克雷白氏肺炎桿菌參與致病的基因-鑑定與特性分析

Abstract

摘要 克雷白氏肺炎桿菌是一種伺機性的病原菌，常在免疫不全的病人體中引起菌血症及尿道感染。我們實驗室藉由美國華盛頓大學在網站上所提供克雷白氏肺炎桿菌MGH48578染色體核酸序列，找到了兩段可能與克雷白氏肺炎桿菌致病性相關的核酸片段。 一段是與Bacillus cereus的hemolysin III基因的胺基酸序列有37﹪一致性的開讀骨架。另一段核酸片段是與Bartonella bacilliformisinvasion protein A：ialA基因的胺基酸序列有40﹪一致性的開讀骨架。首先我們將以聚合脢鏈鎖反應所得到的克雷白氏肺炎桿菌hemolysin III-like（hlyIIIK）基因轉型入大腸桿菌HB101中，分別在以血液平板培養基及抽取細菌的粗萃取液做溶血測試，但均無法觀察到有溶血的現象。接著我們以三個不同長度片段的hlyIIIK基因分別剪接到表現質體pET30及pET32，再分別轉型入大腸桿菌Novablue（DE3）及BL21（DE3）中，都無法看到蛋白質的表現。我們以聚合脢鏈鎖反應得到在同一株克雷白氏肺炎桿菌的ialA-like（ialAK）基因，也將其剪接至表現質體，轉型到大腸桿菌 Novablue（DE3）中大量表現後，得到了不溶的包涵體。之後將此包涵體當成抗原免疫小白鼠，成功的得到了此多株抗體。同時我們以小白鼠為實驗模型進行保護作用的測試發現此蛋白無法增進老鼠對抗克雷白氏肺炎桿菌的能力。我們推測此ialAK基因在克雷白氏肺炎桿菌致病過程中中可能與侵入作用無關，而是與此菌在宿主細胞中的生存有關連。我們進一步利用低溫誘導ialAK基因表現，取得少量可溶性的IalAK蛋白做活性測試，證明此蛋白具有dinucleoside polyphosphatease的活性。最後我們以墨點雜交法分析，發現此兩段基因均存在於所有的本實驗室收集的克雷白氏肺炎桿菌臨床菌株中，顯示這兩段基因在克雷白氏肺炎桿菌生長中可能扮演著必要的角色。

Abstract Klebsiella pneumoniae is an opportunistic pathogen that often causes septicemia and urinary tract infections in immunocompromised patients. According to the genome sequence of K. pneumoniae MGH48578 offered on the net, we have found two contig sequences that may involved in K. pneumoniae pathogenesis. One gene has 37% amino acid identity with the Bacillus cereus hly III gene,the other has 40% amino acid identity with the Bartonella bacilliformi ialA gene. Initially, we transformed the K. pneumoniae hlyIII-like（hlyIIIK）gene isolated by PCR into E.coli HB101. No hemolytic activity was observed both on blood agar and crude extract assay, however. We then cloned the full length and two truncated hlyIIIK genes into plasmids pET30a and pET32a, respectively. None of then were expressed in E.coli. Nevertheless, we have successfully expressed the ialA-like（ialAK）gene in E.coli, which was obtained by PCR with the previously used K. pneumoniae strain as the template. However, insoluble inclusion body was isolated that we can only use to immunize mice. We obtained the polyclonal antibody with appropriate sensitivity and specificity. We went further to use BALB/C mice for protection assay. Unfortunately, the IalAK antibody did not protect the mice from K. pneumoniae infection. The results suggested that the K. pneumoniae ialAK gene is required for its survival in the host cells, but not directly for the invasion. Finally, we tried to overexpress this protein at low temperature for soluble protein. Although only small amount of soluble protein was obtained, we have demostrated clearly that the IalAK possessed dinucleoside polyphophatase activity. The two genes, hlyIIIK and ialAK, were found to present in all laboratory collected K. pneumoniae clinical isolates by dot blot analysis indicating that they may play essential roles in K. pneuminiae infection.