克雷白氏肺炎桿菌CG43絲胺酸/蘇胺酸激酶KpnK的功能性探討

Abstract

近期研究指出，克雷白氏肺炎桿菌之絲胺酸/蘇胺酸激酶KpnK會正向調控細菌抗氧化壓力的反應。本論文探討KpnK在肝膿瘍分離株CG43中是否還扮演其他調控角色，首先，我們發現剔除kpnK基因後會增加CG43對抗巴拉刈的敏感性，而此突變株並沒有如預期的影響其多醣莢膜的生成，且磷酸甘露醣異構酶第98絲胺酸殘基的磷酸化也不受kpnK基因缺損的影響。接著，啟動子活性分析顯示kpnK的表現會受調控胞外結構壓力的雙分子磷酸化系統CpxAR正向影響。有趣的是，kpnK的基因缺失會增加此菌株凝集酵母菌的活性，此暗示第一型線毛的表現量會增加；相對的，此基因剔除對CG43的胞外組成分子包括第三型線毛單位蛋白MrkA或纖維素的生成量卻無顯著影響。另外，為了證明KpnK的酵素活性，還建構了分別帶有KpnK定點突變序列(KpnK-S36A, -D201A, -D217A)的三個pETQ31重組質體，並以Pro-Q Diamond染色分析帶有其中一個重組質體的kpnK基因缺損菌株的總蛋白及重組KpnK蛋白的磷酸化狀態，卻發現二者的磷酸化程度沒有因kpnK基因缺損而改變，此結果暗示單一的定點突變對KpnK的活性影響不大。更進一步以Pro-Q Diamond染色失去自我磷酸化能力的截短KpnK(N-KpnK或C-KpnK)和完整KpnK的重組蛋白，結果都呈現被磷酸化的狀態，這些結果顯示其他激酶可能可以代償KpnK的磷酸化能力。

Klebsiella pneumoniae STK, namely KpnK, has recently been shown to positively regulate the oxidative stress response. The thesis investigates if KpnK plays more regulatory roles in K. pneumoniae CG43, a liver abscess isolate. Firstly, the deletion of kpnK gene was found to increase K. pneumoniae CG43 sensitivity to paraquat, however, the mutant did not exert the changes, as suggested by the previous study, on the CPS production or on the phosphorylation state of ManB-S98, the 98th residue serine of phosphomannosemutase. The subsequent promoter activity analysis demonstrated that kpnK was positively affected by the envelope stress regulation two component system CpxAR. It is interesting to note that the kpnK deletion mutant exhibited an increased mannose-sensitive yeast agglutination activity suggesting an increased expression of type 1 fimbriae, by contrast, no apparent changes of the production of the envelope component type 3 fimbriae pillin MrkA or cellulose. Furthermore, the 3 pETQ31-derived recombinant plasmids with sequences coding for the site-directed mutated KpnK (respectively KpnK-S36A, -D201A, and -D217A) were constructed. The analysis via Pro-Q Diamond staining assay revealed that the total cellular proteins or the recombinant KpnK protein ofkpnK mutant carrying either of the recombinant plasmids had no change of the phosphorylation pattern, suggesting that the single directed-mutation of the KpnK may have little effect on the KpnK enzymatic activity. Furthermore, the recombinant truncated KpnK proteins, N-KpnK and C-KpnK, which had lost the autophosphorylation activity, remained in phosphorylated form as the recombinant KpnK protein after staining by Pro-Q Diamond. These results suggest that other kinases may compensate the phosphorylation activity of KpnK.