探討RcsB在克雷白氏肺炎桿菌CG43中調控抗酸逆境的反應

Abstract

在克雷白氏肺炎桿菌具有厚重莢膜的臨床分離株CG43中，我們發現rcsB基因缺損不僅降低其莢膜生成量也降低了此菌在酸逆境（pH 3-M9培養基）的存活率，而此缺失可被轉殖入一表現rcsB基因的質體所彌補，此暗示RcsB扮演調控酸性逆境反應的角色。經由已定序的CG43基因體中搜尋同源序列，我們發現六個可能被RcsB調控的抗酸基因：其中，yfdX、hdeDB以及hdeB1基因座落於可能的適酸小島(acid fitness island)；此外，以二維蛋白質電泳比較分析以酸處理（pH 4.4-LB一小時）的CG43S3與CG43S3ΔrcsB蛋白體，分離出一個因rcsB缺損而不表現的蛋白點，經串聯質譜分析後鑑定為YfdX蛋白。為了更深入探討RcsB基因在調控抗酸逆境反應的角色，近一年的工作如下:1. 已建構ΔhdeB、ΔhdeD、ΔhdeB1、ΔhdeDB、ΔhdeBΔhdeB1、ΔhdeDBΔhdeB1、ΔhdeDΔyfdX、ΔhdeDBΔyfdX、ΔhdeDBΔhdeDΔhdeB1、ΔrcsBΔyfdX和ΔkvhAΔyfdX基因缺損株。2. 經由酸逆境存活率分析確認AFI基因均參與抗酸反應，而各基因缺損影響為ΔhdeB1＞ΔhdeD＞Δ yfdX＞ΔhdeB；ΔrcsB＞ΔyfdX＞ΔkvhA3. 建構HedB和YfdX的表現系統並純化HedB和YfdX：-免疫兔子取得YfdX多株抗體；以活體外系統分析顯示HedB和YfdX重組蛋白具有伴隨蛋白（chpaerone）活性，而YfdX活性明顯較HedB強。4. 利用LacZ報告系統分析發現hdeDB、hdeB1和yfdX在弱酸、靜置培養下的啟動子活性較高；在靜置或震盪培養下，RcsB和KvhA正向調控AFI基因，而Fur和PhoP只在震盪培養下正向調控AFI基因，另外，rpoS基因缺損會提高hdeDB的表現。5. 西方墨點法分析顯示YfdX在弱酸靜置培養下表現量最大，此時，RcsB和KvhA基因缺損明顯降低其表現，另外在缺鐵情況下，YfdX表現量大幅降低。

In Klebsiella pneumoniae CG43, we have observed that the rcsB deletion not only reduced the bacterial CPS amount but also decreased the bacterial survival under an acid stress treatment (pH 3 in M9). The deleting effect could be complemented by transformation with an RcsB expression plasmid implying a regulatory role in the acid stress response. Homologous gene search in K. pneumoniae CG43 genome revealed six predicted RcsB-dependent acid resistance genes. Among them, yfdX, hdeDB and hdeB1 genes were found to be clustered within the putative AFI (acid fitness island). In addition, comparative proteome analysis using 2D gel electrophoresis of CG43S3 and CG43S3ΔrcsB after the acid adaptation (pH 4.4 in LB for 1 h) revealed a missing protein spot in the rcsB deletion mutant, which later identified as YfdX by tandem mass analysis. The accomplishments for the last 10 months are: 1. The mutants including ΔhdeB, ΔhdeD, ΔhdeB1, ΔhdeDB, ΔhdeBΔhdeB1, ΔhdeDBΔhdeB1, ΔhdeDΔyfdX, ΔhdeDBΔyfdX, ΔhdeDBΔhdeDΔhdeB1, ΔrcsBΔyfdX and ΔkvhAΔyfdX have been constructed.2. Acid stress survival analysis revealed that the AFI genes are all involved in the acid stress response. The order of deleting influences isΔhdeB1＞ΔhdeD＞ΔyfdX＞ΔhdeB； ΔrcsB＞ΔyfdX＞ΔkvhA.3. Overexpression system for the recombinant HdeB and YfdX has been established and the recombinant proteins purified. The recombinant YfdX has been used to immunize rabbit to generate anti-YfdX polyclonal antibody； the recombinant proteins appeared to be able to protect ADH from the extreme acid damage suggesting both are chaperone, while the YfdX exhibited much higher activity than HdeB. 4. Using LacZ reporter system, we have observed that the promoter activity of AFI genes is induced in mild acid and static-cultured condition. RcsB and KvhA positively regulate the promoter activity under both shaking and static-cultured condition, while PhoP and Fur activate the AFI gene expression only under shaking culture. Moreover, the promoter activity of hdeDB was slightly increased by the deletion of rpoS.5. The western blot analysis against anti-YfdX antibody revealed that YfdX expression was increased at the static culture or mild acid but diminished expression in LB addition with iron chelater. In addition, the yfdX expression was abolished in ΔrcsB or ΔkvhA deletion mutants.