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rpoS对鳗弧菌MHK3株Hcp表达和杀菌能力的影响
王小鹿,李杰,李贵阳,唐磊,杨慧超,莫照兰
1.中国海洋大学海洋生命学院 山东 青岛 266003;2.中国水产科学研究院黄海水产研究所 农业农村部海水养殖病害防治重点实验室 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 山东 青岛 266071;3.中国海洋大学三亚海洋研究院 海南 三亚 572000;4.上海海洋大学水产科学国家级实验教学示范中心 上海 201306
摘要:
溶血素共调节蛋白(Hcp)的合成和分泌是六型分泌系统(T6SS)行使功能的重要特征。RNA聚合酶的σ亚基RpoS参与调节细菌的生长和应激反应。为了探究RpoS对鳗弧菌(Vibrio anguillarum) T6SS的调控作用,本研究构建了MHK3∆rpoS突变株,检测了部分表型特征的变化;利用lacZ半乳糖苷酶报告基因检测了突变株hcp1和hcp2在转录水平的变化;进行Western blot并定量分析突变株Hcp在翻译水平的变化;并通过细菌拮抗实验检测了突变株杀菌能力的变化。结果显示,MHK3∆rpoS突变株的生长情况、泳动性、明胶酶活性及酪蛋白酶活性与MHK3野生株相比无显著差异(P>0.05),而平台早期的菌膜形成能力有显著上升(P<0.05);在各生长时期,MHK3∆rpoS的hcp1和hcp2在转录水平的表达量均较MHK3有显著上升(P<0.01),最高时分别为MHK3的1.79倍和1.94倍;在翻译水平上,MHK3∆rpoS在胞内和胞外Hcp的分泌均有显著升高(P<0.05),最高时分别为MHK3的1.59倍和1.31倍;同时,MHK3∆rpoS对大肠杆菌(Escherichia coli) E5的杀菌能力约为MHK3的1%。研究表明,rpoS对鳗弧菌MHK3株的生长情况、泳动性、明胶酶活性及酪蛋白酶活性均无显著调控作用,但对平台早期的菌膜形成能力具有一定的负调控作用,在转录和翻译水平上也负调控Hcp的表达。而在杀菌能力上,rpoS发挥一定的正调控作用。说明菌株的杀菌能力强弱并非直接与Hcp的表达和分泌量正相关。本研究为进一步阐明T6SS的调控机制及其介导的杀菌作用机制提供了新思路,并丰富了其理论基础。
关键词:  鳗弧菌  rpoS  Hcp  T6SS  杀菌
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The effect of rpoS on Hcp expression and bactericidal activity in Vibrio anguillarum MHK3
WANG Xiaolu1,2,3,4, LI Jie3,4, LI Guiyang3,4, TANG Lei1,5, YANG Huichao2,3,4,6, MO Zhaolan1,2,3,4,5
1.College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong 266003, China;2.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences;3.Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs;4.Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, Shandong 266071, China;5.Sanya Ocean Institute, Ocean University of China, Sanya, Hainan 572000, China;6.National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
Abstract:
The type Ⅵ secretion system (T6SS), a protein secretion system generally found in gram-negative bacteria, plays an essential role in virulence, interspecific competition, and environmental adaptability. Hemolysin-coregulated protein (Hcp), an extracellular component of T6SS, is released into the medium and therefore may serve as a marker of a functional T6SS apparatus. RpoS, the σ subunit of RNA polymerase, is involved in the regulation of bacterial growth and stress response. To explore the regulatory effect of rpoS on Vibrio anguillarum T6SS, an MHK3∆rpoS mutant strain was constructed; phenotypic changes were detected using the lacZ reporter gene to construct fusion strains and the changes in hcp1 and hcp2 expression at the transcriptional level were detected using the ONPG (o-nitrophenyl-ß-D-galactopyranoside) method. Western blotting was performed to quantitatively analyze the changes in the mutant Hcp expression at the translational level. The change in the bactericidal activity of the mutant strain was detected via a bacterial antagonistic experiment. The results showed that the growth, mobility, gelatinase activity, and caseinase activity of the MHK3∆rpoS mutant strain showed no significant difference compared with the wild-type strain MHK3 (P>0.05). The biofilm-forming ability significantly increased at the prophase after mutation in rpoS (P<0.05). The transcriptional levels of hcp1 and hcp2 in each growth phase were significantly higher in MHK3∆rpoS than in MHK3 (P<0.01), with highest increases of 1.79-fold and 1.94-fold compared with MHK3, respectively. At the translational level, the secretion of both intracellular and extracellular Hcp in MHK3∆rpoS significantly increased (P<0.05), with highest increases of 1.59-fold and 1.31-fold, respectively. Meanwhile, the bactericidal activity of MHK3∆rpoS against Escherichia coli E5 was about 1% of MHK3. Studies have shown that rpoS has no significant regulatory effect on some phenotypes of MHK3; however, it negatively regulates the biofilm-forming ability at the prophase. This is different from the results of the research on V. anguillarum M3 that posits that rpoS-mediated regulation of the same phenotype in different strains varies. Furthermore, at the levels of transcription and translation, rpoS negatively regulates the expression of Hcp; however, it shows a positive regulatory effect on the bactericidal activity of MHK3. This indicates that the strength of the bactericidal activity of the strain is not directly related to the expression and secretion of Hcp. This study provides innovative ideas and enriches the theoretical basis for further elucidating the regulatory mechanism of T6SS and T6SS-mediated bactericidal activity.
Key words:  Vibrio anguillarum  rpoS  Hcp  T6SS  Bactericidal activity