創傷弧菌YJ016中調控環狀雙鳥嘌呤單磷酸相關的 vva0325-36基因群功能分析

Characterization of the vva0325-36 Gene Cluster Associated with Cyclic-di-GMP Regulation in Vibrio vulnificus YJ016

周莉芳(Li-Fang Chou)

指導教授 ： 張晃猷

國立清華大學/生命科學院/分子醫學研究所/博士(2009年)

https://doi.org/10.6843/NTHU.2009.00255

摘要

利用生物資訊學分析推測，創傷弧菌YJ016第二號染色體上編號vva0325到vva0336的基因群，很可能參與調控分子環狀雙鳥嘌呤單磷酸的代謝。此基因群可轉譯一多重覆片段毒素，一套第一型的分泌系統，以及一包含感應激酶、反應調控蛋白、兩個分別具有GGDEF和EAL功能區域蛋白質的調控系統。已知含有GGDEF功能區域的VVA0326蛋白質及含有EAL功能區域的VVA0328蛋白質，分別具有雙鳥苷酸環化酶及磷酸雙酯鍵分解酶的活性，且調控創傷弧菌體內的環狀雙鳥嘌呤單磷酸之濃度。本研究第一部份首先探討vva0331基因所轉譯一個分子量489 kDa的巨大蛋白質在細菌內的表現位置及生物功能。結果顯示VVA0331蛋白質主要以分泌型式存在，而其產量會受到鐵離子的調控。另外，觀察細菌黏附能力、細胞毒殺分析及老鼠毒力實驗發現創傷弧菌菌株YJ016及VVA0331突變株並無差異。第二部份的研究是分析vva0326~9所轉譯與調控環狀環狀雙鳥嘌呤單磷酸相關的基因產物特性。我們發現剔除vva0326基因的GGDEF區域，會使創傷弧菌的泳動力增加，而減少生物膜的形成及對HEp-2細胞株有較高的毒性能力。然而，剔除vva0328基因的EAL區域之突變菌株，與創傷弧菌菌株YJ016並無明顯差異。另外，分析rtxA、vvhA、vva0331轉錄量及RtxA蛋白質的表現程度顯示這些毒力因子可能受此環狀雙鳥嘌呤單磷酸系統的調控。最後也發現此兩種蛋白質在創傷弧菌菌株YJ016中會有極化分佈的表現。綜合本研究的結果，此環狀雙鳥嘌呤單磷酸系統的調控機制會影響此細菌毒力，相信研究此調控機制將有助於瞭解創傷弧菌的生理與致病機制。

關鍵字

創傷弧菌 ； 多重覆片段毒素 ； 環狀雙鳥嘌呤單磷酸

並列摘要

Bioinformatic analyses reveal a gene cluster (vva0325-vva0336) on the small chromosome of Vibrio vulnificus YJ016 might regulate cyclic-di-GMP levels. The gene cluster encodes a large RTX-like protein (VVA0331), a type I protein secretion system (VVA0332-0336), and a signaling system composing of a sensor kinase (VVA0329), two response regulators with a GGDEF-(VVA0326) and an EAL-domain (VVA0328), respectively. It is hypothesized that they may play roles in pathogenesis through modulating cyclic-di-GMP levels. Previous studies have shown that VVA0326 and VVA0328 possess diguanylate cyclase and phosphodiesterase activity, respectively, in vitro, and regulate intracellular c-di-GMP in V. vulnificus. The first study was to elucidate the subcellular location and functional roles of VVA0331. Results showed VVA0331 existed primarily in a secreted form and regulated by iron. The capabilities of vva0331 mutant strains in adherence and cytotoxicity on cells and virulence in mice were examined in comparison with wild type strain, indicating no difference. The second study was to investigate biological properties of VVA0326 and VVA0328. Deletion in the GGDEF domain of VVA0326 conferred the bacterium a slightly higher swimming motility, less biofilm formation and higher cytotoxicity activity to HEp-2 cells. However, there was no obvious difference between in-frame EAL deletion mutant and the wild type strains. Comparison of the levels of rtxA, vvhA, vva0331 transcripts and RtxA protein in wild type and vva0326 mutant strains indicates that these virulence-associated factors might be regulated by the cyclic-di-GMP signaling system. Furthermore, localization analysis had demonstrated that VVA0326 and VVA0328 were localized at V. vulnificus YJ016 poles by constructing GFP fusion proteins. Taken together, the gene cluster regulated by cyclic-di-GMP may have effects on bacterial virulence in V. vulnificus YJ016. We believe that the findings would be important for understanding the V. vulnificus physiology and pathogenesis mechanism.

並列關鍵字

Vibrio vulnificus ； RTX ； Cyclic di-GMP

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