克雷白氏肺炎桿菌CG43中KvhAS雙分子調控系統的功能分析

Abstract

根據本實驗室過去的EMSA(Electrophoretic mobility shift assay)研究，我們發現KvhAS及其同源雙分子調控系統KvgAS和KvhR可能具有互相調控關係。為了更進一步研究彼此的交互作用，因此構築了一系列的突變株，包括kvhA-kvgA-、kvhA-kvhR-、kvgA-kvhR-及kvhA-kvgA-kvhR-。經過比較這些突變株的生長及菌體低速離心狀態後，將這些突變株分成兩群：第一群菌帶有kvgA或kvhR突變，呈現快速生長及低黏度的表現型；而以老鼠腹膜炎模式分析，刪除kvgA或kvhR會導致老鼠半致死率增加。第二群菌包含kvhA- 和kvhA-kvhR- 突變株，這些突變株呈現和母株LacZ16相同的生長速率及菌體黏度。將含有kvhA的質體轉型入kvhA-kvhR-雙基因突變株，我們發現kvhA-kvhR-突變株會由第二群轉成第一群；且同時含有kvhA及kvhR基因突變時，細菌對人類血清的抵抗能力下降。這些結果顯示KvhA和KvhR之間可能有交互作用，而這些突變株在菌體黏度及致病力表現不同程度的差異，以莢膜多糖體(capsular polysaccharide)為最可能引起變化的因子。因此，我們利用LacZ為報導蛋白，分析莢膜多糖體基因的啟動子在這些突變株的表現差異。結果顯示：莢膜多糖體基因組開放骨架1~2或16~17的啟動子活性在第一群突變株中表現降低，而在kvhR-降低的活性會因為kvhA基因同時突變而回復；然而，開放骨架3~15的啟動子活性在任一突變株中並沒有顯著差異。另外，當壓力調節的主控子RpoS基因突變時，kvhAS的啟動子活性明顯上升，進一步分析發現在kvhAS基因的上游-379至-476片段可能是RpoS調控的區域，而此區也是KvhA的自我調控區域。我們還發現kvhAS 的啟動子活性會隨鎂離子濃度升高而上升，這個結果暗示KvhAS和PhoPQ有交互作用。除此之外，大量表現KvhA蛋白質會改變菌體對不同抗生素的感受性，這結果顯示著KvhA可能調控細菌對藥物的感受性。

Previous EMSA (Electrophoretic mobility shift assay) study in Klebsiella pneumoniae CG43 genome has shown a possibility of the presence of a regulatory circuit in-between the homologous two components (2CS) KvhAS, KvgAS and KvhR. In order to investigate further the regulatory interaction, a series of mutants including kvhA-kvgA-, kvhA-kvhR-, kvgA-kvhR- and kvhA-kvgA-kvhR- derived from K. pneumoniae LacZ16 were constructed. Comparative analysis of the growth and phenotype of the mutants allowed us to classify the mutants into two groups: the Group I carrying either kvgA or kvhR mutation displayed a less mucoid and a faster growth rate by comparing to the parental strain LacZ16. In a mouse peritonitis model, the deletion of either kvgA or kvhR resulted in an increase of LD50. Group II includes kvhA- and kvhA-kvhR- mutants, which exert a similar phenotype and growth rate with that of the parental strain. Complementation of the kvhA-kvhR- with a kvhA containing plasmid converts the mutant into Group I. Only with both kvhA- and kvhR- mutations, the bacteria revealed a decreasing activity of resistance to human serum. The results suggested that a cross-talk is present in-between KvhA and KvhR. Mutations of these genes affect both bacterial mucoidity and virulence at different levels indicating that change of capsular polysaccharide (CPS) is most likely the causing factor. We therefore attempt further to analyze promoters of the CPS encoding genes in the mutants using LacZ as a reporter. The promoter activity of either Porf1-2 or Porf16-17 in Group I mutants appeared to be reduced, which suggests that KvgA and KvhR are the regulators for cps gene expression. The decreasing activity in kvhR- was found to be reversed in kvhA-kvhR-, indicating a direct interaction between KvhA and KvhR. In addition, we have shown that mutation of the global stress regulator RpoS increased the promoter activity of kvhAS. The putative regulatory region of kvhAS by RpoS was localized between -379 and -476 bp upstream of kvhAS, which appeared to contain also the auto-regulating region of KvhA. The promoter activity of kvhAS was found to increase by the addition of MgSO4, suggesting an interaction with PhoPQ regulon. Moreover, changes of resistance activity to antibiotics were noted by overexpression of KvhA in the bacterial, implying its involvement in drug resistance.