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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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2
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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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3
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Sharma SK, Schilke AR, Phan JR, Yip C, Sharma PV, Abel-Santos E, Firestine SM. The design, synthesis, and inhibition of Clostridioides difficile spore germination by acyclic and bicyclic tertiary amide analogs of cholate. Eur J Med Chem 2023; 261:115788. [PMID: 37703709 PMCID: PMC10680100 DOI: 10.1016/j.ejmech.2023.115788] [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: 06/01/2023] [Revised: 08/30/2023] [Accepted: 08/31/2023] [Indexed: 09/15/2023]
Abstract
Clostridioides difficile infection (CDI) is a major identifiable cause of antibiotic-associated diarrhea. In our previous study (J. Med. Chem., 2018, 61, 6759-6778), we have identified N-phenyl-cholan-24-amide as a potent inhibitor of spore germination. The most potent compounds in our previous work are N-arylamides. We were interested in the role that the conformation of the amide plays in activity. Previous research has shown that secondary N-arylamides exist exclusively in the coplanar trans conformation while tertiary N-methyl-N-arylamides exist in a non-planar, cis conformation. The N-methyl-N-phenyl-cholan-24-amide was 17-fold less active compared to the parent compounds suggesting the importance of the orientation of the phenyl ring. To lock the phenyl ring into a trans conformation, cyclic tertiary amides were prepared. Indoline and quinoline cholan-24-amides were both inhibitors of spore germination; however, the indoline analogs were most potent. Isoindoline and isoquinoline amides were inactive. We found that the simple indoline derivative gave an IC50 value of 1 μM, while the 5'-fluoro-substituted compound (5d) possessed an IC50 of 400 nM. To our knowledge, 5d is the most potent known spore germination inhibitor described to date. Taken together, our results indicate that the trans, coplanar conformation of the phenyl ring is required for potent inhibition.
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Affiliation(s)
- Shiv K Sharma
- Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI, 48201, USA
| | - Angel R Schilke
- Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI, 48201, USA
| | - Jacqueline R Phan
- Department of Chemistry and Biochemistry, University of Nevada -Las Vegas, 4505 S. Maryland Pkwy, Las Vegas, NV, 89154, USA
| | - Christopher Yip
- Department of Chemistry and Biochemistry, University of Nevada -Las Vegas, 4505 S. Maryland Pkwy, Las Vegas, NV, 89154, USA
| | - Prateek V Sharma
- Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI, 48201, USA
| | - Ernesto Abel-Santos
- Department of Chemistry and Biochemistry, University of Nevada -Las Vegas, 4505 S. Maryland Pkwy, Las Vegas, NV, 89154, USA
| | - Steven M Firestine
- Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI, 48201, USA.
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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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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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Cheng JKJ, Unnikrishnan M. Clostridioides difficile infection: traversing host-pathogen interactions in the gut. MICROBIOLOGY (READING, ENGLAND) 2023; 169. [PMID: 36848200 DOI: 10.1099/mic.0.001306] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/01/2023]
Abstract
C. difficile is the primary cause for nosocomial infective diarrhoea. For a successful infection, C. difficile must navigate between resident gut bacteria and the harsh host environment. The perturbation of the intestinal microbiota by broad-spectrum antibiotics alters the composition and the geography of the gut microbiota, deterring colonization resistance, and enabling C. difficile to colonize. This review will discuss how C. difficile interacts with and exploits the microbiota and the host epithelium to infect and persist. We provide an overview of C. difficile virulence factors and their interactions with the gut to aid adhesion, cause epithelial damage and mediate persistence. Finally, we document the host responses to C. difficile, describing the immune cells and host pathways that are associated and triggered during C. difficile infection.
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Affiliation(s)
- Jeffrey K J Cheng
- Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, CV4 7AL, UK
| | - Meera Unnikrishnan
- Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, CV4 7AL, UK
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Sundaresan A, Le Ngoc M, Wew MU, Ramkumar V, Raninga P, Sum R, Cheong I. A design of experiments screen reveals that Clostridium novyi-NT spore germinant sensing is stereoflexible for valine and its analogs. Commun Biol 2023; 6:118. [PMID: 36709236 PMCID: PMC9884283 DOI: 10.1038/s42003-023-04496-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2022] [Accepted: 01/17/2023] [Indexed: 01/30/2023] Open
Abstract
Although Clostridium novyi-NT is an anti-cancer bacterial therapeutic which germinates within hypoxic tumors to kill cancer cells, the actual germination triggers for C. novyi-NT are still unknown. In this study, we screen candidate germinants using combinatorial experimental designs and discover by serendipity that D-valine is a potent germinant, inducing 50% spore germination at 4.2 mM concentration. Further investigation revealed that five D-valine analogs are also germinants and four of these analogs are enantiomeric pairs. This stereoflexible effect of L- and D-amino acids shows that spore germination is a complex process where enantiomeric interactions can be confounders. This study also identifies L-cysteine as a germinant, and hypoxanthine and inosine as co-germinants. Several other amino acids promote (L-valine, L-histidine, L-threonine and L-alanine) or inhibit (L-arginine, L-glycine, L-lysine, L-tryptophan) germination in an interaction-dependent manner. D-alanine inhibits all germination, even in complex growth media. This work lays the foundation for improving the germination efficacy of C. novyi-NT spores in tumors.
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Affiliation(s)
- Ajitha Sundaresan
- grid.226688.00000 0004 0620 9198Temasek Life Sciences Laboratory, Singapore, Singapore ,grid.4280.e0000 0001 2180 6431Department of Biological Sciences, National University of Singapore, Singapore, Singapore
| | - Mai Le Ngoc
- grid.4280.e0000 0001 2180 6431NUS High School of Mathematics and Sciences, Singapore, Singapore
| | - Marvell Ung Wew
- grid.4280.e0000 0001 2180 6431NUS High School of Mathematics and Sciences, Singapore, Singapore
| | - Varsha Ramkumar
- grid.4280.e0000 0001 2180 6431NUS High School of Mathematics and Sciences, Singapore, Singapore
| | - Prahlad Raninga
- grid.226688.00000 0004 0620 9198Temasek Life Sciences Laboratory, Singapore, Singapore
| | - Rongji Sum
- grid.226688.00000 0004 0620 9198Temasek Life Sciences Laboratory, Singapore, Singapore ,grid.4280.e0000 0001 2180 6431Department of Biological Sciences, National University of Singapore, Singapore, Singapore
| | - Ian Cheong
- grid.226688.00000 0004 0620 9198Temasek Life Sciences Laboratory, Singapore, Singapore ,grid.4280.e0000 0001 2180 6431Department of Biological Sciences, National University of Singapore, Singapore, Singapore
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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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Abstract
Transmission of bacterial endospores between the environment and people and the following germination in vivo play critical roles in both the deadly infections of some bacterial pathogens and the stabilization of the commensal microbiotas in humans. Our knowledge about the germination process of different bacteria in the mammalian gut, however, is still very limited due to the lack of suitable tools to visually monitor this process. We proposed a two-step labeling strategy that can image and quantify the endospores' germination in the recipient's intestines. Endospores collected from donor's gut microbiota were first labeled with fluorescein isothiocyanate and transplanted to mice via gavage. The recipient mice were then administered with Cyanine5-tagged D-amino acid to label all the viable bacteria, including the germinated endospores, in their intestines in situ. The germinated donor endospores could be distinguished by presenting two types of fluorescent signals simultaneously. The integrative use of cell-sorting, 16S rDNA sequencing, and fluorescence in situ hybridization (FISH) staining of the two-colored bacteria unveiled the taxonomic information of the donor endospores that germinated in the recipient's gut. Using this strategy, we investigated effects of different germinants and pre-treatment interventions on their germination, and found that germination of different commensal bacterial genera was distinctly affected by various types of germinants. This two-color labeling strategy shows its potential as a versatile tool for visually monitoring endospore germination in the hosts and screening for new interventions to improve endospore-based therapeutics.
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Affiliation(s)
- Ningning Xu
- Institute of Molecular Medicine, Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Liyuan Lin
- Institute of Molecular Medicine, Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Yahui Du
- The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, Key Laboratory for Chemical Biology of Fujian Province State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China
| | - Huibin Lin
- Institute of Molecular Medicine, Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Jia Song
- Institute of Molecular Medicine, Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Chaoyong Yang
- Institute of Molecular Medicine, Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China,The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, Key Laboratory for Chemical Biology of Fujian Province State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China,CONTACT Chaoyong Yang
| | - Wei Wang
- Institute of Molecular Medicine, Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China,Wei Wang Institute of Molecular Medicine, Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai200127, China
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10
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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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11
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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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12
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Baloh M, Sorg JA. Clostridioides difficile spore germination: initiation to DPA release. Curr Opin Microbiol 2022; 65:101-107. [PMID: 34808546 PMCID: PMC8792321 DOI: 10.1016/j.mib.2021.11.001] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2021] [Revised: 11/01/2021] [Accepted: 11/04/2021] [Indexed: 02/03/2023]
Abstract
Germination by Clostridioides difficile spores is an essential step in pathogenesis. Spores are metabolically dormant forms of bacteria that resist severe conditions. Work over the last 10 years has elucidated that C. difficile spores germinate thorough a novel pathway. This review summarizes our understanding of C. difficile spore germination and the factors involved in germinant recognition, cortex degradation and DPA release.
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Affiliation(s)
- Marko Baloh
- Department of Biology, Texas A&M University, College Station, TX 77843
| | - Joseph A. Sorg
- Department of Biology, Texas A&M University, College Station, TX 77843,Corresponding author: ph: 979-845-6299,
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13
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Sharma SK, Yip C, Simon MP, Phan J, Abel-Santos E, Firestine SM. Studies on the Importance of the 7α-, and 12α- hydroxyl groups of N-Aryl-3α,7α,12α-trihydroxy-5β-cholan-24-amides on their Antigermination Activity Against a Hypervirulent Strain of Clostridioides (Clostridium) difficile. Bioorg Med Chem 2021; 52:116503. [PMID: 34837818 DOI: 10.1016/j.bmc.2021.116503] [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: 08/10/2021] [Revised: 10/19/2021] [Accepted: 11/02/2021] [Indexed: 10/19/2022]
Abstract
Chenodeoxycholic acid (CDCA) is a natural germination inhibitor for C. difficile spores. In our previous study (J. Med. Chem., 2018, 61, 6759-6778), we identified N-phenyl-3α,7α,12α-trihydroxy-5β-cholan-24-amide as an inhibitor of C. difficile strain R20291 with an IC50 of 1.8 μM. Studies of bile salts on spore germination have shown that chenodeoxycholate, ursodeoxycholate and lithocholate are more potent inhibitors of germination compared to cholate. Given this, we created amide analogs of chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids using amines identified from our previous studies. We found that chenodeoxy- and deoxycholate derivatives were active with potencies equivalent to those for cholanamides. This indicates that only 2 out of the 3 hydroxyl groups are needed for activity and that the alpha stereochemistry at position 7 is required for inhibition of spore germination.
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Affiliation(s)
- Shiv K Sharma
- Department of Pharmaceutical sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48201, United States
| | - Christopher Yip
- Department of Chemistry and Biochemistry, University of Nevada at Las Vegas, 4505 S. Maryland Pkwy., Las Vegas, NV, 89154, United States
| | - Matthew P Simon
- Department of Pharmaceutical sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48201, United States
| | - Jacqueline Phan
- Department of Chemistry and Biochemistry, University of Nevada at Las Vegas, 4505 S. Maryland Pkwy., Las Vegas, NV, 89154, United States
| | - Ernesto Abel-Santos
- Department of Chemistry and Biochemistry, University of Nevada at Las Vegas, 4505 S. Maryland Pkwy., Las Vegas, NV, 89154, United States
| | - Steven M Firestine
- Department of Pharmaceutical sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48201, United States.
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14
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An aniline-substituted bile salt analog protects both mice and hamsters from multiple Clostridioides difficile strains. Antimicrob Agents Chemother 2021; 66:e0143521. [PMID: 34780262 DOI: 10.1128/aac.01435-21] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea. The emergence of hypervirulent C. difficile strains has led to increases in both hospital- and community-acquired CDI. Furthermore, CDI relapse from hypervirulent strains can reach up to 25%. Thus, standard treatments are rendered less effective, making new methods of prevention and treatment more critical. Previously, the bile salt analog CamSA was shown to inhibit spore germination in vitro and protect mice and hamsters from C. difficile strain 630. Here, we show that CamSA was less active at preventing spore germination of other C. difficile ribotypes, including the hypervirulent strain R20291. Strain-specific in vitro germination activity of CamSA correlated with its ability to prevent CDI in mice. Additional bile salt analogs were screened for in vitro germination inhibition activity against strain R20291, and the most active compounds were tested against other strains. An aniline-substituted bile salt analog, (CaPA), was found to be a better anti-germinant than CamSA against eight different C. difficile strains. In addition, CaPA was capable of reducing, delaying, or preventing murine CDI signs in all strains tested. CaPA-treated mice showed no obvious toxicity and showed minor effects on their gut microbiome. CaPA's efficacy was further confirmed by its ability to prevent CDI in hamsters infected with strain 630. These data suggest that C. difficile spores respond to germination inhibitors in a strain-dependent manner. However, careful screening can identify anti-germinants with broad CDI prophylaxis activity.
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Clostridioides difficile SpoVAD and SpoVAE Interact and Are Required for Dipicolinic Acid Uptake into Spores. J Bacteriol 2021; 203:e0039421. [PMID: 34424035 DOI: 10.1128/jb.00394-21] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Clostridioides difficile spores, like the spores from most endospore-forming organisms, are a metabolically dormant stage of development with a complex structure that conveys considerable resistance to environmental conditions, e.g., wet heat. This resistance is due to the large amount of dipicolinic acid (DPA) that is taken up by the spore core, preventing rotational motion of the core proteins. DPA is synthesized by the mother cell, and its packaging into the spore core is mediated by the products of the spoVA operon, which has a variable number of genes, depending on the organism. C. difficile encodes 3 spoVA orthologues, spoVAC, spoVAD, and spoVAE. Prior work has shown that C. difficile SpoVAC is a mechanosensing protein responsible for DPA release from the spore core upon the initiation of germination. However, the roles of SpoVAD and SpoVAE remain unclear in C. difficile. In this study, we analyzed the roles of SpoVAD and SpoVAE and found that they are essential for DPA uptake into the spore, similar to SpoVAC. Using split luciferase protein interaction assays, we found that these proteins interact, and we propose a model where SpoVAC/SpoVAD/SpoVAE proteins interact at or near the inner spore membrane, and each member of the complex is essential for DPA uptake into the spore core. IMPORTANCE C. difficile spore heat resistance provides an avenue for it to survive the disinfection protocols in hospital and community settings. The spore heat resistance is mainly the consequence of the high DPA content within the spore core. By elucidating the mechanism by which DPA is taken up by the spore core, this study may provide insight into how to disrupt the spore heat resistance with the aim of making the current disinfection protocols more efficient at preventing the spread of C. difficile in the environment.
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Onizuka S, Tanaka M, Mishima R, Nakayama J. Cultivation of Spore-Forming Gut Microbes Using a Combination of Bile Acids and Amino Acids. Microorganisms 2021; 9:1651. [PMID: 34442730 PMCID: PMC8401671 DOI: 10.3390/microorganisms9081651] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Revised: 07/28/2021] [Accepted: 07/28/2021] [Indexed: 12/16/2022] Open
Abstract
Spores of certain species belonging to Firmicutes are efficiently germinated by nutrient germinators, such as amino acids, in addition to bile acid. We attempted to culture difficult-to-culture or yet-to-be cultured spore-forming intestinal bacteria, using a combination of bile acids and amino acids. The combination increased the number of colonies that formed on agar medium plated with ethanol-treated feces. The operational taxonomic units of these colonized bacteria were classified into two types. One type was colonized only by the bile acid (BA) mixture and the other type was colonized using amino acids, in addition to the BA mixture. The latter contained 13 species, in addition to 14 species of the former type, which mostly corresponds to anaerobic difficult-to-culture Clostridiales species, including several new species candidates. The use of a combination of BAs and amino acids effectively increased the culturability of spore-forming intestinal bacteria.
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Affiliation(s)
| | | | | | - Jiro Nakayama
- Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Nishi-ku, Fukuoka 819-0395, Japan; (S.O.); (M.T.); (R.M.)
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17
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Yip C, Okada NC, Howerton A, Amei A, Abel-Santos E. Pharmacokinetics of CamSA, a potential prophylactic compound against Clostridioides difficile infections. Biochem Pharmacol 2021; 183:114314. [PMID: 33152344 PMCID: PMC7770080 DOI: 10.1016/j.bcp.2020.114314] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2020] [Revised: 10/28/2020] [Accepted: 10/29/2020] [Indexed: 12/30/2022]
Abstract
Clostridioides difficile infections (CDI) are the leading cause of nosocomial antibiotic-associated diarrhea. C. difficile produces dormant spores that serve as infectious agents. Bile salts in the gastrointestinal tract signal spores to germinate into toxin-producing cells. As spore germination is required for CDI onset, anti-germination compounds may serve as prophylactics. CamSA, a synthetic bile salt, was previously shown to inhibit C. difficile spore germination in vitro and in vivo. Unexpectedly, a single dose of CamSA was sufficient to offer multi-day protection from CDI in mice without any observable toxicity. To study this intriguing protection pattern, we examined the pharmacokinetic parameters of CamSA. CamSA was stable to the gut of antibiotic-treated mice but was extensively degraded by the microbiota of non-antibiotic-treated animals. Our data also suggest that CamSA's systemic absorption is minimal since it is retained primarily in the intestinal lumen and liver. CamSA shows weak interactions with CYP3A4, a P450 hepatic isozyme involved in drug metabolism and bile salt modification. Like other bile salts, CamSA seems to undergo enterohepatic circulation. We hypothesize that the cycling of CamSA between the liver and intestines serves as a slow-release mechanism that allows CamSA to be retained in the gastrointestinal tract for days. This model explains how a single CamSA dose can prevent murine CDI even though spores are present in the animal's intestine for up to four days post-challenge.
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Affiliation(s)
- Christopher Yip
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, 4505 S. Maryland Parkway, Las Vegas, Nevada 89154, United States
| | - Naomi C Okada
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, 4505 S. Maryland Parkway, Las Vegas, Nevada 89154, United States
| | - Amber Howerton
- Department of Physical and Life Sciences, Nevada State College, 1300 Nevada State Drive, Henderson, Nevada, 89002, United States
| | - Amei Amei
- Department of Mathematical Sciences, University of Nevada, Las Vegas, 4505 S. Maryland Parkway, Las Vegas, Nevada 89154, United States
| | - Ernesto Abel-Santos
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, 4505 S. Maryland Parkway, Las Vegas, Nevada 89154, United States.
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Stewart D, Anwar F, Vedantam G. Anti-virulence strategies for Clostridioides difficile infection: advances and roadblocks. Gut Microbes 2020; 12:1802865. [PMID: 33092487 PMCID: PMC7588222 DOI: 10.1080/19490976.2020.1802865] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/05/2020] [Revised: 07/21/2020] [Accepted: 07/23/2020] [Indexed: 02/03/2023] Open
Abstract
Clostridioides difficile infection (CDI) is a common healthcare- and antibiotic-associated diarrheal disease. If mis-diagnosed, or incompletely treated, CDI can have serious, indeed fatal, consequences. The clinical and economic burden imposed by CDI is great, and the US Centers for Disease Control and Prevention has named the causative agent, C. difficile (CD), as an Urgent Threat To US healthcare. CDI is also a significant problem in the agriculture industry. Currently, there are no FDA-approved preventives for this disease, and the only approved treatments for both human and veterinary CDI involve antibiotic use, which, ironically, is associated with disease relapse and the threat of burgeoning antibiotic resistance. Research efforts in multiple laboratories have demonstrated that non-toxin factors also play key roles in CDI, and that these are critical for disease. Specifically, key CD adhesins, as well as other surface-displayed factors have been shown to be major contributors to host cell attachment, and as such, represent attractive targets for anti-CD interventions. However, research on anti-virulence approaches has been more limited, primarily due to the lack of genetic tools, and an as-yet nascent (but increasingly growing) appreciation of immunological impacts on CDI. The focus of this review is the conceptualization and development of specific anti-virulence strategies to combat CDI. Multiple laboratories are focused on this effort, and the field is now at an exciting stage with numerous products in development. Herein, however, we focus only on select technologies (Figure 1) that have advanced near, or beyond, pre-clinical testing (not those that are currently in clinical trial), and discuss roadblocks associated with their development and implementation.
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Affiliation(s)
- David Stewart
- Department of Surgery, University of Arizona, Tucson, AZ, USA
| | - Farhan Anwar
- School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, USA
| | - Gayatri Vedantam
- School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, USA
- Bio5 Institute for Collaborative Research, University of Arizona, Tucson, AZ, USA
- Southern Arizona VA Healthcare System, University of Arizona, Tucson, AZ, USA
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The (p)ppGpp Synthetase RSH Mediates Stationary-Phase Onset and Antibiotic Stress Survival in Clostridioides difficile. J Bacteriol 2020; 202:JB.00377-20. [PMID: 32661079 DOI: 10.1128/jb.00377-20] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Accepted: 07/09/2020] [Indexed: 12/31/2022] Open
Abstract
The human pathogen Clostridioides difficile is increasingly tolerant of multiple antibiotics and causes infections with a high rate of recurrence, creating an urgent need for new preventative and therapeutic strategies. The stringent response, a universal bacterial response to extracellular stress, governs antibiotic survival and pathogenesis in diverse organisms but has not previously been characterized in C. difficile Here, we report that the C. difficile (p)ppGpp synthetase RSH is incapable of utilizing GTP or GMP as a substrate but readily synthesizes ppGpp from GDP. The enzyme also utilizes many structurally diverse metal cofactors for reaction catalysis and remains functionally stable at a wide range of environmental pHs. Transcription of rsh is stimulated by stationary-phase onset and by exposure to the antibiotics clindamycin and metronidazole. Chemical inhibition of RSH by the ppGpp analog relacin increases antibiotic susceptibility in epidemic C. difficile R20291, indicating that RSH inhibitors may be a viable strategy for drug development against C. difficile infection. Finally, transcriptional suppression of rsh also increases bacterial antibiotic susceptibility, suggesting that RSH contributes to C. difficile antibiotic tolerance and survival.IMPORTANCE Clostridioides difficile infection (CDI) is an urgent public health threat with a high recurrence rate, in part because the causative bacterium has a high rate of antibiotic survival. The (p)ppGpp-mediated bacterial stringent response plays a role in antibiotic tolerance in diverse pathogens and is a potential target for development of new antimicrobials because the enzymes that metabolize (p)ppGpp have no mammalian homologs. We report that stationary-phase onset and antibiotics induce expression of the clostridial ppGpp synthetase RSH and that both chemical inhibition and translational suppression of RSH increase C. difficile antibiotic susceptibility. This demonstrates that development of RSH inhibitors to serve as adjuvants to antibiotic therapy is a potential approach for the development of new strategies to combat CDI.
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20
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Aldrete-Tapia JA, Torres JA. Enhancing the Inactivation of Bacterial Spores during Pressure-Assisted Thermal Processing. FOOD ENGINEERING REVIEWS 2020. [DOI: 10.1007/s12393-020-09252-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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21
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Identification of Simplified Microbial Communities That Inhibit Clostridioides difficile Infection through Dilution/Extinction. mSphere 2020; 5:5/4/e00387-20. [PMID: 32727857 PMCID: PMC7392540 DOI: 10.1128/msphere.00387-20] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The gastrointestinal microbiome plays an important role in limiting susceptibility to infection with Clostridioides difficile To better understand the ecology of bacteria important for C. difficile colonization resistance, we developed an experimental platform to simplify complex communities of fecal bacteria through dilution and rapidly screen for their ability to resist C. difficile colonization after challenge, as measured by >100-fold reduction in levels of C. difficile in challenged communities. We screened 76 simplified communities diluted from cultures of six fecal donors and identified 24 simplified communities that inhibited C. difficile colonization in vitro Sequencing revealed that simplified communities were composed of 19 to 67 operational taxonomic units (OTUs) and could be partitioned into four distinct community types. One simplified community could be further simplified from 56 to 28 OTUs through dilution and retain the ability to inhibit C. difficile We tested the efficacy of seven simplified communities in a humanized microbiota mouse model. We found that four communities were able to significantly reduce the severity of the initial C. difficile infection and limit susceptibility to disease relapse. Analysis of fecal microbiomes from treated mice demonstrated that simplified communities accelerated recovery of indigenous bacteria and led to stable engraftment of 19 to 22 OTUs from simplified communities. Overall, the insights gained through the identification and characterization of these simplified communities increase our understanding of the microbial dynamics of C. difficile infection and recovery.IMPORTANCEClostridioides difficile is the leading cause of antibiotic-associated diarrhea and a significant health care burden. Fecal microbiota transplantation is highly effective at treating recurrent C. difficile disease; however, uncertainties about the undefined composition of fecal material and potential long-term unintended health consequences remain. These concerns have motivated studies to identify new communities of microbes with a simpler composition that will be effective at treating disease. This work describes a platform for rapidly identifying and screening new simplified communities for efficacy in treating C. difficile infection. Four new simplified communities of microbes with potential for development of new therapies to treat C. difficile disease are identified. While this platform was developed and validated to model infection with C. difficile, the underlying principles described in the paper could be easily modified to develop therapeutics to treat other gastrointestinal diseases.
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22
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Moore JH, Salahi A, Honrado C, Warburton C, Warren CA, Swami NS. Quantifying bacterial spore germination by single-cell impedance cytometry for assessment of host microbiota susceptibility to Clostridioides difficile infection. Biosens Bioelectron 2020; 166:112440. [PMID: 32745926 DOI: 10.1016/j.bios.2020.112440] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2020] [Revised: 06/18/2020] [Accepted: 07/08/2020] [Indexed: 12/11/2022]
Abstract
The germination of ingested spores is often a necessary first step required for enabling bacterial outgrowth and host colonization, as in the case of Clostridioides difficile (C. difficile) infection. Spore germination rate in the colon depends on microbiota composition and its level of disruption by antibiotic treatment since secretions by commensal bacteria modulate primary to secondary bile salt levels to control germination. Assessment of C. difficile spore germination typically requires measurement of colony-forming units, which is labor intensive and takes at least 24 h to perform but is regularly required due to the high recurrence rates of nosocomial antibiotic-associated diarrhea. We present a rapid method to assess spore germination by using high throughput single-cell impedance cytometry (>300 events/s) to quantify live bacterial cells, by gating for their characteristic electrophysiology versus spores, so that germination can be assessed after just 4 h of culture at a detection limit of ~100 live cells per 50 μL sample. To detect the phenotype of germinated C. difficile bacteria, we utilize its characteristically higher net conductivity versus that of spore aggregates and non-viable C. difficile forms, which causes a distinctive high-frequency (10 MHz) impedance phase dispersion within moderately conductive media (0.8 S/m). In this manner, we can detect significant differences in spore germination rates within just 4 h, with increasing primary bile salt levels in vitro and using ex vivo microbiota samples from an antibiotic-treated mouse model to assess susceptibility to C. difficile infection. We envision a rapid diagnostic tool for assessing host microbiota susceptibility to bacterial colonization after key antibiotic treatments.
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Affiliation(s)
- John H Moore
- Electrical & Computer Engineering, University of Virginia, Charlottesville, VA, 22904, USA
| | - Armita Salahi
- Electrical & Computer Engineering, University of Virginia, Charlottesville, VA, 22904, USA
| | - Carlos Honrado
- Electrical & Computer Engineering, University of Virginia, Charlottesville, VA, 22904, USA
| | | | - Cirle A Warren
- Infectious Diseases, School of Medicine, University of Virginia, VA, 22904, USA
| | - Nathan S Swami
- Electrical & Computer Engineering, University of Virginia, Charlottesville, VA, 22904, USA.
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23
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Shen A. Clostridioides difficile Spores: Bile Acid Sensors and Trojan Horses of Transmission. Clin Colon Rectal Surg 2020; 33:58-66. [PMID: 32104157 DOI: 10.1055/s-0040-1701230] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
The Gram-positive, spore-forming bacterium, Clostridioides difficile is the leading cause of healthcare-associated infections in the United States, although it also causes a significant number of community-acquired infections. C. difficile infections, which range in severity from mild diarrhea to toxic megacolon, cost more to treat than matched infections, with an annual treatment cost of approximately $6 billion for almost half-a-million infections. These high-treatment costs are due to the high rates of C. difficile disease recurrence (>20%) and necessity for special disinfection measures. These complications arise in part because C. difficile makes metabolically dormant spores, which are the major infectious particle of this obligate anaerobe. These seemingly inanimate life forms are inert to antibiotics, resistant to commonly used disinfectants, readily disseminated, and capable of surviving in the environment for a long period of time. However, upon sensing specific bile salts in the vertebrate gut, C. difficile spores transform back into the vegetative cells that are responsible for causing disease. This review discusses how spores are ideal vectors for disease transmission and how antibiotics modulate this process. We also describe the resistance properties of spores and how they create challenges eradicating spores, as well as promote their spread. Lastly, environmental reservoirs of C. difficile spores and strategies for destroying them particularly in health care environments will be discussed.
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Affiliation(s)
- Aimee Shen
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts
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24
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Abhyankar W, Zheng L, Brul S, de Koster CG, de Koning LJ. Vegetative Cell and Spore Proteomes of Clostridioides difficile Show Finite Differences and Reveal Potential Protein Markers. J Proteome Res 2019; 18:3967-3976. [PMID: 31557040 PMCID: PMC6832669 DOI: 10.1021/acs.jproteome.9b00413] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2019] [Indexed: 12/22/2022]
Abstract
Clostridioides difficile-associated infection (CDI) is a health-care-associated infection caused, as the name suggests, by obligate anaerobic pathogen C. difficile and thus mainly transmitted via highly resistant endospores from one person to the other. In vivo, the spores need to germinate into cells prior to establishing an infection. Bile acids and glycine, both available in sufficient amounts inside the human host intestinal tract, serve as efficient germinants for the spores. It is therefore, for better understanding of C. difficile virulence, crucial to study both the cell and spore states with respect to their genetic, metabolic, and proteomic composition. In the present study, mass spectrometric relative protein quantification, based on the 14N/15N peptide isotopic ratios, has led to quantification of over 700 proteins from combined spore and cell samples. The analysis has revealed that the proteome turnover between a vegetative cell and a spore for this organism is moderate. Additionally, specific cell and spore surface proteins, vegetative cell proteins CD1228, CD3301 and spore proteins CD2487, CD2434, and CD0684 are identified as potential protein markers for C. difficile infection.
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Affiliation(s)
- Wishwas
R. Abhyankar
- Department
of Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam Faculty
of Science, Science Park 904, 1098 XH Amsterdam, The Netherlands
- Department
of Mass Spectrometry of Bio-Macromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam Faculty
of Science, Science Park 904, 1098 XH Amsterdam, The Netherlands
| | - Linli Zheng
- Department
of Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam Faculty
of Science, Science Park 904, 1098 XH Amsterdam, The Netherlands
- Department
of Mass Spectrometry of Bio-Macromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam Faculty
of Science, Science Park 904, 1098 XH Amsterdam, The Netherlands
| | - Stanley Brul
- Department
of Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam Faculty
of Science, Science Park 904, 1098 XH Amsterdam, The Netherlands
| | - Chris G. de Koster
- Department
of Mass Spectrometry of Bio-Macromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam Faculty
of Science, Science Park 904, 1098 XH Amsterdam, The Netherlands
| | - Leo J. de Koning
- Department
of Mass Spectrometry of Bio-Macromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam Faculty
of Science, Science Park 904, 1098 XH Amsterdam, The Netherlands
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25
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Oludiran A, Courson DS, Stuart MD, Radwan AR, Poutsma JC, Cotten ML, Purcell EB. How Oxygen Availability Affects the Antimicrobial Efficacy of Host Defense Peptides: Lessons Learned from Studying the Copper-Binding Peptides Piscidins 1 and 3. Int J Mol Sci 2019; 20:E5289. [PMID: 31653020 PMCID: PMC6862162 DOI: 10.3390/ijms20215289] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2019] [Revised: 10/21/2019] [Accepted: 10/22/2019] [Indexed: 02/07/2023] Open
Abstract
The development of new therapeutic options against Clostridioides difficile (C. difficile) infection is a critical public health concern, as the causative bacterium is highly resistant to multiple classes of antibiotics. Antimicrobial host-defense peptides (HDPs) are highly effective at simultaneously modulating the immune system function and directly killing bacteria through membrane disruption and oxidative damage. The copper-binding HDPs piscidin 1 and piscidin 3 have previously shown potent antimicrobial activity against a number of Gram-negative and Gram-positive bacterial species but have never been investigated in an anaerobic environment. Synergy between piscidins and metal ions increases bacterial killing aerobically. Here, we performed growth inhibition and time-kill assays against C. difficile showing that both piscidins suppress proliferation of C. difficile by killing bacterial cells. Microscopy experiments show that the peptides accumulate at sites of membrane curvature. We find that both piscidins are effective against epidemic C. difficile strains that are highly resistant to other stresses. Notably, copper does not enhance piscidin activity against C. difficile. Thus, while antimicrobial activity of piscidin peptides is conserved in aerobic and anaerobic settings, the peptide-copper interaction depends on environmental oxygen to achieve its maximum potency. The development of pharmaceuticals from HDPs such as piscidin will necessitate consideration of oxygen levels in the targeted tissue.
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Affiliation(s)
- Adenrele Oludiran
- Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, USA.
| | - David S Courson
- Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, USA.
| | - Malia D Stuart
- Biology Department, Palomar College, San Marcos, CA 92069, USA.
| | - Anwar R Radwan
- Department of Chemistry, College of William and Mary, Williamsburg, VA 23185, USA.
| | - John C Poutsma
- Department of Chemistry, College of William and Mary, Williamsburg, VA 23185, USA.
| | - Myriam L Cotten
- Department of Applied Science, College of William and Mary, Williamsburg, VA 23185, USA.
| | - Erin B Purcell
- Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, USA.
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Shrestha R, Cochran AM, Sorg JA. The requirement for co-germinants during Clostridium difficile spore germination is influenced by mutations in yabG and cspA. PLoS Pathog 2019; 15:e1007681. [PMID: 30943268 PMCID: PMC6464247 DOI: 10.1371/journal.ppat.1007681] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2018] [Revised: 04/15/2019] [Accepted: 03/05/2019] [Indexed: 12/13/2022] Open
Abstract
Clostridium difficile spore germination is critical for the transmission of disease. C. difficile spores germinate in response to cholic acid derivatives, such as taurocholate (TA), and amino acids, such as glycine or alanine. Although the receptor with which bile acids are recognized (germinant receptor) is known, the amino acid co-germinant receptor has remained elusive. Here, we used EMS mutagenesis to generate mutants with altered requirements for the amino acid co-germinant, similar to the strategy we used previously to identify the bile acid germinant receptor, CspC. Surprisingly, we identified strains that do not require co-germinants, and the mutant spores germinated in response to TA alone. Upon sequencing these mutants, we identified different mutations in yabG. In C. difficile, yabG expression is required for the processing of key germination components to their mature forms (e.g., CspBA to CspB and CspA). A defined yabG mutant exacerbated the EMS mutant phenotype. Building upon this work, we found that small deletions in cspA resulted in spores that germinated in the presence of TA alone without the requirement of a co-germinant. cspA encodes a pseudoprotease that was previously shown to be important for incorporation of the CspC germinant receptor. Herein, our study builds upon the role of CspA during C. difficile spore germination by providing evidence that CspA is important for recognition of co-germinants during C. difficile spore germination. Our work suggests that two pseudoproteases (CspC and CspA) likely function as the C. difficile germinant receptors. Germination by C. difficile spores is one of the very first steps in the pathogenesis of this organism. The transition from the metabolically dormant spore form to the actively-growing, toxin-producing vegetative form is initiated by certain host-derived bile acids and amino acid signals. Despite near universal conservation in endospore-forming bacteria of the Ger-type germinant receptors, C. difficile and related organisms do not encode these proteins. In prior work, we identified the C. difficile bile acid germinant receptor as the CspC pseudoprotease. In this manuscript, we implicate the CspA pseudoprotease as the C. difficile co-germinant receptor. C. difficile cspA is encoded as a translational fusion to cspB. The resulting CspBA protein is processed post-translationally by the YabG protease. Inactivation of yabG resulted in strains whose spores no longer responded to amino acids or divalent cations as co-germinants and germinated in response to bile acid alone. Building upon this, we found that small deletions in the cspA portion of cspBA resulted in spores that could germinate in response to bile acids alone. Our results suggest that two pseudoproteases regulate C. difficile spore germination and provide further evidence that C. difficile spore germination proceeds through a novel spore germination pathway.
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Affiliation(s)
- Ritu Shrestha
- Department of Biology, Texas A&M University, College Station, United States of America
| | - Alicia M. Cochran
- Department of Biology, Texas A&M University, College Station, United States of America
| | - Joseph A. Sorg
- Department of Biology, Texas A&M University, College Station, United States of America
- * E-mail:
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Shrestha R, Sorg JA. Terbium chloride influences Clostridium difficile spore germination. Anaerobe 2019; 58:80-88. [PMID: 30926439 DOI: 10.1016/j.anaerobe.2019.03.016] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2018] [Revised: 03/06/2019] [Accepted: 03/25/2019] [Indexed: 02/06/2023]
Abstract
The germination of Clostridium difficile spores is an important stage of the C. difficile life cycle. In other endospore-forming bacteria, the composition of the medium in which the spores are generated influences the abundance of germination-specific proteins, thereby influencing the sensitivity of the spores towards germinants. In C. difficile media composition on the spores has only been reported to influence the number of spores produced. One of the measures of spore germination is the analysis of the release of DPA from the spore core. To detect DPA release in real time, terbium chloride is often added to the germination conditions because Tb3+ complexes with the released DPA and this can be detected using fluorescence measurements. Although C. difficile spores germinate in response to TA and glycine, recently calcium was identified as an enhancer for spore germination. Here, we find that germination by spores prepared in peptone rich media, such as 70:30, is positively influenced by terbium. We hypothesize that, in these assays, Tb3+ functions similarly to calcium. Although the mechanism(s) causing increased sensitivity of the C. difficile spores that are prepared in peptone rich media to terbium is still unknown, we suggest that the TbCl3 concentration used in the analysis of C. difficile DPA release be carefully titrated so as not to misinterpret future findings.
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Affiliation(s)
- Ritu Shrestha
- Department of Biology, Texas A&M University, College Station, TX, 77843, USA
| | - Joseph A Sorg
- Department of Biology, Texas A&M University, College Station, TX, 77843, USA.
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Bhattacharjee D, Sorg JA. Conservation of the "Outside-in" Germination Pathway in Paraclostridium bifermentans. Front Microbiol 2018; 9:2487. [PMID: 30386321 PMCID: PMC6199464 DOI: 10.3389/fmicb.2018.02487] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2018] [Accepted: 09/28/2018] [Indexed: 12/17/2022] Open
Abstract
Clostridium difficile spore germination is initiated in response to certain bile acids and amino acids (e.g., glycine). Though the amino acid-recognizing germinant receptor is unknown, the bile acid germinant receptor is the germination-specific, subtilisin-like pseudoprotease, CspC. In C. difficile the CspB, CspA, and CspC proteins are involved in spore germination. Of these, only CspB is predicted to have catalytic activity because the residues important for catalysis are mutated in the cspA and cspC sequence. The CspB, CspA, and CspC proteins are likely localized to the outer layers of the spore (e.g., the cortex or the coat layers) and not the inner membrane where the Ger-type germinant receptors are located. In C. difficile, germination proceeds in an “outside-in” direction, instead of the “‘inside-out” direction observed during the germination of Bacillus subtilis spores. During C. difficile spore germination, cortex fragments are released prior to the release of 2,4-dipicolinic acid (DPA) from the spore core. This is opposite to what occurs during B. subtilis spore germination. To understand if the mechanism C. difficile spore germination is unique or if spores from other organisms germinate in a similar fashion, we analyzed the germination of Paraclostridium bifermentans spores. We find that P. bifermentans spores release cortex fragments prior to DPA during germination and the DPA release from the P. bifermentans spore core can be blocked by high concentrations of osmolytes. Moreover, we find that P. bifermentans spores do not respond to steroid-like compounds (unlike the related C. difficile and P. sordellii organisms), indicating that the mere presence of the Csp proteins does permit germination in response to steroid compounds. Our findings indicate that the “outside in” mechanism of spore germination observed in C. difficile can be found in other bacteria suggesting that this mechanism is a novel pathway for endospore germination.
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Affiliation(s)
- Disha Bhattacharjee
- Department of Biology, Texas A&M University, College Station, TX, United States
| | - Joseph A Sorg
- Department of Biology, Texas A&M University, College Station, TX, United States
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Effect of the Synthetic Bile Salt Analog CamSA on the Hamster Model of Clostridium difficile Infection. Antimicrob Agents Chemother 2018; 62:AAC.02251-17. [PMID: 30012758 DOI: 10.1128/aac.02251-17] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2017] [Accepted: 07/01/2018] [Indexed: 12/15/2022] Open
Abstract
Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and has gained worldwide notoriety due to emerging hypervirulent strains and the high incidence of recurrence. We previously reported protection of mice from CDI using the antigerminant bile salt analog CamSA. Here we describe the effects of CamSA in the hamster model of CDI. CamSA treatment of hamsters showed no toxicity and did not affect the richness or diversity of gut microbiota; however, minor changes in community composition were observed. Treatment of C. difficile-challenged hamsters with CamSA doubled the mean time to death, compared to control hamsters. However, CamSA alone was insufficient to prevent CDI in hamsters. CamSA in conjunction with suboptimal concentrations of vancomycin led to complete protection from CDI in 70% of animals. Protected animals remained disease-free at least 30 days postchallenge and showed no signs of colonic tissue damage. In a delayed-treatment model of hamster CDI, CamSA was unable to prevent infection signs and death. These data support a putative model in which CamSA reduces the number of germinating C. difficile spores but does not keep all of the spores from germinating. Vancomycin halts division of any vegetative cells that are able to grow from spores that escape CamSA.
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Sharma SK, Yip C, Esposito EX, Sharma PV, Simon MP, Abel-Santos E, Firestine SM. The Design, Synthesis, and Characterizations of Spore Germination Inhibitors Effective against an Epidemic Strain of Clostridium difficile. J Med Chem 2018; 61:6759-6778. [PMID: 30004695 DOI: 10.1021/acs.jmedchem.8b00632] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Clostridium difficile infections (CDI), particularly those caused by the BI/NAP1/027 epidemic strains, are challenging to treat. One method to address this disease is to prevent the development of CDI by inhibiting the germination of C. difficile spores. Previous studies have identified cholic amide m-sulfonic acid, CamSA, as an inhibitor of spore germination. However, CamSA is inactive against the hypervirulent strain R20291. To circumvent this problem, a series of cholic acid amides were synthesized and tested against R20291. The best compound in the series was the simple phenyl amide analogue which possessed an IC50 value of 1.8 μM, more than 225 times as potent as the natural germination inhibitor, chenodeoxycholate. This is the most potent inhibitor of C. difficile spore germination described to date. QSAR and molecular modeling analysis demonstrated that increases in hydrophobicity and decreases in partial charge or polar surface area were correlated with increases in potency.
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Affiliation(s)
- Shiv K Sharma
- Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences , Wayne State University , 259 Mack Avenue , Detroit , Michigan 48201 , United States
| | - Christopher Yip
- Department of Chemistry and Biochemistry , University of Nevada at Las Vegas , 4505 South Maryland Parkway , Las Vegas , Nevada 89154 , United States
| | | | - Prateek V Sharma
- Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences , Wayne State University , 259 Mack Avenue , Detroit , Michigan 48201 , United States
| | - Matthew P Simon
- Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences , Wayne State University , 259 Mack Avenue , Detroit , Michigan 48201 , United States
| | - Ernesto Abel-Santos
- Department of Chemistry and Biochemistry , University of Nevada at Las Vegas , 4505 South Maryland Parkway , Las Vegas , Nevada 89154 , United States
| | - Steven M Firestine
- Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences , Wayne State University , 259 Mack Avenue , Detroit , Michigan 48201 , United States
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Schäffler H, Breitrück A. Clostridium difficile - From Colonization to Infection. Front Microbiol 2018; 9:646. [PMID: 29692762 PMCID: PMC5902504 DOI: 10.3389/fmicb.2018.00646] [Citation(s) in RCA: 117] [Impact Index Per Article: 16.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2017] [Accepted: 03/19/2018] [Indexed: 12/19/2022] Open
Abstract
Clostridium difficile is the most frequent cause of nosocomial antibiotic-associated diarrhea. The incidence of C. difficile infection (CDI) has been rising worldwide with subsequent increases in morbidity, mortality, and health care costs. Asymptomatic colonization with C. difficile is common and a high prevalence has been found in specific cohorts, e.g., hospitalized patients, adults in nursing homes and in infants. However, the risk of infection with C. difficile differs significantly between these cohorts. While CDI is a clear indication for therapy, colonization with C. difficile is not believed to be a direct precursor for CDI and therefore does not require treatment. Antibiotic therapy causes alterations of the intestinal microbial composition, enabling C. difficile colonization and consecutive toxin production leading to disruption of the colonic epithelial cells. Clinical symptoms of CDI range from mild diarrhea to potentially life-threatening conditions like pseudomembranous colitis or toxic megacolon. While antibiotics are still the treatment of choice for CDI, new therapies have emerged in recent years such as antibodies against C. difficile toxin B and fecal microbial transfer (FMT). This specific therapy for CDI underscores the role of the indigenous bacterial composition in the prevention of the disease in healthy individuals and its role in the pathogenesis after alteration by antibiotic treatment. In addition to the pathogenesis of CDI, this review focuses on the colonization of C. difficile in the human gut and factors promoting CDI.
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Affiliation(s)
- Holger Schäffler
- Division of Gastroenterology, Department of Medicine II, University of Rostock, Rostock, Germany
| | - Anne Breitrück
- Extracorporeal Immunomodulation Unit, Fraunhofer Institute for Cell Therapy and Immunology, Rostock, Germany.,Institute of Medical Microbiology, Virology and Hygiene, University of Rostock, Rostock, Germany
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Zhu D, Sorg JA, Sun X. Clostridioides difficile Biology: Sporulation, Germination, and Corresponding Therapies for C. difficile Infection. Front Cell Infect Microbiol 2018; 8:29. [PMID: 29473021 PMCID: PMC5809512 DOI: 10.3389/fcimb.2018.00029] [Citation(s) in RCA: 91] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2017] [Accepted: 01/23/2018] [Indexed: 12/18/2022] Open
Abstract
Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe, and an important nosocomial pathogen. Due to the strictly anaerobic nature of the vegetative form, spores are the main morphotype of infection and transmission of the disease. Spore formation and their subsequent germination play critical roles in C. difficile infection (CDI) progress. Under suitable conditions, C. difficile spores will germinate and outgrow to produce the pathogenic vegetative form. During CDI, C. difficile produces toxins (TcdA and TcdB) that are required to initiate the disease. Meanwhile, it also produces spores that are responsible for the persistence and recurrence of C. difficile in patients. Recent studies have shed light on the regulatory mechanisms of C. difficile sporulation and germination. This review is to summarize recent advances on the regulation of sporulation/germination in C. difficile and the corresponding therapeutic strategies that are aimed at these important processes.
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Affiliation(s)
- Duolong Zhu
- Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, United States
| | - Joseph A Sorg
- Department of Biology, Texas A&M University, College Station, TX, United States
| | - Xingmin Sun
- Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, United States
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Pickering DS, Vernon JJ, Freeman J, Wilcox MH, Chilton CH. Investigating the effect of supplementation on Clostridioides (Clostridium) difficile spore recovery in two solid agars. Anaerobe 2018; 50:38-43. [PMID: 29408598 DOI: 10.1016/j.anaerobe.2018.01.010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2017] [Revised: 01/22/2018] [Accepted: 01/28/2018] [Indexed: 12/18/2022]
Abstract
BACKGROUND A variety of supplemented solid media are used within Clostridium difficile research to optimally recover spores. Our study sought to investigate different media and additives, providing a method of optimised C. difficile spore recovery. Additionally, due to the results observed in the initial experiments, the inhibitory effects of three amino acids (glycine, l-histidine &l-phenylalanine) on C. difficile spore outgrowth were investigated. METHODS Spores of five C. difficile strains (PCR ribotypes 001,015,020,027,078) were recovered on two commonly used solid media (BHI & CCEY, or cycloserine-cefoxitin egg yolk) supplemented with various concentrations of germinants (taurocholate, glycine & lysozyme). Agar-incorporation minimum inhibitory concentration (MIC) testing was carried out for glycine and taurocholate on vegetative cells and spores of all five strains. Additionally a BHI broth microassay method was utilised to test the growth of C. difficile in the presence of increasing concentrations (0,1,2,3,4%) of three amino acids (glycine,l-histidine,l-phenyalanine). RESULTS CCEY agar alone and BHI supplemented with taurocholate (0.1/1%) provided optimal recovery for C. difficile spores. Glycine was inhibitory to spore recovery at higher concentrations, although these varied between the two media used. In agar-incorporated MIC testing, glycine concentrations higher than 2% (20 g/L) were inhibitory to both C. difficile spore and vegetative cell growth versus the control (mean absorbance = 0.33 ± 0.02 vs 0.12 ± 0.01) (P < 0.001). This indicates a potential mechanism whereby glycine interferes with vegetative cell growth. Further microbroth testing provided evidence of inhibition by two amino acids other than glycine, l-histidine and l-phenylalanine. CONCLUSIONS We provide two media for optimal recovery of C. difficile spores (CCEY alone and BHI supplemented with 0.1/1% taurocholate). CCEY is preferred for isolation from faecal samples. For pure cultures, either CCEY or supplemented BHI agar are appropriate. The inhibitory nature of three amino acids (glycine,l-histidine,l-phenylalanine) to C. difficile vegetative cell proliferation is also highlighted.
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Affiliation(s)
- D S Pickering
- Healthcare Associated Infections Research Group, Molecular Gastroenterology, Leeds Institute for Biomedical and Clinical Sciences, University of Leeds, West Yorkshire, UK.
| | - J J Vernon
- Healthcare Associated Infections Research Group, Molecular Gastroenterology, Leeds Institute for Biomedical and Clinical Sciences, University of Leeds, West Yorkshire, UK
| | - J Freeman
- Microbiology, Leeds Teaching Hospitals Trust, Leeds, UK
| | - M H Wilcox
- Healthcare Associated Infections Research Group, Molecular Gastroenterology, Leeds Institute for Biomedical and Clinical Sciences, University of Leeds, West Yorkshire, UK; Microbiology, Leeds Teaching Hospitals Trust, Leeds, UK
| | - C H Chilton
- Healthcare Associated Infections Research Group, Molecular Gastroenterology, Leeds Institute for Biomedical and Clinical Sciences, University of Leeds, West Yorkshire, UK
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Shrestha R, Sorg JA. Hierarchical recognition of amino acid co-germinants during Clostridioides difficile spore germination. Anaerobe 2018; 49:41-47. [PMID: 29221987 PMCID: PMC5844826 DOI: 10.1016/j.anaerobe.2017.12.001] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2017] [Revised: 11/17/2017] [Accepted: 12/03/2017] [Indexed: 12/15/2022]
Abstract
Bile acids are an important signal for germination of Clostridioides difficile spores; however, the bile acid signal alone is not sufficient. Amino acids, such as glycine, are another signal necessary for germination by C. difficile spores. Prior studies on the amino acid signal required for germination have shown that there is a preference for the amino acid used as a signal for germination. Previously we found that d-alanine can function as a co-germinant for C. difficile spores at 37 °C but not at 25 °C. Here, we tested the ability of other amino acids to act as co-germinants with taurocholate (TA) at 37 °C and found that many amino acids previously categorized as non-co-germinants are co-germinants at 37 °C. Based on the EC50 values calculated for two different strains, we found that C. difficile spores recognize different amino acids with varying efficiencies. Using this data, we ranked the amino acids based on their effect on germination and found that in addition to d-alanine, other D-forms of amino acids are also used by C. difficile spores as co-germinants. Among the different types of amino acids, ones with branched chains such as valine, leucine, and isoleucine are the poorest co-germinants. However, glycine is still the most effective amino acid signal for both strains. Our results suggest that the yet-to-be-identified amino acid germinant receptor is highly promiscuous.
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Affiliation(s)
- Ritu Shrestha
- Department of Biology, Texas A&M University, College Station, TX 77843, United States
| | - Joseph A Sorg
- Department of Biology, Texas A&M University, College Station, TX 77843, United States.
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35
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Hopkins RJ, Wilson RB. Treatment of recurrent Clostridium difficile colitis: a narrative review. Gastroenterol Rep (Oxf) 2018; 6:21-28. [PMID: 29479439 PMCID: PMC5806400 DOI: 10.1093/gastro/gox041] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/21/2017] [Revised: 10/17/2017] [Accepted: 10/31/2017] [Indexed: 12/11/2022] Open
Abstract
Clostridium difficile is a gram-positive, spore-forming, obligate anaerobic bacillus that was originally isolated from the stool of a healthy neonate in 1935. In high-income countries, C. difficile is the most common cause of infectious diarrhoea in hospitalized patients. The incidence of C. difficile infection in the USA has increased markedly since 2000, with hospitalizations for C. difficile infections in non-pregnant adults doubling between 2000 and 2010. Between 20% and 35% of patients with C. difficile infection will fail initial antibiotic treatment and, of these, 40-60% will have a second recurrence. Recurrence of C. difficile infection after initial treatment causes substantial morbidity and is a major burden on health care systems. In this article, current treatments for recurrent C. difficile infection are reviewed and future directions explored. These include the use of antibiotics, probiotics, donor faecal transplants, anion resins, secondary bile acids or anti-toxin antibodies.
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Affiliation(s)
- Roy J Hopkins
- Department of Upper GI Surgery, Liverpool Hospital, Sydney, New South Wales, Australia
| | - Robert B Wilson
- Department of Upper GI Surgery, Liverpool Hospital, Sydney, New South Wales, Australia
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Abstract
Despite being resistant to a variety of environmental insults, the bacterial endospore can sense the presence of small molecules and respond by germinating, losing the specialized structures of the dormant spore, and resuming active metabolism, before outgrowing into vegetative cells. Our current level of understanding of the spore germination process in bacilli and clostridia is reviewed, with particular emphasis on the germinant receptors characterized in Bacillus subtilis, Bacillus cereus, and Bacillus anthracis. The recent evidence for a local clustering of receptors in a "germinosome" would begin to explain how signals from different receptors could be integrated. The SpoVA proteins, involved in the uptake of Ca2+-dipicolinic acid into the forespore during sporulation, are also responsible for its release during germination. Lytic enzymes SleB and CwlJ, found in bacilli and some clostridia, hydrolyze the spore cortex: other clostridia use SleC for this purpose. With genome sequencing has come the appreciation that there is considerable diversity in the setting for the germination machinery between bacilli and clostridia.
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Abstract
Clostridium difficile is the most frequent cause of nosocomial antibiotic-associated diarrhea. The incidence of C. difficile infection (CDI) has been rising worldwide with subsequent increases in morbidity, mortality, and health care costs. Asymptomatic colonization with C. difficile is common and a high prevalence has been found in specific cohorts, e.g., hospitalized patients, adults in nursing homes and in infants. However, the risk of infection with C. difficile differs significantly between these cohorts. While CDI is a clear indication for therapy, colonization with C. difficile is not believed to be a direct precursor for CDI and therefore does not require treatment. Antibiotic therapy causes alterations of the intestinal microbial composition, enabling C. difficile colonization and consecutive toxin production leading to disruption of the colonic epithelial cells. Clinical symptoms of CDI range from mild diarrhea to potentially life-threatening conditions like pseudomembranous colitis or toxic megacolon. While antibiotics are still the treatment of choice for CDI, new therapies have emerged in recent years such as antibodies against C. difficile toxin B and fecal microbial transfer (FMT). This specific therapy for CDI underscores the role of the indigenous bacterial composition in the prevention of the disease in healthy individuals and its role in the pathogenesis after alteration by antibiotic treatment. In addition to the pathogenesis of CDI, this review focuses on the colonization of C. difficile in the human gut and factors promoting CDI.
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Affiliation(s)
- Holger Schäffler
- Division of Gastroenterology, Department of Medicine II, University of Rostock, Rostock, Germany
| | - Anne Breitrück
- Extracorporeal Immunomodulation Unit, Fraunhofer Institute for Cell Therapy and Immunology, Rostock, Germany
- Institute of Medical Microbiology, Virology and Hygiene, University of Rostock, Rostock, Germany
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Alvarado I, Margotta JW, Aoki MM, Flores F, Agudelo F, Michel G, Elekonich MM, Abel-Santos E. Inhibitory effect of indole analogs against Paenibacillus larvae, the causal agent of American foulbrood disease. JOURNAL OF INSECT SCIENCE (ONLINE) 2017; 17:4469416. [PMID: 29117379 PMCID: PMC7206643 DOI: 10.1093/jisesa/iex080] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/09/2017] [Indexed: 06/07/2023]
Abstract
Paenibacillus larvae, a Gram-positive bacterium, causes American foulbrood (AFB) in honey bee larvae (Apis mellifera Linnaeus [Hymenoptera: Apidae]). P. larvae spores exit dormancy in the gut of bee larvae, the germinated cells proliferate, and ultimately bacteremia kills the host. Hence, spore germination is a required step for establishing AFB disease. We previously found that P. larvae spores germinate in response to l-tyrosine plus uric acid in vitro. Additionally, we determined that indole and phenol blocked spore germination. In this work, we evaluated the antagonistic effect of 35 indole and phenol analogs and identified strong inhibitors of P. larvae spore germination in vitro. We further tested the most promising candidate, 5-chloroindole, and found that it significantly reduced bacterial proliferation. Finally, feeding artificial worker jelly containing anti-germination compounds to AFB-exposed larvae significantly decreased AFB infection in laboratory-reared honey bee larvae. Together, these results suggest that inhibitors of P. larvae spore germination could provide another method to control AFB.
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Affiliation(s)
- Israel Alvarado
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV 89154
| | - Joseph W Margotta
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV 89154
| | - Mai M Aoki
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV 89154
| | - Fernando Flores
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV 89154
| | - Fresia Agudelo
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV 89154
| | - Guillermo Michel
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV 89154
| | - Michelle M Elekonich
- Directorate for Biological Sciences, National Science Foundation, 2415 Eisenhower Avenue, Alexandria, VA 22314, and
| | - Ernesto Abel-Santos
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV 89154
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Collins J, Auchtung JM. Control of Clostridium difficile Infection by Defined Microbial Communities. Microbiol Spectr 2017; 5:10.1128/microbiolspec.bad-0009-2016. [PMID: 28936948 PMCID: PMC5736378 DOI: 10.1128/microbiolspec.bad-0009-2016] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2016] [Indexed: 12/11/2022] Open
Abstract
Each year in the United States, billions of dollars are spent combating almost half a million Clostridium difficile infections (CDIs) and trying to reduce the ∼29,000 patient deaths in which C. difficile has an attributed role. In Europe, disease prevalence varies by country and level of surveillance, though yearly costs are estimated at €3 billion. One factor contributing to the significant health care burden of C. difficile is the relatively high frequency of recurrent CDIs. Recurrent CDI, i.e., a second episode of symptomatic CDI occurring within 8 weeks of successful initial CDI treatment, occurs in ∼25% of patients, with 35 to 65% of these patients experiencing multiple episodes of recurrent disease. Using microbial communities to treat recurrent CDI, either as whole fecal transplants or as defined consortia of bacterial isolates, has shown great success (in the case of fecal transplants) or potential promise (in the case of defined consortia of isolates). This review will briefly summarize the epidemiology and physiology of C. difficile infection, describe our current understanding of how fecal microbiota transplants treat recurrent CDI, and outline potential ways that knowledge can be used to rationally design and test alternative microbe-based therapeutics.
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Affiliation(s)
- James Collins
- Alkek Center for Metagenomics and Microbiome Research and Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030
| | - Jennifer M Auchtung
- Alkek Center for Metagenomics and Microbiome Research and Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030
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A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase. Infect Immun 2017; 85:IAI.00347-17. [PMID: 28652311 DOI: 10.1128/iai.00347-17] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2017] [Accepted: 06/20/2017] [Indexed: 12/18/2022] Open
Abstract
The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile, c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability.
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Lee WT, Wu YN, Chen YH, Wu SR, Shih TM, Li TJ, Yang LX, Yeh CS, Tsai PJ, Shieh DB. Octahedron Iron Oxide Nanocrystals Prohibited Clostridium difficile Spore Germination and Attenuated Local and Systemic Inflammation. Sci Rep 2017; 7:8124. [PMID: 28811642 PMCID: PMC5558001 DOI: 10.1038/s41598-017-08387-y] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2017] [Accepted: 07/12/2017] [Indexed: 01/27/2023] Open
Abstract
Clinical management of Clostridium difficile infection is still far from satisfactory as bacterial spores are resistant to many chemical agents and physical treatments. Certain types of nanoparticles have been demonstrated to exhibit anti-microbial efficacy even in multi-drug resistance bacteria. However, most of these studies failed to show biocompatibility to the mammalian host cells and no study has revealed in vivo efficacy in C. difficile infection animal models. The spores treated with 500 µg/mL Fe3-δO4 nanoparticles for 20 minutes, 64% of the spores were inhibited from transforming into vegetative cells, which was close to the results of the sodium hypochlorite-treated positive control. By cryo-electron micro-tomography, we demonstrated that Fe3-δO4 nanoparticles bind on spore surfaces and reduce the dipicolinic acid (DPA) released by the spores. In a C. difficile infection animal model, the inflammatory level triple decreased in mice with colonic C. difficile spores treated with Fe3-δO4 nanoparticles. Histopathological analysis showed a decreased intense neutrophil accumulation in the colon tissue of the Fe3-δO4 nanoparticle-treated mice. Fe3-δO4 nanoparticles, which had no influence on gut microbiota and apparent side effects in vivo, were efficacious inhibitors of C. difficile spore germination by attacking its surface and might become clinically feasible for prophylaxis and therapy.
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Affiliation(s)
- Wei-Ting Lee
- Institute of Basic Medical Sciences, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan
| | - Ya-Na Wu
- Institute of Oral Medicine, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan
| | - Yi-Hsuan Chen
- Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan
| | - Shang-Rung Wu
- Institute of Oral Medicine, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan
| | - Tsai-Miao Shih
- Institute of Oral Medicine, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan
| | - Tsung-Ju Li
- Institute of Basic Medical Sciences, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan
| | - Li-Xing Yang
- Institute of Basic Medical Sciences, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan
| | - Chen-Sheng Yeh
- Department of Chemistry, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan
| | - Pei-Jane Tsai
- Institute of Basic Medical Sciences, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan. .,Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan. .,Center of Infectious Disease and Signaling Research, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan.
| | - Dar-Bin Shieh
- Institute of Basic Medical Sciences, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan. .,Institute of Oral Medicine, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan. .,Department of Stomatology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, 704, Taiwan. .,Advanced Optoelectronic Technology Center and Center for Micro/Nano Science and Technology, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan.
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Kochan TJ, Somers MJ, Kaiser AM, Shoshiev MS, Hagan AK, Hastie JL, Giordano NP, Smith AD, Schubert AM, Carlson PE, Hanna PC. Intestinal calcium and bile salts facilitate germination of Clostridium difficile spores. PLoS Pathog 2017; 13:e1006443. [PMID: 28704538 PMCID: PMC5509370 DOI: 10.1371/journal.ppat.1006443] [Citation(s) in RCA: 66] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2017] [Accepted: 06/03/2017] [Indexed: 12/26/2022] Open
Abstract
Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. C. difficile spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca2+ during C. difficile spore germination and provide direct evidence that intestinal Ca2+ coordinates with bile salts to stimulate germination. Endogenous Ca2+ (released from within the spore) and a putative AAA+ ATPase, encoded by Cd630_32980, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca2+. However, environmental Ca2+ replaces glycine as a co-germinant and circumvents the need for endogenous Ca2+ fluxes. Cd630_32980 is dispensable for colonization in a murine model of C. difficile infection and ex vivo germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca2+ present within the gastrointestinal tract plays a critical role in C. difficile germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (e.g., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI. The anaerobic, spore-forming bacterium Clostridium difficile (C. difficile) is a prominent pathogen in hospitals worldwide and the leading cause of nosocomial diarrhea. Numerous risk factors are associated with C. difficile infections (CDIs) including: antibiotics, advanced age, vitamin D deficiency, and proton pump inhibitors. Antibiotic use disrupts the intestinal microbiota allowing for C. difficile to colonize, however, why these other risk factors increase CDI incidence is unclear. Notably, deficient intestinal calcium absorption (i.e., increased calcium levels) is associated with these risk factors. In this work, we investigate the role of calcium in C. difficile spore germination. C. difficile spores are the infectious particles and they must become metabolically active (germinate) to cause disease. Here, we show that calcium is required for C. difficile germination, specifically activating the key step of cortex hydrolysis, and that this calcium can be derived from either within the spore or the environment. We also demonstrate that intestinal calcium is required for efficient spore germination in vivo, suggesting that intestinal concentrations of other co-germinants are insufficient to induce C. difficile germination. Collectively, these data provide a mechanism that explains the strong clinical correlations between increased intestinal calcium levels and risk of CDI.
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Affiliation(s)
- Travis J. Kochan
- University of Michigan Medical School, Department of Microbiology and Immunology. Ann Arbor, Michigan, United States of America
| | - Madeline J. Somers
- University of Michigan Medical School, Department of Microbiology and Immunology. Ann Arbor, Michigan, United States of America
| | - Alyssa M. Kaiser
- University of Michigan Medical School, Department of Microbiology and Immunology. Ann Arbor, Michigan, United States of America
| | - Michelle S. Shoshiev
- University of Michigan Medical School, Department of Microbiology and Immunology. Ann Arbor, Michigan, United States of America
| | - Ada K. Hagan
- University of Michigan Medical School, Department of Microbiology and Immunology. Ann Arbor, Michigan, United States of America
| | - Jessica L. Hastie
- Center for Biologics Evaluation and Research, US Food and Drug Administration. Silver Spring, Maryland, United States of America
| | - Nicole P. Giordano
- Center for Biologics Evaluation and Research, US Food and Drug Administration. Silver Spring, Maryland, United States of America
| | - Ashley D. Smith
- Center for Biologics Evaluation and Research, US Food and Drug Administration. Silver Spring, Maryland, United States of America
| | - Alyxandria M. Schubert
- Center for Biologics Evaluation and Research, US Food and Drug Administration. Silver Spring, Maryland, United States of America
| | - Paul E. Carlson
- Center for Biologics Evaluation and Research, US Food and Drug Administration. Silver Spring, Maryland, United States of America
| | - Philip C. Hanna
- University of Michigan Medical School, Department of Microbiology and Immunology. Ann Arbor, Michigan, United States of America
- * E-mail:
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43
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Abstract
To survive adverse conditions, some bacterial species are capable of developing into a cell type, the "spore," which exhibits minimal metabolic activity and remains viable in the presence of multiple environmental challenges. For some pathogenic bacteria, this developmental state serves as a means of survival during transmission from one host to another. Spores are the highly infectious form of these bacteria. Upon entrance into a host, specific signals facilitate germination into metabolically active replicating organisms, resulting in disease pathogenesis. In this article, we will review spore structure and function in well-studied pathogens of two genera, Bacillus and Clostridium, focusing on Bacillus anthracis and Clostridium difficile, and explore current data regarding the lifestyles of these bacteria outside the host and transmission from one host to another.
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44
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Shrestha R, Lockless SW, Sorg JA. A Clostridium difficile alanine racemase affects spore germination and accommodates serine as a substrate. J Biol Chem 2017; 292:10735-10742. [PMID: 28487371 DOI: 10.1074/jbc.m117.791749] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2017] [Revised: 05/07/2017] [Indexed: 12/18/2022] Open
Abstract
Clostridium difficile has become one of the most common bacterial pathogens in hospital-acquired infections in the United States. Although C. difficile is strictly anaerobic, it survives in aerobic environments and transmits between hosts via spores. C. difficile spore germination is triggered in response to certain bile acids and glycine. Although glycine is the most effective co-germinant, other amino acids can substitute with varying efficiencies. Of these, l-alanine is an effective co-germinant and is also a germinant for most bacterial spores. Many endospore-forming bacteria embed alanine racemases into their spore coats, and these enzymes are thought to convert the l-alanine germinant into d-alanine, a spore germination inhibitor. Although the C. difficile Alr2 racemase is the sixth most highly expressed gene during C. difficile spore formation, a previous study reported that Alr2 has little to no role in germination of C. difficile spores in rich medium. Here, we hypothesized that Alr2 could affect C. difficile l-alanine-induced spore germination in a defined medium. We found that alr2 mutant spores more readily germinate in response to l-alanine as a co-germinant. Surprisingly, d-alanine also functioned as a co-germinant. Moreover, we found that Alr2 could interconvert l- and d-serine and that Alr2 bound to l- and d-serine with ∼2-fold weaker affinity to that of l- and d-alanine. Finally, we demonstrate that l- and d-serine are also co-germinants for C. difficile spores. These results suggest that C. difficile spores can respond to a diverse set of amino acid co-germinants and reveal that Alr2 can accommodate serine as a substrate.
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Affiliation(s)
- Ritu Shrestha
- From the Department of Biology, Texas A&M University, College Station, Texas 77843
| | - Steve W Lockless
- From the Department of Biology, Texas A&M University, College Station, Texas 77843
| | - Joseph A Sorg
- From the Department of Biology, Texas A&M University, College Station, Texas 77843
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Stoltz KL, Erickson R, Staley C, Weingarden AR, Romens E, Steer CJ, Khoruts A, Sadowsky MJ, Dosa PI. Synthesis and Biological Evaluation of Bile Acid Analogues Inhibitory to Clostridium difficile Spore Germination. J Med Chem 2017; 60:3451-3471. [PMID: 28402634 DOI: 10.1021/acs.jmedchem.7b00295] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Standard antibiotic-based strategies for the treatment of Clostridium difficile infections disrupt indigenous microbiota and commonly fail to eradicate bacterial spores, two key factors that allow recurrence of infection. As an alternative approach to controlling C. difficile infection, a series of bile acid derivatives have been prepared that inhibit taurocholate-induced spore germination. These analogues have been evaluated in a highly virulent NAP1 strain using optical density and phase-contrast microscopy assays. Heterocycle substitutions at C24 were well-tolerated and several tetrazole-containing derivatives were highly potent inhibitors in both assays, with complete inhibition of spore germination observed at 10-25 μM. To limit intestinal absorption, C7-sulfated analogues designed to avoid active and passive transport pathways were prepared. One of these derivatives, compound 21b, was found to be a potent inhibitor of C. difficile spore germination and poorly permeable in a Caco-2 model of intestinal epithelial absorption, suggesting that it is likely to be gut-restricted.
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Affiliation(s)
- Kristen L Stoltz
- Institute for Therapeutics Discovery and Development, Department of Medicinal Chemistry, University of Minnesota , 717 Delaware Street SE, Minneapolis, Minnesota 55414, United States
| | - Raymond Erickson
- BioTechnology Institute, University of Minnesota , 1479 Gortner Avenue, St. Paul, Minnesota 55108, United States
| | - Christopher Staley
- BioTechnology Institute, University of Minnesota , 1479 Gortner Avenue, St. Paul, Minnesota 55108, United States
| | - Alexa R Weingarden
- BioTechnology Institute, University of Minnesota , 1479 Gortner Avenue, St. Paul, Minnesota 55108, United States.,Department of Microbiology and Immunology, University of Minnesota , St. Paul, Minnesota 55108, United States
| | - Erin Romens
- BioTechnology Institute, University of Minnesota , 1479 Gortner Avenue, St. Paul, Minnesota 55108, United States
| | - Clifford J Steer
- Departments of Medicine and Genetics, Cell Biology, and Development, University of Minnesota , VFW Cancer Research Center, 406 Harvard Street, Minneapolis, Minnesota, United States
| | - Alexander Khoruts
- BioTechnology Institute, University of Minnesota , 1479 Gortner Avenue, St. Paul, Minnesota 55108, United States.,Department of Medicine, Division of Gastroenterology, University of Minnesota , Minneapolis, Minnesota 55414, United States.,Center for Immunology, University of Minnesota , Minneapolis, Minnesota 55414, United States
| | - Michael J Sadowsky
- BioTechnology Institute, University of Minnesota , 1479 Gortner Avenue, St. Paul, Minnesota 55108, United States.,Department of Soil, Water & Climate, University of Minnesota , St. Paul, Minnesota 55108, United States
| | - Peter I Dosa
- Institute for Therapeutics Discovery and Development, Department of Medicinal Chemistry, University of Minnesota , 717 Delaware Street SE, Minneapolis, Minnesota 55414, United States
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46
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Chai C, Lee KS, Imm GS, Kim YS, Oh SW. Inactivation of Clostridium difficile spore outgrowth by synergistic effects of nisin and lysozyme. Can J Microbiol 2017; 63:638-643. [PMID: 28346844 DOI: 10.1139/cjm-2016-0550] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Inactivating Clostridium difficile spores is difficult, as they are resistant to heat, chemicals, and antimicrobials. However, this note describes inactivation of C. difficile spore outgrowth by incubation in a solution containing a germinant (1% (m/v) sodium taurocholate), co-germinants (1% (m/v) tryptose and 1% (m/v) NaCl), and natural antimicrobials (20 nmol·L-1 nisin and 0.2 mmol·L-1 lysozyme). Clostridium difficile spores were resistant to nisin and lysozyme but became susceptible during germination and outgrowth triggered and promoted by sodium taurocholate, tryptose, and NaCl. The degree of inactivation of germinated and outgrowing C. difficile spores by both nisin and lysozyme was greater than the sum of that by nisin and lysozyme individually, suggesting synergistic inactivation of C. difficile spores. The germinant, co-germinants, and natural antimicrobials used in this study are safe for human contact and consumption. Therefore, these findings will facilitate the development of a safe and effective method to inactivate C. difficile spore.
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Affiliation(s)
- Changhoon Chai
- a Division of Applied Animal Science, Kangwon National University, Chuncheon 24341, Republic of Korea
| | - Kyung-Soo Lee
- b Department of Food and Nutrition, Kookmin University, Seoul 02707, Republic of Korea
| | - Goo-Sang Imm
- b Department of Food and Nutrition, Kookmin University, Seoul 02707, Republic of Korea
| | - Young Soon Kim
- c Department of Food and Nutrition, Korea University, Seoul 02841, Republic of Korea
| | - Se-Wook Oh
- b Department of Food and Nutrition, Kookmin University, Seoul 02707, Republic of Korea
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Fehér C, Soriano A, Mensa J. A Review of Experimental and Off-Label Therapies for Clostridium difficile Infection. Infect Dis Ther 2017; 6:1-35. [PMID: 27910000 PMCID: PMC5336415 DOI: 10.1007/s40121-016-0140-z] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2016] [Indexed: 12/16/2022] Open
Abstract
In spite of increased awareness and the efforts taken to optimize Clostridium difficile infection (CDI) management, with the limited number of currently available antibiotics for C. difficile the halt of this increasing epidemic remains out of reach. There are, however, close to 80 alternative treatment methods with controversial anti-clostridial efficacy or in experimental phase today. Indeed, some of these therapies are expected to become acknowledged members of the recommended anti-CDI arsenal within the next few years. None of these alternative treatment methods can respond in itself to all the major challenges of CDI management, which are primary prophylaxis in the susceptible population, clinical cure of severe cases, prevention of recurrences, and forestallment of asymptomatic C. difficile carriage and in-hospital spread. Yet, the greater the variety of treatment choices on hand, the better combination strategies can be developed to reach these goals in the future. The aim of this article is to provide a comprehensive summary of these experimental and currently off-label therapeutic options.
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Affiliation(s)
- Csaba Fehér
- Department of Infectious Diseases, Hospital Clínic of Barcelona, Barcelona, Spain.
| | - Alex Soriano
- Department of Infectious Diseases, Hospital Clínic of Barcelona, Barcelona, Spain
- August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Spain
- University of Barcelona, Barcelona, Spain
| | - Josep Mensa
- Department of Infectious Diseases, Hospital Clínic of Barcelona, Barcelona, Spain
- August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Spain
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Dipicolinic Acid Release by Germinating Clostridium difficile Spores Occurs through a Mechanosensing Mechanism. mSphere 2016; 1:mSphere00306-16. [PMID: 27981237 PMCID: PMC5156672 DOI: 10.1128/msphere.00306-16] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2016] [Accepted: 11/14/2016] [Indexed: 12/18/2022] Open
Abstract
Clostridium difficile is transmitted between hosts in the form of a dormant spore, and germination by C. difficile spores is required to initiate infection, because the toxins that are necessary for disease are not deposited on the spore form. Importantly, the C. difficile spore germination pathway represents a novel pathway for bacterial spore germination. Prior work has shown that the order of events during C. difficile spore germination (cortex degradation and DPA release) is flipped compared to the events during B. subtilis spore germination, a model organism. Here, we further characterize the C. difficile spore germination pathway and summarize our findings indicating that DPA release by germinating C. difficile spores occurs through a mechanosensing mechanism in response to the degradation of the spore cortex. Classically, dormant endospores are defined by their resistance properties, particularly their resistance to heat. Much of the heat resistance is due to the large amount of dipicolinic acid (DPA) stored within the spore core. During spore germination, DPA is released and allows for rehydration of the otherwise-dehydrated core. In Bacillus subtilis, 7 proteins are encoded by the spoVA operon and are important for DPA release. These proteins receive a signal from the activated germinant receptor and release DPA. This DPA activates the cortex lytic enzyme CwlJ, and cortex degradation begins. In Clostridium difficile, spore germination is initiated in response to certain bile acids and amino acids. These bile acids interact with the CspC germinant receptor, which then transfers the signal to the CspB protease. Activated CspB cleaves the cortex lytic enzyme, pro-SleC, to its active form. Subsequently, DPA is released from the core. C. difficile encodes orthologues of spoVAC, spoVAD, and spoVAE. Of these, the B. subtilis SpoVAC protein was shown to be capable of mechanosensing. Because cortex degradation precedes DPA release during C. difficile spore germination (opposite of what occurs in B. subtilis), we hypothesized that cortex degradation would relieve the osmotic constraints placed on the inner spore membrane and permit DPA release. Here, we assayed germination in the presence of osmolytes, and we found that they can delay DPA release from germinating C. difficile spores while still permitting cortex degradation. Together, our results suggest that DPA release during C. difficile spore germination occurs though a mechanosensing mechanism. IMPORTANCEClostridium difficile is transmitted between hosts in the form of a dormant spore, and germination by C. difficile spores is required to initiate infection, because the toxins that are necessary for disease are not deposited on the spore form. Importantly, the C. difficile spore germination pathway represents a novel pathway for bacterial spore germination. Prior work has shown that the order of events during C. difficile spore germination (cortex degradation and DPA release) is flipped compared to the events during B. subtilis spore germination, a model organism. Here, we further characterize the C. difficile spore germination pathway and summarize our findings indicating that DPA release by germinating C. difficile spores occurs through a mechanosensing mechanism in response to the degradation of the spore cortex.
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Abstract
Many anaerobic spore-forming clostridial species are pathogenic, and some are industrially useful. Although many are strict anaerobes, the bacteria persist under aerobic and growth-limiting conditions as multilayered metabolically dormant spores. For many pathogens, the spore form is what most commonly transmits the organism between hosts. After the spores are introduced into the host, certain proteins (germinant receptors) recognize specific signals (germinants), inducing spores to germinate and subsequently grow into metabolically active cells. Upon germination of the spore into the metabolically active vegetative form, the resulting bacteria can colonize the host and cause disease due to the secretion of toxins from the cell. Spores are resistant to many environmental stressors, which make them challenging to remove from clinical environments. Identifying the conditions and the mechanisms of germination in toxin-producing species could help develop affordable remedies for some infections by inhibiting germination of the spore form. Unrelated to infectious disease, spore formation in species used in the industrial production of chemicals hinders the optimum production of the chemicals due to the depletion of the vegetative cells from the population. Understanding spore germination in acetone-butanol-ethanol-producing species can help boost the production of chemicals, leading to cheaper ethanol-based fuels. Until recently, clostridial spore germination is assumed to be similar to that of Bacillus subtilis However, recent studies in Clostridium difficile shed light on a mechanism of spore germination that has not been observed in any endospore-forming organisms to date. In this review, we focus on the germinants and the receptors recognizing these germinants in various clostridial species.
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50
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Impact of microbial derived secondary bile acids on colonization resistance against Clostridium difficile in the gastrointestinal tract. Anaerobe 2016; 41:44-50. [PMID: 27163871 DOI: 10.1016/j.anaerobe.2016.05.003] [Citation(s) in RCA: 97] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2016] [Revised: 04/29/2016] [Accepted: 05/03/2016] [Indexed: 02/08/2023]
Abstract
Clostridium difficile is an anaerobic, Gram positive, spore-forming bacillus that is the leading cause of nosocomial gastroenteritis. Clostridium difficile infection (CDI) is associated with increasing morbidity and mortality, consequently posing an urgent threat to public health. Recurrence of CDI after successful treatment with antibiotics is high, thus necessitating discovery of novel therapeutics against this pathogen. Susceptibility to CDI is associated with alterations in the gut microbiota composition and bile acid metabolome, specifically a loss of microbial derived secondary bile acids. This review aims to summarize in vitro, ex vivo, and in vivo studies done by our group and others that demonstrate how secondary bile acids affect the different stages of the C. difficile life cycle. Understanding the dynamic interplay of C. difficile and microbial derived secondary bile acids within the gastrointestinal tract will shed light on how bile acids play a role in colonization resistance against C. difficile. Rational manipulation of secondary bile acids may prove beneficial as a treatment for patients with CDI.
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