军事科学院军事医学研究院 微生物流行病研究所 病原微生物生物安全国家重点实验室，北京 100071
收稿日期：2017-12-28;接收日期：2018-03-16 基金项目：国家重点基础研究发展计划(973计划) (No. 2015CB554202)资助
作者简介：王慧??军事医学研究院微生物流行病研究所研究员，博士生导师。主要从事病原微生物进化、致病机理与防治基础研究，先后主持和承担国家传染病重大专项、国家新药创制重大专项、国家973计划、国家重点研发计划、国家自然科学基金课题20余项。在高致病病原新毒力因子发现、病原菌适应性进化以及感染防治技术方面取得重要进展。研究成果在J Infect Dis、JCI Insight、Sci Reports、Arch Toxicol、Vaccine、Cancer Immuno Immunother等国内外学术期刊发表论文80余篇。获得中国发明专利17项，获得美国、日本、澳大利亚、欧洲等国际发明专利5项。现兼任全军科学技术委员会生物技术专业委员会常委
摘要：毒素-抗毒素(Toxin-Antitoxin，TA)系统广泛存在于原核生物和古细菌的染色体和质粒中。此系统由2个共表达的基因组成，分别编码稳定的毒素蛋白和易降解的抗毒素，毒素通常发挥毒性作用抑制细菌生长，而抗毒素则可中和毒性，二者相互作用对细菌生长状态起精密调节作用。根据TA的组成和抗毒素的性质，目前已经发现有6型TA，这些TA系统在细菌中发挥的作用一直是近年来****们研究的热点，文中对细菌TA的功能研究进展进行了综述。
关键词：毒素-抗毒素系统 环境适应性 持留态细胞 生物被膜 细菌毒力
Functions of bacterial Toxin-Antitoxin systems
Zhili He, Hui Wang
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100071, China
Received: December 28, 2017; Accepted: March 16, 2018
Supported by: National Basic Research Program of China (973 Program) (No. 2015CB554202)
Abstract: TA (Toxin-Antitoxin) systems are widely spread in chromosomes and plasmids of bacteria and archaea. These systems consist of two co-expression genes, encoding stable toxin and sensitive antitoxin, respectively. The toxicity of toxins usually inhibits bacterial growth and antitoxins can neutralize the toxins. Interaction between them would regulate the growth state of bacteria precisely. According to the composition of TA and nature of antitoxin, six types of TA have been found. The role of these TA systems in bacteria has been a hot research topic in recent years. Now, the research status on functions of bacterial TA is reviewed.
Keywords: Toxin-Antitoxin system environmental adaptability persisters biofilm bacterial virulence
毒素-抗毒素系统最初是作为质粒稳定分子被发现的[1]，它们广泛分布在原核生物和古细菌中。TA操纵子编码出一个稳定的毒素和一个易被降解的抗毒素，其中毒素通常为蛋白质如RelE，而抗毒素可以是蛋白质也可以是RNA。随着进一步研究，越来越多染色体TA分子也被发现[2-3]，目前认为染色体TA与维持质粒稳定性无关，而主要通过调节代谢和生长状况来对抗不利环境[4]。质粒TA分子被认为只有唯一功能——执行post- segregational killing (PSK)，杀死不继承父辈TA质粒的子代细胞。但是有研究证明质粒编码的一对TA中，毒素ccdF是一种可持续传代基因[5]。染色体TA分子的功能则表现出多样性，涉及到调节整体水平对抗不利外界环境[6]、参与细胞程序性死亡[7]、对抗质粒编码TA介导的PSK[8]、参与抗生素下持留态细胞的形成[10]、参与生物被膜形成[11]、提高菌株存活率和毒力[12]、对抗噬菌体以及参与菌株定植[13]等多个方向。文中着重于阐述TA在影响细菌环境适应性方向的研究进展，以及其探索性应用与展望。
1 TA的分类在过去的30年里根据抗毒素的性质和TA的组成，总共发现六型TA (TypeⅠ至Type Ⅵ)[14]，如图 1所示。
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| 图 1 六型TA作用模式 Figure 1 Mode of action of the six classes of Toxin-Antitoxin systems. |
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1.1 Ⅰ型TA在这种类型的TA系统，抗毒素是一种sRNA，毒素是蛋白质。抗毒素sRNA能结合毒素的mRNA促进其降解，阻碍毒素翻译[3]。准确地说，Ⅰ型毒素是拥有丰富疏水蛋白的小蛋白(超过60个氨基酸)[15]，而抗毒素编码的RNA是毒素的反义链。对于Ⅰ型TA胞内靶点我们所知甚少，而现在被知晓较多的Ⅰ型TA是hok/sok (hok为毒素，sok为抗毒素)，有趣的是在这个TA系统中被发现存在第3种蛋白mok能协助毒素翻译[16]。在革兰氏阴性菌中鉴定的第一种Ⅰ型TA fst/RNAII也被证明与hok/sok有同样特点，而fst/RNAII陆续在腐生葡萄球菌质粒上、干酪乳杆菌和单核细胞增生李斯特氏菌中被发现。其他Ⅰ型TA，例如大肠杆菌含有ldr/Rdl、tisB/IstR1、bs/Sib、shoB/OhsC和symE/SymR[17-18]，枯草芽孢杆菌含有染色体Ⅰ型TA txpA/RatA[19]。
1.2 Ⅱ型TAⅡ型TA是现在研究最广泛的TA类型，近期更新的毒素-抗毒素数据库TADB 2.0中就收录了105对有实验数据支持的Ⅱ型TA基因座位，还提供了在线预测工具TA finder[9]。Ⅱ型TA中毒素和抗毒素都是蛋白质，相对于稳定的毒素来说，抗毒素更易被降解表现出不稳定性，因为抗毒素含有较少的有序结构，对蛋白水解酶更为敏感[20]。通常情况下，Ⅱ型TA有着其他TA类型没有的特征：1)编码抗毒素蛋白的基因位于毒素蛋白基因上游；2)两个基因共转录；3)两个基因共翻译。在正常生长状况下，毒素和抗毒素以TA复合物状态存在，确保毒素不发挥作用使细胞正常生长。抗毒素会结合在TA操纵子上游覆盖启动子来抑制操纵子表达，而毒素作为辅助抑制剂与抗毒素结合加强这种抑制作用。所以胞内TA复合物的水平受到抗毒素和TA复合物共同调控[10]。一定压力条件下抗毒素被蛋白酶降解后会释放出稳定的毒素蛋白，同时操纵子的抑制解除，持续表达出的抗毒素被继续降解，以上造成的结果就是毒素的积累，毒性效应启动[21]。Ⅱ型TA毒素长度大约在100个氨基酸[22]，分为12个家族，每个家族的胞内靶点不同，发挥不同的生理作用。van Melderen等证明Ⅱ型TA能确保移动遗传元件的安全稳定维护[3]，Engelberg-Kulka等提出了大肠杆菌中mazEF会激活细胞程序性死亡，这样的利他性死亡能帮助细胞压力环境下应对有限的营养物质[23]。大肠杆菌中的hipAB会诱导持留态细胞形成[24-25]来对抗抗生素压力，因为绝大多数抗生素都对生长状态的细菌有杀灭作用，而对处于半休眠的持留态细胞束手无策。MqsAR被证明与生物被膜形成相关[26]，TA的环境适应性一直是科学家关注的重点。而最近Ⅱ型TA与菌株致病性的关系引起了科学家们关注，相关研究也越来越多。
1.3 Ⅲ型TAⅢ型TA系统最初是作为抵抗噬菌体感染被发现的[27]，抗毒素是RNA，能直接作用于毒素蛋白[28]。鉴定出的第一对Ⅲ型TA分子是ToxIN (毒素ToxN，抗毒素ToxI)，它是在腐败果胶杆菌的质粒上被发现的，核糖核酸内切酶是ToxN的靶点。ToxN同源基因在革兰氏阴性菌和革兰氏阳性菌的染色体和质粒上都有发现，且广泛分布于人和动物致病菌、海洋和土壤微生物中[27]。后来发现的TenpI-TenpN和CptI-CptN也参与抵抗噬菌体感染[29]。
1.4 Ⅳ、Ⅴ、Ⅵ型TAⅣ型TA系统中，毒素和抗毒素都是蛋白，但是毒素不是直接与抗毒素形成复合物，而是阻碍毒素作用靶点来抑制毒性效应。例如yeeUV，抗毒素YeeU促进细胞骨架蛋白MerB和FtsZ聚合，让其免受毒素YeeV的抑制作用[30]。唯一报道的Ⅴ型TA是ghoST，毒素蛋白GhoT是一个小的疏水性肽，其表达可导致细胞死亡和持留细菌感染，抗毒素蛋白GhoS不像传统的抗毒素那样在压力条件下不稳定，也不和毒素蛋白的DNA结合抑制转录，而是作为一种序列特异性的核酸内切酶，切割ghoT mRNA，阻止其翻译[31]。后来发现ghoST的表达还受另一对TA msqAR的调控[32]，提示着TA家族之间的相互作用呈现多样性。socAB作为新发现的一类TA系统——Ⅵ型TA出现在我们的视野里[33]，抗毒素SocA作为毒素蛋白SocB的适配器与其连接在一起，与Ⅱ型TA不同的是，SocA并不直接中和SocB的毒性，而是通过特定的蛋白酶促进它的降解。
2 TA的环境适应性TA广泛分布在原核生物和古细菌中，无论在宿主体外还是体内，这些含有TA的原核生物都面临各种各样的生存压力和排斥威胁，那么TA是怎样帮助适应恶劣环境、保证种族延续的呢？
2.1 TA影响致病菌的毒力细菌病原体要承受多重压力，应对宿主定植过程中不断变化的环境，才能成功地感染宿主[34]。在从外部非宿主环境到进入宿主体内的过程中，入侵的微生物要处理多个宿主防御系统，例如肠道病原体在胃内遇到酸性pH值[35]、与肠道菌群的竞争、肠道内其他重要的宿主防御因子抗菌肽(cAMP)和肠细胞分泌的免疫球蛋白[36]。而近几年越来越多的研究证明TA系统参与细菌病原体感染宿主的过程，提高其致病力。2011年Georgiades等意外发现基因组中的TA分子模型与细菌毒力之间存在密切联系[37]。在许多致病菌中发现TA分子广泛分布于可移动遗传元件上，作为毒力岛参与耐药与毒力作用[38]。TA分子通过稳定与维持编码毒力质粒来参与致病菌毒力作用，例如富氏志贺菌中mvpA-mvpt (VapBC)能维持毒力质粒Pmysh 6000的稳定[39]，HigBA帮助普通变形杆菌毒力质粒稳定传代[40]。在不分型流感嗜血杆菌中敲除VapBC后极大减弱了组织毒力损伤，以及动物模型上中耳炎发病率[41]。2012年TA分子ybaJ-hha和yefM-yoeB被证明参与膀胱定植，pasTI参与肾脏定植[42]。次年，Walker等发现sehAB TA分子的缺失能影响老鼠口服途径鼠伤寒沙门氏菌的毒力，但不影响腹腔注射途径感染的毒力，提示sehAB TA分子在鼠伤寒沙门氏菌感染过程的早期阶段发挥作用[43]。Pinel等通过研究sprG1/sprF1 TA分子提出TA模型中的分泌毒素能增强金黄色葡萄球菌的毒力[44]。Ⅱ型TA mazEFSa早年也被认为与金黄色葡萄球菌致病力相关。MazFSa毒素能识别特定的核苷酸序列，参与毒力蛋白SraP的表达调控，同时促进致病菌与宿主细胞的黏附作用[45]。最近报道钩端螺旋体属中的ChpK和MazF毒素能进入宿主细胞质中杀死细胞，同时△hpIK和△mazEF缺失突变株感染显示感染后期的巨噬细胞中细胞坏死数减少[46]。虽然TA分子调控细菌毒力的具体机制还未完全解释清楚，但TA系统的毒素作用已经被科学家们认可。
2.2 TA参与持留态细胞和生物被膜的形成“持留态细胞”是指能够在高浓度的抗生素中生存的细菌。这种现象在生长平台期的菌中更为常见[47]。持留态细胞能使细菌处于半休眠状态，细菌低转录低翻译水平使大部分抗生素对其束手无策。第一对被发现参与持留形成的是E. coli K-12中的TA对hipBA。氨苄青霉素处理下野生株呈现10?5?10?6的持留率，而双突变的hipA菌株(hipA7，能释放HipA毒性)呈现出10?2持留率[48]。在野生株中过量表达HipA毒素也表现出和hipA7相同的持留率[49]。HipA毒素能磷酸化Ser239，导致未修饰的tRNA Glu积累，激活和释放RelA (五磷酸合成酶pppGpp信使)，使pppGpp升高，增加持留水平[50-51]。冗余TA可以增加持留态细胞形成的频率，而随机波动的频率可以自发地打开TA引起细胞正常和持久的双稳态[52]。TA对与生物被膜的关系现今也被广泛关注，有****证明TA基因敲除后能降低生物被膜的形成[10, 25, 53]，同时又有人提出TA分子对生物被膜形成起正向调控作用[54]，而具体机制我们还不得而知。
2.3 TA系统调控GSR细菌应激反应(General stress response, GSR)作为一种可逆状态能帮助细胞在营养匮乏、高氧、强酸和其他不同压力环境下长时间存活。革兰氏阴性菌中rpoS编码的σs是GSR最关键的调控因子。因此生物膜休眠增强和持留细胞生长率显著降低，是生物膜抗生素敏感性降低的主要原因。由于生物被膜的形成，GSR或许直接参与了慢性感染过程。事实证明GSR信号通路确实是在囊性纤维化慢性感染过程中被激活[55]。TA系统中的抗毒素MqsA，能直接抑制压力调节关键蛋白RpoS的表达[56]，阻碍细胞的GSR。MqsA能识别rpoS和csgD操纵子上与mqsRA类似的回文序列[57]并与之结合，而在rpoS操纵子上删除这样的回文序列会使MqsA蛋白无法结合[56]。在高氧压力下，MqsA被蛋白酶Lon降解，导致rpoS去抑制，细胞GSR启动，反向说明了MqsA阻碍细胞的GSR。这是第一个明确的外部压力如何影响基因调控机制，并为TA系统创造了一个重要的新角色。
2.4 其他压力相关TA系统至今为止发现E. coli中至少存在6种RNA酶(MqsR，MazF，RelE，ChpB，YafQ，YoeB)降解不同mRNAs来应对不同压力。这种假设是根据：1)不同压力下激活的TA系统不同，例如YafNO、HigBA、MqsRA TA分子在氨基酸匮乏下被激活，YafNO、HigBA、MqsRA分子在氯霉素(30 μg/mL)处理下激活[58]，YafNO、YafQ/DinJ[59]、TisB/IstR-1[60]、SymE/SymR[61]等分子则参与抗丝裂霉素C和SOS应激，而TisB/IstR-1在环丙沙星(0.1 μg/mL)压力下可被激活[60]。2) MqsR、MazF、YafQ和ChpB分别酶切mRNA GCU[62]、ACA[63]、AAA[59]与ACY (Y=A或者G)位点，它们调控不同的基因功能。此外，有报道证明虽然MazF在抗生素压力下会降解大部分mRNAs，但是未被降解的mRNAs即使含有ACA酶切位点也会编码出特殊蛋白，这些蛋白一部分发挥毒素效应，一部分用来协助细胞生存[64]。
3 TA的应用与展望3.1 作为抗菌药TA广泛存在于原核细胞和古细菌中而不存在于真核细胞中，毒素的利用就能让TA成为很好的抗菌靶点。Ⅱ型TA系统似乎是人工激活毒素复合物最可行的目标。最直接的方法就是破坏或是阻碍TA复合体的形成，或促进抗毒素的降解来释放毒素。然而释放毒素可能会诱导持留态细胞和生物被膜的形成，所以目的TA需谨慎筛选。据报道已有几种肽类能在体外诱导TA系统[65-67]。例如胞外死亡因子EDF (Extracellular death factor)能在胞外激活MazF诱导大肠杆菌死亡[68-71]。EDF是一种NNWNN五肽，通过ClpXP依赖途径从6-磷酸葡萄糖脱氢酶上水解下来[69]并分泌到培养基中。将处于对数生长期的大肠杆菌在富含EDF悬浮物的培养基中培养，或者促进EDF合成，都可以激活MazF[68]和ChpBK[70]，诱导细胞死亡，这种现象能分别被其抗毒素MazE和ChpBI抑制。因此EDF及其同源肽类为新一类抗菌药的研制提供了重要的研究线索。
3.2 作为抗病毒药TA系统另一潜在价值被发现——抗病毒感染。最近含有E. coli mazF基因的逆转录载体构建成功，而mazF是被安插在HIV-1启动子TAR下游并受其调控。HIV感染循环是以病毒Tat蛋白表达开始的，Tat结合TAR序列诱导整个HIV-1基因组转录。被感染的CD4+细胞含有TAR-mazF结构，HIV-1侵入后表达Tat蛋白结合TAR序列后诱导MazF表达，MazF发挥毒性酶切病毒mRNA，阻止HIV-1繁殖，实验在体外进行[72]。另一项研究则在丙型肝炎病毒HCV (Hepatitis C virus)领域中同样证明MazF具有抗病毒潜力。一种多肽链MazF+linker+MazEp (MazE片段)构建成功，其中MazF和MazEp是通过含有NS3酶切位点的延伸链连接在一起的，而NS3是一种病毒丝氨酸蛋白酶，是HCV多聚蛋白质必需的加工酶。含有mazF-linker-mazEp结构的细胞可以正常生长，因为MazF的毒性处于被MazEp抑制状态。当细胞被HCV感染后病毒产生的NS3蛋白酶切断mazF-linker-mazEp中间的linker，MazF毒性释放，阻碍蛋白质合成，根除细胞感染[73]。这种治疗策略也适用于同样依赖蛋白酶的其他病毒，例如HIV。
3.3 作为抗肿瘤药2003年有****提出来自质粒R1的Kid毒素能用来研发抗癌复合物[74]，其中一种方法就是利用肿瘤特定启动子直接打开毒素基因的表达，从而作用于癌细胞[75]。最近，小鼠通过基因工程方法，接入MazF毒素基因后实体瘤发生逆转，然而可能是由于毒素的功能丧失，成功逆转率仅为50%左右[76]。这些发现为抗肿瘤探索路上打开了一扇新的大门。
然而将毒素作为抗菌或抗病毒药物时，我们不得不考虑，既然毒素能被直接作用于患者体内杀死病原体，那么人体内的共生菌群和正常细胞是否也会收到毒素的攻击。或许在真核生物中研制抗菌药物和抗病毒药物时，MazF等毒素的活性和剂量需受到严格把控。
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