Bioengineering of bacteriophage-derived endolysin against Clostridioides difficile
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Issued Date
2022
Copyright Date
2022
Resource Type
Language
eng
File Type
application/pdf
No. of Pages/File Size
xvii, 161 leaves : ill.
Access Rights
open access
Rights
ผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้า
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (Ph.D. (Biochemistry))--Mahidol University, 2022
Suggested Citation
Wichuda Phothichaisri Bioengineering of bacteriophage-derived endolysin against Clostridioides difficile. Thesis (Ph.D. (Biochemistry))--Mahidol University, 2022. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/114205
Title
Bioengineering of bacteriophage-derived endolysin against Clostridioides difficile
Author(s)
Abstract
Clostridioides difficile is a significant cause of antibiotic-associateddiarrhea and pseudomembranous colitis. The extensive use of antibiotics drives the emergence of new antibiotic-resistant C. difficile strains with high virulence and low susceptibility to current antibiotic treatment. Therefore, the development of alternativetherapy is required. Phage endolysin is a peptidoglycan hydrolase enzyme potentially an antibacterial agent for bacterial infection treatment. To apply endolysin fortherapeutic purposes, the fundamental role of the enzyme is needed. In this study, an identical endolysin was identified from two C. difficile phages, ΦHN16-1 and ΦHN50, designated as CD16/50. The sequence analysis revealed a modular architecture of an N-terminal enzymatically active domain (EAD) and a C-terminal cell-wall binding domain(CBD). In vitro characterization confirmed that the EAD possessed bacteriolytic activity while the CBD bound bacterial cell-wall polysaccharide. The EAD alone exhibited lytic activity faster than the full-length, suggesting a CBD-independent activity. The study also showed that the CBD formed a homodimer essential for interaction with cell-wall polymer. Interestingly, the hidden Markov model analysis suggested that the CBD is likely derived from the CWB2 motif of C. difficile cell-wall proteins but exhibits a higher binding affinity to bacterial cell-wall polysaccharides. Finally, endolysin diffusion and sequential cytolytic assays suggested that CBD is required for the endolysin to be trapped into post-lytic bacterial remnants, implying its physiological roles in limiting enzyme diffusion, preserving neighboring host cells, and allowing the phage progeny to initiate new rounds of infection. Collectively, this study showed the role of CBD in endolysin regulation, which may provide an insight into designing potent endolysins against C. difficile.
Degree Name
Doctor of Philosophy
Degree Level
Doctoral degree
Degree Department
Faculty of Science
Degree Discipline
Biochemistry
Degree Grantor(s)
Mahidol University