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Bratkovič T, Zahirović A, Bizjak M, Rupnik M, Štrukelj B, Berlec A. New treatment approaches for Clostridioides difficile infections: alternatives to antibiotics and fecal microbiota transplantation. Gut Microbes 2024; 16:2337312. [PMID: 38591915 PMCID: PMC11005816 DOI: 10.1080/19490976.2024.2337312] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/29/2024] [Accepted: 03/27/2024] [Indexed: 04/10/2024] Open
Abstract
Clostridioides difficile causes a range of debilitating intestinal symptoms that may be fatal. It is particularly problematic as a hospital-acquired infection, causing significant costs to the health care system. Antibiotics, such as vancomycin and fidaxomicin, are still the drugs of choice for C. difficile infections, but their effectiveness is limited, and microbial interventions are emerging as a new treatment option. This paper focuses on alternative treatment approaches, which are currently in various stages of development and can be divided into four therapeutic strategies. Direct killing of C. difficile (i) includes beside established antibiotics, less studied bacteriophages, and their derivatives, such as endolysins and tailocins. Restoration of microbiota composition and function (ii) is achieved with fecal microbiota transplantation, which has recently been approved, with standardized defined microbial mixtures, and with probiotics, which have been administered with moderate success. Prevention of deleterious effects of antibiotics on microbiota is achieved with agents for the neutralization of antibiotics that act in the gut and are nearing regulatory approval. Neutralization of C. difficile toxins (iii) which are crucial virulence factors is achieved with antibodies/antibody fragments or alternative binding proteins. Of these, the monoclonal antibody bezlotoxumab is already in clinical use. Immunomodulation (iv) can help eliminate or prevent C. difficile infection by interfering with cytokine signaling. Small-molecule agents without bacteriolytic activity are usually selected by drug repurposing and can act via a variety of mechanisms. The multiple treatment options described in this article provide optimism for the future treatment of C. difficile infection.
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Affiliation(s)
- Tomaž Bratkovič
- Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
| | - Abida Zahirović
- Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia
| | - Maruša Bizjak
- Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia
| | - Maja Rupnik
- National Laboratory for Health, Environment and Food, Prvomajska 1, Maribor, Slovenia
- Faculty of Medicine, University of Maribor, Maribor, Slovenia
| | - Borut Štrukelj
- Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
- Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia
| | - Aleš Berlec
- Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
- Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia
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2
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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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4
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Hu X, Dong R, Huang S, Zeng Y, Zhan W, Gao X, Tian D, Peng J, Xu J, Wang T, Zhang Y, Wang X, Zhang X, Liu J, Guang B, Yang T. CDBN-YGXZ, a Novel Small-Molecule Drug, Shows Efficacy against Clostridioides difficile Infection and Recurrence in Mouse and Hamster Infection Models. Antimicrob Agents Chemother 2023; 67:e0170422. [PMID: 37052498 PMCID: PMC10190532 DOI: 10.1128/aac.01704-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2022] [Accepted: 03/01/2023] [Indexed: 04/14/2023] Open
Abstract
Clostridioides difficile infection (CDI) causes severe diarrhea and colitis, leading to significant morbidity, mortality, and high medical costs worldwide. Oral vancomycin, a first-line treatment for CDI, is associated with a high risk of recurrence, necessitating novel therapies for primary and recurrent CDI. A novel small-molecule compound, CDBN-YGXZ, was synthesized by modifying the benzene ring of nitazoxanide with lauric acid. The mechanism of action of CDBN-YGXZ was validated using a pyruvate:ferredoxin/flavodoxin oxidoreductase (PFOR) inhibition assay. The efficacy of CDBN-YGXZ was evaluated using the MIC test and CDI infection model in mice and hamsters. Furthermore, metagenomics was used to reveal the underlying reasons for the effective reduction or prevention of CDI after CDBN-YGXZ treatment. The inhibitory activity against PFOR induced by CDBN-YGXZ. MIC tests showed that the in vitro activity of CDBN-YGXZ against C. difficile ranging from 0.1 to 1.5 μg/mL. In the mouse and hamster CDI models, CDBN-YGXZ provided protection during both treatment and relapse, while vancomycin treatment resulted in severe relapse and significant clinical scores. Compared with global effects on the indigenous gut microbiota induced by vancomycin, CDBN-YGXZ treatment had a mild influence on gut microbes, thus resulting in the disappearance or reduction of CDI recurrence. CDBN-YGXZ displayed potent activity against C. difficile in vitro and in vivo, reducing or preventing relapse in infected animals, which could merit further development as a potential drug candidate for treating CDI.
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Affiliation(s)
- Xiaojun Hu
- School of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China
| | - Renhan Dong
- Chengdu Biobel Biotechnology Co., Ltd., Chengdu, Sichuan Province, China
| | - Sheng Huang
- School of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China
| | - Yisheng Zeng
- School of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China
| | - Wei Zhan
- Chengdu Biobel Biotechnology Co., Ltd., Chengdu, Sichuan Province, China
| | - Xiaofang Gao
- School of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China
| | - Dong Tian
- School of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China
| | - Jian Peng
- School of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China
| | - Jiewei Xu
- School of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China
| | - Ting Wang
- School of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China
| | - Yaying Zhang
- School of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China
| | - Xiaohui Wang
- Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan Province, China
| | - Xiaoxia Zhang
- Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan Province, China
| | - Jin Liu
- School of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China
| | - Bing Guang
- School of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China
- Chengdu Biobel Biotechnology Co., Ltd., Chengdu, Sichuan Province, China
| | - Tai Yang
- School of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China
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Budi ND, Godfrey JJ, Safdar N, Shukla SK, Rose WE. Efficacy of Omadacycline or Vancomycin Combined With Germinants for Preventing Clostridioides difficile Relapse in a Murine Model. J Infect Dis 2023; 227:622-630. [PMID: 35904942 PMCID: PMC9978312 DOI: 10.1093/infdis/jiac324] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2022] [Revised: 07/15/2022] [Accepted: 07/27/2022] [Indexed: 11/12/2022] Open
Abstract
BACKGROUND Clostridioides difficile infections (CDI) and recurrence (rCDI) are major health care burdens. Recurrence is likely caused by spores in the gastrointestinal tract that germinate after antibiotic therapy. This murine study explores germinant-antibiotic combinations for CDI. METHODS Previously described murine models were evaluated using C. difficile VPI 10463. The severe model compared omadacycline versus vancomycin in survival, weight loss, clinical scoring, and C. difficile toxin production. The nonsevere model compared these antibiotics with and without germinants (solution of sodium taurocholate, taurine, sodium docusate, calcium gluconate). Additionally, colon histopathology, bile acid analysis, environmental/spore shedding, and 16S sequencing was evaluated. RESULTS In the severe model, omadacycline-treated mice had 60% survival versus 13.3% with vancomycin (hazard ratio [HR], 0.327; 95% confidence interval [CI],.126-.848; P = .015) along with decreased weight loss, and disease severity. In the nonsevere model, all mice survived with antibiotic-germinant treatment versus 60% antibiotics alone (HR, 0.109; 95% CI, .02-.410; P = .001). Omadacycline resulted in less changes in bile acids and microbiota composition. Germinant-treated mice showed no signs of rCDI, spore shedding, or significant toxin production at 15 days. CONCLUSIONS In murine models of CDI, omadacycline improved survival versus vancomycin. Germinant-antibiotic combinations were more effective at preventing rCDI compared to antibiotics alone without inducing toxin production.
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Affiliation(s)
- Noah D Budi
- School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Jared J Godfrey
- Division of Infectious Diseases, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA
- William S. Middleton Memorial Veterans Hospital, Madison, Wisconsin, USA
| | - Nasia Safdar
- Division of Infectious Diseases, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA
- William S. Middleton Memorial Veterans Hospital, Madison, Wisconsin, USA
| | - Sanjay K Shukla
- Center for Precision Medicine Research, Marshfield Clinic Research Institute, Marshfield, Wisconsin, USA
| | - Warren E Rose
- School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA
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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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Bhute SS, Mefferd CC, Phan JR, Ahmed M, Fox-King AE, Alarcia S, Villarama JV, Abel-Santos E, Hedlund BP. A High-Carbohydrate Diet Prolongs Dysbiosis and Clostridioides difficile Carriage and Increases Delayed Mortality in a Hamster Model of Infection. Microbiol Spectr 2022; 10:e0180421. [PMID: 35708337 PMCID: PMC9431659 DOI: 10.1128/spectrum.01804-21] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2021] [Accepted: 05/21/2022] [Indexed: 12/20/2022] Open
Abstract
Studies using mouse models of Clostridioides difficile infection (CDI) have demonstrated a variety of relationships between dietary macronutrients on antibiotic-associated CDI; however, few of these effects have been examined in more susceptible hamster models of CDI. In this study, we investigated the effect of a high-carbohydrate diet previously shown to protect mice from CDI on the progression and resolution of CDI in a hamster disease model, with 10 animals per group. Hamsters fed the high-carbohydrate diet developed distinct diet-specific microbiomes during antibiotic treatment and CDI, with lower diversity, persistent C. difficile carriage, and delayed microbiome restoration. In contrast to CDI protection in mice, most hamsters fed a high-carbohydrate diet developed fulminant CDI including several cases of late-onset CDI, that were not observed in hamsters fed a standard lab diet. We speculate that prolonged high-carbohydrate diet-specific dysbiosis in these animals allowed C. difficile to persist in the gut of the animals where they could proliferate postvancomycin treatment, leading to delayed CDI onset. This study, along with similar studies in mouse models of CDI, suggests some high-carbohydrate diets may promote antibiotic-associated dysbiosis and long-term C. difficile carriage, which may later convert to symptomatic CDI. IMPORTANCE The effects of diet on CDI are not completely known. Here, we used a high-carbohydrate diet previously shown to protect mice against CDI to assess its effect on a hamster model of CDI and paradoxically found that it promoted dysbiosis, C. difficile carriage, and higher mortality. A common thread in both mouse and hamster experimental models was that the high-carbohydrate diet promoted dysbiosis and long-term carriage of C. difficile, which may have converted to fulminant CDI only in the highly susceptible hamster model system. If diets high in carbohydrates also promote dysbiosis and C. difficile carriage in humans, then these diets might paradoxically increase chances of CDI relapse despite their protective effects against primary CDI.
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Affiliation(s)
- Shrikant S. Bhute
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
| | - Chrisabelle C. Mefferd
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
- School of Public Health, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
| | - Jacqueline R. Phan
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
| | - Muneeba Ahmed
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
- College of Osteopathic Medicine, Touro University, Nevada, Henderson, Nevada, USA
| | - Amelia E. Fox-King
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
- College of Osteopathic Medicine, Touro University, Nevada, Henderson, Nevada, USA
| | - Stephanie Alarcia
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
| | - Jacob V. Villarama
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
- Kirk Kerkorian School of Medicine, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
| | - Ernesto Abel-Santos
- Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
- Nevada Institute of Personalized Medicine, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
| | - Brian P. Hedlund
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
- Nevada Institute of Personalized Medicine, University of Nevada, Las Vegas, Las Vegas, Nevada, USA
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8
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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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9
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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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10
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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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11
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Monaghan TM, Seekatz AM, Mullish BH, Moore-Gillon CCER, Dawson LF, Ahmed A, Kao D, Chan WC. Clostridioides difficile: innovations in target discovery and potential for therapeutic success. Expert Opin Ther Targets 2021; 25:949-963. [PMID: 34793686 DOI: 10.1080/14728222.2021.2008907] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2021] [Accepted: 11/17/2021] [Indexed: 10/19/2022]
Abstract
INTRODUCTION Clostridioides difficile infection (CDI) remains a worldwide clinical problem. Increased incidence of primary infection, occurrence of hypertoxigenic ribotypes, and more frequent occurrence of drug resistant, recurrent, and non-hospital CDI, emphasizes the urgent unmet need of discovering new therapeutic targets. AREAS COVERED We searched PubMed and Web of Science databases for articles identifying novel therapeutic targets or treatments for C. difficile from 2001 to 2021. We present an updated review on current preclinical efforts on designing inhibitory compounds against these drug targets and indicate how these could become the focus of future therapeutic approaches. We also evaluate the increasing exploitability of gut microbial-derived metabolites and host-derived therapeutics targeting VEGF-A, immune targets and pathways, ion transporters, and microRNAs as anti-C. difficile therapeutics, which have yet to reach clinical trials. Our review also highlights the therapeutic potential of re-purposing currently available agents . We conclude by considering translational hurdles and possible strategies to mitigate these problems. EXPERT OPINION Considerable progress has been made in the development of new anti-CDI drug candidates. Nevertheless, a greater comprehension of CDI pathogenesis and host-microbe interactions is beginning to uncover potential novel therapeutic targets, which can be exploited to plug gaps in the CDI drug discovery pipeline.
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Affiliation(s)
- Tanya M Monaghan
- NIHR Nottingham Biomedical Research Centre, University of Nottingham, Nottingham, UK
- Nottingham Digestive Diseases Centre, School of Medicine, University of Nottingham, Nottingham, UK
| | - Anna M Seekatz
- Biological Sciences, Clemson University, Clemson, SC, USA
| | - Benjamin H Mullish
- Division of Digestive Diseases, Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London, UK
- Departments of Gastroenterology and Hepatology, St Mary's Hospital, Imperial College Healthcare NHS Trust, London, UK
| | - Claudia C E R Moore-Gillon
- Division of Digestive Diseases, Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London, UK
- Departments of Gastroenterology and Hepatology, St Mary's Hospital, Imperial College Healthcare NHS Trust, London, UK
| | - Lisa F Dawson
- Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, UK
| | - Ammar Ahmed
- NIHR Nottingham Biomedical Research Centre, University of Nottingham, Nottingham, UK
- Nottingham Digestive Diseases Centre, School of Medicine, University of Nottingham, Nottingham, UK
| | - Dina Kao
- Department of Gastroenterology, Zeidler Ledcor Centre, University of Alberta, Edmonton, Alberta, Canada
| | - Weng C Chan
- School of Pharmacy, Biodiscovery Institute, University of Nottingham, University Park, Nottingham, UK
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12
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Aguirre AM, Yalcinkaya N, Wu Q, Swennes A, Tessier ME, Roberts P, Miyajima F, Savidge T, Sorg JA. Bile acid-independent protection against Clostridioides difficile infection. PLoS Pathog 2021; 17:e1010015. [PMID: 34665847 PMCID: PMC8555850 DOI: 10.1371/journal.ppat.1010015] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2021] [Revised: 10/29/2021] [Accepted: 10/07/2021] [Indexed: 12/21/2022] Open
Abstract
Clostridioides difficile infections occur upon ecological / metabolic disruptions to the normal colonic microbiota, commonly due to broad-spectrum antibiotic use. Metabolism of bile acids through a 7α-dehydroxylation pathway found in select members of the healthy microbiota is regarded to be the protective mechanism by which C. difficile is excluded. These 7α-dehydroxylated secondary bile acids are highly toxic to C. difficile vegetative growth, and antibiotic treatment abolishes the bacteria that perform this metabolism. However, the data that supports the hypothesis that secondary bile acids protect against C. difficile infection is supported only by in vitro data and correlative studies. Here we show that bacteria that 7α-dehydroxylate primary bile acids protect against C. difficile infection in a bile acid-independent manner. We monoassociated germ-free, wildtype or Cyp8b1-/- (cholic acid-deficient) mutant mice and infected them with C. difficile spores. We show that 7α-dehydroxylation (i.e., secondary bile acid generation) is dispensable for protection against C. difficile infection and provide evidence that Stickland metabolism by these organisms consumes nutrients essential for C. difficile growth. Our findings indicate secondary bile acid production by the microbiome is a useful biomarker for a C. difficile-resistant environment but the microbiome protects against C. difficile infection in bile acid-independent mechanisms. Secondary bile acid production by the colonic microbiome strongly correlates with an environment that is resistant to C. difficile invasion. However, it remained unclear if these bile acids provided in vivo protection. Here, we show that members of the microbiome that generate secondary bile acids (e.g., C. scindens) protect against C. difficile disease independently of secondary bile acid generation. These results are important because efforts to restore colonization resistance (e.g., FMT or precision bacterial therapy) focus on restoring secondary bile acid generation. Instead, restoring the organisms that produce 5-aminovalerate or consume proline / glycine are more important.
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Affiliation(s)
- Andrea Martinez Aguirre
- Department of Biology, Texas A&M University, College Station, Texas, United States of America
| | - Nazli Yalcinkaya
- Baylor College of Medicine & Texas Children’s Hospital, Houston, Texas, United States of America
| | - Qinglong Wu
- Baylor College of Medicine & Texas Children’s Hospital, Houston, Texas, United States of America
| | - Alton Swennes
- Baylor College of Medicine & Texas Children’s Hospital, Houston, Texas, United States of America
| | - Mary Elizabeth Tessier
- Baylor College of Medicine & Texas Children’s Hospital, Houston, Texas, United States of America
| | - Paul Roberts
- Royal Liverpool and Broadgreen University Hospitals NHS Trust, Liverpool, United Kingdom
| | - Fabio Miyajima
- Royal Liverpool and Broadgreen University Hospitals NHS Trust, Liverpool, United Kingdom
- Oswaldo Cruz Foundation, Ceara branch, Fortaleza, Brazil
| | - Tor Savidge
- Baylor College of Medicine & Texas Children’s Hospital, Houston, Texas, United States of America
| | - Joseph A. Sorg
- Department of Biology, Texas A&M University, College Station, Texas, United States of America
- * E-mail:
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13
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Shen A. Clostridioides difficile Spore Formation and Germination: New Insights and Opportunities for Intervention. Annu Rev Microbiol 2021; 74:545-566. [PMID: 32905755 DOI: 10.1146/annurev-micro-011320-011321] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Spore formation and germination are essential for the bacterial pathogen Clostridioides difficile to transmit infection. Despite the importance of these developmental processes to the infection cycle of C. difficile, the molecular mechanisms underlying how this obligate anaerobe forms infectious spores and how these spores germinate to initiate infection were largely unknown until recently. Work in the last decade has revealed that C. difficile uses a distinct mechanism for sensing and transducing germinant signals relative to previously characterized spore formers. The C. difficile spore assembly pathway also exhibits notable differences relative to Bacillus spp., where spore formation has been more extensively studied. For both these processes, factors that are conserved only in C. difficile or the related Peptostreptococcaceae family are employed, and even highly conserved spore proteins can have differential functions or requirements in C. difficile compared to other spore formers. This review summarizes our current understanding of the mechanisms controlling C. difficile spore formation and germination and describes strategies for inhibiting these processes to prevent C. difficile infection and disease recurrence.
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Affiliation(s)
- Aimee Shen
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA;
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14
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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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15
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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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16
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Differential effects of 'resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination. Biochem J 2020; 477:1459-1478. [PMID: 32242623 PMCID: PMC7200643 DOI: 10.1042/bcj20190875] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2019] [Revised: 03/31/2020] [Accepted: 04/03/2020] [Indexed: 01/02/2023]
Abstract
Clostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of hospital-acquired gastroenteritis. C. difficile infections begin when its spore form germinates in the gut upon sensing bile acids. These germinants induce a proteolytic signaling cascade controlled by three members of the subtilisin-like serine protease family, CspA, CspB, and CspC. Notably, even though CspC and CspA are both pseudoproteases, they are nevertheless required to sense germinants and activate the protease, CspB. Thus, CspC and CspA are part of a growing list of pseudoenzymes that play important roles in regulating cellular processes. However, despite their importance, the structural properties of pseudoenzymes that allow them to function as regulators remain poorly understood. Our recently solved crystal structure of CspC revealed that its pseudoactive site residues align closely with the catalytic triad of CspB, suggesting that it might be possible to ‘resurrect' the ancestral protease activity of the CspC and CspA pseudoproteases. Here, we demonstrate that restoring the catalytic triad to these pseudoproteases fails to resurrect their protease activity. We further show that the pseudoactive site substitutions differentially affect the stability and function of the CspC and CspA pseudoproteases: the substitutions destabilized CspC and impaired spore germination without affecting CspA stability or function. Thus, our results surprisingly reveal that the presence of a catalytic triad does not necessarily predict protease activity. Since homologs of C. difficile CspA occasionally carry an intact catalytic triad, our results indicate that bioinformatic predictions of enzyme activity may underestimate pseudoenzymes in rare cases.
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17
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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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18
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Wang Y, Li J, Zachariah P, Abrams J, Freedberg DE. Relationship between remote cholecystectomy and incident Clostridioides difficile infection. Clin Microbiol Infect 2019; 25:994-999. [PMID: 30583059 PMCID: PMC6589130 DOI: 10.1016/j.cmi.2018.12.016] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2018] [Revised: 12/02/2018] [Accepted: 12/10/2018] [Indexed: 12/18/2022]
Abstract
OBJECTIVES Cholecystectomy (CCY) is associated with increased faecal levels of secondary bile acids. Secondary bile acids confer resistance to Clostridioides difficile infection (CDI, formerly Clostridium difficile infection) in animal studies. This study tested the hypothesis that CCY confers protection against CDI by increasing gut levels of secondary bile acids. METHODS This was a retrospective case-control study. Adults hospitalized between January 2010 and June 2017 at our institution were included. CDI cases were defined as a positive stool PCR followed by anti-CDI treatment and were matched 1:1:1 with two control groups (those who tested negative for CDI and those who were not tested for CDI) by sex, age group, body mass index (BMI), and exposure to antibiotics. CCY was defined as a history of CCY at least 6 months prior to the index C. difficile test or the index admission date in the untested controls. Conditional logistic regression modelling was used to estimate the relationship between remote CCY and risk for CDI. RESULTS The final study population was 7077 (2359 CDI cases, 2359 matched controls without CDI, and 2359 matched controls not tested for CDI). Rates of remote CCY did not differ among the three groups (14.4% vs. 15.5% vs. 14.2%) and this result was unchanged after adjusting for additional clinical factors (adjusted OR 0.90, 95% CI 0.76-1.06 comparing CDI cases vs. matched controls without CDI; adjusted OR 1.04, 95% CI 0.78-1.39 comparing CDI cases vs. matched controls not tested for CDI). CONCLUSIONS There was no association between remote CCY and risk for CDI.
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Affiliation(s)
- Y Wang
- Department of Medicine, Columbia University Medical Center, New York, NY, USA.
| | - J Li
- Department of Medicine, Columbia University Medical Center, New York, NY, USA
| | - P Zachariah
- Department Pediatric Infectious Disease, Columbia University Medical Center, New York, NY, USA
| | - J Abrams
- Department of Medicine, Columbia University Medical Center, New York, NY, USA
| | - D E Freedberg
- Department of Medicine, Columbia University Medical Center, New York, NY, USA
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19
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Rohlfing AE, Eckenroth BE, Forster ER, Kevorkian Y, Donnelly ML, Benito de la Puebla H, Doublié S, Shen A. The CspC pseudoprotease regulates germination of Clostridioides difficile spores in response to multiple environmental signals. PLoS Genet 2019; 15:e1008224. [PMID: 31276487 PMCID: PMC6636752 DOI: 10.1371/journal.pgen.1008224] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2018] [Revised: 07/17/2019] [Accepted: 05/31/2019] [Indexed: 12/18/2022] Open
Abstract
The gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile is thought to use the soluble pseudoprotease, CspC, to detect bile acid germinants. To gain insight into CspC's unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile acid germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile acid-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since mutations in specific residues altered CspC's responsiveness to these different signals, CspC plays a critical role in regulating C. difficile spore germination in response to multiple environmental signals. Taken together, these studies implicate CspC as being intimately involved in the detection of distinct classes of co-germinants in addition to bile acids and thus raises the possibility that CspC functions as a signaling node rather than a ligand-binding receptor.
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Affiliation(s)
- Amy E. Rohlfing
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America
| | - Brian E. Eckenroth
- Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, United States of America
| | - Emily R. Forster
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America
- Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts, United States of America
| | - Yuzo Kevorkian
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America
- Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, United States of America
- Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts, United States of America
| | - M. Lauren Donnelly
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America
- Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, United States of America
- Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts, United States of America
| | - Hector Benito de la Puebla
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America
| | - Sylvie Doublié
- Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, United States of America
| | - Aimee Shen
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America
- * E-mail:
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