Advances on Vibrio parahaemolyticus research in the postgenomic era

Vibrio parahaemolyticus is a leading cause of seafood-borne bacterial gastroenteritis in humans. Since its discovery in 1950, this bacterium has been isolated in widespread outbreaks and in sporadic cases of gastroenteritis worldwide. Although the exotoxin, thermostable direct hemolysin, had been the focus of extensive research on the pathogenicity of V. parahaemolyticus, the whole-genome sequencing of a clinical isolate, RIMD2210633 strain, was a breakthrough in this field. The possession of two sets of gene clusters for type III secretion systems (T3SS1 and T3SS2) was unveiled by that genome project. T3SS is a protein export apparatus that delivers bacterial proteins, called effectors, directly into the host's cytosol, to disrupt host cell function. The subsequent studies have established that T3SS2, which is encoded in an 80 kb pathogenicity island called V. parahaemolyticus pathogenicity island (Vp-PAI), is closely related to enteropathogenicity. Recent functional analyses of Vp-PAI-encoded genes revealed the sophisticated mechanisms in V. parahaemolyticus for sensing the intestinal environment and host cell contact, and a dozen T3SS2-exported proteins encoded in Vp-PAI. In this review, we summarize recent advances in V. parahaemolyticus research regarding the control of the expression of Vp-PAI-encoded genes, structural components and the secretory regulation of T3SS2, and the biological activities of T3SS2-exported effectors. Thus, Vp-PAI-encoded T3SS2 becomes an important key in the postgenomic era to shed light on the enteropathogenic mechanism of V. parahaemolyticus.     

生物膜形成中间状态下副溶血弧菌的基因转录谱分析

【目的】探究生物膜形成中间状态下副溶血弧菌的差异基因表达情况,为今后研究生物膜形成调控机制提供基因信息。【方法】以非生物膜形成条件下为参照,采用Illumina HiSeq测序平台进行转录组测序（RNA sequencing,RNA-seq）研究,分析生物膜形成中间状态下副溶血弧菌的基因表达情况,并采用实时定量PCR （quantitative real-time PCR,qPCR）进行验证。【结果】本研究共获得979个差异显著性表达基因（differentially expressed gene,DEG）,其中下调基因379个,上调基因600个。基因本体（gene ontology,GO）分类分析结果显示,共有363个DEGs注释到分子功能、细胞组分和生物学过程3个一级分类和30个二级分类;京都基因和基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）代谢途径分析结果显示,共有706个DEGs归到37个代谢通路中（Q value<0.05）,差异表达基因主要集中在细胞代谢和转运通路上;蛋白相邻类的聚簇（clusters of orthologous groups,COG）分类结果显示,有888个DEGs可归为20个类别,涉及DEGs最多的为氨基酸转运与代谢、一般功能预测基因、能量产生与转换以及未知功能基因。qPCR验证挑选的DEGs变化趋势均与RNA-seq的结果一致。此外,从转录组数据中共筛选出10个c-di-GMP代谢相关基因、1个侧生鞭毛蛋白基因（lafA）、13个极生鞭毛合成相关基因、6个荚膜多糖合成相关基因、6个胞外多糖合成相关基因、5个IV型菌毛合成相关基因、膜融合蛋白（membrane fusion protein,Mfp）基因（cpsQ-mfpABC）、14个III型分泌系统1（T3SS1）相关基因、14个Vp-PAI基因（1个tdh2和13个T3SS2基因）、3个VI型分泌系统1（T6SS1）相关基因、6个T6SS2基因、45个推定调控子基因和15个推定的外膜蛋白基因。【结论】生物膜形成引起副溶血弧菌基因表达谱发生明显变化,差异表达基因中包含生物膜形成相关基因、关键毒力基因和许多推定调控子基因等,这为后续研究生物膜形成调控机制提供重要信息。     

Distribution of type III secretion systems in Vibrio parahaemolyticus from the northern Gulf of Mexico

Aims: Two well‐characterized Vibrio parahaemolyticus pathogenicity factors – thermostable direct haemolysin (TDH) and TDH‐related haemolysin – are produced by strains containing the tdh and trh genes, respectively. Most strains of V. parahaemolyticus contain two nonredundant type III secretion systems (T3SS), T3SS1 and T3SS2, both of which contribute to pathogenicity. Furthermore, a recent study has revealed two distinct lineages of the V. parahaemolyticus T3SS2: T3SS2α and T3SS2β. The aim of this study was to determine the incidence of these pathogenicity factors in environmental isolates of V. parahaemolyticus. Methods and Results: We collected 130 V. parahaemolyticus isolates (TCBS agar) containing tdh and/or trh (determined by colony hybridization) from sediment, oyster and water in the northern Gulf of Mexico and screened them and 12 clinical isolates (PCR and agarose gel electrophoresis) for pathogenicity factors tdh, trh, T3SS1, T3SS2α and T3SS2β. The majority of potential pathogens were detected in the sediment, including all tdh^?/trh⁺ isolates. T3SS2α components were detected in all tdh⁺/trh ^? isolates and zero of 109 trh⁺ isolates. One T3SS2α gene, vopB2, was found in all tdh⁺/trh^? clinical strains but not in any of the 130 environmental strains. Fluorescence in situ hybridization adapted for individual gene recognition (RING‐FISH) was used to confirm the presence/absence of vopB2. T3SS2β was found in all tdh^?/trh⁺ isolates and in no tdh⁺/trh^? isolates. Conclusions: The combination of haemolysins found in each isolate consistently corresponded to the presence and type of T3SS detected. The vopB2 gene may represent a novel marker for identifying increased virulence among strains. Significance and Impact of the Study: This is the first study to confirm the presence of T3SS2β genes in V. parahaemolyticus strains isolated from the Gulf of Mexico and one of the few that examines the distribution and co‐existence of tdh, trh, T3SS1, T3SS2α and T3SS2β in a large collection of environmental strains.     

Regulation of Vibrio parahaemolyticus T3SS2 gene expression and function of T3SS2 effectors that modulate actin cytoskeleton

Vibrio parahaemolyticus is a leading causative agent of seafood‐borne gastroenteritis worldwide. Most clinical isolates from patients with diarrhoea possess two sets of genes for the type III secretion system (T3SS) on each chromosome (T3SS1 and T3SS2). T3SS is a protein secretion system that delivers effector proteins directly into eukaryotic cells. The injected effectors modify the normal cell functions by altering or disrupting the normal cell signalling pathways. Of the two sets of T3SS genes present in V. parahaemolyticus, T3SS2 is essential for enterotoxicity in several animal models. Recent studies have elucidated the biological activities of several T3SS2 effectors and their roles in virulence. This review focuses on the regulation of T3SS2 gene expression and T3SS2 effectors that specifically target the actin cytoskeleton.     

副溶血弧菌的毒力基因表达调控的分子机制

副溶血弧菌是革兰氏阴性嗜盐细菌,是海洋脊椎动物和无脊椎动物中主要致病菌,也是引起人类急性肠胃炎、败血症和坏死性筋膜炎等疾病的主要病原体。在过去,由副溶血弧菌引起的致病感染在世界范围内有不断增加的趋势。副溶血弧菌的致病性与其自身产生的多种毒力因子有关,这些毒力因子包括粘附因子、脂多糖、溶血素、III型分泌系统、VI型分泌系统、铁摄取系统、蛋白酶、外膜蛋白等。然而,这些毒力因子的表达都受到环境因子以及宿主体内信号因子的调控。副溶血弧菌通过感知外界生存环境的各种信号因子,从而激活体内不同的信号通路,进而诱导不同的毒力因子的表达。本文主要对副溶血弧菌毒力因子表达调控的分子机制进行综述,为更好地理解宿主与病原体的相互作用对副溶血弧菌的致病机制的影响,以及为今后预防和治疗由副溶血弧菌所引起的疾病提供理论参考。     

Presence of genes for type III secretion system 2 in <Emphasis Type="Italic">Vibrio</Emphasis><Emphasis Type="Italic">mimicus</Emphasis> strains

Background  

Vibrios, which include more than 100 species, are ubiquitous in marine and estuarine environments, and several of them e.g. Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus, are pathogens for humans. Pathogenic V. parahaemolyticus strains possess two sets of genes for type III secretion system (T3SS), T3SS1 and T3SS2. The latter are critical for virulence of the organism and be classified into two distinct phylogroups, T3SS2α and T3SS2β, which are reportedly also found in pathogenic V. cholerae non-O1/non-O139 serogroup strains. However, whether T3SS2-related genes are present in other Vibrio species remains unclear.     

Involvement of a PadR regulator PrhP on virulence of Ralstonia solanacearum by controlling detoxification of phenolic acids and type III secretion system

Ralstonia solanacearum can metabolize ferulic acid (FA) and salicylic acid (SA), two representative phenolic acids, to protect it from toxicity of phenolic acids. Here, we genetically demonstrated a novel phenolic acid decarboxylase regulator (PadR)-like regulator PrhP as a positive regulator on detoxification of SA and FA in R. solanacearum. Although the ability to degrade SA and FA enhances the infection process of R. solanacearum toward host plants, PrhP greatly contributes to the infection process besides degradation of SA and FA. Our results from the growth assay, promoter activity assay, RNA-seq and qRT-PCR revealed that PrhP plays multiple roles in the virulence of R. solanacearum: (1) positively regulates expression of genes for degradation of SA and FA; (2) positively regulates expression of genes encoding type III secretion system (T3SS) and type III effectors both in vitro and in planta; (3) positively regulates expression of many virulence-related genes, such as the flagella, type IV pili and cell wall degradation enzymes; and (4) is important for the extensive proliferation in planta. The T3SS is one of the essential pathogenicity determinants in many pathogenic bacteria, and PrhP positively regulates its expression mediated with the key regulator HrpB but through some novel pathway to HrpB in R. solanacearum. This is the first report on PadR regulators to regulate the T3SS and it could improve our understanding of the various biological functions of PadR regulators and the complex regulatory pathway on T3SS in R. solanacearum.     

A CysB regulator positively regulates cysteine synthesis,expression of type III secretion system genes,and pathogenicity in Ralstonia solanacearum

A syringe-like type III secretion system (T3SS) plays essential roles in the pathogenicity of Ralstonia solanacearum, which is a causal agent of bacterial wilt disease on many plant species worldwide. Here, we characterized functional roles of a CysB regulator (RSc2427) in R. solanacearum OE1-1 that was demonstrated to be responsible for cysteine synthesis, expression of the T3SS genes, and pathogenicity of R. solanacearum. The cysB mutants were cysteine auxotrophs that failed to grow in minimal medium but grew slightly in host plants. Supplementary cysteine substantially restored the impaired growth of cysB mutants both in minimal medium and inside host plants. Genes of cysU and cysI regulons have been annotated to function for R. solanacearum cysteine synthesis; CysB positively regulated expression of these genes. Moreover, CysB positively regulated expression of the T3SS genes both in vitro and in planta through the PrhG to HrpB pathway, whilst impaired expression of the T3SS genes in cysB mutants was independent of growth deficiency under nutrient-limited conditions. CysB was also demonstrated to be required for exopolysaccharide production and swimming motility, which contribute jointly to the host colonization and infection process of R. solanacearum. Thus, CysB was identified here as a novel regulator on the T3SS expression in R. solanacearum. These results provide novel insights into understanding of various biological functions of CysB regulators and complex regulatory networks on the T3SS in R. solanacearum.     

Precise region and the character of the pathogenicity island in clinical Vibrio parahaemolyticus strains

In this study, we determined the borders of the pathogenicity island in V. parahaemolyticus RIMD2210633 (Vp-PAI). Vp-PAI has features in common with Tn7 and other related elements at both terminal ends. Our findings indicate that the mobile element with a transposase which contains the DDE motif may have been involved in Vp-PAI formation.     

Hfq regulates the expression of the thermostable direct hemolysin gene in <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Background  

The hfq gene is conserved in a wide variety of bacteria and Hfq is involved in many cellular functions such as stress responses and the regulation of gene expression. It has also been reported that Hfq is involved in bacterial pathogenicity. However, it is not clear whether Hfq regulates virulence in Vibrio parahaemolyticus. To evaluate this, we investigated the effect of Hfq on the expression of virulence-associated genes including thermostable direct hemolysin (TDH), which is considered to be an important virulence factor in V. parahaemolyticus, using an hfq deletion mutant.     

Variability in biofilm formation correlates with hydrophobicity and quorum sensing among Vibrio parahaemolyticus isolates from food contact surfaces and the distribution of the genes involved in biofilm formation

Vibrio parahaemolyticus is one of the leading foodborne pathogens causing seafood contamination. Here, 22 V. parahaemolyticus strains were analyzed for biofilm formation to determine whether there is a correlation between biofilm formation and quorum sensing (QS), swimming motility, or hydrophobicity. The results indicate that the biofilm formation ability of V. parahaemolyticus is positively correlated with cell surface hydrophobicity, autoinducer (AI-2) production, and protease activity. Field emission scanning electron microscopy (FESEM) showed that strong-biofilm-forming strains established thick 3-D structures, whereas poor-biofilm-forming strains produced thin inconsistent biofilms. In addition, the distribution of the genes encoding pandemic clone factors, type VI secretion systems (T6SS), biofilm functions, and the type I pilus in the V. parahaemolyticus seafood isolates were examined. Biofilm-associated genes were present in almost all the strains, irrespective of other phenotypes. These results indicate that biofilm formation on/in seafood may constitute a major factor in the dissemination of V. parahaemolyticus and the ensuing diseases.