新莢膜型克雷伯氏肺炎桿菌二株噬菌體之分析及建構K1莢膜型克雷伯氏肺炎桿菌之報導噬菌體

Abstract

克雷伯氏肺炎桿菌為造成院內感染與社區型感染常見的細菌之一，可引發肺炎、泌尿道感染、敗血症、化膿性肝膿瘍等症狀，並可能伴隨高死亡率。其外圍的莢膜是關鍵的致病因子，能保護菌體免於受到宿主的免疫攻擊，且莢膜的型態與致病力之間具高度的相關性，因此莢膜的分型在臨床治療上扮演重要的角色。 本實驗室以基因分型法分類而獲得新莢膜型克雷伯氏肺炎桿菌(KN6、KN7)，接續自原水中分離出兩株克雷伯氏肺炎桿菌噬菌體，分別為以KN6莢膜型菌株為特異宿主之Can0533-KN6-1，以及以KN7莢膜型為特異宿主的My-17-2-KN7-1，並分析此兩株新莢膜型噬菌體是否帶有莢膜分解酵素。接著經由高通量定序得到兩株噬菌體之基因組序列，長度分別為76,578和70,397 bp，並且在進行分析與比對後，發現Can0533-KN6-1的基因組上有兩段可能為莢膜分解酵素之開放閱讀框架，分別為ORF30以及ORF15；於My-17-2-KN7-1基因組則找到其基因組上可能為莢膜分解酵素之開放閱讀框架ORF12。表現、並純化這三段可能為開放閱讀框架序列所編譯之產物後，發現Can0533-KN6-1的ORF30具有莢膜分解酵素活性，而另外兩段基因之產物則沒有觀察到酵素活性。 於本篇研究中我們另外建構出以克雷伯氏肺炎桿菌為宿主之報導噬菌體，以增加噬菌體分型系統的效益。莢膜型K1菌株在台灣地區是造成化膿性肝膿瘍的主因，於是我們首先建構以莢膜型K1菌株為專一宿主之報導噬菌體。此報導噬菌體以哈維氏弧菌的luxAB基因作為報導基因，利用同源重組方法插入K1莢膜型噬菌體的基因組中，最後獲得冷光酶報導噬菌體NTUH-K2044-K1-1(Plac-luxAB)。測試該報導噬菌體之偵測特異性與敏感性，結果顯示該同源重組報導噬菌體仍保留原噬菌體之特異性，偵測極限則為103 CFU。本研究探討新莢膜型莢膜分解酵素以及報導噬菌體之特性，期望能對克雷伯氏肺炎桿菌感染之診斷與治療有所貢獻。

Klebsiella pneumoniae is one of the pathogens causing nosocomial and community acquired infection. Capsule is the critical virulence factor of K. pneuminiae, and there is strong correlation between the capsular type and pathogenicity. Although there are limitations of traditional serotyping, utilization of genotyping and phage typing methods could support the typing system. Bacteriophage Can0533-KN6-1 and My-17-2-KN7-1 specific for new capsular type K. pneuminiae, KN6 and KN7 respectively, had been isolated from untreated water. The genomic DNA of these two phages was analyzed by high throughput sequencing and alignment of the predicted open reading frame in online database. Two open reading frames on the phage Can0533-KN6-1 genome and one on My-17-2-KN7-1 genome, were recognized as putative capsule depolymerase gene. After expression and purification of the protein encoded by these putative capsule depolymerase genes, only the ORF30 of Can0533-KN6-1 exhibited the enzymatic activity. Furthermore, in order to improve the phage typing system, a luciferase reporter phage specific for capsular type K1 K. pneuminiae was constructed as a model for rapid detection. The luxAB gene from Vibrio harveyi under the promoter of lac operon (Plac) was cloned into the genome of K1 phage as a reporter gene. Bioluminescence signal could be detected during the reporter phage infection, following the addition of substrate. Based on current results of experiments, the original specificity of the phage was retained, and the sensitivity of this reporter phage NTUH-K2044-K1-1(Plac-luxAB) is 103 colony-forming-units. In summary, the characterization of depolymerase and bioluminescence reporter phage in this study is expected to be applicable for diagnosis and treatment of Klebsiella pneumoniae infection.