克雷白氏肺炎桿菌CG43S3的CSS-EAL domain蛋白YoaD的功能性探討

Abstract

細菌中獨特的二級傳訊分子c-di-GMP是一個可抑制細菌運動性但促進黏附與生物膜生成的重要調節因子，由具有GGDEF結構域的二鳥苷酸環化酶(diguanylate cyclase, DGC）環化提升其濃度，而透過具有EAL結構域的磷酸二酯酶（phosphodiesterases, PDE）水解而降低濃度。本篇研究想了解從糖尿病肝膿瘍患者身上分離的克雷白氏肺炎桿菌CG43中YoaD是否與YjcC和MrkJ同樣帶有PDE酵素活性，並分析其生物活性與其活性的調控。經由西方墨點法和剛果紅染色分析發現：剔除yoaD對克雷白氏肺炎桿菌CG43S3的第三型纖毛主要單位蛋白MrkA及胞外多醣的生成沒有明顯影響，進一步在具高濃度c-di-GMP的大腸桿菌BW25113yhjH或克雷白氏肺炎桿菌CG43S3yjcC中以異基因回補方式殖入pRK415-yoaD質體，相較於大腸桿菌BW25113yhjH[pRK415]， yoaD的表現能抑制其胞外多醣生成且增加其游泳能力，而在CG43S3yjcC殖入pRK415-yoaD質體，不僅降低胞外多醣的生成也也抑制了MrkA和生物膜生成，這些結果顯示YoaD具PDE活性。為了證實其酵素活性，進而構築了重組質體pET30b-YoaD-EAL並在大腸桿菌BL21(DE3)表現此重組蛋白，經比較分析粗萃液酵素活性發現重組蛋白YoaD-EAL的PDE活性相對低於YjcC-EAL但高於MrkJ。為了瞭解YoaD是否參與對抗環境壓力反應的調控，進一步以H2O2及pH 2.5來測試YoaD表現對CG43S3yjcC生存率的影響，結果發現yoaD表現能夠提升CG43S3yjcC對抗H2O2或pH 2.5的能力；而為了探討YoaD-CSS motif是否扮演接收外部環境的角色，因此建構了定點突變的重組質體pRK415-YoaD-C109R和pRK415-YjcC-C106R，經回補實驗分析發現YoaD定點突變對克雷白氏肺炎桿菌表現型沒有影響，但YjcC點突變增加其MrkA和胞外多醣的生成；而兩者的點突變皆會降低菌株在H2O2刺激下的存活率；此外，YoaD的表現可增加大腸桿菌XL1-MRP2對P1噬菌體的耐受性。最後，以LacZ作為報告子的啟動子活性分析顯示yoaD的表現可受弱酸和paraquat誘導，而比較yoaD與yjcC啟動子表現發現兩者皆受RpoS正向調控，然而yjcC表現會受弱酸環境所抑制，此暗示弱酸環境可能是決定YoaD 與YjcC二者表現差異的因子。

The ubiquitous second messenger c-di-GMP is a key regulatory factor that interferes with bacterial motility while stimulates its adhesion and biofilm production. The level of c-di-GMP is regulated by diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively represented by the GGDEF-domain and EAL-domain containing proteins. Here, we study in Klebsiella pneumoniae CG43, a liver abscess isolate from diabetic patient, to show if YoaD as well as YjcC and MrkJ carries a PDE activity, and also analyze its biological function and how the activity is regulated. Deletion of yoaD had no apparent effect on the c-di-GMP dependent expression assessed using Western blot analysis of type 3 fimbriae major pilin MrkA production and Congo Red staining for extracellular polysaccharides (EPS) biosynthesis. Therefore, cross-complementation analysis by expression of yoaD in E. coli BW25113yhjH and K. pneumoniae CG43S3yjcC, both exhibiting a high c-di-GMP level phenotype, were subsequently carried out. Compared to E. coli BW25113yhjH[pRK415], E. coli BW25113yhjH[pRK415-yoaD] had reduced the production of EPS but increased the swimming activity. The increased yoaD expression in K. pneumoniae CG43S3yjcC reduced the production of not only EPS but also MrkA and biofilm. The results suggest that YoaD carries a PDE activity. To further demonstrate the enzymatic activity, the recombinant clone pET30b-YoaD-EAL was generated and expressed in E. coli BL21(DE3). The recombinant YoaD-EAL from the cellular crude extracts exerted a PDE activity relatively lower than that of YjcC-EAL but higher than that of MrkJ-EAL. The analysis of survival against oxidative stress or acid stress were then performed to determine if YoaD is involved in stress response regulation. The results showed that yoaD expression in K. pneumoniae CG43S3yjcC increased its survivals when exposed to H2O2 or pH 2.5. To study if the CSS motif of YoaD plays a reception role for the external stress, the site-directed mutation clones pRK415-YoaD-C109R and pRK415-YjcC-C109R were generated. The cross-complementation analysis showed that YoaD-C109R mutation had no apparent change however YjcC106R increased the MrkA and EPS production. Nevertheless, both mutations caused reduction of the survival rates after challenged with H2O2. Besides, expression of YoaD in E. coli XL1-MRP2 was found to increase its resistance to phage P1. Finally, the analysis of promoter activity using LacZ as the reporter showed that yoaD expression was acid and paraquat inducible. Comparative analysis of yoaD and yjcC expression revealed that both expression were decreased by the deletion of rpoS but the expression of yjcC was decreased by weak acid. This implies weak acid may be the determinant for the differential expression of YoaD and YjcC.