生物工程学报  2017, Vol. 33 Issue (9): 1533-1546
http://dx.doi.org/10.13345/j.cjb.170052
中国科学院微生物研究所、中国微生物学会主办
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文章信息

王全民, 马遥, 刘丽钧, 朱军, 刘智
Quanmin Wang, Yao Ma, Lijun Liu, Jun Zhu, Zhi Liu
霍乱弧菌生物被膜发育与环境调控
Biofilm development and environmental determinants in Vibrio cholerae
生物工程学报, 2017, 33(9): 1533-1546
Chinese Journal of Biotechnology, 2017, 33(9): 1533-1546
10.13345/j.cjb.170052

文章历史

Received: February 11, 2017
Accepted: May 2, 2017
霍乱弧菌生物被膜发育与环境调控
王全民, 马遥, 刘丽钧, 朱军, 刘智     
华中科技大学 生命科学与技术学院, 湖北 武汉 430074
收稿日期:2017-02-11; 接收日期:2017-05-02; 网络出版时间:2017-05-17
基金项目:国家自然科学基金(No.81572050)资助
作者简介:刘智 华中科技大学生命科学与技术学院教授, 博士生导师, 中组部"青年千人计划"获得者(第11批)。南京农业大学微生物学系博士, 华中科技大学生物医学工程博士后, 美国宾夕法尼亚大学医学院微生物学系研究助理。研究方向为肠道环境中微生物与宿主相互作用。先后在Proc Natl Acad Sci USA、Nucleic Acids Res、mBoi等国际权威期刊发表SCI论文30多篇, 其中Proc Natl Acad Sci USA 4篇。曾获"国家科学技术进步奖二等"、湖北省自然科学奖和江苏省科学进步奖等, 同时多篇论文多次受到同行专家的专题推荐。回国后在Cell Rep、Mol Microbiol、Scientific Rep等刊物上发表多篇研究论文, 获得国家自然科学基金面上项目及"973"课题资助
摘要:生物被膜状态的霍乱弧菌具有极强的环境适应性和超高的感染性,生物被膜的发育调控研究对霍乱弧菌的宿主感染和环境适应非常重要。本文综述了近年来霍乱弧菌生物被膜研究结果,包括霍乱弧菌生物被膜的组成、发育和环境调控,尤其着重阐述了各种环境因子对霍乱弧菌生物被膜发育的影响,包括细菌自体信号分子、自然环境因子和宿主信号分子。
关键词生物被膜     霍乱弧菌     环境因子    
Biofilm development and environmental determinants in Vibrio cholerae
Wang Quanmin, Ma Yao, Liu Lijun, Zhu Jun, Liu Zhi     
College of Life Science & Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, China
Abstract: Biofilm associated Vibrio cholerae exhibits hypervirulence and supreme fitness against the harsh stresses during its infectious cycle. It is important to study the relationships between the regulation mechanism of V. cholerae biofilm development and its environmental adaption in host niche and aquatic habitat. Here, we summarize the recent advances in V. cholerae biofilm, including biofilm compositions, development and regulation. Particularly, we extensively discuss how V. cholerae fosters its biofilm architecture and assembly via sensing and responding various environmental determinants, such as bacterium self-produced molecules, natural environment components and host factors.
Key words: biofilm     Vibrio cholerae     environmental determinants    

霍乱是人类历史上一类严重的流行性传染病,其典型症状是腹泻和呕吐,造成感染者大量失水甚至死亡。霍乱曾经在世界范围内造成7次大流行[1],至今霍乱依旧危害着人类的生命健康,据WHO资料,每年有300-500万起病例报道,包括10-12万例的致死病例[2]。霍乱弧菌包含O1血清型和非O1血清型两类。O1血清型是造成霍乱流行的病原菌,根据临床症状的差异和主要毒力因子调控的不同,又分为古典型和El Tor型[3]。古典型霍乱弧菌是前6次霍乱流行的病原菌,而El Tor型霍乱弧菌造成了第7次流行[1]

自然生境中,霍乱弧菌主要存活于海水环境中,在富含几丁质的甲壳类水生生物体表形成生物被膜。生物被膜形式的霍乱弧菌能够抵御各种逆境环境,包括氧胁迫、低温、贫营养等,而且能够有效抵御浮游生物的摄食。生物被膜在霍乱弧菌的感染过程中同样起着重要作用,纱布简单过滤能够去除水体中99%的霍乱弧菌[4],在孟加拉国农村65个村庄,约133 000人群中进行的一项实验表明,纱布过滤能使霍乱感染降低48%[5]

自然生境中生物被膜内的霍乱弧菌具有超感染性,发病感染剂量少至悬浮态细菌数量的1/10,乃至1/100[6]。霍乱弧菌一般是通过食道摄入方式进入宿主体内的,生物被膜能够有效抵御宿主胃酸的侵袭,同时在感应肠道信号时,能够有效解离,释放出的霍乱弧菌穿透小肠粘液层,最后定殖到肠道上皮细胞上[7]。霍乱弧菌在宿主肠道内也可以形成生物被膜,这种体内形成的生物被膜相对于悬浮态细菌而言,具有更强的感染性和环境适应性[8]

文中,我们对近年来霍乱弧菌生物被膜的组成和发育过程的研究进展进行综述,主要讨论环境因子对霍乱弧菌生物被膜发育的调控分子机理。这些环境因子包括微生物自身产生的小分子化合物、自然环境信号因子和宿主体内存在的环境因子等。

1 霍乱弧菌生物被膜的化学构成和相关合成基因

霍乱弧菌生物被膜主要构成部分包括表面多糖(VPS)、基质蛋白(Biofilm matrix protein)和胞外DNA (Extracellular DNA,eDNA)。霍乱弧菌生物被膜的50%以上组成部分是表面多糖,表面多糖对于生物被膜三维结构的形成具有决定性的作用[9]。NMR解析霍乱弧菌表面多糖的部分化学结构式是[→4)-α-L-GulpNAcAGly3OAc-(1→4)-β-D-Glcp-(1→4)-α-Glcp-(1→4)-α-D-Galp-(1→]n,其中20%左右的α-D-Glc被替换为α-D-GlcNAc。表面多糖与一些结构未知的物质结合在一起,导致霍乱弧菌表面多糖呈现高粘度的特性[9]。负责合成VPS的基因被分成两个基因簇,其中12个基因定位于vps-1 (vpsUvpsA-K),另外6个定位于vps-2 (vpsL-Q),vpsAvpsL分别是两个操纵子的第一个基因[10]。霍乱弧菌VPS合成主要受VpsR调控,VpsR是细菌双组分信号转导系统(Two-component signal transduction system,TCS)应答调控家族成员,磷酸化修饰的VpsR能够直接结合到vps基因簇的启动子区域,正向调控vps基因的表达[11]。霍乱弧菌的VpsT蛋白属于一类应答调控蛋白,也能通过与vps基因启动子区域结合,直接调控vps基因的表达[12-13]。VpsT对vps基因表达的调控活性需要结合c-di-GMP,但与蛋白质磷酸化修饰无关[13]vpsRvpsT的操纵子区域严重重叠,导致这两个蛋白彼此调控对方的基因表达[14]。霍乱弧菌群体感应核心调控蛋白HapR能够抑制vpsRvpsT的表达[14],同时还可以直接结合到vpsL基因调控序列上,抑制VPS的合成[15]。霍乱弧菌vpsT基因表达还受到毒力调控蛋白AphA和Sigma因子RpoS的正向调控,以及类组蛋白核苷结构蛋白H-NS的负向调控[14, 16-17]。H-NS蛋白是细菌体内的一种丰度很高的蛋白,主要功能是调控核苷的构象和附着在具有富含AT序列和高度弯曲构象特点的启动子上,抑制基因的表达。H-NS还能与vpsLvpsA的启动子区域结合,直接抑制生物被膜VPS的合成[17]。我们的研究还表明,霍乱弧菌转录调控因子VqmA能激发HapR和VqmR的表达,抑制生物膜的合成,vqmR位于vqmA上游,编码小RNA (Small RNA),能够降解vpsT mRNA,从而抑制vpsT的表达[18-19]

基质蛋白包括RbmA、RbmC、Bap1,它们的基因分布在两个vps基因簇之间[20],被认为是生物被膜的骨架,粘合生物被膜中的组成部分,包括介质表面、细菌菌体、VPS、eDNA等。晶体结构显示RbmA蛋白有两个Ⅲ型纤连蛋白(Fibronectin type Ⅲ)折叠位点,用于促进生物被膜的凝聚以及招募细胞粘附到生物被膜中[21-22]。RbmA蛋白是在单层细胞粘附和VPS产生之后开始累积在细胞表面,因此也被认为对于维持液气界面间薄膜状生物被膜(Pellicle)的弹性和皱褶起着重要作用[23]。Bap1蛋白含有4个相互重叠的Vibrio-Colwellia-Bradyrhizobium-Shewanella (VCBS)重复结构域,和4个FG-GAP结构域,前者可能用于细胞之间的粘附,而后者推测用于识别和结合一些诱导物[24-25]。Bap1蛋白对于维持液气界面间薄膜状生物被膜(Pellicle)的机械张力非常重要,同时负责薄膜状生物被膜的疏水特性[23]。霍乱弧菌的RbmC蛋白同样含有4个VCBS结构域,但只含有2个FG-GAP结构域。RbmC蛋白还含有2个羧基端β-prism结构域和2个未知功能的氨基端结构域[26],其中β-prism结构域被认为具有结合糖类物质的特性[27]

霍乱弧菌通过二型分泌系统(Type Ⅱ secretion system,T2S)负责上述3种基质蛋白的分泌[28],而它们在细胞表面的累积是依赖于VPS的存在[24]。基质蛋白表达调控方面的研究较少,生物信息学分析表明,rbmA启动子序列能够被VpsR和VpsT结合,而rbmCbap1的启动子序列只有VpsR识别[12]。有文献报道,霍乱弧菌的小RNA vrrA和rbmC基因的mRNA的5′端非翻译区域相匹配,从而抑制rbmC的表达[29]

胞外DNA (Extracelluar DNA,eDNA)被认为能维持生物被膜的结构稳定性,但其与生物被膜中其他组分相互作用的精细分子机理尚待研究[30]。核酸酶Dns和Xds参与生物被膜中eDNA的降解,调控生物被膜的解离和脱落[30]。研究表明,霍乱弧菌能通过CytR蛋白抑制核苷的摄入和代谢,从而抑制生物被膜的发育[31]

2 霍乱弧菌生物被膜的发育过程

霍乱弧菌生物被膜的发育过程,可以简单分为3个过程:单层细胞粘附、微菌落形成和生物被膜成熟、生物被膜脱落。单层细胞粘附过程中,霍乱弧菌借助鞭毛在介质表面游动,寻找合适的粘附位点,游动同时,霍乱弧菌的甘露糖敏感性凝聚素纤毛(Mannose-sensitive haemagglutinin,MSHA pili)会与无机介质发生粘附。MSHA纤毛与表面介质之间粘附力增强,细菌运动方式由随机漫游(Roaming)形式转变为局限区域性运动(Orbiting),最终完成粘附,进入微菌落形成阶段。霍乱弧菌还可以利用N-乙酰葡萄糖胺结合蛋白GbpA (N-acetylglucosamine-binding protein)[32],以及霍乱毒素共调控纤毛(Toxin coregulated pillus,TCP)[33]与宿主肠道内的黏蛋白或宿主外骨骼上的几丁质层相结合。

微菌落形成过程中,鞭毛合成基因受到抑制,霍乱弧菌开始大量分泌表面多糖(Vibrio polysaccharide,VPS),并在生物被膜成熟的整个过程持续分泌[34]。随后,RbmA也开始在细胞表面累积,使细胞分裂产生的子代细胞(Daughter cell)与亲本细胞(Parental cell)互相粘附[14]。基质蛋白Bap1被分泌到在不同细胞之间的间隙中,并逐渐在临近区域积累,因为Bap1蛋白在亲本细胞(最初粘附在介质表面的细胞)临近的区域浓度最高,因此认为Bap1负责生物被膜与介质表面粘附,同时认为亲本细胞和它们的早期后代主要负责Bap1的合成[24]。随着生物被膜的继续发育和更多的细菌分裂繁殖,基质蛋白RbmC在细胞表面特定的位置分泌,RbmC和Bap1蛋白形成一个包裹细菌菌体的囊膜状结构,这种结构能够随着细菌细胞的分裂而生长变大[24]。除此之外,胞外DNA (Extracellular DNA,eDNA)[30]和外膜颗粒(Outer membrane vesicles,OMV)[35]等也参与生物被膜的构成,这些物质随着细胞分裂逐渐累积,形成动态、柔韧、有序的粘液状物理结构,包裹着聚簇状分布的细菌细胞,共同组成了霍乱弧菌的生物被膜成熟状态[24]

生物被膜发育的最后阶段是生物被膜解离,霍乱弧菌细胞脱离生物被膜结构,开始新一轮生命周期。胞外核酸酶Dns和Xds能够通过降解eDNA调控生物被膜的解离[30]rbmB基因负责编码一个疑似多糖裂解酶,敲除rbmB能够抑制霍乱弧菌生物被膜的生长,因此RbmB被认为可能参与霍乱弧菌生物被膜解离和细菌的释放过程,但该蛋白的酶学活性没有得到试验证实[36]。霍乱弧菌分泌的锌依赖性金属蛋白酶血凝集素/蛋白酶(Hemagglutinin HA/protease) HapA被称作霍乱弧菌生物被膜的解离酶(Detachase),HapA是一种黏蛋白酶(Mucinase),能够降解肠道粘液蛋白[37],降解连接霍乱弧菌与粘液蛋白的GpbA粘附素蛋白[38],以及霍乱弧菌生物被膜中的基质蛋白RbmA[21]

我们的研究结果表明,霍乱弧菌生物被膜中群体感应信号分子CAI-1的浓度比培养基中高10 000倍以上,导致群体感应主要效应蛋白HapR的大量表达[39],进而除了激发hapA基因的表达外[40],还减少胞内c-di-GMP的浓度[41],低浓度的c-di-GMP在霍乱弧菌中能通过激发鞭毛调控蛋白FlrA的活性而促进鞭毛蛋白表达,同时降低VPS的分泌,减轻VPS对鞭毛的束缚,最终达到增强细菌运动能力,利于细菌脱离生物被膜的目的[42]

3 环境因子与生物膜发育调控 3.1 菌体自身信号 3.1.1 群体感应

群体感应是细菌通过识别自身分泌的小分子自体诱导物,调控与群体密度相关基因表达的现象,被称为细菌之间交流的互联网。与其他病原菌不一样,霍乱弧菌中群体感应是抑制生物被膜的形成。霍乱弧菌能分泌两种信号分子CAI-1和AI-2,CAI-1是(S)-3-hydroxytridecan-4-one,一种新型的细菌自体诱导物,主要负责霍乱弧菌之间的信号交流,AI-2分子式是furanosyl borate disaster (2S, 4S)-2-methyl-2, 3, 3, 4-tetrahydroxy tetrahydrofuran borate,被认为用于霍乱弧菌与其他细菌之间的交流[43]。CAI-1和AI-2的受体蛋白分别是CqsS和LuxPQ,归属于组氨酸激酶,在细菌群体低密度时磷酸化LuxO蛋白,进而激发qrr1-4小RNA的合成,这些小RNA能够降解hapR的mRNA[44]。高细菌群体密度情况下,HapR大量表达,可以直接抑制VPS合成基因vpsL[15],VPS调控基因vpsRvpsT的表达[14],也可以通过抑制AphA的表达[16, 45],以及减少c-di-GMP的胞内浓度[15],从而在转录水平和翻译后修饰水平影响VpsT的活性,达到抑制霍乱弧菌生物被膜形成的目的[13, 45]。HapR还可以通过激发蛋白酶HapA的表达,降解生物被膜合成关键蛋白GpbA[38]、RbmA[21],诱导生物被膜解离。我们的研究表明,这种群体感应调控的生物被膜解离对于霍乱弧菌在宿主体内定殖非常重要[7]

3.1.2 c-di-GMP

环化双鸟苷酸(c-di-GMP)是细菌胞内第二信使,参与调控霍乱弧菌游动性和生物被膜形成[46]。c-di-GMP合成代谢由具有GGDEF结构域的双鸟苷酸环化酶(Diguanylate cyclases,DGCs)负责,分解代谢由EAL或HD-GYP结构域的磷酸二酯酶(Phosphodiesterases,PDEs)负责[46]。霍乱弧菌中有31个蛋白具有GGDEF结构域,12个蛋白具有EAL结构域,9个蛋白具有HD-GYP结构域,同时还有10个蛋白同时含有GGDEF和EAL结构域[47]。可以想象,霍乱弧菌对体内c-di-GMP浓度控制非常精密,霍乱弧菌可能利用c-di-GMP浓度的精细改变来调控不同基因的表达。转录组研究表明,VpsR和VpsT能够上调DGCs,抑制PDEs的转录表达[14, 48]。启动子序列分析推测,VpsR能够直接结合c-di-GMP代谢基因cdgAcdgCcdgDvca0165的启动子区域,从而直接调控c-di-GMP的生成[48-49]。与VpsR和VpsT相反,HapR抑制DGCs,上调PDEs的表达[14-15],我们的研究进一步表明HapR能特异性地结合cdgAcdgGvca0080vc2370vc1851vc108650[50]。此外,群体感应系统小RNA Qrr1-4、蛋白酶LonA也参与调控霍乱弧菌体内c-di-GMP的水平[41, 51-52]

提高霍乱弧菌体内c-di-GMP水平会激发编码MSHA纤毛的msh基因,合成表面多糖的vps基因以及其他生物被膜相关基因的转录,同时抑制细菌鞭毛基因的表达[53]。进一步研究表明,小分子c-di-GMP能够结合ATP酶MshE,影响MshA纤毛的产生[54];结合表面多糖主要调控蛋白VpsT和VpsR,影响VPS的合成[13, 55];结合鞭毛合成调控蛋白FlrA,抑制细菌鞭毛的合成[42, 56]。霍乱弧菌中PilZ蛋白也能特异性地和c-di-GMP结合,调控霍乱弧菌生物被膜形成、菌体游动性和毒力[57-58]

3.1.3 (p) ppGpp

鸟苷四磷酸(Guanosin 3′5′-bis(diphosphate),ppGpp)和鸟苷五磷酸(Guanosine 3′-diphosphate 5′-triphosphate,pppGpp)是细菌在营养缺乏时合成的小分子化合物[59]。霍乱弧菌利用RelA、RelV和SpoT催化底物GDP、GTP合成(p)ppGpp,SpoT还负责该类小分子的分解代谢,通过水解反应,将其分解成催化底物和磷酸分子[49, 59-60]。研究表明,vpsR的转录需要上述3种(p)ppGpp合成酶同时存在,而vpsT的转录只是依赖于RelV[59],同时发现,胁迫应答调控因子RpoS也参与(p)ppGpp对霍乱弧菌生物被膜的调控[59]

3.1.4 cAMP

细菌的第二信使分子环磷酸腺苷(cAMP)通常在碳源营养缺乏时,由腺苷环化酶CyaA合成,结合cAMP受体蛋白(cAMP recetor protein,CRP),启动细菌碳源代谢的抑制过程。cAMP在霍乱弧菌中,抑制生物被膜的形成,其作用机制包括直接下调生物被膜合成关键基因rbmArbmCbap1vpsR和其他vps基因[61],抑制c-di-GMP合成相关基因rocScdgAcdgHcdgI[61],激发霍乱弧菌的群体感应系统。cAMP可以直接上调群体感应系统关键调控因子HapR的表达,也可以激发霍乱弧菌群体感应自体诱导物CAI-1的生物合成,间接增强hapR的转录表达[61-62]

3.1.5 甲精胺和精胺

多胺类物质是细胞内广泛分布的有机阴离子[63],霍乱弧菌体内重要的多胺类物质包括甲精胺(Norspermdine)和精胺(Spermdine),它们对生物被膜的调控作用是相反的。甲精胺(Norspermdine)能够促进霍乱弧菌生物被膜的形成,生物合成基因的敲除会极大地降低霍乱弧菌生物被膜的产生[63]。甲精胺的合成酶是NspC,该酶的过表达会导致霍乱弧菌vps基因表达的增强,产生大量的生物被膜[64]。甲精胺的受体蛋白是NspS,两者结合后,能够与含有GGDEF和EAL结构域的内膜蛋白MbaA相互作用,影响MbaA抑制生物被膜的能力[65]。精胺通过底物结合蛋白PotD1进入霍乱弧菌细胞内[66],potD1缺失株生物被膜合成能力极大增强,而外源添加精胺具有显著抑制霍乱弧菌生物被膜产生的作用[66]

3.2 自然环境信号分子 3.2.1 糖类和糖类运输PTS系统

自然状态下,霍乱弧菌生存在营养贫瘠的水体环境中,主要通过高度保守的磷酸烯醇式丙酮酸磷酸转移酶系统(Phosphoenolpyruvate phosphotransferae system,PTS)调控特定碳水化合物的运输和摄取[67]。在PTS系统中,糖的转运和磷酸化途径包含通用组分酶Ⅰ(EI),组氨酸蛋白(Hpr)和糖特异性酶Ⅱ(EII)[68]。在葡萄糖、甘露糖等匮乏的环境中,霍乱弧菌会激发cAMP的合成,形成cAMP-CRP复合物调控营养获取和利用的相关基因的表达,同时激发生物被膜的合成[21, 61]。糖类调控霍乱弧菌生物被膜形成是依赖于PTS系统运输的[68],已经报道有4种独立的PTS系统参与调控霍乱弧菌的生物被膜形成[42, 60]

霍乱弧菌中,PTS系统的关键酶还直接参与生物被膜的合成。例如,磷酸化的EI和HPr能够抑制vps基因的表达[36, 68-69];葡萄糖特异性酶EIIA(Glc)在非磷酸化条件下,直接结合在GGDEF-EAL蛋白CsrD的EAL结构域上,从而影响c-di-GMP的产生[70];EIIA(Glc)激活霍乱弧菌生物被膜合成基因的表达[71],还能够通过结合MshH蛋白,阻止MshH对生物被膜合成的抑制[45]

3.2.2 磷(PhoBR)

霍乱弧菌生存的水体资源很多都是磷素资源贫瘠,在人体小肠环境中,磷元素也是限制营养元素之一[72]。霍乱弧菌通过双组分信号转导系统PhoR/PhoB来应答磷缺乏的刺激,组氨酸激酶PhoR通过PTS系统磷酸化PhoB,磷酸化的PhoB能够直接降低vpsR的表达[73],也可以通过调控c-di-GMP代谢酶AcgAB的表达[65],抑制霍乱弧菌生物被膜的合成。在幼鼠小肠定殖模型中,PhoB对霍乱弧菌生物被膜的抑制调控主要发生在感染后期,因此该调控被认为与霍乱弧菌生物被膜的解离相关[56, 65]

3.2.3 钙离子

环境中钙离子浓度能够调控霍乱弧菌双组分调控系统CarR/CarS的表达,抑制生物被膜vps基因的表达,并且促进生物被膜的解离[74-75]

3.2.4 铁离子

铁缺乏条件下,霍乱弧菌生物被膜合成能力下降,铁离子和铁离子依赖Fur蛋白抑制小RNA RyhB的合成,Microarray结果表明,霍乱弧菌RhyB可能通过调控鞭毛基因的表达或干扰鞭毛依赖的信号途径来影响生物被膜的形成[76]

3.2.5 盐离子浓度和渗透压

水体环境中盐离子浓度和渗透压同样影响霍乱弧菌生物被膜的形成[77]。低盐浓度激发oscR基因转录,产生的转录调控因子OscR抑制vps的表达,促进霍乱弧菌的游动性[78]。相反,在高盐浓度情况下,转录调控因子OscR激发VPS的产生,抑制霍乱弧菌的游动性[79]

3.2.6 冷环境

一般的环境温度为15 ℃或25 ℃,人体肠道为37 ℃,霍乱弧菌在感染周期中,不断循环接触这两种温度。Townsley等的研究结果表明,低温能够增加胞内c-di-GMP的浓度,从而促进霍乱弧菌的生物被膜形成[80]。进一步研究表明,冷休克蛋白CspV参与这一过程[81]。但Stauder等也比较了温度对霍乱弧菌生物被膜形成的影响,发现生物被膜在25 ℃比15 ℃形成得更好,可能与gbpAmshA在25 ℃有较高表达相关[82]。不同实验室结果之间的冲突可能与所用实验菌株不同有关。

3.3 宿主环境信号分子

上文提到,在经过食道进入肠道的感染途径中,霍乱弧菌必须从生物被膜中逃逸,然后穿透小肠粘液层,最终定殖到小肠上皮细胞上[7]。同时,研究表明霍乱弧菌在宿主体内也形成生物被膜,这种体内形成的生物被膜具有超感染性和环境抗逆能力[8]。肠道环境内的许多生理和化学信号参与调控霍乱弧菌生物被膜的发育,包括温度、pH、氧胁迫、渗透压、胆盐等。

我们的研究表明,宿主小肠内的厌氧环境能够通过刺激毒力调控因子AphB235位半胱氨酸的还原,形成AphB分子间的二聚体,毒力调控活性增强[83]。而肠道内的初级胆盐(Taurocholate、Glycocholate等)能够通过刺激膜蛋白TcpP在周质空间内形成分子间的相互作用,提高下游毒力基因的表达[84]。毒力基因的核心调控因子ToxT受到激发后,会抑制MshA纤毛的合成,并通过控制蛋白酶表达,降解已有的MshA,影响生物被膜的形成[85-86]。同时ToxT会激发TCP纤毛的合成,有利于霍乱弧菌与肠道上皮细胞粘附,以及在几丁质介质表面形成生物被膜[33],并认为TCP纤毛参与霍乱弧菌体内微菌落的发育[87]。我们进一步研究发现,小肠粘液蛋白能够抑制霍乱弧菌表面多糖基因vps的表达[88],同时,Bhowmick等证明,小肠粘液蛋白能够刺激霍乱弧菌GbpA的表达,产生的GbpA结合在粘膜蛋白上的寡糖残基,有利于体内生物被膜的形成[89]

我们的研究还发现,霍乱弧菌在穿透小肠粘液层时,伴随着鞭毛断裂,细菌群体感应调控蛋白HapR活性受到抑制,有利于霍乱弧菌毒力和生物被膜基因的表达[90]。动物细胞和宿主实验证明,霍乱弧菌能够感知与小肠细胞的接触,并激发vpsT的表达[91-92]。Klose的研究团队也认为,鞭毛脱落是霍乱弧菌生物被膜形成的信号,并推测霍乱弧菌利用Na离子驱动的鞭毛马达基因作为感应器,识别霍乱弧菌与外界介质的接触[93-94]

肠道胆汁是一个复杂的混合物,包含胆汁酸、胆固醇和许多不饱和脂肪酸等。胆汁提取物和胆酸钠能通过激发VpsR显著促进霍乱弧菌生物被膜的形成[95]。胆酸混合物还能够诱导细胞体内c-di-GMP的浓度来影响生物被膜,这种调控可以被肠道内的碳酸氢钠所中和[96]。我们近期研究发现,肠道内一种主要的胆酸盐(Taurocholate,牛磺酸胆酸盐)能够降解霍乱弧菌生物被膜[97]。有趣的是,最近报道牛磺酸结合态胆酸盐还能抑制霍乱弧菌和绿脓假单胞菌生物被膜[98]。因此,胆酸盐对肠道内霍乱弧菌生物被膜的调控非常复杂和精细。

肠道内的其他物质也参与调控霍乱弧菌生物被膜的发育。例如非特异性人类分泌性免疫球蛋白SIgA能够抑制霍乱弧菌生物被膜的形成,这种抑制作用是SIgA结合寡糖中的甘露糖导致的[99]。人体的肠道微生物能够合成很多吲哚,也作为信号分子激发霍乱弧菌vps基因的表达[100]

4 结论与展望

霍乱弧菌是人类历史上非常重要的一种病原菌,生物被膜的发育对霍乱弧菌的感染和环境适应具有重要的意义。近年来,研究人员对霍乱弧菌生物被膜发育进行了深入的研究。霍乱弧菌生物被膜主要构成成分有霍乱弧菌细菌表面多糖VPS、基质蛋白和eDNA等。霍乱弧菌生物被膜的发育过程中,首先通过鞭毛运动寻找介质表面合适的粘附位点,然后通过MshA纤毛媒介与无机介质表面的粘附,或TCP纤毛和GbpA粘附蛋白,与有机介质表面结合。霍乱弧菌在介质表面形成单层细胞粘附后,VPS大量合成,同时基质蛋白RbmA、Bap1和RbmC也开始合成,共同粘结细菌与介质、细菌与细菌、以及细菌与生物被膜化学成分,形成具有三维立体结构的成熟的生物被膜,eDNA和外膜颗粒OMV也参与霍乱弧菌生物被膜的构成。当霍乱弧菌遇到合适的环境刺激,在蛋白酶和核酸酶等作用下,生物被膜开始解离,释放出的游离细菌有利于霍乱弧菌体内定殖和环境逃逸。霍乱弧菌生物被膜组成物质的合成主要受VpsT和VpsR调控,HapR、H-NS、AphA、VqmA等蛋白也参与其中,它们还互相影响,形成复杂的调控网络。霍乱弧菌的小RNA通过降解或稳定目标基因的mRNA,参与霍乱弧菌对生物被膜发育的调控。

霍乱弧菌能够应答不同的环境信号来调控生物被膜的发育,包括菌体自身产生的信号,例如群体感应信号、c-di-GMP、(p)ppGpp、cAMP和多胺类物质;自然环境信号,例如盐离子浓度和渗透压、糖类物质、磷、钙、铁以及温度等;宿主环境信号,例如肠道细胞粘附、厌氧环境、胆酸盐、肠道粘液蛋白以及肠道环境中宿主和土著微生物分泌的化学物质(吲哚、SIgA等)。

霍乱弧菌生物被膜形成的调控网络得到了较为详尽的研究,但主要集中在研究实验室条件下生物被膜的发育,分子机理研究也主要集中在遗传学方面。考虑到霍乱弧菌是一种肠道致病菌,其发病机制与肠道环境刺激紧密联系,我们建议今后的研究应该聚焦在宿主肠道环境中霍乱弧菌生物被膜的发育过程。

肠道环境中信号分子非常复杂,而且在不同肠道微环境中分布不均匀。例如,Matthew Waldor发现,霍乱弧菌在肠道中的定殖具有位置特异性,在小肠近端霍乱弧菌主要定殖在小肠隐窝(Intestinal crypt)中,而在小肠远端霍乱弧菌均匀定殖在肠上皮细胞上[101]。同时,随着霍乱弧菌的感染,肠道环境会发生动态变化。Andrew Camilli报道,磷反应调控因子PhoB对霍乱弧菌生物被膜和游动性的调控只发生在细菌感染末期[56, 65]。因此,肠道微环境下生物被膜发育的研究需要建立在时空动态变化的基础上。宿主肠道环境包含着多种不同的环境因子能够共同调控霍乱弧菌生物被膜的发育。肠道内胆汁和碳酸氢钠都通过调控霍乱弧菌胞内c-di-GMP浓度,影响生物被膜的合成,胆汁和碳酸氢钠在小肠内分布是不均匀的,因此和其他信号分子一起,构成了霍乱弧菌感应肠道位置的GPS系统[96, 102]。目前,Kim等利用单细胞成像技术发现,遗传背景相同的霍乱弧菌细胞在流体中应答群体感应信号时呈现不同反应,这种反应与霍乱弧菌细胞在流体中的时空分布差异相关[103]

霍乱弧菌会根据不同的环境信号,形成不同特性的生物被膜。例如,霍乱弧菌在淡水中形成VPS依赖性生物被膜,而在海水中形成VPS非依赖性生物被膜[104]。我们近期的研究报道,霍乱弧菌能利用AphB和OhrR精细识别不同的氧化还原电位水平,这种氧化还原电位水平的变化发生在霍乱弧菌在自然环境和宿主环境穿梭的过程中[105-106]。我们相信,霍乱弧菌的生物被膜调控也参与到霍乱弧菌在不同生境之间穿梭的过程中,其具体分子机理的阐述,会对我们深入认识霍乱弧菌以及其他微生物生物被膜发育提供启迪。

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