副溶血性弧菌Ⅲ型分泌系统与Ⅵ型分泌系统的研究进展

副溶血性弧菌(Vibrio parahaemolyticus,VP)是一种常见的革兰氏阴性细菌,主要分布在海洋、海口和河口沉积物中,能够引起人急性肠胃炎、伤口感染和败血症,同时危害水产品养殖业。由该菌引起的食物中毒事件在全世界范围内频繁发生,该菌被认为是微生物性食物中毒的主要病原菌之一。影响副溶血性弧菌致病性的毒力因素包括溶血素、黏附因子、分泌系统等,尤其在分泌系统中,Ⅲ型分泌系统(TypeⅢsecretion systems, T3SS)和Ⅵ型分泌系统(T6SS)与副溶血性弧菌致病性密切相关。T3SS是一种多亚基针状结构,将分泌蛋白通过供体细胞质直接注入受体细胞质;T6SS是一种接触依赖性蛋白分泌装置,将毒素直接注入目标细胞内。通过对Ⅲ型分泌系统和Ⅵ型分泌系统的研究进展进行梳理,从这两种分泌系统的结构出发,并对其功能和调控机制分别进行分析比较,指出了二者在效应蛋白及产生毒素上的不同,找出了二者与副溶血性弧菌定植的关系,以期通过这两者的联系进而深入了解副溶血性弧菌的致病机制,为防治该菌提供科学指导。     

Selectivity of intracellular proteolysis: protein substrates activate the ATP-dependent protease (La)

A critical enzyme in protein breakdown in Escherichia coli is protease La (the lon gene product), which hydrolyzes proteins and adenosine triphosphate (ATP) in a coupled process. The mechanism of this process was studied with fluorogenic tripeptides. Although proteins and peptides are degraded at the same active site, protein substrates enhance the ability of the enzyme to degrade these peptides two- to tenfold. Proteins that are not substrates had little or no effect. Thus, protein substrates must bind to protease La at two sites, the active site and an allosteric site whose occupancy enhances proteolytic activity. This effect did not require that the proteins themselves be degraded. Proteins could induce peptide breakdown even in the absence of ATP, and proteins and ATP had additive effects in stimulating peptidase activity. A multistep cyclical mechanism is proposed in which the binding of the substrate and ATP activates the protease. The enzyme can then cleave a peptide bond, but is inactivated through ATP hydrolysis. Such a mechanism may help account for the selectivity of protein breakdown and prevent inappropriate or excessive proteolysis in vivo.     

The intracellular fate of Salmonella depends on the recruitment of kinesin

Salmonella enterica causes a variety of diseases, including gastroenteritis and typhoid fever. The success of this pathogen depends on its capacity to proliferate within host cells in a membrane-bound compartment. We found that the Salmonella-containing vacuole recruited the plus-end-directed motor kinesin. Bacterial effector proteins translocated into the host cell by a type III secretion system antagonistically regulated this event. Among these effectors, SifA targeted SKIP, a host protein that down-regulated the recruitment of kinesin on the bacterial vacuole and, in turn, controlled vacuolar membrane dynamics.     

A functional screen for the type III (Hrp) secretome of the plant pathogen Pseudomonas syringae

Type III secreted "effector" proteins of bacterial pathogens play central roles in virulence, yet are notoriously difficult to identify. We used an in vivo genetic screen to identify 13 effectors secreted by the type III apparatus (called Hrp, for "hypersensitive response and pathogenicity") of the plant pathogen Pseudomonas syringae. Although sharing little overall homology, the amino-terminal regions of these effectors had strikingly similar amino acid compositions. This feature facilitated the bioinformatic prediction of 38 P. syringae effectors, including 15 previously unknown proteins. The secretion of two of these putative effectors was shown to be type III--dependent. Effectors showed high interstrain variation, supporting a role for some effectors in adaptation to different hosts.     

Pathways of protein secretion in eukaryotes

Protein secretion from cells can take several forms. Secretion is constitutive if proteins are secreted as fast as they are synthesized. In regulated secretion newly synthesized proteins destined for secretion are stored at high concentration in secretory vesicles until the cell receives an appropriate stimulus. When both constitutive and regulated protein secretion can take place in the same cell a mechanism must exist for sorting the correct secretory protein into the correct secretory vesicle. The secretory vesicle must then be delivered to the appropriate region of plasma membrane. Transfection of DNA encoding foreign secretory proteins into regulated secretory cells has provided insight into the specificity of sorting into secretory vesicles.     

The phosphothreonine lyase activity of a bacterial type III effector family

Pathogenic bacteria use the type III secretion system to deliver effector proteins into host cells to modulate the host signaling pathways. In this study, the Shigella type III effector OspF was shown to inactivate mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated kinases 1 and 2 (Erk1/2), c-Jun N-terminal kinase, and p38]. OspF irreversibly removed phosphate groups from the phosphothreonine but not from the phosphotyrosine residue in the activation loop of MAPKs. Mass spectrometry revealed a mass loss of 98 daltons in p-Erk2, due to the abstraction of the alpha proton concomitant with cleavage of the C-OP bond in the phosphothreonine residue. This unexpected enzymatic activity, termed phosphothreonine lyase, appeared specific for MAPKs and was shared by other OspF family members.     

The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles

Many pathogenic bacteria use injectisomes to deliver effector proteins into host cells through type III secretion. Injectisomes consist of a basal body embedded in the bacterial membranes and a needle. In Yersinia, translocation of effectors requires the YopB and YopD proteins, which form a pore in the target cell membrane, and the LcrV protein, which assists the assembly of the pore. Here we report that LcrV forms a distinct structure at the tip of the needle, the tip complex. This unique localization of LcrV may explain its crucial role in the translocation process and its efficacy as the main protective antigen against plague.     

GST-pull down技术筛选毛白杨天冬氨酸蛋白酶PtoAED3互作蛋白

[目的]天冬氨酸蛋白酶属于蛋白水解酶家族,为了解析毛白杨天冬氨酸蛋白酶PtoAED3在植物生长发育中的分子调节机制,利用GST-pull down联合质谱技术,对PtoAED3的互作蛋白进行鉴定和分析.[方法]通过同源克隆获得了毛白杨PtoAED3的CDS序列,构建含GST标签的原核表达载体pGEX-4T-PtoAED...     

黄曲霉野生型及LaeA缺陷型菌株胞外差异蛋白鉴定

【目的】分析黄曲霉野生型产毒株及LaeA缺陷型无毒株在以小麦、玉米为培养基质中生长时的胞外差异蛋白,为进一步明确LaeA因子在黄曲霉产毒调控中的作用提供基础数据。【方法】将小麦及玉米籽粒粉碎后灭菌并加水配制成培养黄曲霉菌的基质,在产毒株(黄曲霉CA14野生型)及无毒株(LaeA缺陷型)培养0、48和72 h时取样,采用SDS-PAGE分别分析黄曲霉毒素B1(AFB1)及胞外差异蛋白,并采用液质联用四极杆飞行时间质谱技术[LC-MS-MS (Q-TOF)]对获得的胞外差异蛋白进行鉴定。【结果】黄曲霉野生型及LaeA缺陷型菌株均能将小麦和玉米基质中的小分子蛋白迅速转化,随着培养时间延长,LaeA缺陷型菌株的胞外蛋白较野生型菌株略少,不产生黄曲霉毒素。产毒株及无毒株胞外差异蛋白条带共有7个,主要为淀粉酶A、碱性蛋白酶、木聚糖酶F3和亮氨酸氨基肽酶A等生理酶类。【结论】黄曲霉产毒株与无毒株的胞外差异蛋白为生理酶类蛋白,该类酶蛋白主要与营养摄入有关,且影响黄曲霉菌丝的生长。     

Salmonella SipA polymerizes actin by stapling filaments with nonglobular protein arms

Like many bacterial pathogens, Salmonella spp. use a type III secretion system to inject virulence proteins into host cells. The Salmonella invasion protein A (SipA) binds host actin, enhances its polymerization near adherent extracellular bacteria, and contributes to cytoskeletal rearrangements that internalize the pathogen. By combining x-ray crystallography of SipA with electron microscopy and image analysis of SipA-actin filaments, we show that SipA functions as a "molecular staple," in which a globular domain and two nonglobular "arms" mechanically stabilize the filament by tethering actin subunits in opposing strands. Deletion analysis of the tethering arms provides strong support for this model.     

Ankyrin repeat proteins comprise a diverse family of bacterial type IV effectors

Specialized secretion systems are used by many bacteria to deliver effector proteins into host cells that can either mimic or disrupt the function of eukaryotic factors. We found that the intracellular pathogens Legionella pneumophila and Coxiella burnetii use a type IV secretion system to deliver into eukaryotic cells a large number of different bacterial proteins containing ankyrin repeat homology domains called Anks. The L. pneumophila AnkX protein prevented microtubule-dependent vesicular transport to interfere with fusion of the L. pneumophila-containing vacuole with late endosomes after infection of macrophages, which demonstrates that Ank proteins have effector functions important for bacterial infection of eukaryotic host cells.     

可可毛色二孢全基因组分泌蛋白的预测及分析

【目的】可可毛色二孢（Lasiodiplodia theobromae）是一种世界性分布的重要植物病原真菌,可引起严重的葡萄溃疡病（Botryosphaeria dieback）,影响果木品质并造成巨大的经济损失。本研究预测并分析可可毛色二孢基因组范围内的分泌蛋白,并明确其基本特征,为该病菌分泌蛋白致病机理的研究打下基础。【方法】依据已公布的可可毛色二孢全基因组序列,利用信号肽预测软件SignalP v5.0、跨膜结构分析软件TMHMM v2.0、细胞器定位分析软件ProtComp v9.0、GPI锚定预测软件big-PI Fungal Predictor和亚细胞器定位分析软件TargetP v2.0生物信息学软件对该菌中的典型分泌蛋白进行筛选。对分泌蛋白N端信号肽的长度、氨基酸使用频率及其切割位点进行统计分析。依据蛋白序列的同源性,应用BLASTP程序对分泌组蛋白进行功能注释分析,预测其生物学功能。采用蔗糖酶缺陷的酵母分泌系统,对所选分泌蛋白的信号肽进行活性检测。利用qRT-PCR方法检测所选分泌蛋白基因在可可毛色二孢侵染葡萄中的表达情况。【结果】在可可毛色二孢全基因组编码蛋白中共筛选获得552个潜在的具有典型信号肽的分泌蛋白,占全基因组预测蛋白总数的4.3%,其编码蛋白长度集中于101—400 aa。信号肽统计分析表明,其信号肽长度以18—20 aa的序列最为集中,信号肽长度为20 aa的蛋白数量最多。信号肽中使用频率最高的氨基酸为丙氨酸;非极性、疏水的氨基酸使用频率最高,占氨基酸总数的60.2%。其信号肽的-3至-1位置上的氨基酸相对保守,切割位点属于A-X-A类型,可被Sp I型信号肽酶识别并切割。336个分泌蛋白具有功能注释,其功能较多集中于细胞壁降解有关的酶类以及致病相关蛋白,并且这些蛋白在分子量、等电点、脂肪族氨基酸指数等方面均存在差异。通过蔗糖酶缺陷的酵母分泌系统证实,挑选的9个分泌蛋白信号肽均具有分泌活性。qRT-PCR检测结果表明,所选分泌蛋白基因在该病菌侵染初期的表达发生变化。【结论】利用生物信息学分析技术从可可毛色二孢全基因组中共预测获得552个经典分泌蛋白。其信号肽氨基酸长度分布广泛,氨基酸组成中非极性、疏水的氨基酸使用频率最高。功能注释主要集中在细胞壁组分降解相关的酶类、致病侵染相关的坏死诱导相关蛋白以及几丁质结合蛋白等。     

Bacterial injectisomes: needle length does matter

Many pathogenic bacteria use a type III secretion nanomachine (an injectisome) to deliver virulence proteins into the cytosol of their eukaryotic host cells. Most injectisomes possess a stiff needlelike structure of a genetically defined length. We found that a minimal needle length was required for efficient functioning of the Yersinia enterocolitica injectisome. This minimal needle length correlated with the length of the major adhesin at the bacterial surface. The needle may be required for triggering type III secretion, and its length may have evolved to match specific structures at the bacterial and host cell surfaces.     

Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm （Bombyx mort） on Day 1 and Day 4 in the 5th Instar Stage

The posterior silk gland （PSG） of silkworm is an important organ where fibroin is synthesized and secreted exclusively. Because fibroin constitutes 75-80% of the silk filament, the mechanism governing fibroin secretion, quality and yield of cocoon can be elucidated by the study on the PSG. Using two-dimensional gel electrophoresis （2-DE） and image analysis system, the changes in the protein composition in the PSG cell were investigated on the day 1 （D1） and day 4 （D4） in the 5th instar stage from five different strains of silkworm （Bombyx mori）. While differences at protein level between days and strains were far less than those observed at the gene level using EST analysis. The change trends in protein composition from D1 to D4 were diverse among the different strains. The results suggest that the secretion of fibroin is regulated by multiple proteins. The site of regulation and the proteins responsible for the regulation vary with the strain, which leads to differences between strains in the capacity of fibroin secretion in the PSG cell.     

枯草芽孢杆菌subtilis str.168双精氨酸途径分泌蛋白预测及功能分析

[目的]对枯草芽孢杆菌subtilis str.168双精氨酸转运途径(Tat)分泌蛋白进行预测和功能分析。[方法]从NCBI中选取枯草芽孢杆菌subtilis str.168基因组注释的蛋白质氨基酸序列,然后用Signal P 4.0、Lipo P、Tat P、TMHMM 2.0软件分析该基因组中双精氨酸途径的分泌蛋白,同时采用COG功能数据库对预测的分泌蛋白进行功能注释和聚类分析。[结果]通过分析发现108个有motif没有酶切位点的Tat信号肽蛋白、25个有酶切位点没有motif的Tat信号肽蛋白、124个既有酶切位点也有motif的Tat信号肽蛋白,其中105个蛋白归为Tat途径的分泌蛋白。[结论]对枯草芽孢杆菌Tat途径分泌蛋白的基因组预测和功能分析,将为分析该菌胞外蛋白的分泌机制打下基础。     

青枯雷尔氏菌致病机制及其相关基因的研究进展

阐述青枯雷尔氏菌致病基因以及它们之间的相互调节。由于青枯雷尔氏菌的复杂性,进而发展了许多青枯雷尔氏菌分子鉴定技术,并且对青枯雷尔氏菌的鉴定逐渐走向快速、便捷和灵敏高的趋势。青枯雷尔氏菌基因组约5.8Mb,具有高(G+C)含量和约5 120个可能的编码基因;它是由3.7Mb的染色体和2.1Mb的大质粒所组成,主要的致病因子有Ⅲ型hrp分泌系统产物、胞外多糖、细胞壁降解酶(包括果胶质酶以及纤维素酶等),其涉及的基因主要包括hrp基因簇、avr基因、毒性基因;青枯雷尔氏菌通过Ⅲ型分泌系统(T3SS)、II型分泌系统(T2SS)等分泌系统将多种毒性因子输送到胞外使寄主植物致病。同时,T3SS和T2SS之间也是相互影响的。上述致病因子的协调作用是由一个复杂的网络调节系统控制的,并以PhcA调节基因的启动和转录为核心,自动而精密地调节有关致病基因的表达及关闭,从而控制细菌的生长状态。