空肠弯曲菌Ⅵ型分泌系统研究进展

Ⅵ型分泌系统(Type Ⅵ Secretion System,T6SS)是一种倒置于细胞膜上的类噬菌体样结构,能够输送效应蛋白并在定殖和生态位建立中发挥作用.近年来,在空肠弯曲菌(Campylobacter jejuni)中发现了T6SS同源基因且能够表达组装成结构完整的T6SS,但T6SS对空肠弯曲菌的毒力影响尚不清...     

Differential regulation of toxoflavin production and its role in the enhanced virulence of Burkholderia gladioli

Burkholderia gladioli is a causal agent of bacterial panicle blight and sheath/grain browning in rice in many countries. Many strains produce the yellow pigment toxoflavin, which is highly toxic to plants, fungi, animals and microorganisms. Although there have been several studies on the toxoflavin biosynthesis system of B. glumae, it is still unclear how B. gladioli activates toxoflavin biosynthesis. In this study, we explored the genomic organization of the toxoflavin system of B. gladioli and its biological functions using comparative genomic analysis between toxoflavin‐producing strains (B. glumae BGR1 and B. gladioli BSR3) and a strain not producing toxoflavin (B. gladioli KACC11889). The latter exhibits normal physiological characteristics similar to other B. gladioli strains. Burkholderia gladioli KACC11889 possesses all the genes involved in toxoflavin biosynthesis, but lacks the quorum‐sensing (QS) system that functions as an on/off switch for toxoflavin biosynthesis. These data suggest that B. gladioli has evolved to use the QS signalling cascade of toxoflavin production (TofI/TofR of QS → ToxJ or ToxR → tox operons) similar to that in B. glumae. However, some strains may have evolved to eliminate toxoflavin production through deletion of the QS genes. In addition, we demonstrate that the toxoflavin biosynthetic system enhances the virulence of B. gladioli. These findings provide another line of evidence supporting the differential regulation of the toxoflavin system in Burkholderia strains.     

The roles of the shikimate pathway genes,aroA and aroB,in virulence,growth and UV tolerance of Burkholderia glumae strain 411gr‐6

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, which is a growing disease problem for rice growers worldwide. In our previous study, some B. glumae strains showed pigmentation phenotypes producing at least two (yellow–green and purple) pigment compounds in casein–peptone–glucose agar medium. The B. glumae strains LSUPB114 and LSUPB116 are pigment‐deficient mutant derivatives of the virulent and pigment‐proficient strain 411gr‐6, having mini‐Tn5gus insertions in aroA encoding 3‐phosphoshikimate 1‐carboxyvinyltransferase and aroB encoding 3‐dehydroquinate synthase, respectively. Both enzymes are known to be involved in the shikimate pathway, which leads to the synthesis of aromatic amino acids. Here, we demonstrate that aroA and aroB are required for normal virulence in rice and onion, growth in M9 minimal medium and tolerance to UV light, but are dispensable for the production of the phytotoxin toxoflavin. These results suggest that the shikimate pathway is involved in bacterial pathogenesis by B. glumae without a significant role in the production of toxoflavin, a major virulence factor of this pathogen.     

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.     

Type III secretion system cluster 3 is required for maximal virulence of Burkholderia pseudomallei in a hamster infection model

Burkholderia pseudomallei, the etiological agent of melioidosis, is an animal pathogen capable of inducing a highly fatal septicemia. B. pseudomallei possesses three type III secretion system (TTSS) clusters, two of which (TTSS1 and TTSS2) are homologous to the TTSS of the plant pathogen Ralstonia solanacearum, and one (TTSS3) is homologous to the Salmonella SPI-1 mammalian pathogenicity island. We have demonstrated that TTSS3 is required for the full virulence of B. pseudomallei in a hamster model of infection. We have also examined the virulence of B. pseudomallei mutants deficient in several putative TTSS3 effector molecules, and found no significant attenuation of B. pseudomallei virulence in the hamster model.     

鲍曼不动杆菌VI型分泌系统溶血素-联合调节蛋白研究进展

鲍曼不动杆菌是一种革兰氏阴性的非发酵致病菌,在医院环境中广泛存在,并且已经成为医院获得性感染的重要病原体之一。近年来,由于抗菌药物的广泛应用,导致多重耐药鲍曼不动杆菌引起的感染和暴发流行,给临床治疗带来了极大的挑战。有研究表明,细菌Ⅵ型分泌系统与细菌的致病性相关。本文综述了鲍曼不动杆菌Ⅵ型分泌系统及主要功能蛋白(溶血素-联合调节蛋白)的研究进展,以期为进一步研究鲍曼不动杆菌的致病机制提供基础。     

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。     

QsvR对副溶血弧菌VI型分泌系统1相关基因的转录调控

【目的】研究调控蛋白QsvR对副溶血弧菌VI型分泌系统1 （type VI secretion system 1,T6SS1）相关基因的转录调控关系。【方法】提取野生株（wild type,WT）和qsvR突变株（ΔqsvR）的总RNA,采用实时定量PCR （quantitative real-time PCR,qPCR）研究QsvR对靶基因的调控关系;进而采用引物延伸法定位靶基因的转录起始位点和核心启动子区,并根据引物延伸产物丰度判断QsvR对靶基因的调控关系;将靶基因的调控区DNA序列克隆入pHRP309质粒中的β-半乳糖苷酶基因上游（LacZ重组质粒）,并将重组质粒转化入WT和ΔqsvR中,通过LacZ报告基因融合试验研究QsvR对靶基因的调控关系;将LacZ重组质粒分别转化入含有pBAD33或pBAD33-qsvR的大肠杆菌100lpir中,进一步采用LacZ报告基因融合试验研究在异体宿主中QsvR对靶基因的调控关系;PCR扩增靶基因调控区DNA序列,同时表达并纯化His-QsvR重组蛋白,采用凝胶阻滞试验（electrophoresis mobility shift assay,EMSA）研究His-QsvR对靶基因调控区DNA序列是否具有直接的结合作用。【结果】qPCR结果显示,与WT相比,ΔqsvR中T6SS1相关基因VP1388 （操纵子VP1388-1390首基因）和hcp1 （操纵子VP1393-1406首基因）的转录水平显著性升高,表明QsvR抑制VP1388和hcp1的转录;引物延伸结果显示VP1388和hcp1各有一个转录起始位点,分别为C （-64）和T （-62）,且它们的转录活性受QsvR的抑制;LacZ报告基因融合试验结果显示QsvR可以抑制副溶血弧菌和EC100lpir中VP1388和hcp1的启动子区转录活性;EMSA结果显示His-QsvR对VP1388和hcp1的启动子区DNA序列具有直接的结合活性。【结论】QsvR对T6SS1相关操纵子VP1388-1390和VP1393-1406的转录具有直接的抑制作用。     

Crystal structure of YwpF from Staphylococcus aureus reveals its architecture comprised of a β‐barrel core domain resembling type VI secretion system proteins and a two‐helix pair

The ywpF gene (SAV2097) of the Staphylococcus aureus strain Mu50 encodes the YwpF protein, which may play a role in antibiotic resistance. Here, we report the first crystal structure of the YwpF superfamily from S. aureus at 2.5‐Å resolution. The YwpF structure consists of two regions: an N‐terminal core β‐barrel domain that shows structural similarity to type VI secretion system (T6SS) proteins (e.g., Hcp1, Hcp3, and EvpC) and a C‐terminal two‐helix pair. Although the monomer structure of S. aureus YwpF resembles those of T6SS proteins, the dimer/tetramer model of S. aureus YwpF is distinct from the functionally important hexameric ring of T6SS proteins. We therefore suggest that the S. aureus YwpF may have a different function compared to T6SS proteins. Proteins 2015; 83:781–788. © 2015 Wiley Periodicals, Inc.     

鼠伤寒沙门氏菌Ⅵ型分泌系统相关基因敲除及其功能特性

Ⅵ型分泌系统(T6SS)是大多数革兰氏阴性细菌中都存在的一种重要的分泌系统,能介导细菌与细菌之间以及细菌和宿主细胞之间的相互作用,溶血素共调节蛋白(Hcp)和缬氨酸甘氨酸重复蛋白G(VgrG)是组成T6SS穿刺装置的重要组分。但鼠伤寒沙门氏菌Ⅵ型分泌系统的Hcp与VgrG在该菌入侵宿主细胞及抗吞噬过程中发挥的作用尚不十分清楚。【目的】本研究旨在利用基因敲除技术构建的鼠伤寒沙门氏菌hcp及vgrg基因缺失株体外接种真核上皮细胞和巨噬细胞,并以其亲本株作为对照,以研究Hcp及VgrG在该菌粘附、侵入上皮细胞及抗吞噬过程中所发挥的作用。【方法】通过优化Red同源重组系统操作过程中各个条件,建立一套快速敲除鼠伤寒沙门氏菌Ⅵ型分泌系统相关基因的操作系统,成功构建鼠伤寒沙门氏菌CVCC541的hcp及vgrg单基因缺失株、双基因缺失株及三基因缺失株,并用Hela细胞接种试验和菌落计数试验,评估不同菌株的粘附和侵袭能力;用小鼠巨噬细胞RAW 264.7接种试验,评估不同菌株的抗吞噬能力。【结果】与亲本株CVCC541粘附侵袭Hela细胞相比,基因缺失株CVCC541Δvgrg、CVCC541Δhcp2Δvgrg和CVCC541Δhcp1Δhcp2Δhcp3的粘附率分别为17.17%±2.1%、14.73%±2.5%和82%±3.7%;CVCC541Δvgrg、CVCC541Δhcp2Δvgrg和CVCC541Δhcp1Δhcp2Δhcp3的侵袭率分别为7.05%±1.05%、6.21%±1.35%和87%±3.25%;与亲本株CVCC541在小鼠巨噬细胞RAW 264.7中的存活相比,基因缺失株CVCC541Δvgrg、CVCC541Δhcp2Δvgrg和CVCC541Δhcp1Δhcp2Δhcp3的存活率分别为15.67%±2.9%、14.47%±1.87%和56.12%±3.48%。【结论】鼠伤寒沙门氏菌Ⅵ型分泌系统VgrG和Hcp对该菌入侵细胞和抗吞噬方面具有重要作用,该研究为鼠伤寒沙门氏菌通过六型分泌系统与宿主细胞相互作用的机制研究奠定了基础。     

Diversifying selection and concerted evolution of a type IV secretion system in Bartonella

We have studied the evolution of a type IV secretion system (T4SS), in Bartonella, which is thought to have changed function from conjugation to erythrocyte adherence following a recent horizontal gene transfer event. The system, called Trw, is unique among T4SSs in that genes encoding both exo- and intracellular components are located within the same duplicated fragment. This provides an opportunity to study the influence of selection on proteins involved in host-pathogen interactions. We sequenced the trw locus from several strains of Bartonella henselae and investigated its evolutionary history by comparisons to other Bartonella species. Several instances of recombination and gene conversion events where detected in the 2- to 5-fold duplicated gene fragments encompassing trwJIH, explaining the homogenization of the anchoring protein TrwI and the divergence of the minor pilus protein TrwJ. A phylogenetic analysis of the 7- to 8-fold duplicated gene coding for the major pilus protein TrwL displayed 2 distinct clades, likely representing a subfunctionalization event. The analyses of the B. henselae strains also identified a recent horizontal transfer event of almost the complete trwL region. We suggest that the switch in function of the T4SS was mediated by the duplication of the genes encoding pilus components and their diversification by combinatorial sequence shuffling within and among genomes. We suggest that the pilus proteins have evolved by diversifying selection to match a divergent set of erythrocyte surface structures, consistent with the trench warfare coevolutionary model.     

Identification and characterization of a type III secretion-associated chaperone in the type III secretion system 1 of Vibrio parahaemolyticus

Vibrio parahaemolyticus causes human gastroenteritis. Genomic sequencing of this organism has revealed that it has two sets of type III secretion systems, T3SS1 and T3SS2, both of which are important for its pathogenicity. However, the mechanism of protein secretion via T3SSs is unknown. A characteristic of many effectors is that they require specific chaperones for efficient delivery via T3SSs; however, no chaperone has been experimentally identified in the T3SSs of V. parahaemolyticus . In this study, we identified candidate T3SS1-associated chaperones from genomic sequence data and examined their roles in effector secretion/translocation and binding to their cognate substrates. From these experiments, we concluded that there is a T3S-associated chaperone, VecA, for a cytotoxic T3SS1-dependent effector, VepA. Further analysis using pulldown and secretion assays characterized the chaperone-binding domain encompassing the first 30–100 amino acids and an amino terminal secretion signal encompassing the first 5–20 amino acids on VepA. These findings will provide a strategy to clarify how the T3SS1 of V. parahaemolyticus secretes its specific effectors.     

Two-component signal transduction systems and regulation of virulence factors in <Emphasis Type=Italic>Xanthomonas</Emphasis>: a perspective

Two-component signal transduction systems (TCSTSs), consisting of a histidine kinase and a response regulator, play a critical role in regulating virulence gene expression in Gram-negative phytopathogenic bacteria Xanthomonas spp.. To date, 12 TCSTS genes have been identified, accounting for approximately 10% of the TCSTS genes in each genome that have been experimentally identified to be related to pathogenesis. These TCSTSs modulate the expression of a number of virulence factors through diverse molecular mechanisms such as interacting with DNA, protein-binding and involvement in second messenger metabolism, which generates a high level of regulatory versatility. Here we summarize the current knowledge in this field and discuss the emerging themes and remaining questions that are important in deciphering the signaling network of TCSTSs in Xanthomonas.