细菌三型分泌系统输出蛋白分泌信号研究进展

自提出三型分泌系统的概念以来,相关分子机制的研究让人们对其有了更深入的了解。与依赖信号肽分泌途径形成鲜明对比的是,蛋白通过细菌三型分泌系统分泌或者转运时没有可识别的保守信号序列。近期对三型分泌蛋白的研究发现了多种可以引导其分泌的分泌信号。本文分别介绍了细菌三型分泌系统的种类,分泌系统分泌蛋白的种类,并着重阐述了分泌信号的分子特性及其机制,以期为新型抗菌药物的研发提供新的思路。     

细菌Ⅴ型分泌系统研究进展

目前已知革兰阴性(G-)细菌的分泌系统至少有5种类型,即Ⅰ～Ⅴ型。其中Ⅴ型分泌系统为G-菌外膜通道转运蛋白系统中最大的一个家族,该系统又称自主转运蛋白系统,它首先通过Sec依赖的分泌通路跨内膜转运,到达外周质间隙后,又通过自身的C端在外膜上形成一个β折叠桶实现跨外膜转运。Ⅴ型分泌系统的分泌装置最为单一,且该系统分泌的蛋白在跨外膜转运过程中似乎不需要能量和辅助因子(蛋白)的参与。随着对运用Ⅴ型分泌系统在G-菌表面展示异源性多肽/蛋白质的深入研究,该系统在生物技术领域已展示出巨大的应用前景。     

细菌Ⅵ型分泌系统效应蛋白的装配及功能的研究进展

蛋白分泌作为细胞之间传递信号的途径之一,在微生物生存竞争中也扮演着重要的角色。革兰氏阴性菌可以通过Ⅵ型分泌系统(type Ⅵ secretion system, T6SS)将效应蛋白传递至胞外或原核和真核微生物中,从而介导微生物间的竞争或宿主-细菌的相互作用,最终建立竞争优势。本文主要总结了T6SS的结构与组成,并重点对效应蛋白的装配以及其与免疫蛋白的作用机制的研究进展进行阐述,为以后靶向T6SS抗菌药物的研制提供新思路。     

青枯菌三型分泌系统研究进展

摘要:青枯菌(Ralstonia solanacearum)可导致多种重要经济作物毁灭性枯萎(bacterial wilt,又称青枯病),是世界上分布最广、危害最严重的十大植物病原细菌之一。注射器状的三型分泌系统(Type III secretion system)是青枯菌的一个决定性致病因子,青枯菌利用T3SS向寄主细胞中注射大量效应蛋白(Type III effectors)来抑制寄 主的免疫反应,从而引起寄主感病。本文围绕近年来有关青枯菌T3SS 遗传特性、表达调控、效应蛋白功能等方面最新进展进行综述,为全面了解青枯菌致病机理和植物细菌病害的防治提供新思路。     

从效应蛋白视角看革兰氏阴性细菌VI型蛋白分泌系统底物转运机理

蛋白质分泌系统是细菌与外界交流的重要工具。革兰氏阴性细菌的Ⅵ型蛋白分泌系统(T6SS)可以转运分泌蛋白至细菌和真核细胞内,在菌间竞争中发挥重要作用,是细菌的一种重要的生存适应性武器。分泌蛋白主要包括起到运载作用的结构蛋白和有细胞毒性的效应蛋白这两类。本文主要从效应蛋白的视角讨论T6SS如何识别并转运效应蛋白的作用机理,回顾了以VgrG和PAAR为端部载体蛋白的转运途径、依赖端部运输的效应蛋白、T6SS伴侣蛋白等重要发现的背景和过程,并综述了T6SS分泌途径的新进展。     

细菌Ⅶ型分泌系统的研究进展

细菌分泌系统参与细菌物质转运,是细菌蛋白或DNA胞外分泌的重要途径,与细菌的生长和致病性密切相关。迄今为止,已发现了Ⅰ～Ⅶ型分泌系统。Ⅰ～Ⅵ型分泌系统存在于革兰阴性菌中,其中Ⅳ型也存在于革兰阳性菌中;Ⅶ型则存在于革兰阳性菌中。Ⅶ型分泌系统是近年来发现的一种特殊分泌系统,能介导病原微生物毒力蛋白分泌,与宿主相互作用,并参与细菌体内锌铁平衡等,在革兰阳性菌的生长代谢及致病过程中发挥重要作用。本文综述细菌Ⅶ型分泌系统的类型、功能及表达调控,以增进对这一新型细菌蛋白分泌机制的认识。     

革兰氏阴性菌Ⅵ型分泌系统及其参与金属离子转运的研究进展

细菌通过其分泌系统将特定的效应蛋白输送到外界环境或进入靶细胞中,从而在细菌和宿主、细菌和微生物群落的相互作用中占据适应性优势。Ⅵ型分泌系统（The type VI secretion system,T6SS）是革兰氏阴性菌中广泛存在的大分子分泌装置,其结构和功能类似于可收缩的噬菌体尾针样,通过细胞间直接接触将细菌各种酶或毒素效应蛋白转运到原核和真核生物中,从而介导细菌间竞争以及对宿主的致病过程。有些效应蛋白还可通过非接触依赖的方式进入胞外环境来帮助细菌获取稀缺金属离子,并且它们对应激条件下细胞内金属稳态的维持至关重要。这篇综述总结了Ⅵ型分泌系统的结构、组装及其分泌的效应蛋白,并重点阐述了Ⅵ型分泌系统在多种金属离子转运机制中作用的研究进展,有助于理解T6SS在细菌间相互作用和细菌感染过程中发挥的重要作用。     

一个细菌的致命之旅——嗜肺军团菌分泌系统及效应蛋白的研究进展

嗜肺军团菌是引起军团菌肺炎以及庞蒂亚克热的革兰氏阴性胞内病原细菌,嗜肺军团菌侵染宿主的主要特点是可以通过其IVB型毒力分泌系统,向宿主细胞内分泌超过150种的底物效应蛋白。通过这些效应蛋白的作用,嗜肺军团菌能够调整宿主细胞的胞内运输途径,改变内外环境来伪装自己的吞噬泡,干扰宿主的细胞周期,抑制宿主细胞的凋亡,从而有效逃避宿主细胞的防御功能,创造出理想的胞内增殖环境。最后,效应蛋白还可以帮助军团菌从宿主细胞中逃逸。目前,嗜肺军团菌已经成为"病原菌-宿主相互作用"的重要研究模型,其毒力分泌系统及其底物效应蛋白的功能也成为细胞微生物学的研究热点。对嗜肺军团菌分泌系统及效应蛋白的研究不仅能够帮助阐明病原细菌的致病机理,还有助于推动对宿主免疫机制的更深层次的研究。文章主要针对嗜肺军团菌的毒力分泌系统,尤其是IVB型分泌系统的结构和功能,以及底物效应蛋白的研究进展进行了综述,向读者展示出一个小小的细菌所拥有的那令人惊叹的、如此狡猾的生存策略和它精致的杀伤武器。     

细菌肽转运蛋白的研究进展

细菌的肽转运蛋白包括3种,寡肽转运蛋白(Oligopeptide permease,Opp)、二肽转运蛋白(Dipeptide permease,Dpp)和二/三肽转运蛋白(Di-and tripeptide permease,Dtp)。Opp和Dpp属于ABC型超家族(ATP-binding cassette superfamily)转运蛋白,利用ATP水解产生的能量实现底物转运。对Opp和Dpp研究最多的是胞外肽结合蛋白OppA和DppA,它们起着最初识别与结合底物的重要作用。Dtp属于主要协助转运蛋白超家族(Major facilitator superfamily,MFS),与质子进行底物共转运。细菌肽转运蛋白的晶体结构解析结合大量的生化数据分析,使得人们对其转运机制有了深入的了解。本文对这三种肽转运蛋白的研究进展分别进行综述。     

革兰氏阴性细菌蛋白分泌系统研究进展

革兰氏阴性细菌由于具有复杂的双层膜结构,其蛋白质分泌能力较差.这使得革兰氏阴性细菌的典型菌株——大肠杆菌作为最常用的受体细胞在生物制药工程和其他生物技术产品生产中受到一定的限制.因此,革兰氏阴性细菌蛋白分泌系统的研究具有重要意义.本文详细地归纳了革兰氏阴性细菌已知的蛋白分泌系统,分别从分泌系统的分泌过程、分泌蛋白类别、...     

Recognition and delivery of effector proteins into eukaryotic cells by bacterial secretion systems

The direct transport of virulence proteins from bacterium to host has emerged as a common strategy employed by Gram-negative pathogens to establish infections. Specialized secretion systems function to facilitate this process. The delivery of 'effector' proteins by these secretion systems is currently confined to two functionally similar but mechanistically distinct pathways, termed type III and type IV secretion. The type III secretion pathway is ancestrally related to the multiprotein complexes that assemble flagella, whereas the type IV mechanism probably emerged from the protein complexes that support conjugal transfer of DNA. Although both pathways serve to transport proteins from the bacterium to host, the recognition of the effector protein substrates and the secretion information contained in these proteins appear highly distinct. Here, we review the mechanisms involved in the selection of substrates by each of these transport systems and secretion signal information required for substrate transport.     

细菌Ⅵ型分泌系统的调控与功能研究进展

Ⅵ型分泌系统(Type Ⅵ Secretion System,T6SS)是近年来研究较多的一种细菌分泌系统,广泛存在于革兰氏阴性菌中,在细菌的毒力、定殖、扩散及竞争遗传中发挥着重要的作用.本文综述了细菌T6SS的结构、调控以及生物学功能的最新研究进展,以期为基于T6SS的抗菌药物研制及细菌感染的诊断与防控提供新思路.     

Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins

Basal resistance in plants is induced by flagellin and several other common bacterial molecules and is implicated in the immunity of plants to most bacteria and other microbes. However, basal resistance can be suppressed by effector proteins that are injected by the type III secretion system (TTSS) of pathogens such as Pseudomonas syringae. This study demonstrates that basal resistance in the leaves of Nicotiana benthamiana is accompanied by reduced vascular flow into minor veins. Reduced vascular flow was assayed by feeding leaves, via freshly excised petioles, with 1% (weight in volume, w/v) neutral red (NR) and then observing differential staining of minor veins or altered levels of extractable dye in excised leaf samples. The reduced vascular staining was localized to tissues expressing basal resistance and was observable when resistance was induced by either the non-pathogen Pseudomonas fluorescens, a TTSS-deficient mutant of P. syringae pv. tabaci, or flg22 (a flagellin-derived peptide elicitor of basal resistance). Nicotiana benthamiana leaf areas expressing basal resistance no longer elicited the hypersensitive response when challenge inoculated with P. syringae pv. tomato DC3000. The reduced vascular staining effect was suppressed by wild-type P. syringae pv. tabaci and P. fluorescens heterologously expressing a P. syringae TTSS and AvrPto1(PtoJL1065). TTSS-proficient P. fluorescens was used to test the ability of several P. syringae pv. tomato DC3000 effectors for their ability to suppress the basal resistance-associated reduced vascular staining effect. AvrE(PtoDC3000), HopM1(PtoDC3000) (formerly known as HopPtoM), HopF2(PtoDC3000) (HopPtoF) and HopG1(PtoDC3000) (HopPtoG) suppressed basal resistance by this test, whereas HopC1(PtoDC3000) (HopPtoC) did not. In summary, basal resistance locally alters vascular function and the vascular dye uptake assay should be a useful tool for characterizing effectors that suppress basal resistance.     

霍乱弧菌Ⅵ型分泌系统的效应蛋白对细菌细胞壁的降解机制

【背景】肽聚糖(Peptidoglycan,PG)是细菌细胞壁的重要组成部分,而霍乱弧菌Ⅵ型分泌系统(Type Ⅵ Secretion System,T6SS)可以分泌具有肽聚糖水解酶活性的效应蛋白到受体细菌中杀死细胞,这类水解酶的作用机制尚未研究清楚。【目的】通过对细菌细胞壁的PG成分进行研究,建立细胞壁PG成分分析方法,并对霍乱弧菌T6SS分泌的2个破坏细胞壁的效应蛋白TseH和VgrG3的作用机制进行解析。【方法】使用显微镜观察TseH和VgrG3异位表达对宿主细菌生长的影响;纯化大肠杆菌细胞壁,使用透射电子显微镜(Transmission Electron Microscope,TEM)观察提纯的细胞壁形态;使用纯化的TseH和VgrG3分解消化PG,利用超高效液相色谱-飞行时间质谱(Ultra-Performance LiquidChromatography-Time-of-FlightMassSpectrometry,UPLC-TOFMS)分析鉴定消化后的产物成分;通过分析结果推导结构。【结果】通过透射电子显微镜观察,发现提纯的PG呈现半透明的薄膜泡状;通过UPLC-TOFMS的分析以及逆向推导,得到了提纯的PG被VgrG3水解酶降解之后的3种主要产物,分别是二糖二肽(Disaccharide,Di)、二糖三肽(Disaccharide Tripeptide,Tri)和二糖四肽(Disaccharide Tetrapeptide,Tetra)。【结论】建立了提纯PG和UPLC-TOFMS分析PG成分的方法,揭示了效应蛋白VgrG3而非TseH可以降解PG多糖链N-乙酰葡糖胺和N-乙酰胞壁酸之间的β(1-4)糖苷键的功能。由于攻击细胞壁的效应蛋白在革兰氏阴性细菌中广泛存在,本研究不仅为鉴定这类重要效应蛋白的功能提供了有效的方法,而且对研究靶向细胞壁的新型抗生素也有重要的指导作用。     

A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a shortage of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here, we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess the function of the type III secretion system encoded by Salmonella pathogenicity island 1. Type III secretion substrate–NanoLuc fusions are readily secreted into the culture supernatant, where they can be quantified by luminometry after removal of bacteria. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanolitre scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed a split NanoLuc-based assay that enables the real-time monitoring of type III secretion-dependent injection of effector–HiBiT fusions into host cells stably expressing the complementing NanoLuc–LgBiT.     

Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness

The Type VI secretion system (T6SS) is a protein translocation nanomachine widespread among Gram‐negative bacteria and used as a means to deliver effectors directly into target bacterial or eukaryotic cells. These effectors have a wide variety of functions within target cells that ultimately help the secreting cell gain a competitive fitness advantage. Here, we discuss the different ways in which these effectors can be delivered by the T6SS and the diverse mechanisms by which they exert their noxious action upon recipient cells. We also highlight the existence of roles for T6SS effectors beyond simply the killing of neighbouring cells.     

植物相关细菌中Ⅵ型分泌系统的功能研究进展北大核心

型分泌系统(typeⅥsecretion system,T6SS)是一种强大的细菌分子武器,它通过将效应蛋白注入原核或真核细胞而介导细菌间竞争并影响宿主的生命活动。T6SS广泛分布于革兰氏阴性菌中,主要存在于变形菌门(Proteobacteria)。尽管T6SS的研究大多集中在动物相关细菌上,但它在植物相关细菌中的作用不能被忽视。本文对植物相关细菌的T6SS进行了较为详细的介绍,主要从T6SS的发现、T6SS在植物相关细菌间竞争中的作用、在细菌与植物互作中的作用以及在植物生物防治中的作用等4个方面综述了最新的研究成果,旨在为今后更好地研究植物相关细菌T6SS的生物学功能及其应用提供指导。