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標題: | 臺灣環境中新分枝桿菌噬菌體之分離及特性分析 Isolating and characterizing novel mycobacteriophages from Taiwan’s environment |
作者: | 蔡理安 Li-An Tsai |
指導教授: | 呂桐睿 Todd L. Lowary |
關鍵字: | 結核病,分枝桿菌,分枝桿菌噬菌體,恥垢分枝桿菌,台灣環境檢體, Tuberculosis,Mycobacterium tuberculosis,Mycobacteriophage,Mycobacterium smegmatis,Taiwan’s environmental samples, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 結核病(Tuberculosis, TB)是由結核桿菌(Mycobacterium tuberculosis, M. tb)感染而造成的疾病。根據世界衛生組織的統計,大約四分之一的世界人口感染結核桿菌。雖然結核病為可治癒的疾病,但是抗藥性菌株的出現讓治療變得困難。因此,尋找替代的治療方法十分重要。其中噬菌體療法(Bacteriophage therapy)是具有潛力的方法之一,它利用分枝桿菌噬菌體(Mycobacteriophage)感染細菌達到治療抗藥性菌株感染的目的。分枝桿菌噬菌體是可感染分枝桿菌(Mycobacteria)的病毒,透過其尾部的受體結合蛋白(Receptor binding protein, RBP)與細菌宿主細胞壁上的受體(Receptor)專一性結合,可將遺傳物質送入細菌細胞中。噬菌體利用宿主的複製機制產生新的病毒顆粒,並在成熟後殺死宿主,釋放新的噬菌體。雖然許多分枝桿菌噬菌體已在過去研究中被分離,但是目前在臺灣並沒有分離的紀錄。臺灣地理位置相對其地區獨立,環境中的分枝桿菌噬菌體也可能較為獨特。本研究利用恥垢分枝桿菌 (Mycobacterium smegmatis, M. smegmatis)作為宿主,欲從土壤中分離分枝桿菌噬菌體,並分析其特性,從而驗證這個假設。自2021年6月至2022年2月,我們已從107個環境檢體中分離19株分枝桿菌噬菌體。其中,17株成功完成定序並經過序列組裝(Genome assembly)分入7個已知的簇(Cluster),包含Cluster A、B、C、E、K、L以及R。我們進一步對5株來自不同簇的噬菌體進行基因註釋(Genome annotation)分析,僅約30%的基因的功能已知。根據分析定序的結果可知來自不同簇噬菌體的基因具有較低的相似度,但某些基因——例如結構相關的基因——具有相似的排列方式。綜合分離的結果,從臺灣分離的分枝桿菌噬菌體都可以被歸入已知的各簇中,並沒有分離出較獨特的分枝桿菌噬菌體,所以無法驗證上述的假設。然而,本實驗只分離少數的分枝桿菌噬菌體,若增加分離的數目,較獨特的噬菌體有可能被發現。另外,改變實驗的條件也有機會分離出新的噬菌體。將來,我們計劃測試分離的噬菌體對於卡介苗(Mycobacterium bovis BCG strain)、從台灣病人分離的結核桿菌,以及非結核分枝桿菌(Non-tuberculosis mycobacterium, NTM)的感染能力,進一步確認這些噬菌體是否具有治療抗藥性結核病的效果。 Tuberculosis (TB) is an airborne transmitted disease caused by Mycobacterium tuberculosis (M. tb). According to the 2022 WHO TB report, about a quarter of the world’s population is infected with M. tb. Although TB is curable, the emergence of drug-resistant strains of M. tb. makes treatment more challenging. Therefore, alternative therapies, such as bacteriophage treatment, are appealing. Mycobacteriophages are viruses that infect mycobacteria. They can specifically recognize the host cell wall then lyse the cell wall after replication. Although many mycobacteria have been reported, at the start of my thesis, none of those reported had been isolated from Taiwan. We wanted to test our hypothesis that, due to its relatively isolated nature as an island, Taiwan may have unique mycobacteriophages. In this study, mycobacteriophages were isolated from environmental samples in Taiwan. Mycobacterium smegmatis (M. smegmatis) was used as an alternative host because it is non-pathogenic and has a similar cell wall to M. tb. Moist topsoil samples were collected from vegetable farms, parks, lawns, flower beds, and the beach. After enrichment, the extracted samples were mixed with M. smegmatis for infection and the double-layer agar method was used to screen for the presence of mycobacteriophages. Nineteen mycobacteriophages were isolated from 107 different samples and a whole-genome sequencing was conducted to determine the identity of the mycobacteriophages. Isolated mycobacteriophages were grouped into seven known mycobacteriophage clusters: Cluster A, B, C, E, K, L and R. Five mycobacteriophages were annotated. Gene function was assigned to around 30% of these genes. The genomes from different clusters have lower sequence similarity. However, the gene organizations between different clusters have high sequence similarity with Siphoviridae, especially for the genes encoding for virion structure and the assembly genes. The isolation results do not support the hypothesis that Taiwan has unique mycobacteriophages. However, it should be noted that only a small number of phages were isolated and characterized and if a larger number of phages were isolated, some new singleton phages might be identified. In addition, using alternate infection conditions (e.g., different host organisms, infection temperatures) may lead to the identification of new phages. In the future, the infectivity of the isolated mycobacteriophages will be tested using M. bovis BCG strain, M. tb strains isolated from TB patients in Taiwan and other non-tuberculosis mycobacterial species as hosts to determine if these mycobacteriophages can be used as a potential treatment for mycobacterial infection, including drug-resistant TB infection. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/87587 |
DOI: | 10.6342/NTU202300347 |
全文授權: | 同意授權(限校園內公開) |
電子全文公開日期: | 2028-02-10 |
顯示於系所單位: | 生化科學研究所 |
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