克雷白氏肺炎桿菌CG43莢膜多醣體的產生和酪胺酸磷酸化作用的研究

Abstract

蛋白質酪胺酸的磷酸化，在真核生物的生理作用中扮演重要的調控角色，長久以來被認知。然而，在原核生物中的功能和重要性卻仍待釐清。本實驗室的研究對象-克雷白氏肺炎桿菌，具有厚實的莢膜，為此菌引起感染的重要因子。先前研究已發現，負責合成莢膜的基因組中含有一個酪胺酸激酶-Yco6和一個去磷酸酶-Yor5。本研究的目標在釐清Yco6和Yor5的交互作用，並分析此作用如何影響克雷白氏菌 CG43 K2血清型的莢膜生成。首先，我們分別建構了yor5和yco6的缺損突變株；進一步利用西方轉印法分析，我們證明兩個突變株的莢膜多醣體（CPS）皆明顯減少；此外，結合西方轉印法和阿爾新藍-銀染法的分析結果，我們發現這兩個基因的缺陷也會影響莢膜多醣體形成時的聚合作用。同時，我們利用大腸桿菌系統來表現和純化Yor5、Yco6和莢膜多醣體基因組中的重組蛋白質，由硝基苯酚磷酸鹽（pNPP）的分解作用，我們證實重組蛋白質Yor5的去磷酸酶活性；而利用體外的去磷酸化實驗，我們也證明Yor5會調控Yco6的磷酸化程度；在尋找Yco6目標蛋白質的磷酸化實驗中，我們初步發現位在莢膜多醣體基因組的Orf1, Orf3,和Orf15及Ugd不被Yco6所磷酸化。更進一步，我們利用免疫沉澱法，在全菌體蛋白質中發現可受Yco6磷酸化的蛋白質，而這些蛋白質仍待分析確認

The phosphorylation of proteins at tyrosine residues has long been known to play a key role in the control of numerous fundamental functions in eukaryotes. In contrast, the biological significance of the protein-tyrosine phosphorylation is not clear in prokaryotes. It has been demonstrated that Yco6 is a tyrosine kinase and Yor5 is a phophatase in the capsule synthesis operon of Klebsiella pneumoniae, an opportunistic pathogen with a heavy capsule as an important virulence factor. In this study, we intend to demonstrate how the Yor5/Yco6 pair affects the capsule production in Klebsiella pneumoniae CG43 of K2 serotype. Firstly, we have constructed respectively yor5 and yco6 deletion mutants. The two mutants appeared to reduce significantly the production of capsular polysaccharides (CPS) as demonstrated by Western blot analysis with an anti-K2 antibody. Using alcian blue-silver stain to analyze the CPS, we have shown that the mutants most likely affect polymerization of the CPS. Secondly, Yor5, Yco6 and some of the genes encoding CPS operon were over-expressed respectively in E. coli and the recombinant proteins purified by a nickel-resin affinity column. The phosphatase activity of Yor5 was demonstrated by its capability to decompose p-nitrophenyl phosphate (PNPP). Using an in vitro dephosphorylation assay, we have shown also that Yor5 can regulate the phosphorlation level of Yco6. Subsequently, in vitro phosphorlation assay showed that Orf1, Orf3 and Orf15 in K2 cps gene cluster and Ugd are not the targets of Yco6. Finally, immunoprecipitation employed to search for the targets of Yco6 has identified several likely target proteins and the identities remain to be verified.