单增李斯特菌PTS系统转运蛋白IIB^(man)介导运动性和感染的功能研究  

Biological function study of the phosphotransferase system (PTS) transporter protein IIB^(man) in mediating motility and infection in Listeria monocytogenes

作　　者：张佳雪 陈绵绵 程丽丽 任耿佳 徐像文 鲁珂蓥 庄楠茜 孙静 宋厚辉 程昌勇 ZHANG Jia-xue;CHEN Mian-mian;CHENG Li-li;REN Geng-jia;XU Xiang-wen;LU Ke-ying;ZHUANG Nan-xi;SUN Jing;SONG Hou-hui;CHENG Chang-yong(Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics&Advanced Technology,Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management,China-Australia Joint Laboratory for Animal Health Big Data Analytics,College of Animal Science and Technology&College of Veterinary Medicine of Zhejiang A&F University,Hangzhou 311300,China;Taizhou Jiaojiang District Agriculture,Rural and Water Resources Bureau,Taizhou 318000,China)

机构地区：[1]浙江农林大学动物科技学院动物医学院/浙江省畜禽绿色生态健康养殖应用技术研究重点实验室/动物健康互联网检测技术浙江省工程研究中心/浙江省动物医学与健康管理国际科技合作基地/中澳动物健康大数据分析联合实验室,浙江杭州311300 [2]台州市椒江区农业农村和水利局,浙江台州318000

出　　处：《中国预防兽医学报》2023年第11期1179-1187,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：浙江省自然科学基金(LQ20C180001、LY23C180002);国家自然科学基金(32002253、31972648、31872620);浙江农林大学创新训练项目(S202210341103);浙江省大学生科技创新活动计划暨新苗人才计划(2023R412020)。

摘　　要：为探究单增李斯特菌(LM)磷酸转移酶系统(PTS)转运蛋白IIB^(man)在LM生长、运动性及其生物学功能,本研究以LM EGD-e株基因组为模板,经PCR扩增iiB^(man)基因的上下游基因片段(不包含iiB^(man)基因),插入质粒pKSV7以构建缺失质粒;以LM EGD-e基因组为模板,采用PCR扩增iiB^(man)基因与启动子片段,插入质粒pIMK2以构建回补质粒,经PCR和测序鉴定后,将缺失质粒转化LM EGD-e感受态细胞,通过氯霉素抗性及42℃压力下使重组质粒与LM EGD-e株的同源片段发生同源重组单交换获得一代同源重组菌株,然后在无抗性培养基中30℃培养使质粒丢失,获得缺失株ΔiiB^(man)。将回补质粒转化ΔiiB^(man)感受态细胞中,通过卡那霉素抗性筛选获得回补株CΔiiB^(man),缺失株和回补株均经PCR和测序鉴定。37℃培养EGD-e、ΔiiB^(man)、CΔiiB^(man)12 h,期间每隔1 h测定OD_(600nm)并绘制生长曲线,分析各菌株生长特性,结果显示,各菌株体外生长趋势相似。将各菌株菌液接种于TSA半固体培养基,培养至24 h和48 h时检测细菌运动圈大小,结果显示ΔiiB^(man)的运动圈直径分别是EGD-e的1.5及1.6倍(P<0.05、P<0.01),CΔiiB^(man)与EGD-e运动圈直径均基本一致。在各菌株培养上清中加入绵羊血,检测各菌株溶血能力,结果显示,ΔiiB^(man)的溶血能力较EGD-e及CΔiiB^(man)增强。将各菌株以MOI 20感染RAW264.7细胞,在感染后2 h、5 h和8 h收集细胞进行点样计数,结果显示,在感染后2 h和5 h时ΔiiB^(man)的胞内增殖能力较EGD-e及CΔiiB^(man)显著增强(P<0.05)。将各菌株以2×10^(5)cfu/只经腹腔注射感染小鼠48 h后剖杀,取肝脏和脾脏研磨并点样计数,结果显示,ΔiiB^(man)在小鼠脏器载菌量较EGD-e及CΔiiB^(man)极显著增多(P<0.001)。上述结果首次表明IIB^(man)不影响LM的生长,但影响LM的运动性及提高LM的体外溶血活性;IIB^(man)参与LM的胞内增殖和宿主脏器定植。本研究为了解LM PTS系统所介导�The aim of this study was to investigate the biological function of the Listeria monocytogenes(LM)phosphotransferase system(PTS)transporter protein IIB^(man)(encoded by iiB^(man)(lmo2002),hereafter referred to as iiB^(man))in bacterial growth,motility and infection.The upstream and downstream DNA fragments of iiB^(man)(without the iiB^(man) gene)were amplified with LM strain EGD-e genome as template,which were ligated to the restriction enzyme cleavage plasmid pKSV7 to construct the deletion plasmid.The iiB^(man) gene and its promoter fragment were amplified and ligated to the restriction enzyme cleavage plasmid pIMK2 to construct the complementary plasmid.The deletion plasmid was transformed into EGD-e competent cells and a homologous recombinant strain was obtained by exchanging the recombinant plasmid with the homologous fragment of EGD-e under chloramphenicol resistance and 42℃pressure.The deletion strainΔiiB^(man) was then obtained by incubation in non-resistant medium at 30℃until loss of the plasmid.The complementary plasmid was transformed intoΔiiB^(man) competent cells and the complementary strain CΔiiB^(man) was obtained by kanamycin resistance screening.EGD-e,ΔiiB^(man) and CΔiiB^(man) were incubated at 37℃for 12 hours and OD_(600nm) was measured at 1 hour intervals.The results showed that the growth curves of these three strains were similar.The bacteria broth was inoculated into TSA semi-solid medium and the size of the bacteria migration circle was observed after 24 hours and 48 hours.The migration circle diameters ofΔiiB^(man) were 1.5 and 1.6 times more than those of EGD-e at 24 hours and 48 hours.The haemolysis was tested by adding sheep blood into the bacteria culture supernatant.The results showed that the haemolysis ofΔiiB^(man) was enhanced compared to EGD-e and CΔiiB^(man).Murine macrophages RAW264.7 were infected with bacteria at MOI 20 and the bacteria in infected cells was harvested for counting at 2 hours,5 hours and 8 hours post infection.Intracellular proliferation ofΔiiB

关 键 词：单增李斯特菌 磷酸转移酶系统 IIB^(man)转运蛋白 运动性 感染 

分 类 号：S852.61[农业科学—基础兽医学]