克雷白氏肺炎桿菌KvgAS雙分子訊息傳遞系統的功能探討

Abstract

克雷白氏肺炎桿菌 (Klebsiella pneumoniae) 是常造成糖尿病人肝膿瘍及院內病人尿道、呼吸道感染的伺機性病原菌。本實驗室先前利用聚合酉每連鎖反應為基礎的基因體刪除雜交法 (PCR-supported genomic subtractive hybridization) 發現在具高毒性的克雷白氏肺炎桿菌CG43 中具有一段特有的訊號傳遞基因組- kvgASQR 。同時在此菌株中另外分離到另一套與kvgAS基因組相似度很高的訊息傳遞基因組- kvhAS 。由序列分析結果發現，其中 KvgA 、 KvhA 和 KvgR 的相似度很高，可能是扮演訊息傳遞系統中調控蛋白的角色; KvgS 和 KvhS 則可能是負責接受外來訊息的感應蛋白; 而 KvgQ 帶有穿越細胞膜的區域，因此我們推測和 KvgS 感應訊息的功能相關。 本實驗為探討 kvgA 所扮演的功能，我們利用 pET 表現系統將 kvgA 大量表現，但所表現出來的重組蛋白為不溶性，因此以 6N 的尿素將蛋白溶解後，利用含硫酸鎳樹脂的管柱加以純化，並以梯度透析方式去除尿素，達到重新摺疊蛋白的目的。接著，利用電泳膠遲滯實驗發現去除磷酸化區域的 KvgA 在此活體外系統中對本身啟動子、 kvgR 和 kvhA 的啟動子均具有專一性結合能力。為找尋可受 KvgA 調控的毒力因子，我們將克雷白氏肺炎桿菌 CG43 中染色體以 BstUI 切斷成 200-500 bp 的核酸片段庫，加入純化的重組 KvgA 蛋白作用後，再與含硫酸鎳樹脂以親和作用吸附已和特定核酸序列專一結合且帶有 His-tag 標定的KvgA，然後利用 PCR 來增幅特定核酸序列片段，最後經由選殖，定序及序列比對結果，得到兩個可能受 KvgA 所調控的序列- PBD1 和 PBD2 ； PBD1與 lysine/cadaverine antiporter membrane protein (cadB)的序列相似，而 PBD2 與 Hemagglutinin-neuramindase (hn) 上游的序列相似。進一步以 Vibro fischeri的螢光酵素作為報告系統，比較不同環境刺激下 kvgAS 的啟動子在克雷白氏肺炎桿菌 CG43-S3 (野生株) 及克雷白氏肺炎桿菌 CG43-S3021 ( kvgS突變株) 的活性，發現在 25℃ 及 H2O2 的存在下，野生株中 kvgAS 啟動子的活性表現量十分高，但突變株在H2O2 的存在下， kvgAS 啟動子的活性卻明顯降低，由這些結果我們推測 kvgAS 可能與細菌抗氧化或壓力的反應有關。最後，我們經由 RT-PCR 和南方轉漬的實驗發現 kvgA 和 kvgS 與博得氏百日咳桿菌的 bvgAS 及大腸桿菌 evgAS 一樣受同一個啟動子所調控。

Klebsiella pneumoniae is an opportunistic pathogen, which causes liver abscess in diabetic patients, and urinary and respiratory tract infections in immunocompromised patients. Two gene clusters, kvgASQR and kvhAS were previously isolated by PCR-supported genomic substractive hybridization from a virulent K. pneumoniae CG43. On the basis of sequence analysis, KvgA, KvhA and KvgR are likely the response regulators of two-component system in the bacteria. KvgS and KvhS may be the proteins for signaling reception of the system. The KvgQ that carries several transmembrane domains is also likely involved in sensing the signaling. In order to study the function role of kvgA, we tried initially to overexpress the kvgA using pET system in Escherichia coli. Several attempts failed to improve the solubililty of the overexpressed protein. Thus, the insoluble protein was solubilized by 6 N urea and purified by His-tag affinity column. Renaturation of the denatured protein was carried out by gradient dialysis. KvgAt that was deleted of the receiver domain showed specific association with p-kvhA, p-kvgR and it’s own promoter by electrophoresis mobility shift assay (EMSA). In searching of the KvgA-regulated virulence factors, genomic DNA of K. pneumoniae CG43 was partially digested with BstUI into a pool of DNA fragments of 200-500 bps. The His-tagged KvgAt was added to the fragmented DNA pool, and the specifically KvgAt associated DNA fragments were then co-isolated by the resin of NiSO4 and PCR amplified. Two clones, PBD1 and PBD2, were obtained and were found to be the E. coli lysine/cadaverine antiporter homolog and mump virus hemagglutinin-neuraminidase homolog, respectively. By using luxAB as the reporter, the expression of p-kvgAS in the wild type CG43-S3 was found to be activated under 25℃ or in the presence of H2O2. In contrast, the p-kvgAS activity was decreased in the kvgS mutant CG43-S3201 by incubating with H2O2. The results suggest that the kvgAS is involved in anti-oxidation and/or anti-stress responses. Finally, RT-PCR and southern blot analysis demonstrated that the kvgAS is organized like an operon as the B. pertussis-bvgAS and E. coli- evgAS.