Detection of type III secretion gene as an indicator for pathogenic Edwardsiella tarda

Aims: To differentiate pathogenic and nonpathogenic Edwardsiella tarda strains based on the detection of type III secretion system (T3SS) gene using polymerase chain reaction (PCR). Methods and Results: Primers were designed to amplify Edw. tarda T3SS component gene esaV, catalase gene katB, haemolysin gene hlyA and 16S rRNA gene as an internal positive control. Genomic DNAs were extracted using a commercial isolation kit from 36 Edw. tarda strains consisting of 18 pathogenic and 18 nonpathogenic strains, and 50 ng of each DNA was used as the template for PCR amplification. PCR was performed with a thermocycler (TaKaRa TP600) in a 25‐μl volume. Products of esaV were detected in all pathogenic strains, but not in nonpathogenic strains; katB was detected in all pathogenic strains and one of nonpathogenic strains; hlyA was not detected in any strains. Conclusions: The detection of esaV gene can be used for the assessment of pathogenic Edw. tarda strains. Significance and Impact of the Study: The strategy using T3SS gene as the virulence indicator provides a useful tool for the clinical assessment of pathogenic Edw. tarda strains and prediction of edwardsiellosis risk in fish culture environments.     

Identification of novel virulence-associated loci in uropathogenic Escherichia coli by suppression subtractive hybridization

To identify novel virulence-associated genes in uropathogenic Escherichia coli (UPEC) strains, a suppression subtractive hybridization strategy was applied to genomic DNA of four clinical UPEC isolates from patients suffering from cystitis or pyelonephritis. The genomic DNA of four isolates (tester strains) was subtracted from the DNA of two different driver strains, the well characterized UPEC strain CFT073 and the non-pathogenic E. coli K-12 strain MG1655. We determined the sequence of 172 tester strain-specific DNA fragments, 86 of which revealed only low or no homology to nucleotide sequences of public databases. We further determined the virulence association of the 86 novel DNA fragments using each DNA fragment as a probe in Southern hybridizations of a reference strain collection consisting of 60 extraintestinal pathogenic E. coli isolates, and 40 non-virulent E. coli strains from stool samples. From this, 19 novel DNA fragments were demonstrated to be significantly associated with virulent strains and thus may represent new virulence traits. Our results support the idea of a considerable genetic variability among UPEC strains and suggest that novel genomic determinants might contribute to virulence of UPEC.     

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VI型分泌系统调节因素的研究进展

细菌的VI型分泌系统(type VI secretion system,T6SS)是一种新发现的分泌系统,在病原菌对宿主黏附、侵入及杀伤等方面均发挥了重要作用。目前的研究主要集中在T6SS在细菌致病、细菌间竞争等作用方面。然而对于其调控因素的研究尚处于初级阶段。对于大多数细菌而言,T6SS的表达并不是恒定的。现已发现温度、渗透压、抗生素、离子等环境因素均可调节T6SS。此外,在分子层面,H-NS蛋白、RpoN转录因子、c-di-GMP等也可发挥对T6SS的调节作用。在这些调控因素的调节下,细菌可以适时地开启或关闭其T6SS的表达,从而更好地感知并适应环境。对T6SS调控因素的研究对于充分认识细菌致病性并进行有效控制至关重要。本文将对调节T6SS的环境因素与调节因子做一综述。     

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。     

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。     

Causalities of war: The connection between type VI secretion system and microbiota

Microbiota niches have space and/or nutrient restrictions, which has led to the coevolution of cooperation, specialisation, and competition within the population. Different animal and environmental niches contain defined resident microbiota that tend to be stable over time and offer protection against undesired intruders. Yet fluxes can occur, which alter the composition of a bacterial population. In humans, the microbiota are now considered a key contributor to maintenance of health and homeostasis, and its alteration leads to dysbiosis. The bacterial type VI secretion system (T6SS) transports proteins into the environment, directly into host cells or can function as an antibacterial weapon by killing surrounding competitors. Upon contact with neighbouring cells, the T6SS fires, delivering a payload of effector proteins. In the absence of an immunity protein, this results in growth inhibition or death of prey leading to a competitive advantage for the attacker. It is becoming apparent that the T6SS has a role in modulating and shaping the microbiota at multiple levels, which is the focus of this review. Discussed here is the T6SS, its role in competition, key examples of its effect upon the microbiota, and future avenues of research.     

Identification of a novel gene, HCR2, in aorta of hypercoagulable rats using suppression subtractive hybridization

Screening and identification of novel genes involved in hypercoagulable state (HCS) is important in understanding the underlying mechanisms of development of atheroslerosis, which implicated in critical vascular diseases such as coronary heart disease and stroke. HCS was induced in rats with high carbohydrate diet. Subtractive hybridization experiments between aorta tissues from hypercoagulable rats and controls showed that a novel cDNA (GenBank Accession No. AY234417), designated as hypercoagulability related gene-2 (HCR2), was highly expressed in aorta tissue. The predicted protein encoded by HCR2 contains 78 amino acids, which has a theoretical isoelectric point (pI) of 8.59 and molecular weight (MW) of 8841.7 based on sequence analysis. Our data suggest that HCR2 may be involved in HCS and identification of the gene may contribute to our understanding of the mechanisms for the production of a hypercoagulable state of several critical clinical disorders.     

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。     

Identification of over-expressed genes in human renal cell carcinoma by combining suppression subtractive hybridization and cDNA library array

To isolate the over-expressed genes in human renal cell carcinoma (RCC) and analyze its molecular basis of carcinogenesis, we used the mRNA from human RCC tissues as tester and that from the matched normal kidney tissues as driver to construct the suppression subtractive hybridization library. 379 of the subtracted clones were arrayed onto a nylon membrane and the over-expressed genes were then screened by hybridizing the filter with radioactively labeled cDNA from RCC and matched normal kidney tissues. 67 clones over-expressed in RCC by a factor of 6 or more were sequenced and its identities were analyzed in GenBank database. 4 clones were previously unknown fragments and 2 clones represent KIAA genes. The rest clones were the known genes and some of them were RCC-related, including vascular endothelial growth factor, vimentin and tissue factor. Most of the known genes were the RCC-related genes previously unknown, including zinc ribbon domain-containing 1 protein (ZNRD1), pituitary tumor transforming gene1 (PTTG1). Northern blot and semi-quantitative RT-PCR confirmed that the mRNA levels of the 3 novel fragments and 1 KIAA and 3 known genes were significantly higher in RCC than in the matched normal kidney tissues. Immunohistochemical and Western blot analysis for PTTG1 and ZNRD1 revealed increased protein level in RCC. The over-expressed genes in RCC are the potential molecular targets for diagnosis and therapy and it is very important to understand the molecular mechanism of RCC through the profile of over-expressed genes.     

Identification of over-expressed genes in human renal cell carcinoma by combining suppression subtractive hybridization and cDNA library array

Renal cell carcinoma (RCC) is a common uro- genital malignancy and often shows odd biological features. RCC accounts for approximately 2% of ma- lignancies worldwide. The incidence of and mortality from RCC have continuously increased during the last 50 years. One third of the patients already have me- tastases when first consulting the doctors. Another 30%—40% of patients develop metastasis after surgi- Identification of over-expressed genes in human RCC 149 cal excision of the pri…     

Diverse toxic effectors are harbored by vgrG islands for interbacterial antagonism in type VI secretion system

The type VI secretion system (T6SS) is considered as one of the key competition strategies by injecting toxic effectors for intestinal pathogens to acquire optimal colonization in host gut, a microenviroment with high-density polymicrobial community where bacteria compete for niches and resources. Enterotoxigenic Escherichia coli (ETEC), a major cause of infectious diarrhea in human and animals, widely encode T6SS clusters in their genomes. In this report, we first identified VT1, a novel amidase effector in ETEC, significantly hydrolyzed D-lactyl-L-Ala crosslinks between N-acetylmuramoyl and L-Ala in peptidoglycan. Further study showed that the VT1/VTI1 effector/immunity pair is encoded within a typical vgrG island, and plays a critical role for the successful establishment of ETEC in host gut. Numerous putative effectors with diverse toxin domains were found by retrieving vgrG islands in pathogenic E. coli, and designated as VT modules. Therein, VT5, a lysozyme-like effector widely encoded in ETEC, was confirmed to effectively kill adjacent cells, suggesting that VT toxin modules may be critical for pathogenic E. coli to seize a significantly competitive advantage for optimal intestinal colonization. To expand our analyses for large-scale search of VT antibacterial effectors based on vgrG island, >200 predicted effectors from 20 bacterial species were found and classified into 11 predicted toxins. This work reports a new retrieval strategy for screening T6SS effectors, and provides an example how pathogenic bacteria antagonize and displace commensal microbiome to successfully colonize in the host niches through a T6SS-dependent manner.     

The type VI protein secretion system contributes to biofilm formation and seed‐to‐seedling transmission of Acidovorax citrulli on melon

The type VI protein secretion system (T6SS) is essential for the virulence of several Gram‐negative bacteria. In this study, we identified a T6SS gene cluster in Acidovorax citrulli, a plant‐pathogenic bacterium that causes bacterial fruit blotch (BFB) of cucurbits. One T6SS cluster, of approximately 25 kb in length and comprising 17 genes, was found in the A. citrulli AAC00‐1 genome. Seventeen A. citrulli mutants were generated, each with a deletion of a single T6SS core gene. There were significant differences in BFB seed‐to‐seedling transmission between wild‐type A. citrulli strain, xjl12, and ΔvasD, ΔimpK, ΔimpJ and ΔimpF mutants (71.71%, 9.83%, 8.41%, 7.15% and 5.99% BFB disease index, respectively). In addition, we observed that these four mutants were reduced in melon seed colonization and biofilm formation; however, they were not affected in virulence when infiltrated into melon seedling tissues. There were no significant differences in BFB seed‐to‐seedling transmission, melon tissue colonization and biofilm formation between xjl12 and the other 13 T6SS mutants. Overall, our results indicate that T6SS plays a role in seed‐to‐seedling transmission of BFB on melon.     

Temperature and Mg2+ sensing by a novel PhoP-PhoQ two-component system for regulation of virulence in Edwardsiella tarda

The PhoP-PhoQ two-component system is commonly used by bacteria to sense environmental factors. Here we show that the PhoP-PhoQ system of Edwardsiella tarda detects changes in environmental temperature and Mg(2+) concentration as well as regulates the type III and VI secretion systems through direct activation of esrB. Protein secretion is activated from 23 to 35 °C or at low Mg(2+) concentrations, but it is suppressed at or below 20 °C, at or above 37 °C, or at high Mg(2+) concentrations. The effects of temperature and Mg(2+) concentration are additive. The PhoQ sensor domain has a low T(m) of 37.9 °C, and it detects temperatures through a conformational change of its secondary structure. Mutation of specific Pro or Thr residues increased the stability of the PhoQ sensor drastically, altering its temperature-sensing ability. The PhoQ sensor detects Mg(2+) concentration through the direct binding of Mg(2+) to a cluster of acidic residues (DDDSAD) and through changes that likely affect its tertiary structure. Here, we describe for the first time the use of PhoP-PhoQ as a temperature sensor for bacterial virulence control.     

Identification and characterization of genes induced for anthocyanin synthesis and chlorophyll degradation in regenerated torenia shoots using suppression subtractive hybridization, cDNA microarrays, and RNAi techniques

Anthocyanin synthesis and chlorophyll degradation in regenerated torenia (Torenia fournieri Linden ex Fourn.) shoots induced by osmotic stress with 7% sucrose were examined to identify the genes regulating the underlying molecular mechanism. To achieve this, suppression subtractive hybridization was performed to enrich the cDNAs of genes induced in anthocyanin-synthesizing and chlorophyll-degrading regenerated shoots. The nucleotide sequences of 1,388 random cDNAs were determined, and these were used in the preparation of cDNA microarrays for high-throughput screening. From 1,056 cDNAs analyzed in the microarrays, 116 nonredundant genes were identified, which were up regulated by 7% sucrose to induce anthocyanin synthesis and chlorophyll degradation in regenerated shoots. Of these, eight genes were selected and RNAi transformants prepared, six of which exhibited anthocyanin synthesis inhibition and/or chlorophyll degradation in their leaf discs. Notably, the RNAi transformants of the glucose 6-phosphate/phosphate translocator gene displayed inhibition both of anthocyanin synthesis and chlorophyll degradation in both leaf discs and regenerated shoots. There was also less accumulation of anthocyanin in the petals, and flowering time was shortened. The genes we identified as being up-regulated in the regenerated torenia shoots may help further elucidate the molecular mechanism underlying the induction of anthocyanin synthesis and chlorophyll degradation.