克雷白氏肺炎桿菌CG43中yfiD基因表現的調控

Abstract

我們利用蛋白質體分析方法探討高致病性克雷白氏肺炎桿菌CG43中KvhAS雙分子系統的調控角色時發現：在弱酸培養後的細菌CG43S3U9451的二維電泳膠片中，有一蛋白質點的強度較CG43S3 U9451kvhAS-膠片中的相對蛋白質點高出許多，經截取、純化此蛋白，再經質譜分析確認此蛋白質點為YfiD。接著以LacZ為報導基因分析yfiD啟動子活性，結果顯示只有在微氧培養的狀態下，yfiD啟動子在□kvhA突變株中的活性減少；有趣的是，□rpoS突變株之yfiD啟動子活性也相對減少；而在截短yfiD啟動子上的一個RpoS結合序列後，受RpoS基因缺損影響的現象即消失。我也利用膠體遲滯電泳分析，證實KvhA直接黏附yfiD啟動子而達到正向轉錄調控的作用。進一步，我還選殖了yfiD基因並將yfiD殖入大腸桿菌表現系統來大量表現YfiD蛋白，並將蛋白純化後注射Balb/c老鼠來取得多株抗體。以西方墨點法分析的結果顯示kvhA的缺損造成YfiD蛋白量的下降，而轉型入KvhA表現質體時，YfiD蛋白表現量即回升；另外，以帶有定點突變的KvhAD52A (持續去磷酸化)或KvhAD52E (持續磷酸化)的質體轉入kvhA的缺損株，我發現KvhA是否被磷酸化會影響yfiD基因的表現調控。yfiD基因缺損株在有氧條件之下，對於酸處理後的感受性提高，而yfiD缺損對細菌的生長並沒有顯著的影響。最後，我構築了YfiDG102A點突變株，證實YfiD的酵素活性參與其酸性逆境表現型的改變。

A proteomic approach has been employed to unravel the regulatory role of the two-component system KvhAS in the highly virulent strain K. pneumoniae CG43. In comparing the two dimensional protein profiles derived from mild acid-growth bacteria, a protein spot highly expressed in the gel of K. pneumoniae CG43S3U9451 but not in that of CG43S3U9451kvhAS- mutant was isolated and later identified as YfiD by mass spectrum analysis. Promoter activity measurement using LacZ as reporter revealed a reduction of PyfiD activity in KvhA- mutant only under a microaerobic environment. Interestingly, deletion of rpoS also decreased yfiD expression. The rpoS deletion effect was no more observed while the putative RpoS-binding element was truncated. Furthermore, EMSA demonstrated a direct binding of KvhA to PyfiD DNA which supporting a positive transcriptional regulation on the expression of yfiD. In the meantime, yfiD encoding gene was cloned and overexpressed in E. coli, and the recombinant YfiD purified to homogeneity to inject Balb/c mice for polyclonal antibody preparation. The analysis of western blotting hybridization against the YfiD polyclonal antibody indicated that the deletion of kvhA reduced the expression of YfiD. The introduction of the plasmid carrying KvhA into the kvhA deletion mutant appeared to enhance the expression of yfiD. The transformation of the plasmid carrying KvhAD52A (constitutively dephosphorylated), or KvhAD52E (constitutively phosphorylated) confirmed the phosphorylation of KvhA is required for the regulation of yfiD expression. Finally, deletion of yfiD was found to increase the bacterial susceptibility to acid treatment under aerobic condition while no effect on the bacterial growth. The YfiDG102A was generated and the involvement of its enzymatic activity in the acid-shock resistance was also demonstrated.