics, Menlo Park, California, USA
| | - Fong Clow
- Summit Therapeutics, Menlo Park, California, USA
| | | | - Erik R Dubberke
- Washington University School of Medicine, St.Louis, Missouri, USA
| | - Mark Wilcox
- Leeds Teaching Hospitals and University of Leeds, School of Medicine, Leeds, United Kingdom
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20
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Di Bella S, Sanson G, Monticelli J, Zerbato V, Principe L, Giuffrè M, Pipitone G, Luzzati R. Clostridioides difficile infection: history, epidemiology, risk factors, prevention, clinical manifestations, treatment, and future options. Clin Microbiol Rev 2024; 37:e0013523. [PMID: 38421181 PMCID: PMC11324037 DOI: 10.1128/cmr.00135-23] [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] [Indexed: 03/02/2024] Open
Abstract
SUMMARYClostridioides difficile infection (CDI) is one of the major issues in nosocomial infections. This bacterium is constantly evolving and poses complex challenges for clinicians, often encountered in real-life scenarios. In the face of CDI, we are increasingly equipped with new therapeutic strategies, such as monoclonal antibodies and live biotherapeutic products, which need to be thoroughly understood to fully harness their benefits. Moreover, interesting options are currently under study for the future, including bacteriophages, vaccines, and antibiotic inhibitors. Surveillance and prevention strategies continue to play a pivotal role in limiting the spread of the infection. In this review, we aim to provide the reader with a comprehensive overview of epidemiological aspects, predisposing factors, clinical manifestations, diagnostic tools, and current and future prophylactic and therapeutic options for C. difficile infection.
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Affiliation(s)
- Stefano Di Bella
- Clinical Department of
Medical, Surgical and Health Sciences, Trieste
University, Trieste,
Italy
| | - Gianfranco Sanson
- Clinical Department of
Medical, Surgical and Health Sciences, Trieste
University, Trieste,
Italy
| | - Jacopo Monticelli
- Infectious Diseases
Unit, Trieste University Hospital
(ASUGI), Trieste,
Italy
| | - Verena Zerbato
- Infectious Diseases
Unit, Trieste University Hospital
(ASUGI), Trieste,
Italy
| | - Luigi Principe
- Microbiology and
Virology Unit, Great Metropolitan Hospital
“Bianchi-Melacrino-Morelli”,
Reggio Calabria, Italy
| | - Mauro Giuffrè
- Clinical Department of
Medical, Surgical and Health Sciences, Trieste
University, Trieste,
Italy
- Department of Internal
Medicine (Digestive Diseases), Yale School of Medicine, Yale
University, New Haven,
Connecticut, USA
| | - Giuseppe Pipitone
- Infectious Diseases
Unit, ARNAS Civico-Di Cristina
Hospital, Palermo,
Italy
| | - Roberto Luzzati
- Clinical Department of
Medical, Surgical and Health Sciences, Trieste
University, Trieste,
Italy
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21
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Osborne MS, Brehm JN, Olivença C, Cochran AM, Serrano M, Henriques AO, Sorg JA. The impact of YabG mutations on C. difficile spore germination and processing of spore substrates. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.06.10.598338. [PMID: 38915615 PMCID: PMC11195116 DOI: 10.1101/2024.06.10.598338] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/26/2024]
Abstract
YabG is a sporulation-specific protease that is conserved among sporulating bacteria. C. difficile YabG processes cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co-germinant receptor, mutations in yabG were found which altered the co-germinants required to initiate spore germination. To understand how these mutations in the yabG locus contribute to C. difficile spore germination, we introduced these mutations into an isogenic background. Spores derived from C. difficile yabG C207A (catalytically inactive), C. difficile yabG A46D, C. difficile yabG G37E, and C. difficile yabG P153L strains germinated in response to TA alone. Recombinantly expressed and purified preproSleC incubated with E. coli lysate expressing wild type YabG resulted in the removal of the pre sequence from preproSleC. Interestingly, only YabGA46D showed any activity towards purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in yabG expression under the mutant promoters were analyzed using a SNAP-tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.
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Affiliation(s)
- Morgan S. Osborne
- Department of Biology, Texas A&M University, College Station, TX 77845, USA
| | - Joshua N. Brehm
- Department of Biology, Texas A&M University, College Station, TX 77845, USA
| | - Carmen Olivença
- Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Alicia M. Cochran
- Department of Biology, Texas A&M University, College Station, TX 77845, USA
| | - Mónica Serrano
- Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Adriano O. Henriques
- Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Joseph A. Sorg
- Department of Biology, Texas A&M University, College Station, TX 77845, USA
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22
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Karnchanapandh K, Sanachai K, Poo-Arporn RP, Rungrotmongkol T. Enhancing bezlotoxumab binding to C. difficile toxin B2: insights from computational simulations and mutational analyses for antibody design. J Biomol Struct Dyn 2024:1-11. [PMID: 38511411 DOI: 10.1080/07391102.2024.2329785] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2023] [Accepted: 03/06/2024] [Indexed: 03/22/2024]
Abstract
Clostridioides difficile infection (CDI) is a significant concern caused by widespread antibiotic use, resulting in diarrhea and inflammation from the gram-positive anaerobic bacterium C. difficile. Although bezlotoxumab (Bez), a monoclonal antibody (mAb), was developed to address CDI recurrences, the recurrence rate remains high, partly due to reduced neutralization efficiency against toxin B2. In this study, we aimed to enhance the binding of Bez to C. difficile toxin B2 by combining computational simulations and mutational analyses. We identified specific mutations in Bez, including S28R, S31W/K, Y32R, S56W and G103D/S in the heavy chain (Hc), and S32F/H/R/W/Y in the light chain (Lc), which significantly improved binding to toxin B2 and formed critical protein-protein interactions. Through molecular dynamics simulations, several single mutations, such as HcS28R, LcS32H, LcS32R, LcS32W and LcS32Y, exhibited superior binding affinities to toxin B2 compared to Bez wild-type (WT), primarily attributed to Coulombic interactions. Combining the HcS28R mutation with four different mutations at residue LcS32 led to even greater binding affinities in double mutants (MTs), particularly HcS28R/LcS32H, HcS28R/LcS32R and HcS28R/LcS32Y, reinforcing protein-protein binding. Analysis of per-residue decomposition free energy highlighted key residues contributing significantly to enhanced binding interactions, emphasizing the role of electrostatic interactions. These findings offer insights into rational Bez MT design for improved toxin B2 binding, providing a foundation for developing more effective antibodies to neutralize toxin B2 and combat-related infections.
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Affiliation(s)
- Kun Karnchanapandh
- Program in Bioinformatics and Computational Biology, Graduate School, Chulalongkorn University, Bangkok, Thailand
| | - Kamonpan Sanachai
- Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand
| | - Rungtiva P Poo-Arporn
- Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, Thailand
| | - Thanyada Rungrotmongkol
- Program in Bioinformatics and Computational Biology, Graduate School, Chulalongkorn University, Bangkok, Thailand
- Center of Excellence in Biocatalyst and Sustainable Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
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23
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Mullowney MW, Fiebig A, Schnizlein MK, McMillin M, Rose AR, Koval J, Rubin D, Dalal S, Sogin ML, Chang EB, Sidebottom AM, Crosson S. Microbially catalyzed conjugation of GABA and tyramine to bile acids. J Bacteriol 2024; 206:e0042623. [PMID: 38174933 PMCID: PMC10810215 DOI: 10.1128/jb.00426-23] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2023] [Accepted: 12/13/2023] [Indexed: 01/05/2024] Open
Abstract
Bile acids (BAs) are cholesterol-derived molecules that aid in digestion and nutrient absorption, regulate host metabolic processes, and influence physiology of the gut microbiota. Both the host and its microbiome contribute to enzymatic modifications that shape the chemical diversity of BAs in the gut. Several bacterial species have been reported to conjugate standard amino acids to BAs, but it was not known if bacteria conjugate BAs to other amine classes. Here, we show that Bacteroides fragilis strain P207, isolated from a bacterial bloom in the J-pouch of a patient with ulcerative colitis pouchitis, conjugates standard amino acids and the neuroactive amines γ-aminobutyric acid (GABA) and tyramine to deoxycholic acid. We extended this analysis to other human gut isolates and identified species that are competent to conjugate GABA and tyramine to primary and secondary BAs, and further identified diverse BA-GABA and BA-tyramine amides in human stool. A longitudinal metabolomic analysis of J-pouch contents of the patient from whom B. fragilis P207 was isolated revealed highly reduced levels of secondary bile acids and a shifting BA amide profile before, during, and after onset of pouchitis, including temporal changes in several BA-GABA amides. Treatment of pouchitis with ciprofloxacin was associated with a marked reduction of nearly all BA amides in the J-pouch. Our study expands the known repertoire of conjugated bile acids produced by bacteria to include BA conjugates to GABA and tyramine and demonstrates that these molecules are present in the human gut. IMPORTANCE BAs are modified in multiple ways by host enzymes and the microbiota to produce a chemically diverse set of molecules that assist in the digestive process and impact many physiological functions. This study reports the discovery of bacterial species that conjugate the neuroactive amines, GABA and tyramine, to primary and secondary BAs. We further present evidence that BA-GABA and BA-tyramine conjugates are present in the human gut, and document a shifting BA-GABA profile in a human pouchitis patient before, during, and after inflammation and antibiotic treatment. GABA and tyramine are common metabolic products of the gut microbiota and potent neuroactive molecules. GABA- and tyramine-conjugated BAs may influence receptor-mediated regulatory mechanisms of humans and their gut microbes, and absorption of these molecules and their entry into enterohepatic circulation may impact host physiology at distal tissue sites. This study defines new conjugated bile acids in the human gut.
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Affiliation(s)
| | - Aretha Fiebig
- Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
| | - Matthew K. Schnizlein
- Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
| | - Mary McMillin
- Duchossois Family Institute, University of Chicago, Chicago, Illinois, USA
| | - Amber R. Rose
- Duchossois Family Institute, University of Chicago, Chicago, Illinois, USA
| | - Jason Koval
- Department of Medicine, University of Chicago, Chicago, Illinois, USA
| | - David Rubin
- Department of Medicine, University of Chicago, Chicago, Illinois, USA
| | - Sushila Dalal
- Department of Medicine, University of Chicago, Chicago, Illinois, USA
| | | | - Eugene B. Chang
- Department of Medicine, University of Chicago, Chicago, Illinois, USA
| | | | - Sean Crosson
- Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
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24
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Gurung B, Stricklin M, Wang S. Gut Microbiota-Gut Metabolites and Clostridioides difficile Infection: Approaching Sustainable Solutions for Therapy. Metabolites 2024; 14:74. [PMID: 38276309 PMCID: PMC10819375 DOI: 10.3390/metabo14010074] [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: 12/23/2023] [Revised: 01/06/2024] [Accepted: 01/19/2024] [Indexed: 01/27/2024] Open
Abstract
Clostridioides difficile (C. difficile) infection (CDI) is the most common hospital-acquired infection. With the combination of a high rate of antibiotic resistance and recurrence, it has proven to be a debilitating public health threat. Current treatments for CDI include antibiotics and fecal microbiota transplantation, which contribute to recurrent CDIs and potential risks. Therefore, there is an ongoing need to develop new preventative treatment strategies for CDI. Notably, gut microbiota dysbiosis is the primary risk factor for CDI and provides a promising target for developing novel CDI therapy approaches. Along with gut microbiota dysbiosis, a reduction in important gut metabolites like secondary bile acids and short-chain fatty acids (SCFAs) were also seen in patients suffering from CDI. In this review study, we investigated the roles and mechanisms of gut microbiota and gut microbiota-derived gut metabolites, especially secondary bile acids and SCFAs in CDI pathogenesis. Moreover, specific signatures of gut microbiota and gut metabolites, as well as different factors that can modulate the gut microbiota, were also discussed, indicating that gut microbiota modulators like probiotics and prebiotics can be a potential therapeutic strategy for CDI as they can help restore gut microbiota and produce gut metabolites necessary for a healthy gut. The understanding of the associations between gut microbiota-gut metabolites and CDI will allow for developing precise and sustainable approaches, distinct from antibiotics and fecal transplant, for mitigating CDI and other gut microbiota dysbiosis-related diseases.
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Affiliation(s)
- Bijay Gurung
- Department of Biomedical Sciences, Ohio University Heritage College of Osteopathic Medicine, Ohio University, Athens, OH 45701, USA; (B.G.); (M.S.)
- Infectious and Tropical Disease Institute, Ohio University, Athens, OH 45701, USA
- Interdisciplinary Molecular and Cellular Biology Program, Ohio University, Athens, OH 45701, USA
| | - Maranda Stricklin
- Department of Biomedical Sciences, Ohio University Heritage College of Osteopathic Medicine, Ohio University, Athens, OH 45701, USA; (B.G.); (M.S.)
- Infectious and Tropical Disease Institute, Ohio University, Athens, OH 45701, USA
| | - Shaohua Wang
- Department of Biomedical Sciences, Ohio University Heritage College of Osteopathic Medicine, Ohio University, Athens, OH 45701, USA; (B.G.); (M.S.)
- Infectious and Tropical Disease Institute, Ohio University, Athens, OH 45701, USA
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25
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Serrano M, Martins D, Henriques AO. Clostridioides difficile Sporulation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2024; 1435:273-314. [PMID: 38175480 DOI: 10.1007/978-3-031-42108-2_13] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2024]
Abstract
Some members of the Firmicutes phylum, including many members of the human gut microbiota, are able to differentiate a dormant and highly resistant cell type, the endospore (hereinafter spore for simplicity). Spore-formers can colonize virtually any habitat and, because of their resistance to a wide variety of physical and chemical insults, spores can remain viable in the environment for long periods of time. In the anaerobic enteric pathogen Clostridioides difficile the aetiologic agent is the oxygen-resistant spore, while the toxins produced by actively growing cells are the main cause of the disease symptoms. Here, we review the regulatory circuits that govern entry into sporulation. We also cover the role of spores in the infectious cycle of C. difficile in relation to spore structure and function and the main control points along spore morphogenesis.
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Affiliation(s)
- Mónica Serrano
- Instituto de Tecnologia Química e Biológica António Xavier, Oeiras, Portugal.
| | - Diogo Martins
- Instituto de Tecnologia Química e Biológica António Xavier, Oeiras, Portugal
| | - Adriano O Henriques
- Instituto de Tecnologia Química e Biológica António Xavier, Oeiras, Portugal
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26
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Fishbein SRS, Mahmud B, Dantas G. Antibiotic perturbations to the gut microbiome. Nat Rev Microbiol 2023; 21:772-788. [PMID: 37491458 DOI: 10.1038/s41579-023-00933-y] [Citation(s) in RCA: 124] [Impact Index Per Article: 62.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/15/2023] [Indexed: 07/27/2023]
Abstract
Antibiotic-mediated perturbation of the gut microbiome is associated with numerous infectious and autoimmune diseases of the gastrointestinal tract. Yet, as the gut microbiome is a complex ecological network of microorganisms, the effects of antibiotics can be highly variable. With the advent of multi-omic approaches for systems-level profiling of microbial communities, we are beginning to identify microbiome-intrinsic and microbiome-extrinsic factors that affect microbiome dynamics during antibiotic exposure and subsequent recovery. In this Review, we discuss factors that influence restructuring of the gut microbiome on antibiotic exposure. We present an overview of the currently complex picture of treatment-induced changes to the microbial community and highlight essential considerations for future investigations of antibiotic-specific outcomes. Finally, we provide a synopsis of available strategies to minimize antibiotic-induced damage or to restore the pretreatment architectures of the gut microbial community.
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Affiliation(s)
- Skye R S Fishbein
- The Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO, USA
- Department of Pathology and Immunology, Division of Laboratory and Genomic Medicine, Washington University School of Medicine, St. Louis, MO, USA
| | - Bejan Mahmud
- The Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO, USA
| | - Gautam Dantas
- The Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO, USA.
- Department of Pathology and Immunology, Division of Laboratory and Genomic Medicine, Washington University School of Medicine, St. Louis, MO, USA.
- Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA.
- Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA.
- Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA.
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27
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Marini E, Olivença C, Ramalhete S, Aguirre AM, Ingle P, Melo MN, Antunes W, Minton NP, Hernandez G, Cordeiro TN, Sorg JA, Serrano M, Henriques AO. A sporulation signature protease is required for assembly of the spore surface layers, germination and host colonization in Clostridioides difficile. PLoS Pathog 2023; 19:e1011741. [PMID: 37956166 PMCID: PMC10681294 DOI: 10.1371/journal.ppat.1011741] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2023] [Revised: 11/27/2023] [Accepted: 10/09/2023] [Indexed: 11/15/2023] Open
Abstract
A genomic signature for endosporulation includes a gene coding for a protease, YabG, which in the model organism Bacillus subtilis is involved in assembly of the spore coat. We show that in the human pathogen Clostridioidesm difficile, YabG is critical for the assembly of the coat and exosporium layers of spores. YabG is produced during sporulation under the control of the mother cell-specific regulators σE and σK and associates with the spore surface layers. YabG shows an N-terminal SH3-like domain and a C-terminal domain that resembles single domain response regulators, such as CheY, yet is atypical in that the conserved phosphoryl-acceptor residue is absent. Instead, the CheY-like domain carries residues required for activity, including Cys207 and His161, the homologues of which form a catalytic diad in the B. subtilis protein, and also Asp162. The substitution of any of these residues by Ala, eliminates an auto-proteolytic activity as well as interdomain processing of CspBA, a reaction that releases the CspB protease, required for proper spore germination. An in-frame deletion of yabG or an allele coding for an inactive protein, yabGC207A, both cause misassemby of the coat and exosporium and the formation of spores that are more permeable to lysozyme and impaired in germination and host colonization. Furthermore, we show that YabG is required for the expression of at least two σK-dependent genes, cotA, coding for a coat protein, and cdeM, coding for a key determinant of exosporium assembly. Thus, YabG also impinges upon the genetic program of the mother cell possibly by eliminating a transcriptional repressor. Although this activity has not been described for the B. subtilis protein and most of the YabG substrates vary among sporeformers, the general role of the protease in the assembly of the spore surface is likely to be conserved across evolutionary distance.
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Affiliation(s)
- Eleonora Marini
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República EAN, Oeiras, Portugal
| | - Carmen Olivença
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República EAN, Oeiras, Portugal
| | - Sara Ramalhete
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República EAN, Oeiras, Portugal
| | - Andrea Martinez Aguirre
- Texas A&M University, Department of Biology, College Station, Texas, United States of America
| | - Patrick Ingle
- Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, University of Nottingham, Nottingham, United Kingdom
| | - Manuel N Melo
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República EAN, Oeiras, Portugal
| | - Wilson Antunes
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República EAN, Oeiras, Portugal
| | - Nigel P Minton
- Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, University of Nottingham, Nottingham, United Kingdom
| | - Guillem Hernandez
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República EAN, Oeiras, Portugal
| | - Tiago N Cordeiro
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República EAN, Oeiras, Portugal
| | - Joseph A Sorg
- Texas A&M University, Department of Biology, College Station, Texas, United States of America
| | - Mónica Serrano
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República EAN, Oeiras, Portugal
| | - Adriano O Henriques
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República EAN, Oeiras, Portugal
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28
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Rubio-Mendoza D, Martínez-Meléndez A, Maldonado-Garza HJ, Córdova-Fletes C, Garza-González E. Review of the Impact of Biofilm Formation on Recurrent Clostridioides difficile Infection. Microorganisms 2023; 11:2525. [PMID: 37894183 PMCID: PMC10609348 DOI: 10.3390/microorganisms11102525] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2023] [Revised: 09/25/2023] [Accepted: 09/29/2023] [Indexed: 10/29/2023] Open
Abstract
Clostridioides difficile infection (CDI) may recur in approximately 10-30% of patients, and the risk of recurrence increases with each successive recurrence, reaching up to 65%. C. difficile can form biofilm with approximately 20% of the bacterial genome expressed differently between biofilm and planktonic cells. Biofilm plays several roles that may favor recurrence; for example, it may act as a reservoir of spores, protect the vegetative cells from the activity of antibiotics, and favor the formation of persistent cells. Moreover, the expression of several virulence genes, including TcdA and TcdB toxins, has been associated with recurrence. Several systems and structures associated with adhesion and biofilm formation have been studied in C. difficile, including cell-wall proteins, quorum sensing (including LuxS and Agr), Cyclic di-GMP, type IV pili, and flagella. Most antibiotics recommended for the treatment of CDI do not have activity on spores and do not eliminate biofilm. Therapeutic failure in R-CDI has been associated with the inadequate concentration of drugs in the intestinal tract and the antibiotic resistance of a biofilm. This makes it challenging to eradicate C. difficile in the intestine, complicating antibacterial therapies and allowing non-eliminated spores to remain in the biofilm, increasing the risk of recurrence. In this review, we examine the role of biofilm on recurrence and the challenges of treating CDI when the bacteria form a biofilm.
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Affiliation(s)
- Daira Rubio-Mendoza
- Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey 64460, Mexico; (D.R.-M.); (H.J.M.-G.); (C.C.-F.)
| | - Adrián Martínez-Meléndez
- Facultad de Ciencias Químicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza 66455, Mexico;
| | - Héctor Jesús Maldonado-Garza
- Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey 64460, Mexico; (D.R.-M.); (H.J.M.-G.); (C.C.-F.)
| | - Carlos Córdova-Fletes
- Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey 64460, Mexico; (D.R.-M.); (H.J.M.-G.); (C.C.-F.)
| | - Elvira Garza-González
- Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey 64460, Mexico; (D.R.-M.); (H.J.M.-G.); (C.C.-F.)
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29
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Alenezi T, Fu Y, Alrubaye B, Alanazi T, Almansour A, Wang H, Sun X. Potent Bile Acid Microbial Metabolites Modulate Clostridium perfringens Virulence. Pathogens 2023; 12:1202. [PMID: 37887718 PMCID: PMC10610205 DOI: 10.3390/pathogens12101202] [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: 08/24/2023] [Revised: 09/19/2023] [Accepted: 09/26/2023] [Indexed: 10/28/2023] Open
Abstract
Clostridium perfringens is a versatile pathogen, inducing diseases in the skin, intestine (such as chicken necrotic enteritis (NE)), and other organs. The classical sign of NE is the foul smell gas in the ballooned small intestine. We hypothesized that deoxycholic acid (DCA) reduced NE by inhibiting C. perfringens virulence signaling pathways. To evaluate the hypothesis, C. perfringens strains CP1 and wild-type (WT) HN13 and its mutants were cultured with different bile acids, including DCA and isoallolithocholic acid (isoalloLCA). Growth, hydrogen sulfide (H2S) production, and virulence gene expression were measured. Notably, isoalloLCA was more potent in reducing growth, H2S production, and virulence gene expression in CP1 and WT HN13 compared to DCA, while other bile acids were less potent compared to DCA. Interestingly, there was a slightly different impact between DCA and isoalloLCA on the growth, H2S production, and virulence gene expression in the three HN13 mutants, suggesting possibly different signaling pathways modulated by the two bile acids. In conclusion, DCA and isoalloLCA reduced C. perfringens virulence by transcriptionally modulating the pathogen signaling pathways. The findings could be used to design new strategies to prevent and treat C. perfringens-induced diseases.
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Affiliation(s)
- Tahrir Alenezi
- Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA; (T.A.); (Y.F.); (B.A.); (T.A.); (A.A.); (H.W.)
- Cell and Molecular Biology, University of Arkansas, Fayetteville, AR 72701, USA
- College of Medical Applied Sciences, The Northern Border University, Arar 91431, Saudi Arabia
| | - Ying Fu
- Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA; (T.A.); (Y.F.); (B.A.); (T.A.); (A.A.); (H.W.)
| | - Bilal Alrubaye
- Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA; (T.A.); (Y.F.); (B.A.); (T.A.); (A.A.); (H.W.)
| | - Thamer Alanazi
- Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA; (T.A.); (Y.F.); (B.A.); (T.A.); (A.A.); (H.W.)
- Cell and Molecular Biology, University of Arkansas, Fayetteville, AR 72701, USA
| | - Ayidh Almansour
- Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA; (T.A.); (Y.F.); (B.A.); (T.A.); (A.A.); (H.W.)
- Cell and Molecular Biology, University of Arkansas, Fayetteville, AR 72701, USA
| | - Hong Wang
- Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA; (T.A.); (Y.F.); (B.A.); (T.A.); (A.A.); (H.W.)
- Cell and Molecular Biology, University of Arkansas, Fayetteville, AR 72701, USA
| | - Xiaolun Sun
- Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA; (T.A.); (Y.F.); (B.A.); (T.A.); (A.A.); (H.W.)
- Cell and Molecular Biology, University of Arkansas, Fayetteville, AR 72701, USA
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30
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Ribis JW, Melo L, Shrestha S, Giacalone D, Rodriguez EE, Shen A, Rohlfing A. Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop. mSphere 2023; 8:e0000523. [PMID: 37338207 PMCID: PMC10449524 DOI: 10.1128/msphere.00005-23] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2023] [Accepted: 04/21/2023] [Indexed: 06/21/2023] Open
Abstract
Clostridioides difficile infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation co-germinants in the small intestine. While bile acid germinants are essential for C. difficile spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, are essential for inducing germination, while another proposes that either co-germinant class can induce germination. The former model is based on the finding that spores defective in releasing large stores of internal Ca2+ in the form of calcium dipicolinic acid (CaDPA) cannot germinate when germination is induced with bile acid germinant and amino acid co-germinant alone. However, since the reduced optical density of CaDPA-less spores makes it difficult to accurately measure their germination, we developed a novel automated, time-lapse microscopy-based germination assay to analyze CaDPA mutant germination at the single-spore level. Using this assay, we found that CaDPA mutant spores germinate in the presence of amino acid co-germinant and bile acid germinant. Higher levels of amino acid co-germinants are nevertheless required to induce CaDPA mutant spores to germinate relative to WT spores because CaDPA released by WT spores during germination can function in a feedforward loop to potentiate the germination of other spores within the population. Collectively, these data indicate that Ca2+ is not essential for inducing C. difficile spore germination because amino acid and Ca2+ co-germinant signals are sensed by parallel signaling pathways. IMPORTANCE Clostridioides difficile spore germination is essential for this major nosocomial pathogen to initiate infection. C. difficile spores germinate in response to sensing bile acid germinant signals alongside co-germinant signals. There are two classes of co-germinant signals: Ca2+ and amino acids. Prior work suggested that Ca2+ is essential for C. difficile spore germination based on bulk population analyses of germinating CaDPA mutant spores. Since these assays rely on optical density to measure spore germination and the optical density of CaDPA mutant spores is reduced relative to WT spores, this bulk assay is limited in its capacity to analyze germination. To overcome this limitation, we developed an automated image analysis pipeline to monitor C. difficile spore germination using time-lapse microscopy. With this analysis pipeline, we demonstrate that, although Ca2+ is dispensable for inducing C. difficile spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores.
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Affiliation(s)
- John W. Ribis
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA
- Graduate School of Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts, USA
| | - Luana Melo
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA
| | - Shailab Shrestha
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA
- Graduate School of Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts, USA
| | - David Giacalone
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA
- Graduate School of Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts, USA
| | | | - Aimee Shen
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA
| | - Amy Rohlfing
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA
- Tufts University, Boston, Massachusetts, USA
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Kadowaki R, Tanno H, Maeno S, Endo A. Spore-forming properties and enhanced oxygen tolerance of butyrate-producing Anaerostipes spp. Anaerobe 2023; 82:102752. [PMID: 37301503 DOI: 10.1016/j.anaerobe.2023.102752] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2023] [Revised: 05/25/2023] [Accepted: 06/06/2023] [Indexed: 06/12/2023]
Abstract
OBJECTIVES Butyrate producing bacteria are promising candidates for next-generation probiotics. However, they are extremely sensitive to oxygen, which is a significant obstacle to their inclusion in food matrices in a viable form. The present study characterized the spore-forming properties and stress tolerance of human gut butyrate-producing Anaerostipes spp. METHODS Spore formation properties in six species of Anaerostipes spp. were studied by in vitro and in silico tests. RESULTS Spores were observed from the cells of three species using microscopic analyses, while the remaining three did not form spores under the tested conditions. Spore-forming properties were confirmed by an ethanol treatment. The spores of Anaerostipes caccae were tolerant to oxygen and survived for 15 weeks under atmospheric conditions. Spores tolerated heat stress at 70 °C, but not at 80 °C. An in silico analysis of the conservation of potential sporulation signature genes revealed that the majority of human gut butyrate-producing bacteria were classified as potential spore formers. Comparative genomics revealed that three spore-forming Anaerostipes spp. specifically possessed the spore formation-related genes of bkdR, sodA, and splB, which may be key genes for different sporulation properties in Anaerostipes spp. CONCLUSIONS The present study demonstrated the enhanced stress tolerance of butyrate producing Anaerostipes spp. for future probiotic application. Presence of specific gene(s) are possibly keys for sporulation in Anaerostipes spp.
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Affiliation(s)
- Ren Kadowaki
- Department of Food, Aroma and Cosmetic Chemistry, Faculty of Bioindustry, Tokyo University of Agriculture, 099-2493, Hokkaido, Japan
| | - Hiroki Tanno
- Department of Food, Aroma and Cosmetic Chemistry, Faculty of Bioindustry, Tokyo University of Agriculture, 099-2493, Hokkaido, Japan
| | - Shintaro Maeno
- Graduate School of Medical Sciences, Kyushu University, 812-8582, Fukuoka, Japan
| | - Akihito Endo
- Department of Food, Aroma and Cosmetic Chemistry, Faculty of Bioindustry, Tokyo University of Agriculture, 099-2493, Hokkaido, Japan; Department of Nutritional Science and Food Safety, Faculty of Applied Bioscience, Tokyo University of Agriculture, 156-8502, Tokyo, Japan.
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Lyu F, Zhang T, Gui M, Wang Y, Zhao L, Wu X, Rao L, Liao X. The underlying mechanism of bacterial spore germination: An update review. Compr Rev Food Sci Food Saf 2023; 22:2728-2746. [PMID: 37125461 DOI: 10.1111/1541-4337.13160] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2022] [Revised: 03/22/2023] [Accepted: 04/01/2023] [Indexed: 05/02/2023]
Abstract
Bacterial spores are highly resilient and universally present on earth and can irreversibly enter the food chain to cause food spoilage or foodborne illness once revived to resume vegetative growth. Traditionally, extensive thermal processing has been employed to efficiently kill spores; however, the relatively high thermal load adversely affects food quality attributes. In recent years, the germination-inactivation strategy has been developed to mildly kill spores based on the circumstance that germination can decrease spore-resilient properties. However, the failure to induce all spores to geminate, mainly owing to the heterogeneous germination behavior of spores, hampers the success of applying this strategy in the food industry. Undoubtedly, elucidating the detailed germination pathway and underlying mechanism can fill the gap in our understanding of germination heterogeneity, thereby facilitating the development of full-scale germination regimes to mildly kill spores. In this review, we comprehensively discuss the mechanisms of spore germination of Bacillus and Clostridium species, and update the molecular basis of the early germination events, for example, the activation of germination receptors, ion release, Ca-DPA release, and molecular events, combined with the latest research evidence. Moreover, high hydrostatic pressure (HHP), an advanced non-thermal food processing technology, can also trigger spore germination, providing a basis for the application of a germination-inactivation strategy in HHP processing. Here, we also summarize the diverse germination behaviors and mechanisms of spores of Bacillus and Clostridium species under HHP, with the aim of facilitating HHP as a mild processing technology with possible applications in food sterilization. Practical Application: This work provides fundamental basis for developing efficient killing strategies of bacterial spores in food industry.
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Affiliation(s)
- Fengzhi Lyu
- College of Food Science and Nutritional Engineering, National Engineering Research Center for Fruit and Vegetable Processing, Key Laboratory of Fruit and Vegetable Processing of Ministry of Agriculture and Rural Affairs, Beijing Key Laboratory for Food Non-Thermal Processing, China Agricultural University, Beijing, China
| | - Tianyu Zhang
- College of Food Science and Nutritional Engineering, National Engineering Research Center for Fruit and Vegetable Processing, Key Laboratory of Fruit and Vegetable Processing of Ministry of Agriculture and Rural Affairs, Beijing Key Laboratory for Food Non-Thermal Processing, China Agricultural University, Beijing, China
| | - Meng Gui
- Fisheries Science Institute Beijing Academy of Agriculture and Forestry Sciences, Beijing, China
| | - Yongtao Wang
- College of Food Science and Nutritional Engineering, National Engineering Research Center for Fruit and Vegetable Processing, Key Laboratory of Fruit and Vegetable Processing of Ministry of Agriculture and Rural Affairs, Beijing Key Laboratory for Food Non-Thermal Processing, China Agricultural University, Beijing, China
| | - Liang Zhao
- College of Food Science and Nutritional Engineering, National Engineering Research Center for Fruit and Vegetable Processing, Key Laboratory of Fruit and Vegetable Processing of Ministry of Agriculture and Rural Affairs, Beijing Key Laboratory for Food Non-Thermal Processing, China Agricultural University, Beijing, China
| | - Xiaomeng Wu
- College of Food Science and Nutritional Engineering, National Engineering Research Center for Fruit and Vegetable Processing, Key Laboratory of Fruit and Vegetable Processing of Ministry of Agriculture and Rural Affairs, Beijing Key Laboratory for Food Non-Thermal Processing, China Agricultural University, Beijing, China
| | - Lei Rao
- College of Food Science and Nutritional Engineering, National Engineering Research Center for Fruit and Vegetable Processing, Key Laboratory of Fruit and Vegetable Processing of Ministry of Agriculture and Rural Affairs, Beijing Key Laboratory for Food Non-Thermal Processing, China Agricultural University, Beijing, China
| | - Xiaojun Liao
- College of Food Science and Nutritional Engineering, National Engineering Research Center for Fruit and Vegetable Processing, Key Laboratory of Fruit and Vegetable Processing of Ministry of Agriculture and Rural Affairs, Beijing Key Laboratory for Food Non-Thermal Processing, China Agricultural University, Beijing, China
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33
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Yip C, Phan JR, Abel-Santos E. Mechanism of germination inhibition of Clostridioides difficile spores by an aniline substituted cholate derivative (CaPA). J Antibiot (Tokyo) 2023; 76:335-345. [PMID: 37016015 PMCID: PMC10406169 DOI: 10.1038/s41429-023-00612-3] [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: 02/15/2023] [Revised: 02/28/2023] [Accepted: 03/07/2023] [Indexed: 04/06/2023]
Abstract
Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea and has been declared an urgent threat by the CDC. C. difficile forms dormant and resistant spores that serve as infectious vehicles for CDI. To cause disease, C. difficile spores recognize taurocholate and glycine to trigger the germination process. In contrast to other sporulating bacteria, C. difficile spores are postulated to use a protease complex, CspABC, to recognize its germinants. Since spore germination is required for infection, we have developed anti-germination approaches for CDI prophylaxis. Previously, the bile salt analog CaPA (an aniline-substituted cholic acid) was shown to block spore germination and protect rodents from CDI caused by multiple C. difficile strains and isolates. In this study, we found that CaPA is an alternative substrate inhibitor of C. difficile spore germination. By competing with taurocholate for binding, CaPA delays C. difficile spore germination and reduces spore viability, thus diminishing the number of outgrowing vegetative bacteria. We hypothesize that the reduction of toxin-producing bacterial burden explains CaPA's protective activity against murine CDI. Previous data combined with our results suggests that CaPA binds tightly to C. difficile spores in a CspC-dependent manner and irreversibly traps spores in an alternative, time-delayed, and low yield germination pathway. Our results are also consistent with kinetic data suggesting the existence of at least two distinct bile salt binding sites in C. difficile spores.
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Affiliation(s)
- Christopher Yip
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, NV, 89154, USA
| | - Jacqueline R Phan
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, NV, 89154, USA
| | - Ernesto Abel-Santos
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, NV, 89154, USA.
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34
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Janardhanan J, Kim C, Qian Y, Yang J, Meisel J, Ding D, Speri E, Schroeder V, Wolter W, Oliver A, Mobashery S, Chang M. A dual-action antibiotic that kills Clostridioides difficile vegetative cells and inhibits spore germination. Proc Natl Acad Sci U S A 2023; 120:e2304110120. [PMID: 37155891 PMCID: PMC10193928 DOI: 10.1073/pnas.2304110120] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2023] [Accepted: 03/29/2023] [Indexed: 05/10/2023] Open
Abstract
Clostridioides difficile infection (CDI) is the most lethal of the five CDC urgent public health treats, resulting in 12,800 annual deaths in the United States alone [Antibiotic Resistance Threats in the United States, 2019 (2019), www.cdc.gov/DrugResistance/Biggest-Threats.html]. The high recurrence rate and the inability of antibiotics to treat such infections mandate discovery of new therapeutics. A major challenge with CDI is the production of spores, leading to multiple recurrences of infection in 25% of patients [C. P. Kelly, J. T. LaMont, N. Engl. J. Med. 359, 1932-1940 (2008)], with potentially lethal consequence. Herein, we describe the discovery of an oxadiazole as a bactericidal anti-C. difficile agent that inhibits both cell-wall peptidoglycan biosynthesis and spore germination. We document that the oxadiazole binds to the lytic transglycosylase SleC and the pseudoprotease CspC for prevention of spore germination. SleC degrades the cortex peptidoglycan, a critical step in the initiation of spore germination. CspC senses germinants and cogerminants. Binding to SleC is with higher affinity than that to CspC. Prevention of spore germination breaks the nefarious cycles of CDI recurrence in the face of the antibiotic challenge, which is a primary cause of therapeutic failure. The oxadiazole exhibits efficacy in a mouse model of recurrent CDI and holds promise in clinical treatment of CDI.
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Affiliation(s)
- Jeshina Janardhanan
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN46556
| | - Choon Kim
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN46556
| | - Yuanyuan Qian
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN46556
| | - Jingdong Yang
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN46556
| | - Jayda E. Meisel
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN46556
| | - Derong Ding
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN46556
| | - Enrico Speri
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN46556
| | - Valerie A. Schroeder
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN46556
| | - William R. Wolter
- Freimann Life Sciences Center, University of Notre Dame, Notre Dame, IN46556
| | - Allen G. Oliver
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN46556
| | - Shahriar Mobashery
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN46556
| | - Mayland Chang
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN46556
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35
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Yadegar A, Pakpoor S, Ibrahim FF, Nabavi-Rad A, Cook L, Walter J, Seekatz AM, Wong K, Monaghan TM, Kao D. Beneficial effects of fecal microbiota transplantation in recurrent Clostridioides difficile infection. Cell Host Microbe 2023; 31:695-711. [PMID: 37167952 PMCID: PMC10966711 DOI: 10.1016/j.chom.2023.03.019] [Citation(s) in RCA: 22] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/13/2023]
Abstract
Fecal microbiota transplantation (FMT) is highly effective in preventing recurrent Clostridioides difficile infection (rCDI). However, the mechanisms underpinning its clinical efficacy are incompletely understood. Herein, we provide an overview of rCDI pathogenesis followed by a discussion of potential mechanisms of action focusing on the current understanding of trans-kingdom microbial, metabolic, immunological, and epigenetic mechanisms. We then outline the current research gaps and offer methodological recommendations for future studies to elevate the quality of research and advance knowledge translation. By combining interventional trials with multiomics technology and host and environmental factors, analyzing longitudinally collected biospecimens will generate results that can be validated with animal and other models. Collectively, this will confirm causality and improve translation, ultimately to develop targeted therapies to replace FMT.
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Affiliation(s)
- Abbas Yadegar
- Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Sepideh Pakpoor
- School of Engineering, University of British Columbia, Kelowna, BC, Canada
| | - Fathima F Ibrahim
- National Institute for Health Research Nottingham Biomedical Research Centre, University of Nottingham, Nottingham, UK; Nottingham Digestive Diseases Centre, School of Medicine, University of Nottingham, Nottingham, UK
| | - Ali Nabavi-Rad
- Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Laura Cook
- Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, VIC, Australia
| | - Jens Walter
- School of Microbiology, Department of Medicine and APC Microbiome Ireland, University College Cork, Cork, Ireland
| | - Anna M Seekatz
- Department of Biological Sciences, Clemson University, Clemson, SC, USA
| | - Karen Wong
- Division of Gastroenterology, Department of Medicine, University of Alberta, Edmonton, AB, Canada
| | - Tanya M Monaghan
- National Institute for Health Research Nottingham Biomedical Research Centre, University of Nottingham, Nottingham, UK; Nottingham Digestive Diseases Centre, School of Medicine, University of Nottingham, Nottingham, UK.
| | - Dina Kao
- Division of Gastroenterology, Department of Medicine, University of Alberta, Edmonton, AB, Canada.
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36
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Liggins M, Ramírez Ramírez N, Abel-Santos E. Comparison of sporulation and germination conditions for Clostridium perfringens type A and G strains. Front Microbiol 2023; 14:1143399. [PMID: 37228374 PMCID: PMC10203408 DOI: 10.3389/fmicb.2023.1143399] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2023] [Accepted: 04/14/2023] [Indexed: 05/27/2023] Open
Abstract
Clostridium perfringens is a spore forming, anaerobic, Gram-positive bacterium that causes a range of diseases in humans and animals. C. perfringens forms spores, structures that are derived from the vegetative cell under conditions of nutrient deprivation and that allows survival under harsh environmental conditions. To return to vegetative growth, C. perfringens spores must germinate when conditions are favorable. Previous work in analyzing C. perfringens spore germination has produced strain-specific results. Hence, we analyzed the requirements for spore formation and germination in seven different C. perfringens strains. Our data showed that C. perfringens sporulation conditions are strain-specific, but germination responses are homogenous in all strains tested. C. perfringens spores can germinate using two distinct pathways. The first germination pathway (the amino acid-only pathway or AA) requires L-alanine, L-phenylalanine, and sodium ions (Na+) as co-germinants. L-arginine is not a required germinant but potentiates germination. The AA pathway is inhibited by aromatic amino acids and potassium ions (K+). Bicarbonate (HCO3-), on the other hand, bypasses potassium-mediated inhibition of C. perfringens spore germination through the AA pathway. The second germination pathway (the bile salt / amino acid pathway or BA) is more promiscuous and is activated by several bile salts and amino acids. In contrast to the AA pathway, the BA pathway is insensitive to Na+, although it can be activated by either K+ or HCO3-. We hypothesize that some C. perfringens strains may have evolved these two distinct germination pathways to ensure spore response to different host environments.
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Affiliation(s)
- Marc Liggins
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV, United States
| | - Norma Ramírez Ramírez
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV, United States
- Departamento de Biología, Universidad de Guanajuato, Guanajuato, Mexico
| | - Ernesto Abel-Santos
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV, United States
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37
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Icho S, Ward JS, Tam J, Kociolek LK, Theriot CM, Melnyk RA. Intestinal bile acids provide a surmountable barrier against C. difficile TcdB-induced disease pathogenesis. Proc Natl Acad Sci U S A 2023; 120:e2301252120. [PMID: 37126691 PMCID: PMC10175849 DOI: 10.1073/pnas.2301252120] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2023] [Accepted: 04/04/2023] [Indexed: 05/03/2023] Open
Abstract
Intestinal bile acids play an essential role in the Clostridioides difficile lifecycle having been shown in vitro to modulate various aspects of pathogenesis, including spore germination, vegetative growth, and more recently the action of the primary virulence determinant, TcdB. Here, we investigated whether physiological levels of the total pool of intestinal bile acids in mice and humans protect against TcdB action. Small molecules extracted from the lumenal contents of the small intestine, cecum, colon, and feces were found to inhibit TcdB in accordance with the differential amounts of total bile acids in each compartment. Extracts from antibiotic-treated and germ-free mice, despite harboring dramatically altered bile acid profiles, unexpectedly also prevented TcdB-induced cell rounding to similar extents. We show that protection, however, is surmountable and can be overcome at higher doses of TcdB-typical to those seen during severe C. difficile infection-suggesting that the protective properties of intestinal bile acids are operant primarily under low to moderate toxin levels. Taken together, these findings demonstrate a role for intestinal bile acids in attenuating virulence, provide insights into asymptomatic carriage of toxigenic C. difficile, and inform strategies to manipulate bile acid levels for therapeutic benefit.
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Affiliation(s)
- Simoun Icho
- Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, ONM5G 0A4
- Department of Biochemistry, University of Toronto, Toronto, ONM5S 1A8
| | - Jennifer S. Ward
- Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, ONM5G 0A4
- Department of Biochemistry, University of Toronto, Toronto, ONM5S 1A8
| | - John Tam
- Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, ONM5G 0A4
| | - Larry K. Kociolek
- Ann & Robert H. Lurie, Children’s Hospital of Chicago, Chicago, IL60611
- Department of Pediatrics, Division of Pediatric Infectious Diseases, Northwestern University Feinberg School of Medicine, Chicago, IL60611
| | - Casey M. Theriot
- Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC27606
| | - Roman A. Melnyk
- Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, ONM5G 0A4
- Department of Biochemistry, University of Toronto, Toronto, ONM5S 1A8
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38
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Gao Y, Amon JD, Artzi L, Ramírez-Guadiana FH, Brock KP, Cofsky JC, Marks DS, Kruse AC, Rudner DZ. Bacterial spore germination receptors are nutrient-gated ion channels. Science 2023; 380:387-391. [PMID: 37104613 PMCID: PMC11154005 DOI: 10.1126/science.adg9829] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2023] [Accepted: 03/29/2023] [Indexed: 04/29/2023]
Abstract
Bacterial spores resist antibiotics and sterilization and can remain metabolically inactive for decades, but they can rapidly germinate and resume growth in response to nutrients. Broadly conserved receptors embedded in the spore membrane detect nutrients, but how spores transduce these signals remains unclear. Here, we found that these receptors form oligomeric membrane channels. Mutations predicted to widen the channel initiated germination in the absence of nutrients, whereas those that narrow it prevented ion release and germination in response to nutrients. Expressing receptors with widened channels during vegetative growth caused loss of membrane potential and cell death, whereas the addition of germinants to cells expressing wild-type receptors triggered membrane depolarization. Therefore, germinant receptors act as nutrient-gated ion channels such that ion release initiates exit from dormancy.
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Affiliation(s)
- Yongqiang Gao
- Department of Microbiology, Harvard Medical ScF(2hool, 77 Avenue Louis Pasteur, Boston MA 02115
| | - Jeremy D. Amon
- Department of Microbiology, Harvard Medical ScF(2hool, 77 Avenue Louis Pasteur, Boston MA 02115
- Present Address: Moderna Genomics, 200 Technology Square, Cambridge MA 02139
| | - Lior Artzi
- Department of Microbiology, Harvard Medical ScF(2hool, 77 Avenue Louis Pasteur, Boston MA 02115
- Present Address: Evolved By Nature, 196 Boston Ave, Medford MA 02155
| | | | - Kelly P. Brock
- Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston MA 02115
- Present Address: Kernal Biologics, 238 Main Street, Cambrdige MA 02142
| | - Joshua C. Cofsky
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston MA 02115
| | - Deborah S. Marks
- Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston MA 02115
| | - Andrew C. Kruse
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston MA 02115
| | - David Z. Rudner
- Department of Microbiology, Harvard Medical ScF(2hool, 77 Avenue Louis Pasteur, Boston MA 02115
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Etifa P, Rodríguez C, Harmanus C, Sanders IMJG, Sidorov IA, Mohammed OA, Savage E, Timms AR, Freeman J, Smits WK, Wilcox MH, Baines SD. Non-Toxigenic Clostridioides difficile Strain E4 (NTCD-E4) Prevents Establishment of Primary C. difficile Infection by Epidemic PCR Ribotype 027 in an In Vitro Human Gut Model. Antibiotics (Basel) 2023; 12:435. [PMID: 36978302 PMCID: PMC10044524 DOI: 10.3390/antibiotics12030435] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2022] [Revised: 02/13/2023] [Accepted: 02/14/2023] [Indexed: 02/25/2023] Open
Abstract
Clostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human recurrent CDI. In this study, we evaluated the efficacy of metronidazole-resistant NTCD-E4 in preventing CDI facilitated by a range of antimicrobials in an in vitro human gut model. NTCD-E4 spores (at a dose of 107) were instilled 7 days before a clinical ribotype (RT) 027 (at the same dose) strain (210). In separate experiments, four different antimicrobials were used to perturb gut microbiotas; bacterial populations and cytotoxin production were determined using viable counting and Vero cell cytotoxicity, respectively. RT027 and NTCD-E4 proliferated in the in vitro model when inoculated singly, with RT027 demonstrating high-level cytotoxin (3-5-log10-relative units) production. In experiments where the gut model was pre-inoculated with NTCD-E4, RT027 was remained quiescent and failed to produce cytotoxins. NTCD-E4 showed mutations in hsmA and a gene homologous to CD196-1331, previously linked to medium-dependent metronidazole resistance, but lacked other metronidazole resistance determinants. This study showed that RT027 was unable to elicit simulated infection in the presence of NTCD-E4 following stimulation by four different antimicrobials. These data complement animal and clinical studies in suggesting NTCD offer prophylactic potential in the management of human CDI.
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Affiliation(s)
- Perezimor Etifa
- Department of Food and Nutritional Sciences, School of Chemistry, Food and Pharmacy, Reading RG6 6DZ, UK
| | - César Rodríguez
- Facultad de Microbiología & CIET, Universidad de Costa Rica, San Pedro 11501-2060, Costa Rica
| | - Céline Harmanus
- Leiden University Medical Center, Department of Medical Microbiology, Albinusdreef, P.O. Box 9600, 2300 RC Leiden, The Netherlands
| | - Ingrid M. J. G. Sanders
- Leiden University Medical Center, Department of Medical Microbiology, Albinusdreef, P.O. Box 9600, 2300 RC Leiden, The Netherlands
| | - Igor A. Sidorov
- Leiden University Medical Center, Department of Medical Microbiology, Albinusdreef, P.O. Box 9600, 2300 RC Leiden, The Netherlands
| | - Olufunmilayo A. Mohammed
- Department of Clinical, Pharmaceutical and Biological Sciences, School of Life and Medical Sciences, University of Hertfordshire, Hatfield AL10 9AB, UK
| | - Emily Savage
- Department of Clinical, Pharmaceutical and Biological Sciences, School of Life and Medical Sciences, University of Hertfordshire, Hatfield AL10 9AB, UK
| | - Andrew R. Timms
- Department of Clinical, Pharmaceutical and Biological Sciences, School of Life and Medical Sciences, University of Hertfordshire, Hatfield AL10 9AB, UK
| | - Jane Freeman
- Healthcare Associated Infections Research Group, Leeds Institute of Medical Research, University of Leeds, Leeds LS2 9JT, UK
- Department of Microbiology, Leeds Teaching Hospitals NHS Trust, Leeds LS1 3EX, UK
| | - Wiep Klaas Smits
- Leiden University Medical Center, Department of Medical Microbiology, Albinusdreef, P.O. Box 9600, 2300 RC Leiden, The Netherlands
- Centre for Microbial Cell Biology, Einsteinweg 55, 2333 CC Leiden, The Netherlands
| | - Mark H. Wilcox
- Healthcare Associated Infections Research Group, Leeds Institute of Medical Research, University of Leeds, Leeds LS2 9JT, UK
- Department of Microbiology, Leeds Teaching Hospitals NHS Trust, Leeds LS1 3EX, UK
| | - Simon D. Baines
- Department of Clinical, Pharmaceutical and Biological Sciences, School of Life and Medical Sciences, University of Hertfordshire, Hatfield AL10 9AB, UK
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Soldavini Pelichotti PC, Cejas D, Fernández-Caniggia L, Trejo FM, Pérez PF. Characterization of a Clostridioides difficile ST-293 isolate from a recurrent infection in Argentina. Rev Argent Microbiol 2023:S0325-7541(22)00102-X. [PMID: 36599754 DOI: 10.1016/j.ram.2022.09.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2022] [Revised: 06/30/2022] [Accepted: 09/27/2022] [Indexed: 01/03/2023] Open
Abstract
Clostridioides difficile is an opportunistic spore-forming pathogen responsible for antibiotic-associated diarrhea in humans. C. difficile produces two main toxins: TcdA and TcdB as well as a third toxin named binary toxin (CDT) that is also involved in virulence. The present study aimed at characterizing the C. difficile isolate ALCD3 involved in a relapse episode of nosocomial infection. Molecular characterization showed that isolate ALCD3 belongs to toxinotype 0/v and the MLST analysis demonstrated allelic profile adk:91, atpA:1, dxr:2, glyA: 1, recA:27, sodA: 1 and tpi:1 which corresponds to ST293 (MLST clade: 1). During growth, isolate ALCD3 showed an early increase in the sporulation ratio as well as maximal values of heat resistant forms after 2 days of incubation. Both sporulation kinetics and production of heat resistant forms were faster for isolate ALCD3 than for the reference strain VPI 10463. Germination in the presence of the natural germinant taurocholate was faster for isolate ALCD3 than for strain VPI 10463, which indicates that isolate ALCD3 starts cortex hydrolysis earlier than strain VPI 10463. Furthermore, the co-germinant glycine, induces rapid release of dipicolinic acid (DPA) in isolate ALCD3. These findings indicate that isolate ALCD3 is particularly efficient in both sporulation and germination. The present work represents the first report of the circulation of C. difficile ST293 in Argentina. The ability of isolate ALCD3 to produce toxins and its high sporulation/germination capacity are key features compatible with a microorganism with high dissemination potential and the possibility of inducing recurrent infections.
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Affiliation(s)
- P Cecilia Soldavini Pelichotti
- Cátedra de Microbiología, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Calle 47 y 115, La Plata, Argentina; Centro de Investigación y Desarrollo en Criotecnología de Alimentos, CCT La Plata, CONICET-UNLP, 47 y 116 (s/n), La Plata B1900AJI, Argentina
| | - Daniela Cejas
- Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Instituto de Investigaciones en Bacteriología y Virología Molecular (IBaViM), Ciudad Autónoma de Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina
| | - Liliana Fernández-Caniggia
- Laboratorio de Microbiología, Hospital Alemán, Av. Pueyrredón 1640, Ciudad Autónoma de Buenos Aires, Argentina
| | - Fernando M Trejo
- Cátedra de Microbiología, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Calle 47 y 115, La Plata, Argentina
| | - Pablo F Pérez
- Cátedra de Microbiología, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Calle 47 y 115, La Plata, Argentina; Centro de Investigación y Desarrollo en Criotecnología de Alimentos, CCT La Plata, CONICET-UNLP, 47 y 116 (s/n), La Plata B1900AJI, Argentina.
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Aguirre AM, Adegbite AO, Sorg JA. Clostridioides difficile bile salt hydrolase activity has substrate specificity and affects biofilm formation. NPJ Biofilms Microbiomes 2022; 8:94. [PMID: 36450806 PMCID: PMC9712596 DOI: 10.1038/s41522-022-00358-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2022] [Accepted: 11/14/2022] [Indexed: 12/02/2022] Open
Abstract
The Clostridioides difficile pathogen is responsible for nosocomial infections. Germination is an essential step for the establishment of C. difficile infection (CDI) because toxins that are secreted by vegetative cells are responsible for the symptoms of CDI. Germination can be stimulated by the combinatorial actions of certain amino acids and either conjugated or deconjugated cholic acid-derived bile salts. During synthesis in the liver, cholic acid- and chenodeoxycholic acid-class bile salts are conjugated with either taurine or glycine at the C24 carboxyl. During GI transit, these conjugated bile salts are deconjugated by microbes that express bile salt hydrolases (BSHs). Here, we surprisingly find that several C. difficile strains have BSH activity. We observed this activity in both C. difficile vegetative cells and in spores and that the observed BSH activity was specific to taurine-derived bile salts. Additionally, we find that this BSH activity can produce cholate for metabolic conversion to deoxycholate by C. scindens. The C. scindens-produced deoxycholate signals to C. difficile to initiate biofilm formation. Our results show that C. difficile BSH activity has the potential to influence the interactions between microbes, and this could extend to the GI setting.
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Affiliation(s)
| | | | - Joseph A Sorg
- Department of Biology, Texas A&M University, College Station, TX, 77843, USA.
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Forster ER, Yang X, Tai AK, Hang HC, Shen A. Identification of a Bile Acid-Binding Transcription Factor in Clostridioides difficile Using Chemical Proteomics. ACS Chem Biol 2022; 17:3086-3099. [PMID: 36279369 PMCID: PMC10518218 DOI: 10.1021/acschembio.2c00463] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
Clostridioides difficile is a Gram-positive anaerobic bacterium that is the leading cause of hospital-acquired gastroenteritis in the US. In the gut milieu, C. difficile encounters microbiota-derived, growth-inhibiting bile acids that are thought to be a significant mechanism of colonization resistance. While the levels of certain bile acids in the gut correlate with susceptibility to C. difficile infection, their molecular targets in C. difficile remain unknown. In this study, we sought to use chemical proteomics to identify bile acid-interacting proteins in C. difficile. Using photoaffinity bile acid probes and chemical proteomics, we identified a previously uncharacterized MerR family protein, CD3583 (now BapR), as a putative bile acid-sensing transcription regulator. Our data indicate that BapR specifically binds to and is stabilized by lithocholic acid (LCA) in C. difficile. Although loss of BapR did not affect C. difficile's sensitivity to LCA, ΔbapR cells elongated more in the presence of LCA compared to wild-type cells. Transcriptomics revealed that BapR regulates several gene clusters, with the expression of the mdeA-cd3573 locus being specifically de-repressed in the presence of LCA in a BapR-dependent manner. Electrophoretic mobility shift assays revealed that BapR directly binds to the mdeA promoter region. Because mdeA is involved in amino acid-related sulfur metabolism and the mdeA-cd3573 locus encodes putative transporters, we propose that BapR senses a gastrointestinal tract-specific small molecule, LCA, as an environmental cue for metabolic adaptation.
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Affiliation(s)
- Emily R Forster
- Graduate School of Biomedical Sciences, Tufts University, Boston, Massachusetts 02111, United States
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, United States
| | - Xinglin Yang
- Department of Immunology and Microbiology, Scripps Research, La Jolla, California 92037, United States
| | - Albert K Tai
- Department of Immunology, Tufts University School of Medicine, Boston, Massachusetts 02111, United States
- Data Intensive Studies Center, Tufts University, Medford, Massachusetts 02155, United States
| | - Howard C Hang
- Department of Immunology and Microbiology, Scripps Research, La Jolla, California 92037, United States
- Department of Chemistry, Scripps Research, La Jolla, California 92037, United States
| | - Aimee Shen
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, United States
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Abstract
Clostridioides difficile spores are the infective form for this endospore-forming organism. The vegetative cells are intolerant to oxygen and poor competitors with a healthy gut microbiota. Therefore, in order for C. difficile to establish infection, the spores have to germinate in an environment that supports vegetative growth. To initiate germination, C. difficile uses Csp-type germinant receptors that consist of the CspC and CspA pseudoproteases as the bile acid and cogerminant receptors, respectively. CspB is a subtilisin-like protease that cleaves the inhibitory propeptide from the pro-SleC cortex lytic enzyme, thereby activating it and initiating cortex degradation. Though several locations have been proposed for where these proteins reside within the spore (i.e., spore coat, outer spore membrane, cortex, and inner spore membrane), these have been based, mostly, on hypotheses or prior data in Clostridium perfringens. In this study, we visualized the germination and outgrowth process using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and used immunogold labeling to visualize key germination regulators. These analyses localize these key regulators to the spore cortex region for the first time. IMPORTANCE Germination by C. difficile spores is the first step in the establishment of potentially life-threatening C. difficile infection (CDI). A deeper understanding of the mechanism by which spores germinate may provide insight for how to either prevent spore germination into a disease-causing vegetative form or trigger germination prematurely when the spore is either in the outside environment or in a host environment that does not support the establishment of colonization/disease.
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Gut Microbiota Composition Associated with Clostridioides difficile Colonization and Infection. Pathogens 2022; 11:pathogens11070781. [PMID: 35890026 PMCID: PMC9322938 DOI: 10.3390/pathogens11070781] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2022] [Revised: 07/01/2022] [Accepted: 07/05/2022] [Indexed: 12/12/2022] Open
Abstract
Clostridioides difficile is an anaerobic Gram-positive and spore-forming bacterium. The majority of C. difficile strains produce two toxins, A and B, associated with the development of acute diarrhea and/or colitis. In this review, two situations are distinguished: C. difficile infection (CDI) and asymptomatic colonization (AC). The main objective of this review is to explore the available data related to the link between the gut microbiota and the development of CDI. The secondary aim is to provide more information on why some people colonized with toxigenic C. difficile develop an infection while others show no signs of disease. Several factors, such as the use of antibiotics and proton pump inhibitors, hospitalization, and age, predispose individuals to C. difficile colonization and/or C. difficile infection. The gut microbiota of people with AC showed decreased abundances of Prevotella, Alistipes, Bacteroides, Bifidobacterium, Dorea, Coprococcus, and Roseburia. The gut microbiota of people suffering from CDI showed reductions in the abundances of Lachnospiraceae, Ruminococcaceae, Blautia spp., Prevotella spp., Dialister spp., Bifidobacterium spp., Roseburia spp., Anaerostipes spp., Faecalibacterium spp. and Coprococcus spp., in comparison with healthy people. Furthermore, increases in the abundances of Enterococcaceae and Enterococcus were associated with C. difficile infection.
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Functional and Metagenomic Evaluation of Ibezapolstat for Early Evaluation of Anti-Recurrence Effects in Clostridioides difficile Infection. Antimicrob Agents Chemother 2022; 66:e0224421. [PMID: 35862742 PMCID: PMC9380534 DOI: 10.1128/aac.02244-21] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Reduction of Clostridioides difficile infection (CDI) recurrence is an essential endpoint for CDI-directed antibiotic development that is often not evaluated until Phase III trials. The purpose of this project was to use a functional and metagenomic approach to predict the potential anti-CDI recurrence effect of ibezapolstat, a DNA polymerase IIIC inhibitor, in clinical development for CDI. As part of the Phase I ibezapolstat clinical study, stool samples were collected from 22 healthy volunteers, who were given either ibezapolstat or vancomycin. Stool samples were evaluated for microbiome changes and bile acid concentrations. Ibezapolstat 450 mg and vancomycin, but not ibezapolstat 300 mg, showed statistically significant changes in alpha diversity over time compared to that of a placebo. Beta diversity changes confirmed that microbiota were significantly different between study groups. Vancomycin had a more wide-ranging effect on the microbiome, characterized by an increased proportion of Gammaproteobacteria. Ibezapolstat demonstrated an increased proportion of Actinobacteria, including the Bifidobacteriaceae family. Using a linear regression analysis, vancomycin was associated with significant increases in primary bile acids as well as primary:secondary bile acid ratios. An overabundance of Enterobacteriaceae was most highly correlated with primary bile acid concentrations (r = 0.63; P < 0.0001). Using Phase I healthy volunteer samples, beneficial changes suggestive of a lower risk of CDI recurrence were associated with ibezapolstat compared to vancomycin. This novel omics approach may allow for better and earlier prediction of anti-CDI recurrence effects for antibiotics in the clinical development pipeline.
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Shulpekova Y, Zharkova M, Tkachenko P, Tikhonov I, Stepanov A, Synitsyna A, Izotov A, Butkova T, Shulpekova N, Lapina N, Nechaev V, Kardasheva S, Okhlobystin A, Ivashkin V. The Role of Bile Acids in the Human Body and in the Development of Diseases. MOLECULES (BASEL, SWITZERLAND) 2022; 27:molecules27113401. [PMID: 35684337 PMCID: PMC9182388 DOI: 10.3390/molecules27113401] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/14/2022] [Revised: 05/13/2022] [Accepted: 05/23/2022] [Indexed: 11/28/2022]
Abstract
Bile acids are specific and quantitatively important organic components of bile, which are synthesized by hepatocytes from cholesterol and are involved in the osmotic process that ensures the outflow of bile. Bile acids include many varieties of amphipathic acid steroids. These are molecules that play a major role in the digestion of fats and the intestinal absorption of hydrophobic compounds and are also involved in the regulation of many functions of the liver, cholangiocytes, and extrahepatic tissues, acting essentially as hormones. The biological effects are realized through variable membrane or nuclear receptors. Hepatic synthesis, intestinal modifications, intestinal peristalsis and permeability, and receptor activity can affect the quantitative and qualitative bile acids composition significantly leading to extrahepatic pathologies. The complexity of bile acids receptors and the effects of cross-activations makes interpretation of the results of the studies rather difficult. In spite, this is a very perspective direction for pharmacology.
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Affiliation(s)
- Yulia Shulpekova
- Sechenov First Moscow State Medical University (Sechenov University), 119435 Moscow, Russia; (Y.S.); (M.Z.); (P.T.); (I.T.); (N.L.); (V.N.); (S.K.); (A.O.); (V.I.)
| | - Maria Zharkova
- Sechenov First Moscow State Medical University (Sechenov University), 119435 Moscow, Russia; (Y.S.); (M.Z.); (P.T.); (I.T.); (N.L.); (V.N.); (S.K.); (A.O.); (V.I.)
| | - Pyotr Tkachenko
- Sechenov First Moscow State Medical University (Sechenov University), 119435 Moscow, Russia; (Y.S.); (M.Z.); (P.T.); (I.T.); (N.L.); (V.N.); (S.K.); (A.O.); (V.I.)
| | - Igor Tikhonov
- Sechenov First Moscow State Medical University (Sechenov University), 119435 Moscow, Russia; (Y.S.); (M.Z.); (P.T.); (I.T.); (N.L.); (V.N.); (S.K.); (A.O.); (V.I.)
| | - Alexander Stepanov
- Biobanking Group, Branch of Institute of Biomedical Chemistry “Scientific and Education Center”, 119435 Moscow, Russia; (A.S.); (A.I.); (T.B.)
| | - Alexandra Synitsyna
- Biobanking Group, Branch of Institute of Biomedical Chemistry “Scientific and Education Center”, 119435 Moscow, Russia; (A.S.); (A.I.); (T.B.)
- Correspondence: ; Tel.: +7-499-764-98-78
| | - Alexander Izotov
- Biobanking Group, Branch of Institute of Biomedical Chemistry “Scientific and Education Center”, 119435 Moscow, Russia; (A.S.); (A.I.); (T.B.)
| | - Tatyana Butkova
- Biobanking Group, Branch of Institute of Biomedical Chemistry “Scientific and Education Center”, 119435 Moscow, Russia; (A.S.); (A.I.); (T.B.)
| | | | - Natalia Lapina
- Sechenov First Moscow State Medical University (Sechenov University), 119435 Moscow, Russia; (Y.S.); (M.Z.); (P.T.); (I.T.); (N.L.); (V.N.); (S.K.); (A.O.); (V.I.)
| | - Vladimir Nechaev
- Sechenov First Moscow State Medical University (Sechenov University), 119435 Moscow, Russia; (Y.S.); (M.Z.); (P.T.); (I.T.); (N.L.); (V.N.); (S.K.); (A.O.); (V.I.)
| | - Svetlana Kardasheva
- Sechenov First Moscow State Medical University (Sechenov University), 119435 Moscow, Russia; (Y.S.); (M.Z.); (P.T.); (I.T.); (N.L.); (V.N.); (S.K.); (A.O.); (V.I.)
| | - Alexey Okhlobystin
- Sechenov First Moscow State Medical University (Sechenov University), 119435 Moscow, Russia; (Y.S.); (M.Z.); (P.T.); (I.T.); (N.L.); (V.N.); (S.K.); (A.O.); (V.I.)
| | - Vladimir Ivashkin
- Sechenov First Moscow State Medical University (Sechenov University), 119435 Moscow, Russia; (Y.S.); (M.Z.); (P.T.); (I.T.); (N.L.); (V.N.); (S.K.); (A.O.); (V.I.)
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Liu Y, Zhang S, Zhou W, Hu D, Xu H, Ji G. Secondary Bile Acids and Tumorigenesis in Colorectal Cancer. Front Oncol 2022; 12:813745. [PMID: 35574393 PMCID: PMC9097900 DOI: 10.3389/fonc.2022.813745] [Citation(s) in RCA: 41] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2021] [Accepted: 03/21/2022] [Indexed: 01/11/2023] Open
Abstract
Colorectal cancer (CRC) is one of the most common and deadly cancers in the world and is a typical inflammatory tumor. In recent years, the incidence of CRC has been increasing year by year. There is evidence that the intake of high-fat diet and overweight are associated with the incidence of CRC, among which bile acids play a key role in the pathogenesis of the disease. Studies on the relationship between bile acid metabolism and the occurrence of CRC have gradually become a hot topic, improving the understanding of metabolic factors in the etiology of colorectal cancer. Meanwhile, intestinal flora also plays an important role in the occurrence and development of CRC In this review, the classification of bile acids and their role in promoting the occurrence of CRC are discussed, and we highlights how a high-fat diet affects bile acid metabolism and destroys the integrity of the intestinal barrier and the effects of gut bacteria.
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Affiliation(s)
- Yujing Liu
- Institute of Digestive Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Shengan Zhang
- Institute of Digestive Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Wenjun Zhou
- Institute of Digestive Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Dan Hu
- Department of Internal Medicine of Chinese Medicine, Shanghai Pudong New Area Hospital of Traditional Chinese Medicine, Shanghai, China
| | - Hanchen Xu
- Institute of Digestive Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Guang Ji
- Institute of Digestive Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
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Phothichaisri W, Chankhamhaengdecha S, Janvilisri T, Nuadthaisong J, Phetruen T, Fagan RP, Chanarat S. Potential Role of the Host-Derived Cell-Wall Binding Domain of Endolysin CD16/50L as a Molecular Anchor in Preservation of Uninfected Clostridioides difficile for New Rounds of Phage Infection. Microbiol Spectr 2022; 10:e0236121. [PMID: 35377223 PMCID: PMC9045149 DOI: 10.1128/spectrum.02361-21] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Accepted: 03/20/2022] [Indexed: 01/21/2023] Open
Abstract
Endolysin is a phage-encoded cell-wall hydrolase which degrades the peptidoglycan layer of the bacterial cell wall. The enzyme is often expressed at the late stage of the phage lytic cycle and is required for progeny escape. Endolysins of bacteriophage that infect Gram-positive bacteria often comprises two domains: a peptidoglycan hydrolase and a cell-wall binding domain (CBD). Although the catalytic domain of endolysin is relatively well-studied, the precise role of CBD is ambiguous and remains controversial. Here, we focus on the function of endolysin CBD from a recently isolated Clostridioides difficile phage. We found that the CBD is not required for lytic activity, which is strongly prevented by the surface layer of C. difficile. Intriguingly, hidden Markov model analysis suggested that the endolysin CBD is likely derived from the CWB2 motif of C. difficile cell-wall proteins but possesses a higher binding affinity to bacterial cell-wall polysaccharides. Moreover, the CBD forms a homodimer, formation of which is necessary for interaction with the surface saccharides. Importantly, endolysin diffusion and sequential cytolytic assays showed that CBD of endolysin is required for the enzyme to be anchored to post-lytic cell-wall remnants, suggesting its physiological roles in limiting diffusion of the enzyme, preserving neighboring host cells, and thereby enabling the phage progeny to initiate new rounds of infection. Taken together, this study provides an insight into regulation of endolysin through CBD and can potentially be applied for endolysin treatment against C. difficile infection. IMPORTANCE Endolysin is a peptidoglycan hydrolase encoded in a phage genome. The enzyme is attractive due to its potential use as antibacterial treatment. To utilize endolysin for the therapeutic propose, understanding of the fundamental role of endolysin becomes important. Here, we investigate the function of cell-wall binding domain (CBD) of an endolysin from a C. difficile phage. The domain is homologous to a cell-wall associating module of bacterial cell-wall proteins, likely acquired during phage-host coevolution. The interaction of CBD to bacterial cell walls reduces enzyme diffusion and thereby limits cell lysis of the neighboring bacteria. Our findings indicate that the endolysin is trapped to the cell-wall residuals through CBD and might serve as an advantage for phage replication. Thus, employing a CBD-less endolysin might be a feasible strategy for using endolysin for the treatment of C. difficile infection.
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Affiliation(s)
- Wichuda Phothichaisri
- Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand
| | | | - Tavan Janvilisri
- Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand
| | - Jirayu Nuadthaisong
- Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand
| | - Tanaporn Phetruen
- Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand
| | - Robert P. Fagan
- School of Biosciences, Florey Institute, University of Sheffield, Sheffield, United Kingdom
| | - Sittinan Chanarat
- Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand
- Laboratory of Molecular Cell Biology, Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok, Thailand
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Alves F, Nunes A, Castro R, Sequeira A, Moreira O, Matias R, Rodrigues JC, Silveira L, Gomes JP, Oleastro M. Assessment of the Transmission Dynamics of Clostridioides difficile in a Farm Environment Reveals the Presence of a New Toxigenic Strain Connected to Swine Production. Front Microbiol 2022; 13:858310. [PMID: 35495679 PMCID: PMC9050547 DOI: 10.3389/fmicb.2022.858310] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2022] [Accepted: 03/21/2022] [Indexed: 01/05/2023] Open
Abstract
The recent increase in community-acquired Clostridioides difficile infections discloses the shift in this bacterium epidemiology. This study aimed at establishing a transmission network involving One Health components, as well as assessing the zoonotic potential and genomic features of dominant clones. Samples were collected from different compartments of animal, human and environmental origin, from an animal production unit. C. difficile isolates were characterized for toxigenic profile by multiplex-PCR, while genetic diversity was evaluated by PCR-ribotyping and whole genome-based analysis. The overall C. difficile prevalence was 37.2% (70/188), and included samples from environmental (58.3%, 35/60) and animal (31.5%, 35/111) compartments; human samples (n = 17) taken from healthy workers were negative. A predominant clone from RT033 was found in almost 90% of the positive samples, including samples from all compartments connected to the pig production unit, with core-genome single nucleotide variant (SNV)-based Analysis supporting a clonal transmission between them (mean distance of 0.1 ± 0.1 core-SNVs). The isolates from this clone (herein designated PT RT033) were positive for all C. difficile toxin genes (tcdA, tcdB, cdtA/cdtB). The phyloGenetic positioning of this clone was clearly distinct from the classical RT033 cluster, suggesting a different evolutionary route. This new clone shares genomic features with several RTs from the clade 5 Sequence Type (ST) 11, including a complete pathogenicity locus (PaLoc) that is more similar to the one found in toxigenic strains and contrasting to the less virulent classical RT033 (tcdA-, tcdB-, cdtA + /cdtB +). The presence of a tcdA gene truncated into two ORFs, not previously described, requires further evaluation concerning toxin functionality. We hypothesize that the unique combination of genetic elements found in the PT RT033 clone may contribute to host tropism and environmental dissemination and maintenance. This study constitutes the first report of a toxigenic RT033 clone and adds to the overall knowledge on Clade 5 sequence type 11, considered the C. difficile evolutionary lineage with the highest zoonotic potential. The presence of this clone in all compartments associated with the pig production unit suggests a transmission chain involving these animals and contributes to unveil the role played by animal and environmental reservoirs in this pathogen epidemiology.
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Affiliation(s)
- Frederico Alves
- Infectious Diseases Department, National Institute of Health Dr. Ricardo Jorge (INSA), Lisbon, Portugal
| | - Alexandra Nunes
- Infectious Diseases Department, National Institute of Health Dr. Ricardo Jorge (INSA), Lisbon, Portugal
- Faculty of Veterinary Medicine, Lusófona University, Lisbon, Portugal
- CBIOS – Lusófona University Research Centre for Biosciences & Health Technologies, Lisbon, Portugal
| | - Rita Castro
- Infectious Diseases Department, National Institute of Health Dr. Ricardo Jorge (INSA), Lisbon, Portugal
| | - António Sequeira
- National Zootechnical Station, National Institute for Agrarian and Veterinarian Research, Santarém, Portugal
| | - Olga Moreira
- National Zootechnical Station, National Institute for Agrarian and Veterinarian Research, Santarém, Portugal
| | - Rui Matias
- Infectious Diseases Department, National Institute of Health Dr. Ricardo Jorge (INSA), Lisbon, Portugal
| | - João Carlos Rodrigues
- Infectious Diseases Department, National Institute of Health Dr. Ricardo Jorge (INSA), Lisbon, Portugal
| | - Leonor Silveira
- Infectious Diseases Department, National Institute of Health Dr. Ricardo Jorge (INSA), Lisbon, Portugal
| | - João Paulo Gomes
- Infectious Diseases Department, National Institute of Health Dr. Ricardo Jorge (INSA), Lisbon, Portugal
- Faculty of Veterinary Medicine, Lusófona University, Lisbon, Portugal
| | - Mónica Oleastro
- Infectious Diseases Department, National Institute of Health Dr. Ricardo Jorge (INSA), Lisbon, Portugal
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Hazleton KZ, Martin CG, Orlicky DJ, Arnolds KL, Nusbacher NM, Moreno-Huizar N, Armstrong M, Reisdorph N, Lozupone CA. Dietary fat promotes antibiotic-induced Clostridioides difficile mortality in mice. NPJ Biofilms Microbiomes 2022; 8:15. [PMID: 35365681 PMCID: PMC8975876 DOI: 10.1038/s41522-022-00276-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2019] [Accepted: 02/21/2022] [Indexed: 11/15/2022] Open
Abstract
Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea, and emerging evidence has linked dietary components with CDI pathogenesis, suggesting that dietary modulation may be an effective strategy for prevention. Here, we show that mice fed a high-fat/low-fiber “Western-type” diet (WD) had dramatically increased mortality in a murine model of antibiotic-induced CDI compared to a low-fat/low-fiber (LF/LF) diet and standard mouse chow controls. We found that the WD had a pro- C. difficile bile acid composition that was driven in part by higher levels of primary bile acids that are produced to digest fat, and a lower level of secondary bile acids that are produced by the gut microbiome. This lack of secondary bile acids was associated with a greater disturbance to the gut microbiome with antibiotics in both the WD and LF/LF diet compared to mouse chow. Mice fed the WD also had the highest level of toxin TcdA just prior to the onset of mortality, but not of TcdB or increased inflammation. These findings indicate that dietary intervention to decrease fat may complement previously proposed dietary intervention strategies to prevent CDI in high-risk individuals.
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Affiliation(s)
- Keith Z Hazleton
- Department of Pediatrics, Section of Gastroenterology, Hepatology and Nutrition. University of Colorado, Denver Anschutz Medical Campus, Aurora, CO, 80045, USA.,Digestive Health Institute, Children's Hospital Colorado, Aurora, CO, 80045, USA.,Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, University of Arizona, Tucson, AZ, 85719, USA
| | - Casey G Martin
- Department of Immunology and Microbiology, University of Colorado, Denver Anschutz Medical Campus, Aurora, CO, 80045, USA
| | - David J Orlicky
- Department of Pathology, University of Colorado, Denver Anschutz Medical Campus, Aurora, CO, 80045, USA
| | - Kathleen L Arnolds
- Department of Immunology and Microbiology, University of Colorado, Denver Anschutz Medical Campus, Aurora, CO, 80045, USA
| | - Nichole M Nusbacher
- Department of Medicine, Division of Biomedical Informatics and Personalized Medicine, University of Colorado, Denver Anschutz Medical Campus, Aurora, CO, 80045, USA
| | - Nancy Moreno-Huizar
- Department of Medicine, Division of Biomedical Informatics and Personalized Medicine, University of Colorado, Denver Anschutz Medical Campus, Aurora, CO, 80045, USA
| | - Michael Armstrong
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA
| | - Nichole Reisdorph
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA
| | - Catherine A Lozupone
- Department of Medicine, Division of Biomedical Informatics and Personalized Medicine, University of Colorado, Denver Anschutz Medical Campus, Aurora, CO, 80045, USA.
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