克雷白氏肺炎桿菌CG43 CSS-EAL domain蛋白YlaB、YoaD、16830及Rtn之功能性探討

Abstract

環狀雙鳥苷酸是許多細菌的二級傳訊分子，負責調控纖維素的合成、泳動能力、生物膜生成以及毒力因子的表達；此二級傳訊分子的濃度由雙鳥苷酸環化酶（diguanylate cyclase; DGC）及磷酸二酯酶（phosphodiesterase; PDE）所調節，雙鳥苷環化酶通常具有高度保留的GGDEF胺基酸編碼，磷酸二酯酶則具有EAL domain或HD-GYP編碼。先前研究已知具有CSS-EAL結構域蛋白YjcC具有PDE活性、其表現會受巴拉刈所誘導，yjcC基因缺損會降低克雷白氏肺炎桿菌CG43抗氧化壓力能力、提高第三型纖毛的表現；另外，已知僅具EAL 結構域的MrkJ蛋白也有PDE活性，且其基因缺損會明顯提高第三型纖毛的表現。經分析已解碼的CG43基因體發現除了yjcC外，另有四個可轉譯CSS-EAL結構域蛋白的基因ylaB、yoaD、rtn與16830，本論文旨在探討克雷白氏肺炎桿菌CG43中帶有 CSS-EAL domain蛋白YlaB、YoaD、16830及Rtn之功能性。首先，利用回補yjcC或mrkJ的基因缺損的實驗顯示：只有表現YlaB或YoaD，才能降低yjcC或mrkJ基因剔除株的MrkA蛋白生成、纖維素合成及生物膜生成，此結果暗示YlaB和YoaD（而非Rtn或16830）具有PDE活性；另外，在yjcC基因剔除菌中表現YlaB或YoaD，均能提高其抗氧化壓力反應，顯示此二蛋白可能與YjcC的N-端CSS結構域類似具有接收氧化壓力的能力。同時，透過LacZ報告系統分析啟動子活性發現：PyalB與PyoaD活性可受酸誘導，而Prtn活性在csgD基因剔除菌中被活化。最後，比較分析CG43S3與yaoD基因剔除菌表現型差異，結果發現MrkA生成及生物膜生成均因yaoD基因剔除而增加，再度確認YoaD具有PDE的活性。

Cyclic di GMP (c-di-GMP) is used in many bacteria as a second messenger for the regulation of cellulose synthesis, motility, biofilm formation and the expression of virulence factors. The level of c-di-GMP is regulated by diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively represented by the GGDEF-domain and EAL (or HD-GYP)-domain containing proteins. Previous study has shown that the CSS-EAL protein YjcC exerts a PDE activity which is paraquat-inducible, and the gene deletion reduced the survivals of Klebsiella pneumoniae CG43 upon the oxidative stress but increase the type 3 fimbriae expression. Besides, the EAL domain protein MrkJ also carries PDE activity and the deletion apparently increased the expression of type 3 fimbriae in K. pneumoniae CG43S3. Analysis of the sequenced genome of K. pneumoniae CG43 revealed that in addition to yjcC, there are 4 other CSS-EAL protein encoding genes ylaB, yoaD, 16830, and rtn. The thesis intends to characterize the functional role of YlaB, YoaD, 16830, and Rtn. Firstly, the complementation analysis revealed that the MrkA pilin production, cellulose biosynthesis and biofilm formation of ΔyjcC or ΔmrkJ could be reduced by introducing in the mutant with the plasmid expressing YlaB or YoaD (but not Rtn nor 16830). In addition, the expression of YlaB or YoaD inΔyjcC could increase the resistant activity to oxidative stress implying their N-terminal CSS-motif carry similar reception structure as that of YjcC. In the meantime, the promoter activity measurement via a LacZ reporter system showed that the promoter activity of ylaB and yoaD are acid inducible and rtn expression is increased by the deletion of csgD. At last, the comparative phenotype analysis of CG43S3 and ΔyoaD mutant revealed that MrkA pilin production, cellulose biosynthesis and biofilm formation are all increased by the deletion of yoaD which further confirms the PDE activity of YoaD.