The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile
ABSTRACT Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacteria that is one of the leading causes of antibiotic-associated diarrhea. The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immu...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-04-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02704-21 |
| _version_ | 1828406944234209280 |
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| author | Qingshuai Zhou Fengqin Rao Zhenghong Chen Yumei Cheng Qifang Zhang Jie Zhang Zhizhong Guan Yan He Wenfeng Yu Guzhen Cui Xiaolan Qi Wei Hong |
| author_facet | Qingshuai Zhou Fengqin Rao Zhenghong Chen Yumei Cheng Qifang Zhang Jie Zhang Zhizhong Guan Yan He Wenfeng Yu Guzhen Cui Xiaolan Qi Wei Hong |
| author_sort | Qingshuai Zhou |
| collection | DOAJ |
| description | ABSTRACT Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacteria that is one of the leading causes of antibiotic-associated diarrhea. The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. We constructed a cwp66 gene deletion mutant using Clustered Regularly Interspaced Short Palindromic Repeats Cpf1 (CRISPR-Cpf1) system. The phenotypic and transcriptomic changes of the Δcwp66 mutant compared with the wild-type (WT) strain were studied. The deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the Δcwp66 mutant is more sensitive than the WT strain to clindamycin, ampicillin, and erythromycin but more resistant than the latter to vancomycin and metronidazole. Moreover, mannitol utilization capability in the Δcwp66 mutant was lost. Comparative transcriptomic analyses indicated that (i) 22.90-fold upregulation of cwpV gene and unable to express gpr gene were prominent in the Δcwp66 mutant; (ii) the cwp66 gene was involved in vancomycin resistance of C. difficile by influencing the expression of d-Alanine-d-Alanine ligase; and (iii) the mannose/fructose/sorbose IIC and IID components were upregulated in Δcwp66 mutant. The present work deepens our understanding of the contribution of the cwp66 gene to cell adhesion, stress tolerance, antibiotic resistance, and mannitol transportation of C. difficile. IMPORTANCE The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. The current study provides direct evidence that the cwp66 gene serves as a major adhesion in C. difficile, and also suggested that deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the antibiotic resistance and carbon source utilization profiles of the Δcwp66 mutant were significantly changed. These phenotypes were detrimental to the survival and pathogenesis of C. difficile in the human gut and may shed light on preventing C. difficile infection. |
| first_indexed | 2024-12-10T11:18:44Z |
| format | Article |
| id | doaj.art-3f93aeb349fc41f7abe52cfd74302c41 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-12-10T11:18:44Z |
| publishDate | 2022-04-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-3f93aeb349fc41f7abe52cfd74302c412022-12-22T01:51:03ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972022-04-0110210.1128/spectrum.02704-21The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficileQingshuai Zhou0Fengqin Rao1Zhenghong Chen2Yumei Cheng3Qifang Zhang4Jie Zhang5Zhizhong Guan6Yan He7Wenfeng Yu8Guzhen Cui9Xiaolan Qi10Wei Hong11Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, ChinaKey Laboratory of Endemic and Ethnic Diseases, Ministry of Education & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, ChinaKey Laboratory of Microbiology and Parasitology of Education Department of Guizhou, School of Basic Medical Science, Guizhou Medical University, Guiyang, Guizhou, ChinaDepartment of Critical Care Medicine, the Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou, ChinaKey Laboratory of Endemic and Ethnic Diseases, Ministry of Education & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, ChinaState Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, ChinaKey Laboratory of Endemic and Ethnic Diseases, Ministry of Education & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, ChinaKey Laboratory of Endemic and Ethnic Diseases, Ministry of Education & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, ChinaKey Laboratory of Endemic and Ethnic Diseases, Ministry of Education & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, ChinaKey Laboratory of Microbiology and Parasitology of Education Department of Guizhou, School of Basic Medical Science, Guizhou Medical University, Guiyang, Guizhou, ChinaKey Laboratory of Endemic and Ethnic Diseases, Ministry of Education & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, ChinaKey Laboratory of Endemic and Ethnic Diseases, Ministry of Education & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, ChinaABSTRACT Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacteria that is one of the leading causes of antibiotic-associated diarrhea. The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. We constructed a cwp66 gene deletion mutant using Clustered Regularly Interspaced Short Palindromic Repeats Cpf1 (CRISPR-Cpf1) system. The phenotypic and transcriptomic changes of the Δcwp66 mutant compared with the wild-type (WT) strain were studied. The deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the Δcwp66 mutant is more sensitive than the WT strain to clindamycin, ampicillin, and erythromycin but more resistant than the latter to vancomycin and metronidazole. Moreover, mannitol utilization capability in the Δcwp66 mutant was lost. Comparative transcriptomic analyses indicated that (i) 22.90-fold upregulation of cwpV gene and unable to express gpr gene were prominent in the Δcwp66 mutant; (ii) the cwp66 gene was involved in vancomycin resistance of C. difficile by influencing the expression of d-Alanine-d-Alanine ligase; and (iii) the mannose/fructose/sorbose IIC and IID components were upregulated in Δcwp66 mutant. The present work deepens our understanding of the contribution of the cwp66 gene to cell adhesion, stress tolerance, antibiotic resistance, and mannitol transportation of C. difficile. IMPORTANCE The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. The current study provides direct evidence that the cwp66 gene serves as a major adhesion in C. difficile, and also suggested that deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the antibiotic resistance and carbon source utilization profiles of the Δcwp66 mutant were significantly changed. These phenotypes were detrimental to the survival and pathogenesis of C. difficile in the human gut and may shed light on preventing C. difficile infection.https://journals.asm.org/doi/10.1128/spectrum.02704-21Clostridioides difficileCRISPR-Cpf1cell wall protein 66 (Cwp66)phenotypic analysistranscriptome analysis |
| spellingShingle | Qingshuai Zhou Fengqin Rao Zhenghong Chen Yumei Cheng Qifang Zhang Jie Zhang Zhizhong Guan Yan He Wenfeng Yu Guzhen Cui Xiaolan Qi Wei Hong The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile Microbiology Spectrum Clostridioides difficile CRISPR-Cpf1 cell wall protein 66 (Cwp66) phenotypic analysis transcriptome analysis |
| title | The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile |
| title_full | The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile |
| title_fullStr | The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile |
| title_full_unstemmed | The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile |
| title_short | The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile |
| title_sort | cwp66 gene affects cell adhesion stress tolerance and antibiotic resistance in clostridioides difficile |
| topic | Clostridioides difficile CRISPR-Cpf1 cell wall protein 66 (Cwp66) phenotypic analysis transcriptome analysis |
| url | https://journals.asm.org/doi/10.1128/spectrum.02704-21 |
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