副溶血弧菌海产品和临床分离株的表型及溶血素相关基因分析

副溶血弧菌是(Vibrio parahaemolyticus)常见的食源性病原菌,可污染多种水产品,并引起人的食物中毒,其致病性与溶血素密切相关,如直接耐热溶血素(TDH)、TDH-相关溶血素(TRH)、不耐热溶血素(TLH)。用PCR方法对分离自浙江省部分地区的副溶血弧菌临床和海产品分离株的3种溶血素基因进行检测。结果表明,所有副溶血弧菌菌株均可检测到tlh基因;11株临床分离株均检测到tdh基因,而42株水产品分离株中只有1株检出tdh基因,携带tdh的分离株神奈川试验(KP)均为阳性。所有分离菌株中均未检测到trh基因以及其尿素酶试验呈阴性,由此可知trh基因可能与尿素酶基因连锁。副溶血弧菌分离株中致病性相关毒力因子TDH的阳性率极低,然而副溶血弧菌性食物中毒发生率较高,它们之间的关系及其发病机制还有待深入研究。     

Cloning and characterization of a gene encoding a new thermostable hemolysin from Vibrio parahaemolyticus

A new thermostable hemolysin (delta-VPH) gene was cloned from a Kanagawa-negative Vibrio parahaemolyticus strain into vector pBR322 in Escherichia coli K12. The nucleotide and amino acid sequences had no homology with those of the thermostable direct hemolysin (TDH) which causes the Kanagawa phenomenon, and of the thermolabile hemolysin (TLH) of V. parahaemolyticus. The gene was present in all V. parahaemolyticus strains tested and also in one strain of V. damsela.     

Gut SCP is an immune-relevant molecule involved in the primary immunological memory or pattern recognition in the amphioxus Branchiostoma belcheri

To understand the role of calcium-binding proteins of invertebrates in immunological response, amphioxus sarcoplasmic calcium-binding protein (SCP) was investigated in the present study. Following gene cloning, recombinant protein expression and purification and antibody preparation, the expression and alteration of SCP in the response to bacterial challenge were detected using Western blotting. SCP was not detected in the branchia, humoral fluid, gonad or in the gut of wounded animals, but it was abundant in muscle and appeared in the gut of healthy animals using Vibrio parahaemolyticus immunization and challenge. Furthermore, whether gut SCP possessed anamnestic response was investigated using cross-immune challenge between Gram-positive and -negative bacteria. Gut SCP showed stronger anamnestic activity or pattern-recognition in response to Gram-negative bacterium V. parahaemolyticus than Gram-positive bacterium Staphylococcus aureus. The response was faster and more species-specific to V. parahaemolyticus, whereas it was slower and longer to S. aureus. The reason why the response showed significant difference between Gram-positive and -negative bacteria awaits investigation. These results indicate that gut SCP is an immune-relevant molecule involved in the primary immunological memory or pattern recognition in the amphioxus Branchiostoma belcheri.     

Vibrio VopQ induces PI3-kinase-independent autophagy and antagonizes phagocytosis

Vibrio parahaemolyticus is a Gram-negative bacterium responsible for gastroenteritis acquired from the consumption of contaminated shellfish. This bacterium harbours two type III secretion systems, one on each chromosome. The type III secretion system on chromosome I induces cell death by a temporally controlled sequence of events that is caspase-independent and first involves induction of autophagy, followed by cellular rounding, and finally cellular lysis. VopQ is a type III secreted effector that is necessary for the induction of autophagy as mutant strains lacking VopQ are attenuated in their ability to induce autophagy during infection. VopQ is sufficient to induce rapid autophagy as demonstrated by microinjection of recombinant VopQ into GFP-LC3 HeLa cells. Our results demonstrate that VopQ is both necessary and sufficient for induction of autophagy during V. parahaemolyticus -mediated cell death and this effect is independent of phosphatidylinositol-3-kinases but requires Atg5. Furthermore, induction of VopQ-mediated autophagy prevents recruitment of the necessary cellular machinery required for phagocytosis of V. parahaemolyticus during infection. These data provide important insights into the mechanism used by V. parahaemolyticus to cause disease.     

VHH/TLH溶血素基因在海洋弧菌中分布的研究

哈维氏弧菌(V.harveyi)的VHH溶血素是对海水养殖鱼类的潜在致病因子。哈维氏弧菌的VHH溶血素基因与副溶血弧菌(V.parahaemolyticus)的TLH热不稳定性溶血素基因具有高度相似性,其氨基酸序列的相似性达到85.6 %。根据哈维氏弧菌vhhA溶血素基因序列,合成一个地高辛标记的VHH基因探针,利用其进行Southern Blot ,检测VHH溶血素基因在57株弧菌(包括26株国际标准菌株,20株哈维氏弧菌,11株副溶血弧菌)中的分布情况。结果显示,VHH基因探针与13株弧菌标准菌株有强杂交信号,包括2株溶藻胶弧菌(V.alginolyticus) ,2株哈维氏弧菌以及1株霍氏格里蒙菌(Grimontia hollisae) ,坎贝氏弧菌(V.campbellii) ,辛辛那提弧菌(V.cincinatiensis) ,费氏弧菌(V.fischeri) ,拟态弧菌(V.mimicus) ,飘浮弧菌(V.natriegens) ,副溶血弧菌,解蛋白弧菌(V.proteolyticus)和火神弧菌(V.logei)。与6株弧菌标准菌株有弱杂交信号,包括鳗弧菌(V.anguillarum) ,河口弧菌(V.aestuarianus) ,美人鱼发光杆菌(Photobacterium damselae subsp.damselae) ,河弧菌(V.fluvialis) ,弗尼斯弧菌(V.furnissii)和创伤弧菌(V.vulnificus) ,而另外7株弧菌标准菌株中无杂交信号。所有的哈维氏弧菌菌株至少含有一条杂交带,其中菌株VIB645 , VIB 648和SF-1分别含有2条杂交带。11株副溶血弧菌中均含有一条杂交带。上述数据表明,vhh/tlh溶血素基因广泛分布于弧菌中,尤其是哈维氏弧菌相关菌株和费氏弧菌相关菌株中。另外对鳗弧菌VIB 72 ,坎贝氏弧菌VIB 285 ,飘浮弧菌VIB 299和哈维氏弧菌VIB 647的vhh/tlh溶血素基因进行克隆并测序,其氨基酸序列与VHH溶血素和TLH溶血素氨基酸序列的同源性分别为67 %~99 %和69 %~91 %。对vhh/tlh溶血素基因在弧菌中的分布研究,将有助于进一步确定这类溶血素基因在病原弧菌致病性中的作用。     

Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

BackgroundChimeric antigen receptor (CAR) T cell therapy has yielded impressive clinical results in hematological malignancies and is a promising approach for solid tumor treatment. However, toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CARs bearing a low- and high-affinity single-chain variable fragment (scFv) binding to a similar epitope and cross-reactive with murine GPC3.ResultsWhere the high-affinity CAR-T cells were toxic in vivo, the low-affinity CAR maintained cytotoxic function against antigen-positive tumor cells but did not show toxicity against normal tissues. High-affinity CAR-induced toxicity was caused by on-target, off-tumor binding, based on the observation that higher doses of the high-affinity CAR-T caused toxicity in non–tumor-bearing mice and accumulated in organs with low expression of GPC3. To explore another layer of controlling CAR-T toxicity, we developed a means to target and eliminate CAR-T cells using anti-TNF-α antibody therapy after CAR-T infusion. The antibody was shown to function by eliminating early antigen-activated, but not all, CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from antitumor efficacy with only a minor loss in tumor control. By exploring additional traits of the CAR-T cells after activation, we identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that eliminated early activated CAR-T following antigen engagement in vivo.ConclusionsBy combining the reduced-affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.     

Genetic analyses of the putative O and K antigen gene clusters of pandemic Vibrio parahaemolyticus

Pandemic V. parahaemolyticus strains have rapidly changed their serotypes, but its determinants, especially K antigen, and the genes involved in serotype have been an open question. The purpose of this study was to gain insights into these points. Although V. parahaemolyticus is known to be lacking O-side chain on its lipopolysaccharide, and O antigens are thought to be represented by core OS, the genome sequence of V. parahaemolyticus O3:K6 strain RIMD2210633 suggests that this bacterium potentially synthesizes O-side chain. To explore possible relatedness between this O-side chain biosynthesis gene cluster, which is similar in the serotypes of Vibrio cholerae, and of V. parahaemolyticus, we amplified both core OS and O-side chain gene clusters of the strains belonging to various serotypes of V. parahaemolyticus by long PCR and performed PCR RFLP analyses. The results of our RFLP analyses suggest that the core OS biosynthesis gene cluster is related to the O antigens of pandemic V. parahaemolyticus and that the putative O-side chain gene cluster is related to K antigens of pandemic V. parahaemolyticus. We then determined the sequence of these regions of a pandemic O4:K68 strain, and compared it with the corresponding sequence of RIMD2210633. In addition, PCR analysis showed the putative O4 and K68 antigen gene clusters are unique to the strains belonging to the O4 and K68 serotype respectively. The data implies that the pandemic O4:K68 V. parahaemolyticus strain emerged from the pandemic O3:K6 strain by replacement of the putative O and K antigen gene clusters.     

Gut CaVP is an innate immune protein against bacterial challenge in amphioxus Branchiostoma belcheri

The importance of calcium-binding proteins in immune response of vertebrates is determined, but whether they have the role in invertebrates is largely unknown. In the present study, phylogenetic analysis indicated that calcium vector protein (CaVP), a protein unique to amphioxus, shared 68% similarity in amino acid sequence with human and mouse calmodulin (CaM). CaVP cDNA was cloned into a bacterial vector pET-32a, and its His-tagged fusion protein was produced in Eschherichia coli cells (BL21). The recombinant CaVP was purified by Ni-NTA column and SDS-PAGE, and then utilized for antibody preparing. The prepared antibodies could recognize amphioxus CaVP with high specificity. Further analysis by Western blotting showed that CaVP was detected in muscle and humoral fluid of normal animals and appeared in gut of bacterial immunized or challenged amphioxus. Interestingly, gut CaVP was significantly higher in a healthy sub-group than a wounded sub-group post bacterial challenge. This response was detected strongly in immunization and challenge by the same Gram-negative bacterium Vibro parahaemolyticus and weakly in immunization by V.?parahaemolyticus and then challenge by Gram-negative Aeromonas hydrophila, whereas no any feedback was found in immunization by V.?parahaemolyticus and challenge by Gram-positive Staphylococcus aureus. These findings indicate the importance of gut CaVP in response to bacterial challenge.     

副溶血性弧菌Vibrio parahaemolyticus O3:K6大流行克隆的溯源

副溶血性弧菌(Vibrio Parahaemolyticus)是一种革兰氏阴性嗜盐性海洋细菌。1950年从日本一次暴发性食物中毒中首次分离发现。作为一种食源性人鱼共患致病菌,副溶血性弧菌在全球的河口、海洋和沿海广泛传播,由其引起的食物中毒已跃居其它病原菌之首。副溶血性弧菌在进化过程中通过基因重组和基因水平转移逐渐改善其对环境的适应性,因而与其它所有致病微生物相比,副溶血性弧菌的基因型和血清型都具有高度的多样性。本文就副溶血性弧菌,特别是1996年后在世界范围内出现的O3:K6新血清型流行株(形成所谓的O3:K6大流行克隆Pandemic clone)的发现及流行特征、变异分子流行病学特征、在我国的分布及研究进展进行综述,以期为O3:K6大流行克隆的溯源提供更多依据。     

副溶血弧菌的Ⅲ型分泌系统

摘要：副溶血弧菌是一种嗜盐性革兰氏阴性短杆菌,主要引起食物中毒性肠胃炎,还可引起水生动物疾病。除了耐热直接溶血毒素和耐热直接溶血相关毒素外,近年发现的副溶血弧菌两套Ⅲ型分泌系统也与该菌的致病性密切相关。Ⅲ型分泌系统1位于染色体1上,主要贡献对宿主细胞的细胞毒性,介导宿主细胞的自体吞噬作用,最后导致细胞死亡。Ⅲ型分泌系统2位于位于染色体2的毒力岛上,具有肠毒性。本文扼要介绍副溶血弧菌Ⅲ型分泌系统的组成、功能及相关转录调控机制。     

Characterization by PCR of Vibrio parahaemolyticus isolates collected during the 1997-1998 Chilean outbreak

Between November 1997 and April 1998, several human gastroenteritis cases were reported in Antofagasta, a city in the north of Chile. This outbreak was associated with the consumption of shellfish, and the etiologic agent responsible was identified as Vibrio parahaemolyticus. This was the first report of this bacterium causing an epidemic in Chile. V. parahaemolyticus was the only pathogenic bacterium isolated from patient stools and from shellfish samples. These isolates were analyzed by polymerase chain reaction (PCR) amplification of the pR72H gene, a species-specific sequence. Based on the pR72H gene amplification pattern, at least three different isolates of V. parahaemolyticus were found. Two isolates (named amplicons A and C) generated PCR products of approximately 400 bp and 340 bp respectively, while another type of isolate designated B, did not generate a PCR product, regardless of which method of DNA purification was used. Sequence analysis of the amplicons A and C shows that they have an 80 bp and 183 bp conserved region at the 5' end of the gene. However, both isolates have different sequences at their 3' terminus and are also different from the pR72H sequence originally reported. Using this PCR assay we demonstrated that these three isolates were found in clinical samples as well as in shellfish. The warm seawater caused by the climatological phenomena "El Ni?o" perhaps favored the geographic dispersion of the bacterium (bacterial bloom) occurring in Antofagasta that occurred during that time of year.     

VopV, an F-actin-binding type III secretion effector, is required for Vibrio parahaemolyticus-induced enterotoxicity

Vibrio parahaemolyticus, a Gram-negative halophilic bacterium that causes acute gastroenteritis in humans, is characterized by two type III secretion systems (T3SS), namely T3SS1 and T3SS2. T3SS2 is indispensable for enterotoxicity but the effector(s) involved are unknown. Here, we identify VopV as a critical effector that is required to mediate V. parahaemolyticus T3SS2-dependent enterotoxicity. VopV was found to possess multiple F-actin-binding domains and the enterotoxicity caused by VopV correlated with its F-actin-binding activity. Furthermore, a T3SS2-related secretion system and a vopV homologous gene were also involved in the enterotoxicity of a non-O1/non-O139 V. cholerae strain. These results indicate that the F-actin-targeting effector VopV is involved in enterotoxic activity of T3SS2-possessing bacterial pathogens.     

Genetic and physical characterization of proBA genes of the marine bacterium Vibrio parahaemolyticus

Intracellular proline pools have been implicated in the halotolerance of many organisms. To examine this relationship in a moderately halotolerant marine bacterium, Vibrio parahaemolyticus, proline biosynthesis genes were cloned in various plasmids. Some genetic and structural properties of those genes were examined. Subcloning showed that about 3.1 kilobases of V. parahaemolyticus DNA could complement proA and proB but not proC mutations of Escherichia coli. The same fragment would also complement some Pro- mutants of V. parahaemolyticus. Gamma-delta insertion mutagenesis of this subcloned fragment indicated that proB and proA genes of V. parahaemolyticus might be transcribed from different promoters. Two other genes, phoE and gpt, which map closely to the proBA genes in E. coli, were also found to be in close proximity to the proBA genes of V. parahaemolyticus.     

Genetic and physical characterization of proBA genes of the marine bacterium Vibrio parahaemolyticus.

Intracellular proline pools have been implicated in the halotolerance of many organisms. To examine this relationship in a moderately halotolerant marine bacterium, Vibrio parahaemolyticus, proline biosynthesis genes were cloned in various plasmids. Some genetic and structural properties of those genes were examined. Subcloning showed that about 3.1 kilobases of V. parahaemolyticus DNA could complement proA and proB but not proC mutations of Escherichia coli. The same fragment would also complement some Pro- mutants of V. parahaemolyticus. Gamma-delta insertion mutagenesis of this subcloned fragment indicated that proB and proA genes of V. parahaemolyticus might be transcribed from different promoters. Two other genes, phoE and gpt, which map closely to the proBA genes in E. coli, were also found to be in close proximity to the proBA genes of V. parahaemolyticus.     

Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters

Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10(4) CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh(+) and trh(+) strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.     

Studies on pathogenic Vibrio parahaemolyticus during a warm weather season in the Seto Inland Sea,Japan

Vibrio parahaemolyticus is a potentially pathogenic bacterium, occurring naturally in estuarine and marine environments throughout the world. The incidence of this organism in an aquatic environment depends upon many ecofactors. Sea water and organic material were collected during the warm weather season from a coast of the Seto Inland Sea, Japan, and analysed to determine V. parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 99% of samples were positive for V. parahaemolyticus with densities of 3 to >1400 cells per 100 ml of water or 10 g of organic samples by the most-probable-number (MPN)-PCR technique, but only 76.6% were positive by the conventional MPN culture technique, with densities ranging from 3 to >1400 cells per 100 ml of water or 10 g of organics. Furthermore, the tdh and trh genes were positive in 41.5% and 8.5% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN culture procedure. The difference in detection between the MPN culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors.     

基于组合胶体金纳米颗粒的免疫层析试纸条快速可视化检测海产品中副溶血性弧菌

本研究以野生型副溶血性弧菌(Vibrio parahaemolyticus)为免疫原制备单克隆抗体,并以组合胶体金纳米粒子(CP-AuNPs)为抗体标记探针,开发了一种基于双抗体夹心原理的可视化快速检测副溶血性弧菌的免疫层析试纸条方法.该方法被应用于梅子鱼、鲜虾、白蛤等海产品中副溶血性弧菌的检测,具有特异性强、检测时间...     

The multiple identities of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a ubiquitous marine bacterium and human pathogen. The organism possesses multiple cell types appropriate for life under different circumstances. The swimmer cell, with a single polar flagellum, is adapted to life in liquid environments. The polar flagellum is powered by the sodium motive force and can propel the bacterium at fast speeds. The swarmer cell, propelled by many proton-powered lateral flagella, can move through highly viscous environments, colonize surfaces, and form multicellular communities which sometimes display highly periodic architecture. Signals that induce differentiation to the surface-adapted cell type are both physical and chemical in nature. Surface-induced gene expression may aid survival, whether attached to inanimate surfaces or in a host organism. Genetic rearrangements create additional phenotypic versatility, which is manifested as variable opaque and translucent colony morphotypes. Discovery that a LuxR homolog controls the opaque cell type implicates intercellular signaling as an additional survival strategy. The alternating identities of V. parahaemolyticus may play important roles in attachment and detachment, how bacterial populations adapt to growth on surfaces, form structured communities, and develop biofilms.     

添加有扩增内标的副溶血弧菌PCR检测方法的建立

摘要：【目的】发掘副溶血弧菌特异性更强的检测靶点,并人工构建扩增内标,建立可以有效避免假阴性的新PCR检测体系。【方法】利用生物信息学方法,从副溶血弧菌（Vibrio parahaemolyticus）基因组DNA中发掘特异性很高的序列,并设计相应的特异性引物,人工构建扩增内标,建立PCR检测体系。【结果】本研究发掘得到的序列vp1332特异性很强,经检索,该序列是编码ABC转运子接合蛋白组分的基因片段,根据此序列设计一对特异检测引物（vp1332L/vp1332R）,同时,构建了扩增内标,并建立了PCR检测体系。利用该体系对296株副溶血弧菌和33株非副溶血弧菌进行检测,结果显示,所有以副溶血弧菌为模板的PCR反应均可扩增到一条343 bp的特异片段,而模板来源于非副溶血弧菌的则只能扩增到一条499 bp的扩增内标片段。灵敏度实验表明,该PCR反应体系的检测灵敏度为1.6×102 cfu/mL。人工污染实验表明,起始染菌量为1.24 cfu/25 g样品时经8 h增菌,即可检测到副溶血弧菌。实际样品检测结果也证实该方法的有效性。【结论】本研究建立的PCR反应体系能特异地检测副溶血弧菌,并可有效地排除假阴性,提高检测准确率。