克雷白氏肺炎桿菌抗碲基因tehB的功能研究

Abstract

克雷白氏肺炎桿菌是一種伺機性的病原菌，常引起免疫不全病人的菌血症及尿道感染。我們實驗室藉分析美國華盛頓大學於網站上所提供的克雷白氏肺炎桿菌MGH78578染色體核酸序列，發現一段可能與致病性相關的核酸片段；這段核酸片段包含一開讀骨架與Eikenella corrodens的紅血球凝集蛋白Hag1的胺基酸序列有47% 的一致性；而與大腸桿菌的抗碲基因tehB的胺基酸更有高達77% 的一致性。因此我們以聚合酉每連鎖反應由高致病性的克雷白氏肺炎桿菌CG43中選殖了抗碲基因，並將此開讀骨架命名為tehB。經由序列比對，大腸桿菌tehB和E. corrodens Hag1及克雷白氏肺炎桿菌tehB基因均具有三段決定硫-腺甘甲硫胺酸-非核甘酸類的甲基轉移酉每活性的保留胺基酸序列。 為了探討這段開讀骨架的生物活性，我們首先將tehB基因剪接到表現載體pET30c，再轉型入大腸桿菌Nova blue (DE3)以表現大量的蛋白質，我們嘗試過各種方法後仍然得到不溶性的蛋白，最後藉由尿素將此不溶性蛋白溶解，接著以His-tag親和管柱純化並經透析再折疊，最後得到了可溶性蛋白。除了證明此蛋白具甲基轉移酉每的活性外，我們還將此蛋白免疫小白鼠並成功的得到具專一性的多株抗體，進一步以西方墨點法分析發現TehB蛋白很可能位於克雷白氏肺炎桿菌的細胞膜上。同時，我們以小老鼠為實驗模型，結果發現此蛋白無法增進老鼠對抗克雷白氏肺炎桿菌的能力。最後，我們利用基因取代法經由同源互換建構了克雷白氏肺炎桿菌CG43S3 tehB突變株。結果除了tehB突變株CG43S3T1外，我們還得到一株菌落型態較不平滑的突變株CG43S3T2。進一步比較野生株與tehB突變株的紅血球凝集活性和抗碲活性，我們發現野生株與T1突變株對紅血球凝集的能力沒有差異，而T2突變株凝集效果明顯較佳。而野生株與突變株CG43S3T1在抗碲活性上和對BALB/C小鼠的毒性上都沒有明顯的差別。克雷白氏肺炎桿菌CG43帶有一約200 kb的毒性質體pLVPK，我們發現去除此質體的菌株KpCG43-101抗碲活性明顯下降，由目前已定序pLVPK完成的序列，發現有抗碲的基因組terZABCDEF坐落於上。因此，我們認為決定抗碲活性的主要因子位於pLVPK，而tehB基因可能間接參與抗碲的作用。

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 MGH78578 offered on the net, we have identified an ORF that may be involved in K. pneumoniae pathogenesis. The deduced amino acid sequence of the tehB showed a 47 % identity with the sequence of Eikenella corrodens hemagglutinin hag1 gene, and a 77 % identity with that of Escherichia coli tehB. We have isolated the tellurite resistance gene, namely tehB, from K. pneumoniae CG43 by PCR cloning and the sequence determined. The three conserved motifs identified in the AdoMet-dependent non-nucleic acid methytransferase family were also found in the TehB. The tehB was subsequently subcloned into a pET expression vector and over expressed in E. coli. The over expressed protein, however, resulted mostly in an insoluble form. Denaturation using urea and refolding of the purified protein by His-tag affinity column significantly improved its solubility. The purified TehB was shown to carry an AdoMet-dependent methytransferase activity. A polyclonal antibody was generated by immunizing mouse with the purified protein and the specificity demonstrated. The TehB was detected in K. pneumoniae membrane fraction using the specific antibody. We went further using the purified TehB to immunize BALB/C mice for protection assay. Unfortunately, the TehB antibody did not protect the mice from K. pneumoniae infection. Finally, two morphologically different K. pneumoniae CG43S3 tehB deletion mutants CG43S3T1 and CG43S3T2 were obtained and the deletion demonstrated by southern analysis. There was no difference detected in hema -gglutination activity between the wild type strain and T1 mutant. The T2 mutant , however, showed an apparent hemagglutination activity. The tehB deletion appeared no major effect on the bacterial tellurite resistance or LD50 with a mouse peritonitis model. Curing of the large virulence-associated plasmid pLVPK was found to obviously increaseing of the tellurite susceptibility of K. pneumoniae CG43, indicating that the tellurite resistance factor is encoded by pLVPK. The finding of a tellurite resistance associated gene cluster terZABCDEF in the available contig sequence of pLVPK further supports the notion that the pLVPK carries the major determinant of tellurite resistance.