分離及鑑定一金黃色葡萄球菌噬菌體之溶菌酶

Abstract

金黃色葡萄球菌屬革蘭氏陽性球菌，在近10年台灣醫療院所常見的感染症病源常居前10名，層出不窮的抗藥性菌株也顯示需要可替代或輔助傳統療程的新策略。噬菌體為可辨認細菌表面構造，並感染、殺死菌株的病毒，故有治療細菌感染症的潛力。本實驗目的為分離、鑑定宿主範圍廣、殺菌能力亦佳的金黃色葡萄球菌噬菌體，表現、純化具抑菌活性的溶菌蛋白（lytic proteins），並討論其應用於醫療用途的可能性。我們自13種水源分離出9株噬菌體，從其中選擇øS4-1，與MRSA菌株S. aureus SA055進行噬菌體殺菌試驗（killing assay），其於MOI = 1×10^3時可將菌株存活率下降至57.9%，MOI＝1×10^4時則可下降至1.6%。限制片段長度多型性（restriction fragment length polymorphism）顯示øS4-1的切割圖譜未與之前所發現的噬菌體的重複，顯示其為尚未被我們分離的噬菌體。全基因體定序（whole genome sequencing，WGS）顯示øS4-1屬於尾狀噬菌體目（Caudovirales）的Rountreeviridae科，與比對到最為相似的噬菌體PSa3相似度為92.82%。爾後挑選øS4-1基因上三種可能具溶菌蛋白活性的蛋白進行表現，分別命名為øS4-1 endolysin、øS4-1 tail protein-1及øS4-1 minor tail protein，且經數據庫比對後，相似蛋白的研究顯示øS4-1 minor tail protein可能具有溶菌酶之活性。øS4-1 endolysin在破菌後僅分佈於離心的沉澱物，並未分布在上清液中，øS4-1 tail protein-1具高水溶性，但不能夠結合上磁珠，故此兩蛋白無法完成純化。øS4-1 minor tail protein則可被表現與純化，且在點試驗中對數種金黃色葡萄球菌株皆具抑菌效果。然而使用該蛋白與噬菌體øS4-1混合，共同執行殺菌試驗後，發現相較於只有噬菌體的組別，添加øS4-1 minor tail protein並不能降低SA055的存活率。本實驗所分離之噬菌體øS4-1，以及所純化出的øS4-1 minor tail protein在多株金黃色葡萄球菌株，包含數株MRSA上皆表現出抑菌效果，顯示他們具有廣泛的宿主範圍。與他種溶菌蛋白共同使用，或與抗生素做合併治療，皆有機會提升治療效果。因此噬菌體øS4-1，以及所純化出的øS4-1 minor tail protein對輔助現有療法，或提供替代療法上仍具有發展潛力。

Staphylococcus aureus, a Gram-positive bacterium, has been among the top 10 common pathogens for causing infections over the past decade in Taiwan. The proliferating emergence of antibiotic-resistant strains also indicates the urgency to develop alternative therapies to strengthen traditional antibiotic treatments. Bacteriophages are viruses that can specifically recognize bacterial surface structures, infect them, and eventually lysis the host strains, thus they are potential for treating bacterial infections. This study aims to isolate and characterize S. aureus phages which bear a broad host range and good bactericidal activity, purify their hypothetical lytic proteins, and discuss their potential applications for the implications in medicine. We isolated 9 phages from 13 sewage samples and selected øS4-1 for conducting killing assays against S. aureus SA055. At an MOI of 1×10^3, øS4-1 could reduce the survival rate of the bacterial strain to 57.9% and reduced to 1.6% at an MOI of 1×10^4. The result of restriction fragment length polymorphism did not repeat with other phages isolated by our lab, indicating that øS4-1 may be a novel phage to our laboratory. Results of whole genome sequencing (WGS) indicated that øS4-1 belongs to the Rountreeviridae family from Caudovirale, and shared 92.82% identity with PSa3, the most closely matched phage in the NCBI database. Among all the sequences of øS4-1, we selected three hypothetical lytic protein sequences for further expression and named them as øS4-1 endolysin, tail protein-1, and øS4-1 minor tail protein. Research about one similar protein of øS4-1 minor tail protein indicated that it may possess endolysin activity. After protein expression, øS4-1 endolysin did not distribute in the centrifuged supernatant but only distributed in precipitate after cell lysis. Tail protein-1 exhibited high solubility but couldn't bind to magnetic beads. Therefore, these two proteins could not carry out the purification process. The øS4-1 minor tail protein can be expressed and purified successfully, and it also exhibited bacteriostatic effects against several S. aureus strains in the result of the spot test. However, after combining øS4-1 minor tail protein with phage øS4-1 for killing assay, the result showed that the addition of øS4-1 minor tail protein couldn’t reduce the survival rate of SA055 compared to the phage-only group. The isolated phage øS4-1 and the purified øS4-1 minor tail protein both can present anti-microbial activity among multiple S. aureus strains, including several MRSA strains, suggesting that they have a broad host spectrum. If combined with other lytic proteins or antibiotics, it has the potential to enhance therapeutic effects. Therefore, phage øS4-1 and the øS4-1 minor tail protein still have potential applications for serving as alternative therapies for antibiotics treatment or as adjuncts for existing therapies.