雙分子系統反應調節因子RcsB在克雷白氏 肺炎桿菌CG43中所扮演的角色

Abstract

當細菌感受到外界環境壓力後，雙分子調控系統（Two-component system）的偵測激酶RcsC和RcsD將訊號傳遞給下游之反應調控蛋白RcsB，磷酸化該蛋白的第56個氨基酸天冬氨酸鹽（Aspartate）並使RcsB活化。 過去研究顯示，RcsB與RcsA形成雙合體來調控細菌莢膜多醣體的基因表現。在肝膿瘍克雷白氏肺炎桿菌分離株CG43中，rcsB基因的缺失可導致莢膜多醣體的生合成、第三型線毛的表現和抗酸能力明顯下降。本論文中，我們擬探討RcsB的磷酸化是否會影響莢膜多醣體、第三型線毛和抗酸能力相關基因的表現。本研究首先構築了兩個定點突變的RcsB，分別為RcsB-D56A和RcsB-D56E。RcsB-D56A模擬無法接受磷酸根的RcsB；而RcsB-D56E則是模擬持續磷酸化狀態的RcsB。CG43S3ΔrcsB回補pRK415-RcsB-D56A和pRK415-RcsB結果相近，均提高莢膜多醣體的生合成和細菌的抗酸能力。相對地，回補pRK415-RcsB-D56E則恢復了第三型線毛的主要單元蛋白MrkA的表現量。此結果暗示著RcsB的磷酸化影響此蛋白和DNA或是其他蛋白質的結合，進而決定它的調控目標。我們以西方點墨分析發現RcsB-D56A正向調控酸逆境伴隨蛋白，而啟動子活性分析顯示其對抗酸相關的蛋白YfdX的為轉錄層面的影響。同時，我們藉大腸桿菌來大量表現RcsB的N端並純化之後免疫兔子後取得多株抗體，有趣的是，我們發現此多株抗體只能偵測RcsB和RcsB-D56A卻無法辨識RcsB-D56E，此暗示RcsB-D56E殘基改變可能影響其結構而致失去抗體辨識的抗原決定部位 (epitope)。不過，西方墨點法顯示RcsB可受酸誘導表現，而此抗體將可以用於免疫共沉澱找出RcsB協同作用的蛋白質。這些結果暗示著RcsB的磷酸化狀態對於克雷白氏肺炎桿菌CG43的各種致病方式的調控扮演著非常重要的角色，並且可能會是其致病的關鍵點，往後RcsB的協同作用蛋白的發現將可以幫助我們進一步去更瞭解RcsB的調控路徑。

The two-component system (TCS) response regulator RcsB is activated by phosphorylation at Asp56 upon receiving stress signals transferred from the sensor kinase RcsC and RcsD. RcsB has been demonstrated to be required for CPS (capsular polysaccharide) gene expression through heterodimer forming with RcsA. In Klebsiella pneumoniae CG43, a liver abscess isolate, deletion of rcsB reduced the CPS expression, type 3 fimbriae pilin MrkA production, and acid stress response. This study, investigates if RcsB phosphorylation plays a role in regulating CPS production, MrkA expression, and acid stress response. Two site-directed mutants RcsB-D56A which is unable to accept the signal transferred, and RcsB-D56E, a phosphorylation mimetic form of RcsB, have been generated. Comparing with CG43S3ΔrcsB[pRK415], CG43S3ΔrcsB[pRK415-RcsB-D56A] and CG43S3ΔrcsB[pRK415-RcsB] exhibit an increased the CPS production and acid survival rate. By contrast, CG43S3ΔrcsB[pRK415-RcsB-D56E] restored the MrkA production. These findings suggest that the phosphorylation status of RcsB affects its DNA binding or protein binding activity thereafter determines its regulation targets. Western blot analysis showed that RcsB-D56A positively regulates the acid stress chaperone YfdX expression and promoter activity assay suggested that the RcsB-mediated regulation is at the transcriptional level. In addition, the N-terminal region of RcsB was overexpressed in E. coli and the protein was purified to immunize rabbit for polyclonal antibody generation. The raised antibody could recognize RcsB and RcsB-D56A but not RcsB-D56E. This result implies that the amino acid D56E substitution of RcsB alters the protein conformation, and hence the loss of the specific epitope for the antibody. Western blot analysis also revealed that RcsB was acid inducible and co-immunoprecipitation analysis would be feasible for the identification of the RcsB interacting proteins. The findings indicated that the phosphorylation status of RcsB might be the key of the regulation of different virulence factors in K. pneumoniae CG43, and further identification of the RcsB-interacting protein will help toward understanding the regulatory pathway of RcsB.