綠膿桿菌噬菌體其內溶素的純化與功能特性分析

Abstract

綠膿桿菌是一種伺機性病原菌，常引發院內感染，並廣泛存在於水源、土壤以及下水道、水塔等潮濕環境中。由於綠膿桿菌會產生生物膜（biofilm）及莢膜（capsule）等致病因子，導致細菌難以徹底清除，大幅降低抗生素治療效果，進而引發抗藥性問題，為臨床治療帶來重大挑戰。因此，尋找新的治療方法成為當前研究的重點。噬菌體（bacteriophage）是一種能夠感染細菌的病毒，並且具有宿主特異性，噬菌體除了能夠直接裂解細菌外，還能編碼多種具有抗菌活性的酵素，例如內溶素（endolysin），因此，本研究希望從噬菌體中篩選並鑑定具有抗菌活性的酵素，以作為開發新型抗菌藥物的潛在候選物，為對抗抗藥性細菌提供新的解決方案。 本研究首先從醫院廢水中分離出噬菌體，並通過一系列實驗方法進行純化與鑑定。首先使用噬菌斑試驗（plaque assay）確認噬菌體的活性與型態，透過點試驗（spot test）確認有廣泛的綠膿桿菌宿主範圍，同時為了開發噬菌體衍生之酵素，優先挑選具有分泌酵素潛力的雙圈型態噬菌斑，並進行限制性片段長度多型性（restriction fragment length polymorphism）分析與全基因體序列分析，最終確定該噬菌體為新型噬菌體，命名為P28。將P28序列經過註解（annotation）分析，分別找到可能具有內溶素活性序列：CDS_183與CDS_223， 以及具有降解多醣活性的序列CDS_221。CDS_183 之M6片段與CDS_223（後稱為M144）經體外殺菌活性試驗（In vitro killing assay）證明內溶素在EDTA協同下可有效清除細菌，在100 μg/mL M6的作用下，綠膿桿菌菌株PA054的存活率幾乎為0%；在100 μg/mL M144的作用下，存活率為0.48%。而CDS_221可能帶有解聚酶活性，1250 μg/mL之CDS_221能有效將莢膜多醣以及其他多醣類降解成小片段多糖。因此證明P28帶有具發展藥物潛力的兩種殺菌蛋白，與一種可能具有破壞多醣類的酵素，未來可應用於協同抗生素一同清除病原菌。

P. aeruginosa is an opportunistic pathogen that frequently causes nosocomial infections. It is commonly found in water sources, soil, sewers, water tanks, and similar environments. Due to its ability to form biofilms and produce virulence factors like the capsule structure, the bacteria become difficult to eradicate, reducing the efficacy of antibiotics and contributing to the rise of antibiotic resistance. This presents a critical challenge for clinical treatment, highlighting the urgent need for alternative therapies. Bacteriophages are viruses that can infect bacteria with high host specificity. In addition to directly lysing bacteria, bacteriophages can encode various enzymes with antibacterial activity, such as endolysins. Therefore, this study aims to screen and characterize antibacterial enzymes from bacteriophages as potential candidates for the development of novel antimicrobial agents, providing new solutions to combat antibiotic-resistant bacteria. In this study, a bacteriophage was isolated from hospital wastewater, purified, and characterized through a series of experiments. Plaque assays were used to confirm the activity and morphology of the bacteriophage, and spot tests demonstrated a broad host range against P. aeruginosa. To develop phage-derived enzymes, double-ring plaques, which indicate potential enzyme secretion, were prioritized for further study. Restriction fragment length polymorphism analysis and whole-genome sequencing revealed that the phage is a novel strain, named P28. The sequences potentially possessing endolysin activity are identified as CDS_183 and CDS_223, while the sequence with polysaccharide-degrading activity is identified as CDS_221. In vitro bactericidal assays showed that the M6 fragment of CDS_183 and CDS_223（hereafter referred to as M144） exhibited effective endolysin activity in the presence of EDTA, eliminating the P. aeruginosa strain PA054. At a 100 μg/mL concentration, M6 achieved an almost 0% survival rate of PA054, while M144 resulted in a survival rate of 0.48%. CDS_221 may possess depolymerase activity by effectively degrading capsular polysaccharides into smaller polysaccharide fragments at 1250 μg/mL. This proves that P28 contains two bactericidal proteins with potential for drug development, as well as an enzyme that may degrade polysaccharides, which could be applied in the future to assist antibiotics in eliminating pathogens.