K88ac~+和K88ad~+产肠毒素大肠杆菌hlyA基因缺失株的构建及相关功能初步分析
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摘要
为研究产肠毒素大肠杆菌(ETEC)溶血素hlyA基因的相关生物学特性,本研究通过Red同源重组的方法构建了ETEC标准株K88ac+C83902和K88ad+C83903的hlyA基因缺失株。体外生长试验和酵解试验表明:缺失株C83902△hlyA和C83903△hlyA生长速度及生长周期各个阶段的特性与相应野生株相比无差异,其分解发酵糖类和氨基酸的能力也未发生改变。小鼠感染试验显示经野生株和回补株感染的小鼠致死率高,而缺失株不致死,表明hlyA基因可以增强ETEC对ICR小鼠的致病力。从感染小鼠的十二指肠、空肠、回肠分离感染的靶细菌,平板计数和PCR检测表明,ETEC在小鼠回肠的黏附能力强于在十二指肠和空肠,并且基因缺失株黏附能力比野生株显著降低。本研究首次对ETEC溶血素hlyA基因进行研究,为进一步阐释α-溶血素与机体相互作用的分子机制和ETEC的致病机制,以及预防ETEC感染提供了相关的实验依据。
In this study, we successfully constructed hlyA gene deletion mutants of K88ac+and K88ad+enterotoxigenic Escherichia coli(ETEC) using the Red-based recombination system, and then compared the differences of biological characteristics between the wild type strains and the mutants. In vitro assays indicated that no significant difference characters were found between wild type strains and △hlyA mutants regarding the aspects of growth rate and sugar fermentations. The mice experimental infections revealed higher death rates occurred in mice groups inoculated with the wild type or complemented strains, but no death in groups with △hlyA mutants, which suggested hlyA was able to increase the virulence of ETEC in mice. However, accoding to the bacteria load recovered from the duodenum, jejunum and ileum in the artificially infected mice, the adherence of ETEC to the ileum was stronger than those to the duodenum or jejunum, and the adherence of hlyA mutant was significantly decreased compared with wild type strain. It is the first time to reveal the role of hlyA gene encoding the hemolysin in ETEC, and provide the basis to explain the molecular mechanisms of interaction between the hemolysin and host cells, pathogenic mechanisms of ETEC.
In this study, we successfully constructed hlyA gene deletion mutants of K88ac+and K88ad+enterotoxigenic Escherichia coli(ETEC) using the Red-based recombination system, and then compared the differences of biological characteristics between the wild type strains and the mutants. In vitro assays indicated that no significant difference characters were found between wild type strains and △hlyA mutants regarding the aspects of growth rate and sugar fermentations. The mice experimental infections revealed higher death rates occurred in mice groups inoculated with the wild type or complemented strains, but no death in groups with △hlyA mutants, which suggested hlyA was able to increase the virulence of ETEC in mice. However, accoding to the bacteria load recovered from the duodenum, jejunum and ileum in the artificially infected mice, the adherence of ETEC to the ileum was stronger than those to the duodenum or jejunum, and the adherence of hlyA mutant was significantly decreased compared with wild type strain. It is the first time to reveal the role of hlyA gene encoding the hemolysin in ETEC, and provide the basis to explain the molecular mechanisms of interaction between the hemolysin and host cells, pathogenic mechanisms of ETEC.
引文
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[7]Cavalieri S J,Snyder I S.Cytotoxic activity of partially purified Escherichia coli alpha hemolysin[J].J Med Microbiol,1982,15:11-21.
[8]Snith H W.The haemolysins of Escherichia coli[J].J Pathol Bacteriol,1963,85:197-211.
[9]Beutin L,Montenegro M.Enterohaemolysin and shiga-like genes in E.coli[J].Vet Rec,1990,127:316.
[10]Dhakal B K,Mulvey M A.The UPEC pore forming toxinα-hemolysin triggers proteolysis of host proteins to disrupt cell adhesion,inflammatory and survival pathways[J].Cell Host Microbe,2012,11(1):58-69.
[2]周立雄,张兆山.菌α-溶血素分泌系统基因表达调控研究进展[J].生物技术通讯,2004,04:374-378.
[3]Gentschev I,Dietrich G,Goebel W.The E.coliα-hemolysin secretion system and its use in vaccine development[J].Trends in Microbiol,2002,10(1):39-45.
[4]朱春红,孙晓庆,何素芬,等.基于Red同源重组和高效自杀性载体系统构建肠炎沙门氏菌突变株方法的比较[J].中国预防兽医学报,2009,31(2):81-98.
[5]Datsenko K A,Wanner B L.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J].Proc Natl Acad Sci USA,2000,97(12):6640-6645.
[6]Allen K P,Randolph M M,Fleckenstein J M.Importance of heat-labile enterotoxin in colonization of the adult mouse small intestine by human enterotoxigenic Escherichia coli strains[J].Infect Immun,2006,74(2):869-875.
[7]Cavalieri S J,Snyder I S.Cytotoxic activity of partially purified Escherichia coli alpha hemolysin[J].J Med Microbiol,1982,15:11-21.
[8]Snith H W.The haemolysins of Escherichia coli[J].J Pathol Bacteriol,1963,85:197-211.
[9]Beutin L,Montenegro M.Enterohaemolysin and shiga-like genes in E.coli[J].Vet Rec,1990,127:316.
[10]Dhakal B K,Mulvey M A.The UPEC pore forming toxinα-hemolysin triggers proteolysis of host proteins to disrupt cell adhesion,inflammatory and survival pathways[J].Cell Host Microbe,2012,11(1):58-69.