绵羊肺源大肠杆菌cyaA基因缺失对小鼠肺泡巨噬细胞感染的影响  

Effects of cyaA gene deletion in sheep lung-derived Escherichia coli on murine alveolar macrophage infection

作　　者：操义恒 王子杰 王巧梅 党士荣 张丽媛 马雪 张乾义[3] 周霞[1] 黄新[2] 张星星[2] 吴桐忠[2] 钟发钢 CAO Yi-heng;WANG Zi-jie;WANG Qiao-mei;DANG Shi-rong;ZHANG Li-yuan;MA Xue;ZHANG Qian-yi;ZHOU Xia;HUANG Xin;ZHANG Xing-xing;WU Tong-zhong;ZHONG Fa-gang(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;State Key Laboratory for Sheep Genetic Improvement and Healthy Production,Xinjiang Academy of Agricultural and Reclamation Science,Shihezi 832000,China;China Institute of Veterinary Drugs Control,Beijing 100081,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]新疆农垦科学院省部共建绵羊遗传改良与健康养殖国家重点实验室,新疆石河子832000 [3]中国兽医药品监察所,北京100081

出　　处：《中国预防兽医学报》2023年第3期295-301,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：新疆兵团重点领域科技攻关计划项目(2019AB029);兵团重点领域科技攻关计划(2021AB012);新疆兵团国际科技合作计划(2019BC004);新疆农垦科学院优秀中青年人才培养引导计划专项(SKLSGIHP2021A02)。

摘　　要：为研究cyaA基因编码的腺苷酸环化酶(AC)在绵羊肺源大肠杆菌XJ10感染小鼠肺泡巨噬细胞(MH-S)中的作用,本研究以绵羊肺源大肠杆菌XJ10野毒株(XJ10WT)为对照,通过荧光定量PCR检测XJ10 cyaA基因缺失株(XJ10ΔcyaA)在感染MH-S细胞过程中与黏附侵袭相关的motA、crl、fimH、pap基因的m RNA转录水平,结果显示,与XJ10WT相比,XJ10ΔcyaA在感染MH-S细胞过程中与黏附侵袭相关的motA、crl、fimH、pap基因的m RNA转录水平在感染后0和6 h时均极显著降低(P<0.001),crl和pap基因的m RNA转录水平分别在2 h和12 h时均极显著上调(P<0.001)。进一步通过吉姆萨染色观察XJ10WT和XJ10ΔcyaA黏附MH-S细胞情况,采用细胞和细菌共培养方式分析XJ10ΔcyaA对MH-S细胞的黏附及侵袭能力,并通过CCK-8法分析细菌对MH-S细胞增殖的抑制情况。结果显示,XJ10ΔcyaA和XJ10WT在感染后3 h黏附MH-S细胞的活菌数量达峰值并持续至5 h,随后逐渐下降,在感染后1 h~5 h的时间段内,XJ10ΔcyaA黏附MH-S细胞的活菌数量均极显著低于XJ10WT(P<0.001)。XJ10ΔcyaA和XJ10WT侵袭MH-S细胞的活菌数量随着感染时间的延长逐渐升高,5 h时达到峰值,后持续下降,XJ10ΔcyaA在感染后1 h~10 h侵袭MH-S细胞的活菌数量均显著或极显著低于XJ10WT。在感染MH-S细胞后的3 h~6 h,XJ10ΔcyaA对细胞增殖的抑制率均极显著低于XJ10WT(P<0.001)。上述结果首次表明,cyaA基因表达的AC可以显著上调绵羊肺源E.coli与黏附侵袭相关基因的转录水平,并参与绵羊肺源E.coli对MH-S细胞的黏附与侵袭,抑制MH-S细胞增殖。本研究为绵羊肺源E.coli致病机制的研究奠定了基础。In order to study the effects of cyaA gene encoding adenylate cyclase(AC)on the adhesion and invasion by sheep lung-derived E.coli XJ10 to murine alveolar macrophages(MH-S),the mRNA transcription levels of motA,crl,fimH and pap genes related to adhesion and invasion by XJ10 cyaA gene deletion strain(XJ10ΔcyaA)were detected by quantitative real-time PCR,with sheep lung-derived E.coli XJ10 wildtype strain(XJ10WT)as a control.The results showed that compared with XJ10WT,the mRNA transcription levels of motA,crl,fimH and pap genes related to adhesion and invasion in XJ10ΔcyaA infected MH-S cells were significantly decreased at 0 and 6 hours,while the mRNA transcription levels of crl and pap genes were significantly up-regulated at 2 hours and 12 hours.The adhesion of XJ10WT and XJ10ΔcyaA to MH-S cells was observed by Giemsa staining.The adhesion and invasion ability of XJ10ΔcyaA to MH-S cells was analyzed by cell and bacterial co-culture,and the inhibition of bacteria on MH-S cell proliferation was analyzed by CCK-8 method.The results showed that the number of viable bacteria adhered to MH-S cells by XJ10ΔcyaA and XJ10WT reached saturation at 3 hours and lasted for 5 hours,and then decreased gradually.The number of viable bacteria adhered to MH-S cells by XJ10ΔcyaA was significantly lower than that by XJ10WT during 1 hours-5 hours.The number of viable bacteria invading MH-S cells of XJ10ΔcyaA and XJ10WT gradually increased as time went by,peaked at 5 hours,and then continued to decrease.The number of viable bacteria invading MH-S cells of XJ10ΔcyaA during 1 hours-10 hours was significantly lower than that of XJ10WT.At 3 hours-6 hours after infection of MH-S cells,the cell proliferation inhibition rate of XJ10ΔcyaA was significantly lower than that of XJ10WT.This study showed that AC expressed by cyaA gene could significantly increase the transcription level of genes related to adhesion and invasion of sheep lung-derived E.coli,participate in the adhesion and invasion of MH-S cells,and inhibit the proliferati

关 键 词：绵羊肺源大肠杆菌 cyaA基因 小鼠肺泡巨噬细胞 黏附及侵袭 细胞增殖抑制率 

分 类 号：S855.1[农业科学—临床兽医学]