克雷白氏肺炎桿菌CG43中KvhAS雙分子調控系統的特性分析

Abstract

我們利用在高致病性克雷白氏肺炎桿菌 CG43中的一組可能與致病相關的雙分子調控系統kvgASQR ( Klebsiella virulence gene)與華盛頓大學所公佈的克雷白氏肺炎桿菌MGH78578的基因體序列比對，發現有一段基因與kvgAS有很高的一致性。我們以克雷白氏肺炎桿菌 CG43的染色體為模板經聚合酉每連鎖反應選殖得此基因序列，並將它命名為kvhAS ( kvg homologue )。我們以點墨法分析，結果發現實驗室收集的克雷白氏肺炎桿菌臨床分離株都帶有此基因組；此外，kvhAS的月安基酸序列和博德氏百日咳桿菌的bvgAS毒力因子調控基因組，和大腸桿菌的evgAS多重抗藥調控基因組有很高的相似度。 為了瞭解kvhAS在克雷白氏肺炎桿菌CG43中的功能，我們首先證明kvhAS是受同一個啟動子所調控的基因組；另外，我們分別利用電泳膠遲滯實驗，以及以galU為報導基因的啟動子活性測試證明kvhAS能夠正向地自我調控。而經由同源互換的方法，我們成功構築了四個kvhAS的突變株：CG43-S3-kvhAS-，CG43-S3-kvhS-，CG43-S3-U9451-kvhAS-，和CG43-S3-U9451-kvhS-。為了探討kvhAS和克雷白氏肺炎桿菌致病的相關性，我們以老鼠腹膜炎的模式比較野生株與kvhAS突變株對老鼠半致死率的影響，結果發現kvhAS突變株的半致死率有些微的增加。而我們也發現攝氏四十四度的熱刺激會活化kvhAS的啟動子，使galU大量表現。由於許多雙分子訊息傳遞系統都能就近調控其周圍的基因，我們分析克雷白氏肺炎桿菌MGH78578坐落於kvhAS周圍的序列發現dha 調節子的基因，三個抗酸相關的基因，一個和evgA所調控的yfdX有很高相似度的基因，和一些功能未知的開讀骨架；我們進而分析CG43包含kvhAS片段的噬菌體株，發現CG43和MGH78578 kvhAS基因組周圍的基因組成和排列相同。經由電泳膠遲滯實驗，我們發現磷酸化後的His6-KvhA也能和kvhAS基因周圍的兩個包含啟動子的片段有專一性的結合。我們分析所有能和His6-KvhA專一結合的核酸序列，發現它們都具有許多重複的AT序列、回文序列（palindrome）和A、T高度聚集的區域。為了再找到可能受kvhAS調控的基因，我們分別使用鎳離子螯合樹酯與洋菜膠電泳進行直接結合的實驗，卻無法利用KvhA和核酸序列結合的特性搜尋到和其專一結合的啟動子區域。

A sequence exhibiting high homology with the kvgAS (Klebsiella virulence gene) which has been previously identified in the highly virulent strain K. pneumoniae CG43 were isolated by PCR-based cloning from CG43 and designated kvhAS (kvg homolog). The kvhAS was demonstrated to prevail in all the strains analyzed by dot-blotting hybridization. In addition, KvhAS share common motifs, especially the residues near the biological active sites, with the virulence-associated two-component system BvgAS and the multidrug resistance-associated two-component system EvgAS in B. pertussis and E. coli respectively. To investigate the functional role of kvhAS in K. pneumoniae CG43, the operon structure of kvhAS was verified. In addition, the positively auto-regulatory mechanism was verified both in vitro by electrophoretic mobility shift assay and in vivo by the galU reporter system. The four kvhAS mutants, CG43-S3-kvhAS-, CG43-S3-kvhS-, CG43-S3-U9451-kvhAS-, and CG43-S3-U9451-kvhS-, were constructed by gene replacement through homologous recombination. To determine whether kvhAS is a virulence determinant in CG43 pathogenesis, a mouse peritonitis model was used to determine LD50 of the wild type CG43-S3 and the isogenic kvhAS mutant, and a slight increase of LD50 of the kvhAS mutant was observed. Moreover, heat shock (44℃) was found to activate the PkvhAS activity. While a lot of two-component systems were found to regulate the physically linked genes, the genes adjacent to the kvhAS were analyzed and annotated. The genes of dha regulon, the putative acid-resistance genes, yfdXL which show a high homology with the evgAS-regulated yfdX, and several ORFs with unknown function were identified downstream or upstream of kvhAS. The identical gene organization of kvhAS and the physically linked genes were demonstrated by analysis of the recombinant phage DNA containing kvhAS. The specific binding between the phosphorylated His6-KvhA and the adjacent promoter-containing regions were demonstrated by electrophoretic mobility shift assay. The sequences specifically bound by the phosphorylated His6-KvhA were analyzed, and the features of the 5 sequences were observed. They all contain multiple repeats of T and A, 6~8 bp of palindromes, and AT-concentrated regions. To further identify the KvhA-regulated genes, the direct-binding experiments coupled with nickel-chelating resin or agarose gel electrophoresis were carried out, but found to fail to fish out the His6-KvhA bound target promoters.