克雷白氏肺炎桿菌CG43中參與莢膜多醣體生合成之核心蛋白Wza、Yor5、Yco6和Wzx的功能性研究

Abstract

克雷白氏肺炎桿菌為一株伺機性引起區內感染疾病的格蘭氏陰性菌，其外部包覆著由多醣體所組成的厚莢膜。此一莢膜可以讓細菌逃避細胞的吞噬作用以及避免被血清因子所毒殺。克雷白氏肺炎桿菌的莢膜多醣體被認為和大腸桿菌的第一類莢膜多醣體之生合成與組合途徑相類似，由位於cps基因組中的主要因子所調控，這些主要因子包括了Wzi、Wza、Yor5、Yco6、Wzx和Wzy。本研究的主要目標為證明其中四個主要因子－Wza、Yor5、Yco6和Wzx－的生物功能性。在實驗室之前的研究中，證明了無論剔除酪胺酸去磷酸酶或是酪胺酸激酶的基因，yor5或yco6皆會明顯地減少其原本所具有的黏性和莢膜多醣體的量。在此研究之中，我們亦構築了剔除了wza或wzx的突變株。其中剔除了位在外膜上的多醣體輸送蛋白wza，會使得莢膜多醣體之聚合體無法運送到細菌表面，並且進而回饋抑制上游的生合成途徑，造成整體生成量明顯地下降。而在wzx突變株之中，雖然莢膜多醣體的總量同樣下降，但是仍然有少量的聚合體存在，這部分的多醣體合成作用可能不需要經由Wzx來調控。此外，剔除yor5和剔除yco6這兩個突變株皆完全地喪失了莢膜多醣體的聚合體，而這也證明了由Yco6和Yor5所調控的酪胺酸磷酸化作用對於莢膜多醣體的生成與聚合作用來說相當重要。經由體外磷酸化試驗，我們證實了克雷白氏肺炎桿菌和大腸桿菌的蛋白質酪胺酸激酶－Yco6和Wzc－都能夠將克雷白氏肺炎桿菌中的Ugd、Gnd、ManB和ManC這四個酵素磷酸化，而這些酵素和莢膜多醣體醣類核甘酸前驅物的生成相關。而Ugd和Gnd的磷酸化對於其酵素活性則有明顯的提升效果。另外，以蛋白質酪胺酸去磷酸酶Yor5來進行體外去磷酸化試驗則發現，Gnd和ManC可以徹底地去磷酸化，而KpUgd則是部分被去磷酸化。相反地，ManB則是完全不受Yor5影響。以上實驗結果明確地證實了這些主要因子對於克雷白氏肺炎桿菌的莢膜多醣體之生合成與組合作用的重要性和其功能性角色。

Klebsiella pneumoniae, an opportunistic gram-negative bacterium causing community-acquired diseases, are mostly encapsulated by a considerable thick polysaccharidic capsule acting to protect the bacteria from phagocytosis and prevent from killing by serum factors. The capsular polysaccharide (CPS) is made from a similar biosynthetic and assembly pathway to that of Escherichia coli group 1 CPS. The core elements encoded in cps gene cluster include Wzi, Wza, Yor5, Yco6, Wzx and Wzy. Our previous study has demonstrated that deletion of either yor5, encoding a protein-tyrosine phosphatase, or yco6, coding for a protein-tyrosine kinase, in K. pneumoniae CG43 reduced significantly the bacterial mucoidy and CPS content. In this study, both wza- and wzx- mutants were generated and the deletion effects were analyzed and compared. Deletion of wza, encoding an outer membrane polysaccharide export protein, or wzx, coding for a flippase, appeared to affect also the mucoidy and reduce the CPS content. Polymeric CPS were blocked in periplasma in wza- mutants and resulted in feedback inhibition of total CPS synthesis. In contrast, some polymeric CPS was observed on cell surface in the wzx- mutant suggesting an alternative translocation system for oligosaccharides through the cytoplasmic membrane. Nonetheless, to demonstrate further a role of tyrosine phosphorylation, several recombinant clones were generated and the proteins, including Yor5, Yco6, Ugd, Gnd, ManB and ManC that have been shown to be required for regulation and synthesis of CPS sugar nucleotide precursors, were purified. Via in vitro phosphorylation assay, the recombinant Yco6 appeared to be able to phosphorylate the enzymes Ugd, Gnd, ManB and ManC, and the phosphorylation increased the enzymatic activities of Ugd and Gnd. Moreover, the phosphorylated Ugd, Gnd and ManC, but not the ManB, could be dephosphorylated by the recombinant Yor5.