探討 Sigma 因子 RpoS 與 RpoN 在創傷弧菌　YJ016　致病過程中的調控機轉

Regulatory Roles of the Sigma Factors RpoS and RpoN in the Pathogenesis of Vibrio vulnificus YJ016

陳俊良(Chun-Liang Chen)

指導教授 ： 詹明修 ； 賴怡琪

中山醫學大學/醫學院/醫學研究所/碩士(2005年)

https://doi.org/10.6834/CSMU.2005.00060

摘要

創傷弧菌（Vibrio vulnificus）是能經傷口及食物感染人體且具高度致死率的伺機性感染病源菌（opportunistic pathogen），主要造成原發型敗血症及傷口化膿潰爛。 創傷弧菌由海水入侵人體後，必須因應環境改變與宿主免疫壓力，表現特定致病基因以成功造成感染。 在調控致病相關基因表現的因子當中，sigma factors S（RpoS）是細菌抗壓反應的主要因子之一，而 sigma factor N（RpoN）對具鞭毛的細菌而言，是調控運動性表現的主要因子。 為深入了解 RpoS 及 RpoN 等 sigma 因子在創傷弧菌 YJ016 致病過程中的調控機轉，我們以同源重組（homologous recombination）技術建構 rpoS 缺失（YJΔS）、rpoN 缺失（YJΔN）及 rpoS/rpoN 雙重缺失（YJΔNS）的突變菌株。 YJΔS、YJΔN、YJΔNS 等突變株在不同培養條件下的生長能力與野生型 YJ016 間無顯著的差異，但 rpoS 基因缺失的 YJΔS 與 YJΔNS 突變株，無法在 TCBS 培養基上發酵蔗糖產酸，而且亦失去對血液培養基的溶血能力。 此外 YJΔS 在半固態培養基游走（swarming）的速度略快於 YJ016，在固態培養基表面顯現邊緣呈鋸齒狀的菌落，不同於野生型的圓滑邊緣。 西方墨點轉漬法與穿透式電子顯微鏡觀察的結果顯示 rpoN 基因缺乏使創傷弧菌因無法合成鞭毛結構蛋白 FlaA 而無鞭毛構造，導致 YJΔN 及 YJΔNS 突變株喪失游走能力，顯示 rpoN 基因在鞭毛合成上的重要性。 rpoS 基因的缺失，使 YJΔS 在 30℃ 的 LB 及 TCG 培養後的生物膜生成量，為其他菌株的 2 ~ 5 倍多，且完全喪失 caseinase 的活性。 以 MOI=10 菌量的不同創傷弧菌株感染人類大腸表皮細胞 Caco-2 細胞 2 小時發現 YJ016 有顯著細胞毒殺性，YJΔS 次之，而 YJΔN 及 YJΔNS 的毒殺能力明顯降低。 因為此種胞殺性需要細菌跟細胞直接接觸，所以缺失 rpoN 基因的突變菌株因失去鞭毛的運動能力使得菌落化 Caco-2 細胞的能力下降，而影響其細胞毒殺力。 皮下感染的小鼠動物模式中， rpoS 基因缺失使創傷弧菌在感染初期 24 小時內的毒力降低，YJΔS 的半致死菌量約為 YJ016 的 4 倍。 究竟 RpoS 與 RpoN 在創傷弧菌感染 Caco-2 細胞時扮演何種調控角色? RpoS 與 RpoN 對創傷弧菌游走能力的調控機制為何? 由於 RpoS 與 RpoN 在 E. coli 及 V. cholerae 內可調控許多不同基因的表達，我們將針對創傷弧菌運動相關與胞殺能力相關的基因，以蛋白質體學方法分析不同菌株間的蛋白表現圖譜，試圖勾勒 RpoS 與 RpoN 在創傷弧菌致病過程中之調控機轉。

關鍵字

創傷弧菌 YJ016 ； RpoS ； RpoN ； Caco-2細胞 ； 鞭毛蛋白 ； 線毛蛋白

並列摘要

Vibrio vulnificus is an opportunistic pathogen that causes serious wound infection and fulminant septicemia with a high mortality rate in humans. On entering the host from ocean, V. vulnificus must sense the microenvironments and respond by altering gene expression patterns via alternative sigma factors, such as RpoS and RpoN, to establish a successful infection in humans. To elucidate the virulence mechanism of V. vulnificus YJ016, rpoS and rpoN deletion mutants (YJΔS and YJΔN) and the double mutant (YJΔNS) were initially constructed with allelic exchange technique. Compared to their parental strain YJ016, these mutants had no detectable defect in in-vitro growth; however, the caseinase and hemolytic activities as well as the sucrose fermentation on TCBS were found to be lost in YJΔS. Intriguingly, YJΔS displayed significantly different motility with an increased velocity of swimming on the 0.325 % agar and a radial swarming pattern on solid surfaces. Based on the results obtained from Western blotting and TEM observation, the deletion of rpoN blocked the synthesis of flagellin and thus the formation of flagellum in YJΔN and YJΔNS. Furthermore, the production of biofilm was twofold more in YJΔS than that other mutant strains after a 24 h-incubation in the LB medium at 30oC, and fivefold more with the incubation in TCG medium. The deletion of rpoS gene in V. vulnificus YJ016 resulted in a slightly decrease in adhering ability to the Caco-2 cells as assessed by competition assay and the LD50 of YJΔS was fourfold more than that of YJ016 in the first 24-hr infection period in the BALB/c mice model generated with a subcutaneous route. Finally, the mutant phenotypes of YJΔS and YJΔN strains observed were completed by providing the rpoS or rpoN gene in-trans. What might be the role of RpoS and RpoN in the infection in Caco-2 cell model and in the regulation of motility for V. vulnificus as well? RpoS and RpoN had been shown to function as both a positive and a negative regulator of expression of different proteins in E. coli and V. cholera. We are trying to figure out the underlying regulatory mechanism by RpoS and RpoN in V. vulnificus, by using a proteomics approach to determine the expression profiling of motility and cytotoxicity associated genes in a panel of mutants generated.

並列關鍵字

Vibrio vulnificus YJ016 ； RpoS ； RpoN ； Caco-2 cells ； flagellin ； pilin

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