鼠伤寒沙门菌ST1920株cpxR基因缺失株的构建及其对小鼠LD_(50)的影响
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摘要
为了探索鼠伤寒沙门菌双组分信号系统CpxAR的应答调节蛋白基因cpxR对小鼠半数致死量(LD_(50))的影响,为下一步研究CpxAR系统对鼠伤寒沙门菌致病性的调控机制提供基础。本研究通过ERIC-PCR、MLST分型技术筛选流行克隆株JS_分,应用噬菌体转导技术构建基因缺失突变株JS_分△cpxR,并分别测定了小鼠腹腔注射JS_标、JS_标△cpxR、JS_分、JS_分△cpxR后的LD_(50)。结果显示:89株鼠伤寒沙门菌分为11个ERIC型、4个ST型,流行性克隆株为ST1920型(新的ST型)。LD_(50)结果显示,JS_标、JS_标△cpxR、JS_分、JS_分△cpxR对小鼠的LD_(50)分别为5.79×108、7.85×108、1.91×107、3.35×108 CFU。其中ST1920型代表株(JS_分)的LD_(50)较JS_标低约30.31倍,JS_标△cpxR突变株的LD_(50)比其亲本株JS_标升高了1.36倍,JS_分△cpxR突变株LD_(50)比其亲本株JS_分升高了17.54倍。结果表明cpxR基因缺失株毒力均明显下降,cpxR基因与鼠伤寒沙门菌毒力密切相关,对致病性有重要影响。
To explore the influence of cpxR gene of two-component signaling system in Salmonella enterica serovar typhimurium on the median lethal dose(LD_(50))in mice and to provide the foundation for further research on its regulatory mechanism of Salmonellatyphimurium pathogenicity.This study screened the epidemic strain JSc(clinical isolates)by ERIC-PCR,the multi-locus sequence typing(MLST)and constructed the cpxR gene deletion mutant which named as JSc△cpxR by P22 phage transduction.Then the lethal dose(LD_(50))of JSs(standard strains),JSs△cpxR,JSc,JSc△cpxR strains in mice were determined.The results showed that 89 Salmonella enterica serovar typhimurium strains were grouped into 11 and 4patterns by ERIC-PCR and MLST,respectively.The ST of 33 isolate was identified as newly discovered ST-1920;The LD_(50) of JSs,JSs△cpxR,JSc,JSc△cpxR strains in mice were 5.79×108 CFU,7.85×108 CFU,1.91×107 CFU,3.35×108 CFU,respectively.The LD_(50) of the representative strain(JSc)of the new ST1920 decreased by about 30.31 times compared with that of JSs.The LD_(50) of JSs△cpxR increased by about 1.36 times compared with that of its parent strain,while the LD_(50) of JSc△cpxR increased by about 17.54 times compared with that of its parent strain,suggesting that the virulence of cpxR gene deletion strain become less virulent than that of the parent strain.In conclusion,cpxR gene is closely related to the pathogenicity of S.enterica serovar typhimurium and have important effects on the pathogenicity of S.enterica serovar typhimurium.
To explore the influence of cpxR gene of two-component signaling system in Salmonella enterica serovar typhimurium on the median lethal dose(LD_(50))in mice and to provide the foundation for further research on its regulatory mechanism of Salmonellatyphimurium pathogenicity.This study screened the epidemic strain JSc(clinical isolates)by ERIC-PCR,the multi-locus sequence typing(MLST)and constructed the cpxR gene deletion mutant which named as JSc△cpxR by P22 phage transduction.Then the lethal dose(LD_(50))of JSs(standard strains),JSs△cpxR,JSc,JSc△cpxR strains in mice were determined.The results showed that 89 Salmonella enterica serovar typhimurium strains were grouped into 11 and 4patterns by ERIC-PCR and MLST,respectively.The ST of 33 isolate was identified as newly discovered ST-1920;The LD_(50) of JSs,JSs△cpxR,JSc,JSc△cpxR strains in mice were 5.79×108 CFU,7.85×108 CFU,1.91×107 CFU,3.35×108 CFU,respectively.The LD_(50) of the representative strain(JSc)of the new ST1920 decreased by about 30.31 times compared with that of JSs.The LD_(50) of JSs△cpxR increased by about 1.36 times compared with that of its parent strain,while the LD_(50) of JSc△cpxR increased by about 17.54 times compared with that of its parent strain,suggesting that the virulence of cpxR gene deletion strain become less virulent than that of the parent strain.In conclusion,cpxR gene is closely related to the pathogenicity of S.enterica serovar typhimurium and have important effects on the pathogenicity of S.enterica serovar typhimurium.
引文
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[2]Lamont S J,Kaiser M G,Liu W.Candidate genes for resistance to Salmonella enteritidis colonization in chickens as detected in a novel genetic cross[J].Vet Immunol Immunopathol,2002,87:423-428.
[3]Mariani P,Barrow P A,Cheng H H,et al.Localization to chicken chromosome 5of a novel locus determining salmonellosis resistance[J].Immunogenetics,2001,53:786-791.
[4]Van Diemen P M,Kreukniet M B,Galina L,et al.Characterization of a resource population of pigs screened for resistance to salmonellosis[J].Vet Immunol Immunopathol,2002,88:183-196.
[5]谢懋英,赖婧,马立才,等.肉鸡产业链中沙门菌流行情况及其耐药性[J].中国兽医学报,2014,34(11):1790-1794.
[6]Hunke S,Keller R,Muller V S.Signal integration by the Cpx-envelope stress system[J].FEMS Microbiol Lett,2012,326:12-22.
[7]Dong J,Iuchi S,Kwan H S,et al.The deduced aminoacid sequence of the cloned cpxR gene suggests the protein is the cognate regulator for the membrane sensor,CpxA,in a two-component signal transduction system of Escherichia coli[J].Gene,1993,136(1):227-230.
[8]Acosta N,Pukatzki S,Raivio T L.The Cpx system regulates virulence gene expression in Vibrio cholerae[J].Infect Immun,2015,83(6):2396-2408.
[9]Humphreys S,Rowley G,Stevenson A,et al.Role of the two-component regulator cpxAR in the virulence of Salmonella enterica serotype typhimurium[J].Infect Immun,2004,72(8):4654-4661.
[10]Shanmugasundaram M,Radhika M,Murali H S,et al.Detection of Salmonella enterica serovar Typhimurium by selective amplification of fliC,fljB,iroB,invA,rfbJ,STM2755,STM4497genes by polymerase chain reaction in a monoplex and multiplex format[J].World J Microbiol Biotechnol,2009,25:1385-1394.
[11]Si W,Wang X,Liu H,et al.Physiology,pathogenicity and immunogenicity of live,attenuated Salmonella enterica serovar Enteritidis mutants in chicks[J].Microb Pathog,2015,83-84:6-11.
[12]Campioni F,Moratto Bergamini A M,Falco J P.Genetic diversity,virulence genes and antimicrobial resistance of Salmonella enteritidis isolated from food and humans over a 24-year period in Brazil[J].Food Microbiol,2012,32(2):254-264.
[13]Litrup E,Torpdahl M,Malorny B,et al.Association between phylogeny,virulence potential and serovars of Salmonella enterica[J].Infect Genet Evol,2010,10(7):1132-1139.
[14]承慧.胸膜肺炎放线杆菌CpxA/R双组份调控系统基因缺失突变株构建及生物学特性研究[D].湖北武汉:华中农业大学,2013.
[15]Main-Hester K L,Colpitts K M,Thomas G A.Coordinate regulation of Salmonella pathogenicity island 1(SPI1)and SPI4in Salmonella enterica serovar Typhimurium[J].Infect Immun,2008,76(3):1024-1035.
[16]Jubelin G,Vianney A,Beloin C,et al.CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli[J].J Bacteriol,2005,187(6):2038-2049.
[17]郑禹,冷静,卞钊.curli菌毛致败血症脓毒症的研究进展[J].中国医学文摘:内科学,2006,27(3):193-195.
[18]Dorel C O,Vidal C,Prigent-Combaret C,et al.Involvement of the Cpx signal transduction pathway of E.coli in biofilm formation[J].FEMS Microbiol Lett,1999,178(1):169-175.
[19]Moubareck C,Brémont S,Conroy M C,et al.GES-11,aNo-vel integron-Associated GES variantiin Acinetobacterbau-mannii[J].Antimicrob Agents Chemother,2009,53(8):3579-3581.
[20]Nagamatsu K,Hannan T J,Guest R L,et al.Dysregulation of Escherichia coliα-hemolysin expression alters the course of acute and persistent urinary tract infection[J].Proc Natl Acad Sci USA,2015,112(8):871-880.