河弧菌中群体感应系统相关基因及信号分子检测  

Prevalence of quorum sensing(QS)systems and its signal molecules production in Vibrio fluvialis

作　　者：胡晓 刁保卫[2] 李杰[2] 王卉[1] 梁未丽[2] HU Xiao;DIAO Baowei;LI Jie;WANG Hui;LIANG Weili(College of Life Sciences,Nanjing Agricultural University,Nanjing 210095,China;National Institute for CommunicableDisease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]南京农业大学生命科学学院,江苏南京210095 [2]中国疾病预防控制中心传染病预防控制所,北京102206

出　　处：《南京农业大学学报》2019年第4期682-688,共7页Journal of Nanjing Agricultural University

基　　金：国家重点研发计划项目(2017YFC1601503);国家自然科学基金项目(81772242)

摘　　要：[目的]本文旨在研究河弧菌在30和37℃条件下群体感应信号分子CAI-1、AI-2和N-酰基高丝氨酸内酯(AHL)的动态变化水平以及群体感应系统相关基因在不同来源河弧菌菌株中的分布及信号分子产生情况。[方法]制备待测菌株的无菌培养上清液,分别用群体感应信号分子CAI-1、AI-2和AHL特异性检测菌株MM920、BB170和KYC55上清液中相应的信号分子。PCR检测不同来源河弧菌中群体感应系统组成的相关基因。通过测定偶氮酪蛋白的方法检测HapA蛋白酶活性。[结果]在30和37℃的培养条件下,河弧菌参考菌株VF85003菌液上清液中CAI-1、AI-2和AHL信号分子水平均随菌体密度的增加而升高。相同菌体密度时,37℃培养上清液中的CAI-1和AI-2水平高于30℃培养的,但AHL水平均低于30℃培养的。对70株不同来源的河弧菌菌株进行PCR检测,发现其中45株河弧菌含有全部群体感应系统组成的相关基因;45株菌株上清液中大多数的CAI-1、AI-2和AHL信号分子水平分布在100~1000的范围内,但并非所有菌株都具有这3种信号分子。45株河弧菌中有48.89%的菌株HapA蛋白酶活性检测为阳性,51.11%的菌株检测结果为阴性。[结论]VF85003培养上清液中CAI-1、AI-2和AHL信号分子水平呈密度依赖性升高,AHL信号分子的表达水平在温暖的环境(30℃)温度下较宿主温度(37℃)下更高,而CAI-1、AI-2信号分子则相反,在宿主温度(37℃)条件表达更高,提示其在河弧菌致病性和环境适应性生存方面的不同作用。群体感应系统组成基因的分布、3种信号分子表达水平和毒力因子金属蛋白酶HapA活性存在菌株间差异。[Objectives] The paper aimed to analyze the dynamic changes of quorum sensing(QS)signal molecules CAI-1, AI-2 and AHL in Vibrio fluvialis at 30 and 37 ℃,the prevalence of QS system genes and the production of signal molecules in V.fluvialis isolates from different sources,and provide basis for further understanding the role of QS systems in the pathogenicity and environmental fitness of V.fluvialis .[Methods] Cell free culture supernatant was prepared. QS signal molecules CAI-1,AI-2 and AHL in the supernatant were detected by using the specific bioassay reporter strains MM920,BB170 and KYC55,respectively. The presence of QS system genes in V.fluvialis isolates from different sources were detected by polymerase chain reaction(PCR). The activity of HapA protease activity was measured by an azocasein assay.[Results] The levels or concentrations of CAI-1,AI-2 and AHL molecules displayed cell density dependent pattern at 30 ℃and 37 ℃ in V.fluvialis reference stain VF85003. At the same cell density,the levels of CAI-1 and AI-2 in the supernatant at 37 ℃ were higher than those at 30 ℃,but the level of AHL at 37 ℃ was lower than 30 ℃. PCR screening showed that 45 V.fluvialis strains out of 70 isolates contained all tested QS-related genes,and the majority of 45 isolates produced medium to high levels of CAI-1,AI-2 and AHL signals ranging from 100 to 1 000,while not all strains generated all three signal molecules. For the HapA protease,a virulence factor which was activated by QS system,48.89% of the 45 tested strains were positive for HapA protease activity,and 51.11% were negative.[Conclusions] The dynamic levels of CAI-1,AI-2 and AHL QS signal molecules in the culture supernatant of VF85003 increased in a cell density-dependent way. Generally,the level of AHL signal molecule at warm environmental temperature(30 ℃)was higher than that at the host temperature(37 ℃). On the contrary,the production of CAI-1,AI-2 signal molecules at the host temperature(37 ℃)was higher than 30 ℃,which suggested the di

关 键 词：河弧菌 群体感应 CAI-1/AI-2 AHL 金属蛋白酶HapA 

分 类 号：Q935[生物学—微生物学]