本文主要研究内容
作者任方哲（2019）在《空肠弯曲菌FlhF调节鞭毛合成分子机制及其对细菌毒力的影响分析》一文中研究指出：空肠弯曲菌是一种重要的食源性人兽共患病原菌,其可通过动物、食物、水、牛奶等传播给人,并引发以腹泻为主的急性胃肠炎等症状。近年来弯曲菌感染案例在世界各地普遍呈上升趋势,给公共卫生和食品安全带来严重威胁。鞭毛在空肠弯曲菌致病过程中发挥重要作用,其不仅介导细菌运动力,帮助黏附、侵袭细胞,定植宿主,还参与分泌毒力蛋白以及逃避宿主天然免疫应答;另外部分鞭毛因子还被发现可以协同调节该菌其他毒力基因表达,使得鞭毛成为空肠弯曲菌重要的致病因子。FlhF是近年来在极鞭毛细菌中发现调控鞭毛生成数目和定位的关键蛋白,FlhF在不同细菌鞭毛合成过程中发挥不一样的效应;在空肠弯曲菌中,FlhF突变使得细菌完全失去鞭毛结构以及运动力,但其作用机制目前还未阐明。本研究利用表达谱芯片技术在全基因水平分析了 FlhF所影响的代谢通路及其效应,并在鞭毛系统中探究了其所发挥的功能,同时还就FlhF突变对空肠弯曲菌毒力以及致腹泻能力的影响进行了分析;研究结果为深入解析FlhF的作用机制及其与该菌毒力的偶联性提供了依据。1.空肠弯曲菌flhF突变株全基因转录表达谱比较分析应用荧光定量PCR方法评价在不同生长阶段的空肠弯曲菌鞭毛基因的相对表达量,选取鞭毛基因表达相对旺盛的时间点进行表达谱芯片实验,分析FlhF在全基因水平的影响;对所获得的差异基因进行聚类以及富集分析,确定FlhF突变主要映射的代谢通路;收集空肠弯曲菌鞭毛以及其他毒力系统基因,分析FlhF在鞭毛合成通路内的效应,以及其对该菌其他毒力系统所发挥的协同调节作用。研究结果显示在体外培养20 h时空肠弯曲菌的生长状态良好,大部分鞭毛基因的相对表达量较高。对比空肠弯曲菌野生株和flhF突变株的表达谱数据,差异基因聚类分析表明FlhF突变后对空肠弯曲菌基因表达谱产生了明显影响;以差异倍数大于2,p值小于0.01设为阈值进行筛选,总计获得10个差异表达基因,对差异基因进行Gene Ontology和Pathway分析,发现鞭毛及其介导的运动力是显著受影响的通路。在鞭毛通路中进行深入分析发现FlhF在鞭毛系统中发挥一种总调节子功能,其突变后64%的鞭毛相关基因都呈现下调表达;该效应从鞭毛合成早期就开始体现,并持续到鞭毛合成晚期;将鞭毛基因按照其功能相关性以及转录级联层次进行分类,显著性分析结果表明鞭毛丝相关基因以及Ⅲ类鞭毛基因的转录相较其他基因是显著下调表达的。空肠弯曲菌鞭毛基因散布于基因组中,利用基因表达谱数据,本研究发现FlhF突变协同使该菌60%的趋化基因、51.4%的荚膜多糖基因、31.8%的脂寡糖基因、16.7%的铁离子摄取基因、52%的黏附侵袭基因以及39.2%的定植基因呈现下调表达;显著性分析表明FlhF对空肠弯曲菌定植、黏附侵袭和趋化基因表达的影响要显著高于铁离子摄取、脂寡糖和荚膜多糖成分。2.FlhF影响空肠弯曲菌鞭毛合成分子机制研究测定FlhF的相对表达量并观察其亚细胞定位,分析FlhF蛋白的生物学特性;利用荧光示踪技术研究FlhF对其他鞭毛亚单位胞内定位的介导能力,分析空肠弯曲菌鞭毛合成被阻断的机制;通过免疫共沉淀实验结合GSTpull-down验证,筛选并分析FlhF蛋白在胞内可能的作用靶点及其互作结构域;利用凝胶迁移实验(Electrophoretic mobility shift assay,EMSA)探究FlhF是否具有转录调控功能。结果显示FlhF在空肠弯曲菌中是以Ⅰ类鞭毛基因的形式持续性表达的,FlhF主要定位于空肠弯曲菌鞭毛初始着生的细胞极端内膜组分中,说明其可能在鞭毛合成早期发挥作用。FlhF突变后FlhB-GFP和FlgB-GFP均不能再定位于细胞极端,表明鞭毛输出系统组装以及运输功能均受到明显影响,空肠弯曲菌鞭毛组装从而被阻断在合成早期。互作蛋白筛选结果表明FlhF可能与鞭毛组装、趋化、信号识别颗粒、转录翻译、代谢相关蛋白存在功能联系,进一步验证发现FlhF在胞内与FlhG,CheA,DnaK和GlyA有直接相互作用;FlhF蛋白自身可以分为B、N、G三个结构域,结果显示N结构域是FlhF与其他蛋白互作的结构基础;FlhG被发现与FlhF的B结构域有最强的结合作用;GlyA与FlhF二聚体一样通过与其NG结构域结合来互作;CheA和DnaK被发现只与FlhF的N结构域结合。在鞭毛合成三个级联系统中随机选取10个基因的启动子序列进行EMSA验证,发现FlhF可以与调节鞭毛基因转录的sigma因子(σ70和σ28)的启动子序列结合,说明FlhF可以作为转录因子直接调控鞭毛基因转录,影响Ⅰ类和Ⅲ类鞭毛基因的表达。3.FlhF突变对空肠弯曲菌毒力以及致腹泻能力的影响利用分子生物学方法构建flhF突变株以及回复株,评价FlhF突变对空肠弯曲菌毒力以及致腹泻能力的影响。结果显示与野生株相比,flhF突变株只能在接种处生长,完全失去了运动力;其正常鞭毛表型从94%下降为0%,并且不能在电子显微镜视野中观察到鞭毛结构。FlhF突变后空肠弯曲菌的自凝集以及生物被膜形成能力显著降低;利用三种趋化物对菌株的趋化性进行分析,发现flhF突变株在DL-苹果酸和α酮戊二酸诱导体系中的趋化性显著低于野生株。在对Caco-2细胞的黏附、侵袭过程中,FlhF突变使得突变株的相对黏附、侵袭率分别下降为野生株的20.15%和2.90%;通过离心处理尽量排除运动力影响因素后,其相对黏附率可回复至74.78%,但对侵袭能力的修复却没有明显效果,仍只有野生株水平的3.09%;说明flhF突变株对细胞的黏附缺陷主要由运动力丧失导致,而侵袭能力的降低更可能是由鞭毛缺失引起。以鸡和小鼠分别代表禽类和哺乳类研究了 FlhF突变对空肠弯曲菌定植能力的影响。在攻毒后1天,flhF突变株在鸡除结肠外的所有肠道组织中的定植量均显著低于野生株,而在小鼠中则未观察到显著性差异,说明FlhF对空肠弯曲菌在不同宿主中建立初始定植的影响不一;在攻毒后7天,发现空肠弯曲菌野生株可以在鸡和小鼠中持续定植,而flhF突变株则全部被小鼠清除出体外,在鸡中的情况也很相似,flhF突变株仅在一只鸡的盲肠中被分离到,细菌载量也只有104CFU(Colony forming units)/g;说明FlhF对于空肠弯曲菌在宿主体内持续定植的重要性。通过空肠弯曲菌感染仔兔动物模型比较了不同菌株之间的致腹泻能力的差异,结果显示在攻毒后1,2,3天flhF突变株感染仔兔引发的腹泻严重程度均低于野生株。
Abstract
kong chang wan qu jun shi yi chong chong yao de shi yuan xing ren shou gong huan bing yuan jun ,ji ke tong guo dong wu 、shi wu 、shui 、niu nai deng chuan bo gei ren ,bing yin fa yi fu xie wei zhu de ji xing wei chang yan deng zheng zhuang 。jin nian lai wan qu jun gan ran an li zai shi jie ge de pu bian cheng shang sheng qu shi ,gei gong gong wei sheng he shi pin an quan dai lai yan chong wei xie 。bian mao zai kong chang wan qu jun zhi bing guo cheng zhong fa hui chong yao zuo yong ,ji bu jin jie dao xi jun yun dong li ,bang zhu nian fu 、qin xi xi bao ,ding zhi su zhu ,hai can yu fen bi du li dan bai yi ji tao bi su zhu tian ran mian yi ying da ;ling wai bu fen bian mao yin zi hai bei fa xian ke yi xie tong diao jie gai jun ji ta du li ji yin biao da ,shi de bian mao cheng wei kong chang wan qu jun chong yao de zhi bing yin zi 。FlhFshi jin nian lai zai ji bian mao xi jun zhong fa xian diao kong bian mao sheng cheng shu mu he ding wei de guan jian dan bai ,FlhFzai bu tong xi jun bian mao ge cheng guo cheng zhong fa hui bu yi yang de xiao ying ;zai kong chang wan qu jun zhong ,FlhFtu bian shi de xi jun wan quan shi qu bian mao jie gou yi ji yun dong li ,dan ji zuo yong ji zhi mu qian hai wei chan ming 。ben yan jiu li yong biao da pu xin pian ji shu zai quan ji yin shui ping fen xi le FlhFsuo ying xiang de dai xie tong lu ji ji xiao ying ,bing zai bian mao ji tong zhong tan jiu le ji suo fa hui de gong neng ,tong shi hai jiu FlhFtu bian dui kong chang wan qu jun du li yi ji zhi fu xie neng li de ying xiang jin hang le fen xi ;yan jiu jie guo wei shen ru jie xi FlhFde zuo yong ji zhi ji ji yu gai jun du li de ou lian xing di gong le yi ju 。1.kong chang wan qu jun flhFtu bian zhu quan ji yin zhuai lu biao da pu bi jiao fen xi ying yong ying guang ding liang PCRfang fa ping jia zai bu tong sheng chang jie duan de kong chang wan qu jun bian mao ji yin de xiang dui biao da liang ,shua qu bian mao ji yin biao da xiang dui wang cheng de shi jian dian jin hang biao da pu xin pian shi yan ,fen xi FlhFzai quan ji yin shui ping de ying xiang ;dui suo huo de de cha yi ji yin jin hang ju lei yi ji fu ji fen xi ,que ding FlhFtu bian zhu yao ying she de dai xie tong lu ;shou ji kong chang wan qu jun bian mao yi ji ji ta du li ji tong ji yin ,fen xi FlhFzai bian mao ge cheng tong lu nei de xiao ying ,yi ji ji dui gai jun ji ta du li ji tong suo fa hui de xie tong diao jie zuo yong 。yan jiu jie guo xian shi zai ti wai pei yang 20 hshi kong chang wan qu jun de sheng chang zhuang tai liang hao ,da bu fen bian mao ji yin de xiang dui biao da liang jiao gao 。dui bi kong chang wan qu jun ye sheng zhu he flhFtu bian zhu de biao da pu shu ju ,cha yi ji yin ju lei fen xi biao ming FlhFtu bian hou dui kong chang wan qu jun ji yin biao da pu chan sheng le ming xian ying xiang ;yi cha yi bei shu da yu 2,pzhi xiao yu 0.01she wei yu zhi jin hang shai shua ,zong ji huo de 10ge cha yi biao da ji yin ,dui cha yi ji yin jin hang Gene Ontologyhe Pathwayfen xi ,fa xian bian mao ji ji jie dao de yun dong li shi xian zhe shou ying xiang de tong lu 。zai bian mao tong lu zhong jin hang shen ru fen xi fa xian FlhFzai bian mao ji tong zhong fa hui yi chong zong diao jie zi gong neng ,ji tu bian hou 64%de bian mao xiang guan ji yin dou cheng xian xia diao biao da ;gai xiao ying cong bian mao ge cheng zao ji jiu kai shi ti xian ,bing chi xu dao bian mao ge cheng wan ji ;jiang bian mao ji yin an zhao ji gong neng xiang guan xing yi ji zhuai lu ji lian ceng ci jin hang fen lei ,xian zhe xing fen xi jie guo biao ming bian mao si xiang guan ji yin yi ji Ⅲlei bian mao ji yin de zhuai lu xiang jiao ji ta ji yin shi xian zhe xia diao biao da de 。kong chang wan qu jun bian mao ji yin san bu yu ji yin zu zhong ,li yong ji yin biao da pu shu ju ,ben yan jiu fa xian FlhFtu bian xie tong shi gai jun 60%de qu hua ji yin 、51.4%de jia mo duo tang ji yin 、31.8%de zhi gua tang ji yin 、16.7%de tie li zi she qu ji yin 、52%de nian fu qin xi ji yin yi ji 39.2%de ding zhi ji yin cheng xian xia diao biao da ;xian zhe xing fen xi biao ming FlhFdui kong chang wan qu jun ding zhi 、nian fu qin xi he qu hua ji yin biao da de ying xiang yao xian zhe gao yu tie li zi she qu 、zhi gua tang he jia mo duo tang cheng fen 。2.FlhFying xiang kong chang wan qu jun bian mao ge cheng fen zi ji zhi yan jiu ce ding FlhFde xiang dui biao da liang bing guan cha ji ya xi bao ding wei ,fen xi FlhFdan bai de sheng wu xue te xing ;li yong ying guang shi zong ji shu yan jiu FlhFdui ji ta bian mao ya chan wei bao nei ding wei de jie dao neng li ,fen xi kong chang wan qu jun bian mao ge cheng bei zu duan de ji zhi ;tong guo mian yi gong chen dian shi yan jie ge GSTpull-downyan zheng ,shai shua bing fen xi FlhFdan bai zai bao nei ke neng de zuo yong ba dian ji ji hu zuo jie gou yu ;li yong ning jiao qian yi shi yan (Electrophoretic mobility shift assay,EMSA)tan jiu FlhFshi fou ju you zhuai lu diao kong gong neng 。jie guo xian shi FlhFzai kong chang wan qu jun zhong shi yi Ⅰlei bian mao ji yin de xing shi chi xu xing biao da de ,FlhFzhu yao ding wei yu kong chang wan qu jun bian mao chu shi zhao sheng de xi bao ji duan nei mo zu fen zhong ,shui ming ji ke neng zai bian mao ge cheng zao ji fa hui zuo yong 。FlhFtu bian hou FlhB-GFPhe FlgB-GFPjun bu neng zai ding wei yu xi bao ji duan ,biao ming bian mao shu chu ji tong zu zhuang yi ji yun shu gong neng jun shou dao ming xian ying xiang ,kong chang wan qu jun bian mao zu zhuang cong er bei zu duan zai ge cheng zao ji 。hu zuo dan bai shai shua jie guo biao ming FlhFke neng yu bian mao zu zhuang 、qu hua 、xin hao shi bie ke li 、zhuai lu fan yi 、dai xie xiang guan dan bai cun zai gong neng lian ji ,jin yi bu yan zheng fa xian FlhFzai bao nei yu FlhG,CheA,DnaKhe GlyAyou zhi jie xiang hu zuo yong ;FlhFdan bai zi shen ke yi fen wei B、N、Gsan ge jie gou yu ,jie guo xian shi Njie gou yu shi FlhFyu ji ta dan bai hu zuo de jie gou ji chu ;FlhGbei fa xian yu FlhFde Bjie gou yu you zui jiang de jie ge zuo yong ;GlyAyu FlhFer ju ti yi yang tong guo yu ji NGjie gou yu jie ge lai hu zuo ;CheAhe DnaKbei fa xian zhi yu FlhFde Njie gou yu jie ge 。zai bian mao ge cheng san ge ji lian ji tong zhong sui ji shua qu 10ge ji yin de qi dong zi xu lie jin hang EMSAyan zheng ,fa xian FlhFke yi yu diao jie bian mao ji yin zhuai lu de sigmayin zi (σ70he σ28)de qi dong zi xu lie jie ge ,shui ming FlhFke yi zuo wei zhuai lu yin zi zhi jie diao kong bian mao ji yin zhuai lu ,ying xiang Ⅰlei he Ⅲlei bian mao ji yin de biao da 。3.FlhFtu bian dui kong chang wan qu jun du li yi ji zhi fu xie neng li de ying xiang li yong fen zi sheng wu xue fang fa gou jian flhFtu bian zhu yi ji hui fu zhu ,ping jia FlhFtu bian dui kong chang wan qu jun du li yi ji zhi fu xie neng li de ying xiang 。jie guo xian shi yu ye sheng zhu xiang bi ,flhFtu bian zhu zhi neng zai jie chong chu sheng chang ,wan quan shi qu le yun dong li ;ji zheng chang bian mao biao xing cong 94%xia jiang wei 0%,bing ju bu neng zai dian zi xian wei jing shi ye zhong guan cha dao bian mao jie gou 。FlhFtu bian hou kong chang wan qu jun de zi ning ji yi ji sheng wu bei mo xing cheng neng li xian zhe jiang di ;li yong san chong qu hua wu dui jun zhu de qu hua xing jin hang fen xi ,fa xian flhFtu bian zhu zai DL-ping guo suan he αtong wu er suan you dao ti ji zhong de qu hua xing xian zhe di yu ye sheng zhu 。zai dui Caco-2xi bao de nian fu 、qin xi guo cheng zhong ,FlhFtu bian shi de tu bian zhu de xiang dui nian fu 、qin xi lv fen bie xia jiang wei ye sheng zhu de 20.15%he 2.90%;tong guo li xin chu li jin liang pai chu yun dong li ying xiang yin su hou ,ji xiang dui nian fu lv ke hui fu zhi 74.78%,dan dui qin xi neng li de xiu fu que mei you ming xian xiao guo ,reng zhi you ye sheng zhu shui ping de 3.09%;shui ming flhFtu bian zhu dui xi bao de nian fu que xian zhu yao you yun dong li sang shi dao zhi ,er qin xi neng li de jiang di geng ke neng shi you bian mao que shi yin qi 。yi ji he xiao shu fen bie dai biao qin lei he bu ru lei yan jiu le FlhFtu bian dui kong chang wan qu jun ding zhi neng li de ying xiang 。zai gong du hou 1tian ,flhFtu bian zhu zai ji chu jie chang wai de suo you chang dao zu zhi zhong de ding zhi liang jun xian zhe di yu ye sheng zhu ,er zai xiao shu zhong ze wei guan cha dao xian zhe xing cha yi ,shui ming FlhFdui kong chang wan qu jun zai bu tong su zhu zhong jian li chu shi ding zhi de ying xiang bu yi ;zai gong du hou 7tian ,fa xian kong chang wan qu jun ye sheng zhu ke yi zai ji he xiao shu zhong chi xu ding zhi ,er flhFtu bian zhu ze quan bu bei xiao shu qing chu chu ti wai ,zai ji zhong de qing kuang ye hen xiang shi ,flhFtu bian zhu jin zai yi zhi ji de mang chang zhong bei fen li dao ,xi jun zai liang ye zhi you 104CFU(Colony forming units)/g;shui ming FlhFdui yu kong chang wan qu jun zai su zhu ti nei chi xu ding zhi de chong yao xing 。tong guo kong chang wan qu jun gan ran zai tu dong wu mo xing bi jiao le bu tong jun zhu zhi jian de zhi fu xie neng li de cha yi ,jie guo xian shi zai gong du hou 1,2,3tian flhFtu bian zhu gan ran zai tu yin fa de fu xie yan chong cheng du jun di yu ye sheng zhu 。
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