副溶血弧菌毒力相关因子与临床致病机制

作者:陈晓 陈瑜
作者单位:浙江大学医学院附属第一医院检验科 2022-11-10

陈瑜,教授,博士生导师。现任浙江大学医学院附属第一医院检验中心主任,浙江省临床体外诊断技术研究重点实验室主任,浙江大学检验医学研究所所长,传染病诊治国家重点实验室副主任。国家“万人计划”科技创新领军人才。中国医师协会检验医师分会常委兼人工智能与检验医学专业委员会(学组)主任委员,中华医学会检验分会委员,浙江省检验学会主任委员。主要研究方向:感染性疾病发病机制研究及新型实验诊断技术研发。主持国家重大专项、国家973、863以及国家自然基金等课题23项,获国家科技进步一等奖、中华医学科技奖一等奖、浙江省自然科学奖一等奖、教育部技术发明二等奖等8项。在国际顶级期刊《柳叶刀》等发表SCI论文150余篇,获国家发明专利16项,主编参编专著23部。


陈晓,主任技师,长期从事病原体变异变迁规律研究;专业擅长感染性疾病病原体鉴定和耐药检测。主持国家科技重大专项和国家自然科学基金等多项课题;发表SCI文章20余篇,获国家发明专利授权4项,获教育部科学技术发明二等奖,参编《全国临床检验操作规程》等专著。


【摘要】副溶血弧菌已成为全球沿海国家食源性疾病和感染性腹泻的最主要病原菌,主要引起散发或聚集性暴发的急性胃肠炎,在免疫力低下人群可进展为败血症和坏死性筋膜炎,严重者甚至死亡。副溶血弧菌的致病性是多种毒力因子共同作用的结果,本文对副溶血弧菌的溶血毒素、Ⅲ型分泌系统、Ⅵ型分泌系统、摄铁系统、粘附因子和蛋白酶等相关毒力因子的研究现状进行综述,旨在为副溶血弧菌的防控和临床治疗提供理论支撑。


【关键词】副溶血弧菌;毒力因子;致病机制


副溶血弧菌(Vibrio parahaemolyticus,VP)是一种革兰阴性嗜盐弧菌,广泛分布于海洋、盐湖及鱼虾贝类等海产品中。副溶血弧菌可导致水产养殖动物疾病,给水产养殖业造成巨大的经济损失。人类主要通过海水暴露、生食或半生食海产品而感染,通常引起散发或聚集性暴发的急性胃肠炎,也可引起伤口感染和败血症,严重者甚至死亡。随着全球气候变暖,海水温度上升,以及人类活动的日益频繁,副溶血弧菌引起的食源性疾病已逐渐成为世界范围的公共卫生问题[1-3]。因此,对副溶血弧菌的研究受到越来越多的重视。


副溶血弧菌感染包括粘附、侵袭、增殖和释放毒素等过程。这些过程与副溶血弧菌产生的多种毒力相关因子密切相关,包括溶血毒素、分泌系统、粘附因子、摄铁系统、脂多糖和蛋白酶等[4]。随着生物化学、测序、基因组学和蛋白组学技术的不断发展,以及合适动物模型的建立,人们对副溶血弧菌的毒力因子和致病机制有了更深入认识。为系统了解副溶血弧菌致病机制的研究现状,本文对其主要毒力相关因子进行论述,旨在为副溶血弧菌的防控和临床治疗提供理论支撑。


一、溶血毒素(Helmolysin)


溶血素是一种能引起红细胞破裂并释放出血红蛋白的外毒素。副溶血弧菌可以产生三种不同的溶血素,分别是耐热直接溶血素(thermostable direct hemolysin,TDH)、耐热直接溶血素相关溶血素(thermostable direct hemolysin-related hemolysin,TRH)和不耐热溶血素(Thermolabile hemolisin,TLH),分别由tdh、trh和tlh编码[4]。分子流行病学研究显示,大部分临床分离株携带tdh和/或trh,而环境分离株较少携带[5-7]。TDH和TRH被认为是副溶血弧菌的主要致病因子。


TDH是一种耐热的成孔毒素,由156个氨基酸组成,分子量为46kD,在溶液中以四聚体形式存在[8-9]。TDH具有溶血、细胞毒性和肠毒性等生物学活性。TDH与红细胞膜上GT1神经节苷脂受体相结合,在磷脂双分子层中形成跨膜孔,细胞膜外的水和离子自由进入红细胞,导致红细胞的膨胀和破裂[10-11]。TDH可以从细胞外部和内部诱导其细胞毒性,并通过细胞凋亡杀死细胞[12-13]。高浓度TDH使肠上皮细胞钙离子浓度升高,激活细胞膜上CaCC通道,导致肠细胞内氯离子分泌增加,引起水样泻[14-16]。跨膜调节蛋白ToxR和ToxS[13, 17]、转录调节蛋白VtrA和VtrB[18],可促进TDH的表达;而转录调节因子CalR[19]和Hfq[20]抑制tdh转录和表达。此外,TDH表达受环境因素的影响,如甘露醇、高浓度氯化钠、胆汁酸和氨基酸可以促进TDH的产生[21]。


TRH由189个氨基酸组成,分子量为48kDa,60℃加热10min即可灭活,是一种热不稳定毒素。TRH由trh1和trh2两个基因编码,两者的相似度为84%。TRH和TDH的氨基酸序列具有67%的相似度,两者具有相似的生物学活性[22-24]。TLH是一种非典型磷脂酶,它只能在卵磷脂存在的情况下发挥溶血作用[25]。模拟人体肠道的环境时,tlh的表达量大幅度增加,其功能及致病机制尚不清楚[26]。TLH在临床和环境分离菌株中都普遍存在,且高度保守,可用于副溶血弧菌分子鉴定的靶基因。


二、Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)


T3SS由30-40kb大小的基因区域编码,通常以毒力岛(pathogenicity island,PAI)的形式存在于细菌的质粒或染色体。T3SS是由结构蛋白、转运蛋白、效应蛋白和分子伴侣等三十多种蛋白质共同组成的类似于“注射器”的跨膜装置,主要由横跨细菌内和外膜的基体、聚合并延伸到细胞外的针状结构以及激活T3SS活性的顶端结构组成[4, 27]。T3SS能有效将细菌效应蛋白注射到宿主细胞中,操纵细胞的多种信号转导通路,从而破坏宿主细胞正常功能[28]。


副溶血弧菌有2套T3SS,分别位于两条大小不一的染色体上,即T3SS1和T3SS2。T3SS1存在于所有副溶血弧菌,基因簇包含42个基因,与耶尔森菌T3SS的相似度最高;其主要功能是引起细胞毒性、影响生物膜的形成和运动性,有助于副溶血性弧菌在环境中的生存[29-30]。T3SS1表达主要受相互作用的ExsA、ExsC、ExsD和ExsE蛋白组成的级联调节系统调控,其中ExsA属于AraC转录激活因子家族的成员,是激活T3SS1基因转录的主要调控因子[31]。转录调节因子HlyU和核酸相关DNA结合蛋白H-NS可以通过调节exsA的表达来调节 T3SS1[31-32]。此外,跨膜调节蛋白ToxR和调节因子CalR协同负向调控T3SS1部分基因的表达[33]。改变外界环境,如合适的培养温度(30℃)、提高Mg2+浓度和降低Ca2+浓度,可以促进T3SS1表达,但其分子机制尚不清楚[34]。宿主产生的亚硝酸盐通过半胱氨酸86位点的S-亚硝基化激活组氨酸激酶传感器VbrK,抑制其正调节子exsC,导致T3SS1操纵子的下调[35]。


目前,T3SS1已鉴定四种效应蛋白,分别为VopQ、VopS、VPA0450和VopR。VopQ是T3SS1介导对真核细胞发挥毒性最重要的蛋白。研究表明,VopQ能与溶酶体膜上的V-ATPase结合,改变溶酶体中的质子浓度,促进溶酶体裂解和自噬囊泡形成,导致宿主细胞自噬和细胞毒性[36]。VopQ还可以通过激活MAPKs信号通路中的p38、JNK和ERK,促进IL-8分泌[37]。VopS是一种翻译后调节蛋白,其活性主要依赖于C端的Fic结构域,可介导Rho GTP酶(RhoA、Rac1和Cdc42)的腺苷酸化,导致其无法与下游底物结合,阻断肌动蛋白细胞骨架的信号级联,导致细胞骨架塌陷和细胞变圆[38-39]。VPA0450是一种肌醇磷酸酶,能水解质膜上4,5-二磷酸磷脂酰肌醇的磷酸基团,破坏肌动蛋白细胞骨架和质膜之间的连接,导致质膜内气泡的形成、细胞的变圆和溶解[40]。VopR通过N端磷酸肌醇结构域与宿主细胞膜上的磷酸肌醇结合,可促进其它T3SS效应蛋白进入宿主细胞后的正确折叠,该蛋白的功能有待进一步阐明[41]。


T3SS2基因簇位于副溶血弧菌80kb致病岛内,仅存在于具有神奈川现象(KP+)的临床分离株,这与其它细菌T3SS的相似度较低,T3SS2进化为2个分枝,即T3SS2α和T3SS2β;其功能是参与细菌的定植及细胞炎症的负调控反应,有利于宿主体内病原菌的免疫逃避过程[29-30]。T3SS2的表达受VtrA-VtrB调节级联反应的调控。VtrA通过与其上游区域结合正向调节vtrB转录,而VtrB可激活T3SS2基因的转录表达[42-43]。ToxR在 T3SS2基因表达的调节中也发挥作用,ToxR可能通过增强VtrA能力或与VtrA之间的相互作用,导致 VtrB表达的激活并进一步增加T3SS2的表达[44]。T3SS2的转录调节机制目前尚未完全揭示。


目前已发现T3SS2的8个效应蛋白,包括VopA、VopT、VopZ、VopC、VopL、VopO、VopV和VPA1380。VopA、VopT和VopZ通过抑制MAPKs信号通路的不同成分来抑制免疫反应。VopC、VopL、VopV和VopO直接或间接的以肌动蛋白细胞骨架为作用靶点。VopA属于耶尔森氏菌外蛋白(YopJ)家族成员,是一种乙酰基转移酶,通过乙酰化修饰MAPK激酶催化环中的丝氨酸/苏氨酸或赖氨酸残基,阻断磷酸化位点或影响ATP与磷酸化位点结合,切断激酶的磷酸化,从而抑制MAPKs信号传导[45-46]。新近研究发现VopA蛋白可以抑制肠上皮细胞的正常迁移和凋亡,增强副溶血性弧菌对肠上皮细胞的粘附,从而促进细菌的增殖和感染[47]。VopT与铜绿假单胞菌中的2种效应蛋白ExoT和ExoS具有相似的ADP-核糖基转移酶结构域,能够ADP核糖基化修饰低分子量G蛋白Ras,从而调控MAPKs信号途径[48]。VopZ是副溶血弧菌肠道致病所必需的效应蛋白,它抑制TGF-β活化激酶1(TAK1)的激活,进而抑制下游MAPKs和NF-κB信号通路。敲除VopZ的38~62位氨基酸序列的副溶血性弧菌突变株,可减少TAK1失活和肠道液体积聚[49]。VopC含有与大肠杆菌的细胞毒性坏死因子(CNF)和博德特氏菌属的皮肤坏死毒素(DNT)相似的催化结构域,具有脱酰胺酶/转谷氨酰胺酶活性。通过脱酰胺作用,激活Rac1和Cdc42,导致肌动蛋白细胞骨架的变化,从而促进副溶血弧菌入侵到非吞噬的宿主细胞[50-51]。VopL是肌动蛋白成核剂,通过N-端3个Wiskott-Aldrich同源区2结构域和独特的C-端结构域使肌动蛋白丝成核。VopL还可以中和宿主细胞中由烟酰胺二核苷酸磷酸(NADPH)氧化酶复合物产生的活性氧(ROS),以促进细菌在宿主细胞中的存活[52-53]。VopO与ras同源基因家族成员A(RhoA)的鸟嘌呤核苷酸交换因子 H1(GEF-H1)结合,GEF-H1从微管中解离,激活下游RhoA-Rho相关激酶(ROCK)通路,导致肌动蛋白应力纤维的形成,从而破坏肠上皮屏障[54]。VopV是一个关键的效应蛋白,是副溶血弧菌肠道定植以及乳兔模型中肠道积液所必需的因子。VopV通过其C-端结构域和长重复序列,与F-肌动蛋白和和细丝蛋白结合,导致F肌动蛋白的堆积,从而引起肠毒性[55]。然而,VopV与肌动蛋白或细丝蛋白的结合如何导致腹泻仍有待阐明。VPA1380与多种细菌毒素中的半胱氨酸蛋白酶结构域(cysteine protease domain,CPD)高度相似,与其他的细菌毒素的CPD一样,VPA1380在酵母中表达时需要肌醇磷酸己糖(IP6)作为激活剂来介导细胞毒性[56]。但是,VPA1380在宿主细胞中的具体功能尚不明确。目前,T3SS基因簇中仍有大量基因的功能未被鉴定;且已知的效应蛋白是否存在相互作用以及它们是如何协调发挥作用均不清楚。


三、Ⅵ型分泌系统(Type 6 secretion system, T6SS)


T6SS在结构和功能上与T3SS相似,主要通过针尖样分泌装置将细菌效应蛋白注射入宿主细胞而发挥生物学效应。副溶血弧菌含有两套T6SS,即T6SS1和T6SS2。T6SS1主要存在于临床分离株,具有细胞粘附和抑菌活性,与环境适应性相关;T6SS2存在于所有副溶血弧菌中,具有细胞粘附活性,可能与细菌毒力相关[57-58]。T6SS相关基因的转录受群体感应(quorum sensing,QS)系统的调节[59]。OpaR和AphA是QS的核心调节因子,分别在高细菌密度和低细菌密度下发挥调控作用。低密度时,AphA激活,从而抑制T6SS1和T6SS2相关基因的转录;高密度时,OpaR激活,抑制T6SS1的转录,但能正调节T6SS2相关基因的表达[60-61]。核酸结合蛋白H-NS对T6SS1和T6SS2相关基因的表达均具有抑制作用[62-63]。CalR是H-NS的拮抗因子,可通过竞争H-NS与靶基因启动子区域的结合位点来激活T6SS2基因的转录[64]。目前对副溶血弧菌T6SSs的研究仍处于起始阶段。


四、粘附因子(Adhesion factors)


病原菌粘附是感染的首要条件。副溶血弧菌与粘附相关的主要因子有血凝素、烯醇化酶、MAM-7和T6SS2等。甘露糖敏感血凝素(Mannose-sensitive hemagglutinin,MSHA)对多糖(如唾液酸和GM1神经节苷脂)具有高亲和力,促进副溶血弧菌在肠上皮细胞的定植[65]。烯醇化酶是一种与纤溶酶原结合的表面暴露蛋白,可以促进细菌对Hep-2细胞的粘附[66]。粘附因子MAM-7可以与宿主细胞膜上的纤连蛋白和膜磷脂酸结合组装成三分子聚合物介导细菌的粘附,抑制MAM-7表达可以降低副溶血性弧菌的粘附性和毒力[67]。CalR可以通过调节T6SS2的表达来调节副溶血性弧菌的粘附[64]。RpoN是一种调控鞭毛合成的调节蛋白,可能影响副溶血性弧菌的定植[68]。对细菌粘附机制的深入了解,可以为控制副溶血性弧菌在宿主中的定植和中断感染提供新的视角。


五、铁摄取系统(Iron uptake system)


金属离子在副溶血性弧菌基因表达的调控中起重要作用。高钙和低铁生长条件刺激细菌群体行为和T3SS转录[69]。致病性弧菌获取铁的途径主要有两种:一是产生外毒素破坏红细胞,释放血红蛋白;另一种是生产低分子铁螯合剂,对血红素中的铁离子具有高亲和力[70-71]。弧菌铁蛋白是由副溶血弧菌产生的一种能够螯合铁离子形成铁-铁载体复合物的蛋白,该复合物能够与病原菌细胞外膜的铁载体受体PvuA1和PvuA2结合,再与内膜的ABC-PvuBCDE转运系统结合,将铁-弧菌素转运至胞内,进行铁同化[72-73]。TonB系统在整个铁转运过程中提供能量。副溶血性弧菌可分泌一种铁还原蛋白,将铁离子还原为二价铁;还可以通过分泌蛋白酶来降解血红蛋白,从人体中获取铁[74]。此外,一些外膜蛋白受体(如肠杆菌素受体VctA、IrgA,血红素受体HutA、HutR)对铁的获取很重要。


六、蛋白酶和脂多糖等其它毒力因子


分泌到细胞外的一些蛋白酶与副溶血性弧菌的发病机制密切相关。Lee等人从tdh- trh- 副溶血性弧菌临床分离株中获得了一种蛋白酶A,该蛋白对细胞有明显的毒性作用,对红细胞有裂解活性,还可导致组织溶血[75]。此外,PrtA是一种丝氨酸蛋白酶,发挥溶血活性和细胞毒性[76]。脂多糖是细菌内毒素的主要物质。副溶血性弧菌脂多糖对鼠腹膜巨噬细胞有影响,增加脂多糖剂量,腹腔巨噬细胞的RNA含量和溶酶体活性显著增加[77]。弧菌外膜蛋白位于外膜表面,可以与细胞外部广泛相互作用,既是重要的毒力因子也是免疫原。


七、小结与展望


副溶血性弧菌具有多种毒力因子,主要表现为溶血活性、细胞毒性和肠道毒性,导致宿主胃肠炎、败血症甚至死亡。副溶血性弧菌的致病性是多种毒力因子共同作用的结果,需要综合考虑各种毒力因子的相互作用和调控网络。加强外界环境信号调节毒力因子表达的机制研究,将促进我们对副溶血性弧菌致病性的了解。此外,临床工作中时常分离到不携带已知毒力因子的副溶血弧菌,这提示有可能存在着新的毒力因子,值得进一步研究。深入探索副溶血弧菌的致病机理,将有助于预防和治疗副溶血性弧菌引起的感染。


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