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Item Open Access Bioengineering of bacteriophage-derived endolysin against Clostridioides difficile(Mahidol University, 2022) Wichuda Phothichaisri; Sittinan Chanarat; Surang Chankhamhaengdecha; Tavan JanvilisriClostridioides 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.Item Open Access Production of co-expressed double-stranded RNA and virus-like particle in a novel escherichia coli strain for protection against white spot syndrome virus in shrimp(Mahidol University, 2021) Kitti Wuthisathid; Ornchuma Itsathitphaisarn; Thawatchai Chaijarasphong; Kallaya Sritunyalucksana; Charoonroj Chotwiwatthanakun; Monsicha SomritWhite spot syndrome virus (WSSV) causes significant economic losses to shrimp aquaculture worldwide. In severe cases, WSSV can lead to a 100% shrimp mortality rate within five days. The aquaculture industry requires antiviral strategies. Currently, dsRNA-based platforms are among the promising tools for triggering an antiviral response in shrimp through an RNA interference (RNAi) pathway. The main obstacle to utilizing the RNAi strategy in the shrimp aquaculture industry is the lack of a low-cost, efficient, and practical delivery approach. Recently, virus-like particles (VLPs) have been used as nanocarriers to deliver dsRNA into shrimp tissues. The VLP-based delivery system extends the half-life of the targeted dsRNA, provides broad tissue tropism, and enhances shrimp’s innate immunity. Recombinant proteins and dsRNA are traditionally expressed in two different E. coli strains, a protease-deficient BL21(DE3) strain, and an RNase III-deficient HT115(DE3) strain, respectively. To reduce the production cost of dsRNA-based treatment, VLPs and dsRNA were simultaneously expressed in a novel E. coli strain which is both protease- and RNase III-deficient, constructed via P1 phage transduction. The results revealed that the newly engineered E. coli strain could be harnessed to co-express MrNV-VLP and dsRNA-VP28. Co-expression of VLPs and dsRNA in the same cell is feasible. This platform could serve as a basis for future cost-effective and streamlined production of shrimp antiviral therapeutics.