- 學位論文
克雷伯氏肺炎桿菌K5莢膜型之噬菌體的醣解酵素活性與宿主專一性
Glycosidase activity and host specificity of Bacteriophage K5 of Klebsiella pneumoniae
摘要
克雷伯氏肺炎桿菌是造成院內與社區感染的常見菌株，莢膜為其重要的致病因子之一，莢膜又被稱為K抗原(K antigen)，莢膜分型一直以來都是流行病學了解菌株與疾病之間的唯一方法，也可能有助於疫苗的研發。傳統以來一直都是利用血清法來進行莢膜分型，然而血清分型法敏感度與專一性都不佳，因此先前有研究利用噬菌體來進行克雷伯氏肺炎桿菌部分莢膜分型，發現可感染克雷伯氏肺炎桿菌的噬菌體，尾端通常帶有莢膜醣類分解酵素(Capsule depolymerase)，然而先前研究並無核酸序列或病毒檢體留存。因此本實驗室重新研究使用噬菌體所帶有的莢膜醣類分解酵素來區分莢膜型的可能，我們在分離對莢膜具有專一性噬菌體過程中，發現有許多噬菌體能感染多種莢膜型的克雷伯氏肺炎桿菌。因此本論文研究目的希望找出噬菌體能感染多種莢膜型的原因。實驗結果發現，從水源分離出來能夠感染K5莢膜型的噬菌體，共有四株ΦK5-1、ΦK5-2、ΦK5-3(A44)、ΦK5-4(A44)。而其中ΦK5-2與ΦK5-4(A44)並不具專一性，能感染多種莢膜型。此外，透過高通量定序，完成了ΦK5-2與ΦK5-4(A44)基因體定序，經過基因序列的比對，發現ΦK5-2與ΦK5-4(A44)，帶有多種莢膜醣類分解酵素使噬菌體能夠感染多種莢膜型。從這兩株噬菌體，共得到了四種可能的酵素基因序列，經由選殖表現載體進行蛋白質表現與純化。實驗結果證實這些基因產物為K8莢膜醣類分解酵素K5-4(A44)_ORF23、K5莢膜醣類分解酵素K5-4(A44)_ORF24與K30/K69莢膜醣類分解酵素K5-2_ORF26。另外，利用化學藥劑產生的突變株Φ2-K5-2，證實了失去莢膜醣類分解酵素，會影響噬菌體的宿主範圍。而這些莢膜醣類分解酵素也具有專一性，利用這些莢膜醣類分解酵素進行分型，其敏感度比噬菌體本身效果來得好。未來期望能夠找出所有莢膜醣類分解酵素基因，建立完成出一套新的克雷伯氏肺炎桿菌莢膜分型系統。
並列摘要
Klebsiella pneumoniae is a common cause of community-acquired and nosocomial infections. It has polysaccharide capsule that is a major pathogenicity factor. Capsule was also called K antigen. Determination of the K type has long been the preferred method for the investigation of epidemiological relationships among strains and was also possibly helpful to the vaccine development. In the past, serotyping can be employed to determine the K type. Moreover, the anti-sera had limited specificity and sensitivity in the previous reports. Therefore, previous studies reported that use of bacteriophages can determine part of capsule types. They found that bacteriophages infected K. pneumoniae often carried the capsule depolymerase in the tail. However, these phages were no longer available and there was no sequencing result in previous studies. Therefore, our lab tried to study capsule depolymerase which come from capsular-type-specific bacteriophages and established a new method for capsular typing. Capsular-type-specific bacteriophages for K. pneumonia were isolated in our lab. But we also found that many bacteriophages can infect more than one capsular types of K. pneumoniae. Therefore, the aim of this research is to determine why the bacteriophage can infect more than one capsular types of K. pneumoniae. We isolate four bacteriophages which could infect K. pneumoniae strains with K5 capsule type from untreated water, ΦK5-1, Φ K5-2, ΦK5-3(A44) and ΦK5-4(A44). ΦK5-2 and ΦK5-4(A44) infected more than one capsular type strains. By high-throughput DNA sequencing, we completed genome sequence of ΦK5-2 and ΦK5-4(A44). By comparison with NCBI BLAST databases, we found bacteriophage encodes two different glycosidases, allowing it to infect two capsule type strains. From these bacteriophages, we got four glycosidases sequence. We expressed and purified these glycosidases to demonstrate their function. In our results, K5-4(A44)_ORF23 was a glycosidase to digest K8 capsule. K5-4(A44)_ORF24 was a glycosidase to digest K5 capsule. K5-2_ORF26 was a glycosidase to digest K30 and K69 capsules. By mutagenesis, we found that the mutant Φ2-K5-2 which lost glycosidase activity changed its host range. Capsular typing by these glycosidases was more specific and sensitive than by bacteriophages. In the future, we hope a glycosidases typing system covered all capsular types will be established.