Expression of an iron-regulated hemolysin by Edwardsiella tarda

Abstract The ability of Edwardsiella tarda to hemolyse red blood cells was investigated. Most E. tarda strains (> 80%) produced a hemolysin when assayed by either an agar overlay or contact-dependent hemolysis technique. This activity was cell-associated (CAH) and not released into the culture supernatant under routine conditions. When quantified, E. tarda strains significantly produced 30–40-fold higher levels of hemolytic activity against guinea pig, sheep, or rabbit erythrocytes than either E. hoshinae or E. ictaluri . When grown under iron restricted-conditions in the presence of ethylenediamine di( o -hydroxyphenylacetic acid), hemoglubin, hematin and hemin were found to stimulate growth in both liquid and agar bioassays. Hemolysin activity could be released from selected E. tarda strains when grown in L broth supplemented with EDDA; hemolytic activity was 3- to > 40-fold under these conditions when compared to L broth alone. Preliminary characterization of the hemolysin of strain ET-13 indicates that it is a heat-labile protein with active sulphydryl and thiol groups. These results indicate that, in addition to its invasive capabilities, E. tarda produces a hemolysin which is at least partially regulated by the relative availability of iron and may play a role in human disease.     

迟缓爱德华菌溶血相关基因的测序和初步的功能分析

溶血素是迟缓爱德华菌（Edwardsiella tarda简称ET）的重要致病因子。用鸟枪法从ET－12菌的染色体中克隆到1株含有溶血活性的克隆子,经测定其序列的大小为4264bp,和已报道的ET两种溶血素基因无同源性,其中开放阅读框3（ORF3）424bp序列和伤寒沙门氏菌溶血调控基因（slyA0序列有68％同源性。含有完整的ORF3的亚克隆子有溶血性,而卡那霉素基因插入ORF3内的酶切位点,其转化子无溶血性,斑点杂交和Southern blot证实,该基因片段来源于供体菌ET－12,而且也存在于其他ET菌染色体上,但这些ET菌表型不一定溶血。含有溶血相关基因重组质粒的大肠杆菌（Escherichia coli简称E.coli）JM109、E.coli LE392有溶血现象。含有溶血相关基因重组质粒的ET菌,不一定有溶血现象。该基因不是溶血结构基因,而是溶血相关基因。     

迟缓爱德华氏菌(Edwardsiella tarda)产生的二聚和三聚吲哚类生物碱

为了开发利用病原菌资源, 研究迟缓爱德华氏菌 (Edwardsiella tarda) 的次生代谢产物, 采用凝胶柱色谱、反相高效液相色谱等分离纯化手段, 从该菌发酵产物的乙酸乙酯提取物中分离得到5个吲哚类生物碱1-5。结合其理化性质和波谱学特征, 化合物1-5的结构依次鉴定为: bis(1H-indol-3-yl)phenylmethane (1)、1,1-bis(1H-indol-3-yl)-2-phenylethane (2)、(2S)-3,3-bis (1H-indol-3-yl)propane-1,2-diol (3)、1H,1￠H,1￠￠H-3,2￠:3￠,3￠￠-terindole (4) 和(3,2￠:2￠,3￠￠- terindolin)-3￠-one (5), 其中化合物1、2和4为新天然产物。利用药敏纸片法、二倍稀释法和SRB法, 对5个单体化合物的抗菌和细胞毒活性进行了初步评价。其中化合物1和3对产气杆菌有微弱的抑制作用, 最小抑菌浓度(MIC)均为0.125 g/L。     

Phenotypic diversity of Edwardsiella tarda isolated from different origins

Aims: To evaluate the diversity of phenotypic characteristics among isolates of Edwardsiella tarda from various origins. Methods and results: A total of 10 E. tarda strains were investigated on biological characteristics including flagella formation, bacterial motility, biofilm formation, extracellular protein and plasmid profiles. All the E. tarda strains (including two previous recognized as nonflagellation strains) were proven to have an average of 1–7 peritrichous flagella with the precise number positively correlated with motility and biofilm formation. All the E. tarda strains exhibited similar protein profiles except ET2034, LMG2793 and ET080814, which lacked the three major bands of approximately 18, 21 and 55 kDa. E. tarda with the same geographic location shared similar plasmid profiles. Conclusions:  Edwardsiella tarda strains exhibited diversities in phenotypic characteristics that may be linked to differences in geographic location or host origin. In addition, the number of flagella is essential for bacterial motility and biofilm formation. Significance and Impact of the Study: This is the first report demonstrating the difference in flagella formation between E. tarda strains, which may broaden the understanding of flagellation trait at intra‐species level. Furthermore, evaluation of virulence‐associated characteristics can provide useful information for unveiling the diverse pathogenic mechanisms of E. tarda.     

Isolation of an unusual strain of Edwardsiella tarda from turbot and establish a PCR detection technique with the gyrB gene

Aims:  The aim of this study was to report an unusual Edwardsiella tarda and develop an effective method to identify this bacterium.
Methods and Results:  During the spring and summer of 2006, an epizootic occurred among cultured turbot ( Scophthalmus maximus ) in Qingdao, China. A gram-negative, rod-shaped bacterium (designated as LTB-4) was isolated from the infected fish, and was proved to be virulent to turbot. Based on the 16S rDNA sequencing and phenotypic tests, the bacterial pathogen was identified as E. tarda. Unlike those commonly described E. tarda strains, no flagellate was observed. Partial gyrB genes were amplified from E. tarda using the universal primers of gyrB genes and sequenced. The polymerase chain reaction (PCR) primers for the gyrB gene were designed specific to E. tarda . It revealed positive amplification of the gyrB fragment in E. tarda , whereas other bacterial species were negative. In addition, the technique enabled the recognition of E. tarda from diseased fish.
Conclusions:  The isolate was identified as E. tarda without flagellate and an effective method was developed to identify E. tarda based on using the gyrB gene as a taxonomic marker.
Significance and Impact of the Study:  The unusual E. tarda was first reported in China and the PCR allowed the rapid and sensitive detection of E. tarda.     

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.     

迟缓爱德华氏菌中甘油醛-3-磷酸脱氢酶的胞外分泌调控

【目的】迟缓爱德华氏菌甘油醛-3-磷酸脱氢酶(GAPDH)是糖酵解途径中关键酶之一,前期研究证实是一种广谱性抗原,可作为水产养殖细菌病免疫防治中疫苗的开发靶点。本文探究迟缓爱德华氏菌甘油醛-3-磷酸脱氢酶的胞外分泌机制。【方法】通过Western blot和ELISA方法考察迟缓爱德华氏菌经典分泌系统缺失株GAPDH胞外分泌情况;使用ELISA方法对迟缓爱德华氏菌突变体文库的GAPDH胞外分泌进行了大规模筛查,并结合q RT-PCR对筛查得到的插入失活株进行了表达分析。【结果】经典分泌系统与GAPDH的胞外分泌存在一定相关性。突变体文库的大规模筛查得到两株GAPDH分泌量明显增加的插入失活株Δesr A和Δesr C,这两个基因的失活会导致GAPDH的胞外分泌量显著上调。【结论】迟缓爱德华氏菌GAPDH的胞外分泌受Esr A和Esr C负调控。     

迟钝爱德华氏菌RNA提取及痕量基因组DNA去除方法探究

迟钝爱德华氏菌EIB202是一类细胞壁结构特殊的革兰氏阴性菌,高质量RNA提取相对较难。为了从转录组水平研究这类致病菌的致病机理,需要摸索有效的RNA提取及RNA样品中痕量基因组DNA去除方法。对常规RNA提取步骤进行改进,增加PBS清洗、反复冻融及较高浓度溶菌酶处理等步骤;另外,利用小体系基因组DNA去除系统,Mg2+与Mn2+协同激活DNase I去除RNA样品中基因组DNA污染。利用优化方法提取的RNA在质量及浓度(1 740 ng/μL)方面均有了显著改善,并建立了一套完全去除RNA样品中痕量基因组DNA污染的程序。     

迟钝爱德华氏菌菌蜕系统的构建

菌蜕系统（Bacterial Ghost,BG）的形成是利用噬菌体PhiX174的裂解蛋白E在革兰阴性菌细胞膜形成一个跨膜孔道结构,使细菌胞内物质由孔道排出而引起死亡。这种基因灭活的过程不引起细菌表面结构的任何理化变性,因此生成的细菌空壳具有与活菌相同功能的膜抗原结构,可诱导机体的体液免疫和细胞免疫应答。检测和比较了在铁调控启动子PyncE和温度调控启动子PR/cI控制下的E基因对迟钝爱德华氏菌菌蜕系统（EBG）的生成效率。结果显示,2种启动子均能成功生成EBG,电镜下可观察到细菌两端有直径约为80～400 nm的孔洞。传统菌蜕系统所用的热启动子在诱导后3 h开始裂解,8 h后细菌停止死亡;而新型铁诱导启动子在诱导后2 h细菌即完全停止生长。本研究为将来开发菌蜕载体疫苗防治爱德华氏菌症奠定了基础。     

鲫源迟缓爱德华氏菌的分离鉴定及其毒力基因的检测

【目的】探明上海某养殖场的异育银鲫(Carassius auratus gibelio)发生死亡的病因,研究致病菌分类地位和毒力基因携带情况。【方法】通过病原菌的筛查和回感试验,确定发病原因,对16S rRNA基因、gyrB和rpoB管家基因测序,建立病原菌系统进化树,结合API-32E细菌鉴定系统对菌株的生理生化进行鉴定,综合判定致病菌的种类及其分类地位;根据已报道的7个毒力基因fimA、citC、gadB、mukF、katB、esrB和sodB序列,设计引物进行PCR扩增,研究病原菌毒力基因携带情况。【结果】致病菌GY15是导致异育银鲫发病的原因,GY15的16S rRNA、gyrB和rpoB基因与已报道的迟缓爱德华氏菌(Edwardsiella tarda)相似性在99%以上,构建系统进化树和API鉴定确定该菌株为E.tarda,腹腔注射回感可导致异育银鲫死亡,半致死浓度(LD_(50))为4.26×105 CFU/m L;已报道的7种毒力基因在该致病菌中均能被检测到;药敏试验结果显示,该菌对恩诺沙星、氟苯尼考和氧氟沙星等15种药物敏感,对新霉素、四环素和复方新诺明等18种药物表现为耐药。【结论】首次报道E.tarda可感染异育银鲫,它对异育银鲫的养殖造成威胁。     

应用绿色荧光蛋白标记迟缓爱德华菌感染斑马鱼

目的建立斑马鱼模型研究迟缓爱德华菌的致病性及感染途径。方法应用绿色荧光蛋白标记迟缓爱德华菌,追踪观察其感染斑马鱼的动力学过程及病理组织学变化。结果病理组织学检查以肝脏水肿变性,肝细胞萎缩、坏死、脱落,脾脏散在增生性结节、充血、水肿、淋巴细胞大量缺失等病变为主;感染后,该菌先后在斑马鱼肠道、鳃和皮肤中定植。结论斑马鱼可作为研究迟缓爱德华菌致病性的动物模型。肠道、鳃和皮肤可能是迟缓爱德华菌先后感染斑马鱼的主要途径。     

Analysis of 16S-23S intergenic spacer regions of the rRNA operons in Edwardsiella ictaluri and Edwardsiella tarda isolates from fish

AIMS: To analyse interspecies and intraspecies differences based on the 16S-23S rRNA intergenic spacer region (ISR) sequences of the fish pathogens Edwardsiella ictaluri and Edwardsiella tarda. METHODS AND RESULTS: The 16S-23S rRNA spacer regions of 19 Edw. ictaluri and four Edw. tarda isolates from four geographical regions were amplified by PCR with primers complementary to conserved sequences within the flanking 16S-23S rRNA coding sequences. Two products were generated from all isolates, without interspecies or intraspecific size polymorphisms. Sequence analysis of the amplified fragments revealed a smaller ISR of 350 bp, which contained a gene for tRNA(Glu), and a larger ISR of 441 bp, which contained genes for tRNA(Ile) and tRNA(Ala). The sequences of the smaller ISR of different Edw. ictaluri isolates were essentially identical to each other. Partial sequences of larger ISR from several Edw. ictaluri isolates also revealed no differences from the one complete Edw. ictaluri large ISR sequence obtained. The sequences of the smaller ISR of Edw. tarda were 97% identical to the Edw. ictaluri smaller ISR and the larger ISR were 96-98% identical to the Edw. ictaluri larger ISR sequence. The Edw. tarda isolates displayed limited ISR sequence heterogeneity, with > or =97% sequence identity among isolates for both small and large ISR. CONCLUSIONS: There is a high degree of size and sequence similarity of 16S-23S ISR both among isolates within Edw. ictaluri and Edw. tarda species and between the two species. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results confirm a close genetic relationship between Edw. ictaluri and Edw. tarda and the relative homogeneity of Edw. ictaluri isolates compared with Edw. tarda isolates. Because no differences were found in ISR sequences among Edw. ictaluri isolates, sequence analysis of the ISR will not be useful to distinguish isolates of Edw. ictaluri. However, we identified restriction sites that differ between ISR sequences of Edw. ictaluri and Edw. tarda, which will be useful in distinguishing the two species.     

Overexpression of mltA in Edwardsiella tarda reduces resistance to antibiotics and enhances lethality in zebra fish

Aims: The aim of this study was to investigate the role of membrane‐bound lytic murein transglycosylase A (MltA) in a bacterial fish pathogen Edwardsiella tarda. Methods and Results: An mltA in‐frame deletion mutant (ΔmltA) and an mltA overexpression strain (mltA⁺) of Edw. tarda were constructed through double‐crossover allelic exchange and by transformation of a low‐copy plasmid carrying the intact mltA into the ΔmltA mutant, respectively. Either inactivation or overexpression of MltA in Edw. tarda resulted in elevated sensitivity to β‐lactam antibiotics and lower viability in oligotrophic or high osmotic environment than wild‐type strain. Autolysis induced by EDTA was reduced in ΔmltA strain, while mltA⁺ strain was virtually flimsy, indicating that MltA is responsible for the lysis effect. Moreover, mltA⁺ strain exhibited significant increases in lipopolysaccharide (LPS) biosynthesis and virulence to zebra fish compared with wild‐type strain. Conclusions: The results indicated that MltA plays essential roles in β‐lactam antibiotics and environmental stresses resistance, autolysis, LPS biosynthesis and pathogenicity of Edw. tarda. This is the first report that MltA has a virulence‐related function in Edw. tarda. Significance and Impact of the Study: This study provided useful information for further studies on pathogenesis of Edw. tarda.     

拟态弧菌溶血素基因的克隆测序与生物信息学分析

目的对拟态弧菌安徽分离株HX4(V.mimicusHX4株)的全长溶血素基因(vmh)进行克隆测序和生物信息学分析,为表达溶血素蛋白(VMH)奠定基础。方法采用PCR法扩增V.mimicusHX4菌株全长vmh基因,将其克隆至pMD18-Tvector并进行测序,应用生物信息学软件分析vmh基因的同源性及其编码蛋白的分子特征。结果V.mimicusHX4菌株vmh基因全长序列2235 bp,编码由744个氨基酸组成的分子量约为82.85 kDa的VMH蛋白。V.mimicusHX4菌株vmh基因的核苷酸序列和氨基酸序列与参考株相应序列的同源性分别介于98.9%~99.1%和96.6%~97.3%。VMH蛋白N端前25个氨基酸组成信号肽,7~27位氨基酸之间存在一个跨膜区域,蛋白二级结构中无规卷曲含量最高,达39.52%,其次为α-螺旋和β-折叠,分别占25.81%和26.75%,β转角含量最低,仅占7.93%。VMH蛋白含有多个T细胞和B细胞抗原表位,同时存在T、B细胞抗原表位的区域最有可能位于肽链第86~95、193~211、419~440和459~501位区段。结论拟态弧菌VMH蛋白是一种高度保守的毒素蛋白,对HX4菌株vmh基因及其编码蛋白信息特征的了解,有助于进一步表达VMH蛋白。     

Evaluation of the selective and differential ET medium for detection of Edwardsiella tarda in aquaculture systems

Aims: Edwardsiella tarda is an important pathogen in aquaculture where it can cause serious losses. A rapid detection of it is vital to minimize the mortalities caused by this disease, and in this work, the effectiveness of the selective differential Edw. tarda medium (ET) was evaluated for the diagnosis of edwardsiellosis as well as for its possible use in epidemiological studies. Methods and Results: ET medium was evaluated in parallel with the commercial Salmonella–Shigella agar (SS), which is usually employed for the selective isolation of enteric bacilli. Moreover, two general media (TSA‐1 and MA) were employed as a control. The results obtained showed that ET is distinctly selective for the isolation of Edw. tarda, allowing its recovery from mixed cultures and natural samples as a unique species. In contrast, although colonies of Edw. tarda could be clearly distinguishable in SS because of the appearance of a characteristic black centre, other enteric and nonenteric bacterial species were also able to grow on this medium. Conclusions: We recommend ET agar as an useful medium for the primary isolation of Edw. tarda from aquaculture samples. Significance and Impact of the Study: The results obtained support ET medium as the most appropriate to develop epidemiological studies of edwardsiellosis in aquaculture and permits an earlier diagnosis of this important disease.     

Green fluorescent protein-tagged Edwardsiella tarda reveals portal of entry in fish

The application of green fluorescent protein (GFP) to identify the portal(s) of entry of bacterial pathogens in animal hosts was studied using the fish pathogen Edwardsiella tarda and blue gourami, Trichogaster trichopterus. An immersion challenge model was utilized to mimic natural infection conditions in fish. Gastrointestinal tract, gills and the body surface of fish were found to be the sites of entry of virulent E. tarda (PPD130/91) by histological and infection kinetics studies. On the other hand, avirulent E. tarda (PPD125/87) was mainly found in the gastrointestinal tract, and the bacterial population in tissue declined over a period of 7 days.     

Comparative analysis of the roles of catalases KatB and KatG in the physiological fitness and pathogenesis of fish pathogen Edwardsiella tarda

Aims: The aim of this study was to reveal functional redundancy and variation of the two catalases KatB and KatG in Edwardsiella tarda. Methods and Results: Genome sequencing of fish pathogen Edw. tarda EIB202 reveals that it contains two genes putatively encoding catalases, katB (ETAE_1368) and katG (ETAE_0889). Under free‐living conditions, single disruption in katB or katG resulted in no growth impairment, whereas double mutation of the two genes led to moderate decrease in growth, indicating that these two catalases were together essential for the physiological fitness by dissipating the endogenous H₂O₂. katG mutant exhibited much more elevated sensitivity to exogenous H₂O₂ than katB mutant did, indicating that KatG was quasi‐essential in detoxifying external reactive oxygen species (ROS) in Edw. tarda EIB202. Further comparative analysis indicated that katB or katG disruption showed different effects on the virulence‐related processes of Edw. tarda such as haemolysin production, bile and serum resistance, as well as the internalization within fish epithelial cells. Moreover, both of the katB and katG mutants exhibited incapacity to replicate in murine macrophage J774 cell model, although the deficiency was seen much severe for ΔkatB/katG mutant. With regard to in vivo virulence, katB and katG mutants displayed delayed lethality and increased LD₅₀ values for zebrafish. Conclusions: KatB and KatG in Edw. tarda serve for the physiological fitness, and pathogenesis related the bacterial survival in macrophage and in vivo of fish. Significance and Impact of the Study: Counteracting ROS for systemic infection, Edw. tarda catalase KatG and KatB merits as potential targets for attenuated live vaccine construction.     

Search for live attenuated vaccine candidate against edwardsiellosis by mutating virulence-related genes of fish pathogen Edwardsiella tarda

Aims: The aims of this study were to construct and evaluate the live attenuated vaccine against edwardsiellosis on zebra fish model. Methods and Results: In this study, the deletion mutant of aroC gene for the biosynthesis of chorismic acid in Edwardsiella tarda EIB202 was firstly constructed by allelic exchange strategy. According to the genome information, 19 double mutants and one multiple mutant were successively constructed by deleting virulence‐associated genes based on the ΔaroC mutant. Zebra fish model was used to assay the virulence of the mutants by intramuscular (i.m.) injection. Fourteen mutants were significantly attenuated with accumulated mortality ranged from 0 to 63% (P < 0·05). The zebra fish vaccinated with ΔaroC, ΔaroCΔesrC, ΔaroCΔslyA and ΔaroCΔeseBCDΔesaC via i.m. injection showed ideal protection, resulting in relative per cent survival (RPS) of 68·3, 71·3, 80·1 and 81% against subsequent challenge with the wild‐type Edw. tarda EIB202. Conclusions: ΔaroCΔeseBCDΔesaC behaved a low virulence and the highest RPS on zebra fish model. When the zebra fish were vaccinated with ΔaroCΔeseBCDΔesaC via injection, the expression of immune‐related factors including IgM and MHC II was up‐regulated. Significance and Impact: The mutant ΔaroCΔeseBCDΔesaC might serve as an effective live attenuated vaccine against edwardsiellosis.     

Identification of novel putative virulence factors, adhesin AIDA and type VI secretion system, in atypical strains of fish pathogenic Edwardsiella tarda by genomic subtractive hybridization

Edwardsiella tarda, which is known to be the causative agent of edwardsiellosis in freshwater and marine fish, has two motility phenotypes. Typical strains exhibiting motility are isolated mainly from freshwater fish and Japanese flounder. Atypical strains exhibiting non-motility are isolated mainly from marine fish, with the exception of Japanese flounder. Subtractive hybridization was performed to identify genomic differences between these two phenotypes. Two fragments which showed homology to potential virulence factors were isolated from atypical strains: the autotransporter adhesin AIDA and a component of T6SS. We analysed DNA sequences of about 5 kbp containing these fragments and identified two partial ORF, and ORF encoding for other components of T6SS. The predicted amino acid sequences showed remarkably low homology to components of T6SS reported in the typical E. tarda strain PPD130/91. Furthermore, the organization of these ORF was different from the gene cluster of the typical E. tarda strain. AIDA and T6SS may therefore be associated with different pathogenicity in typical and atypical E. tarda hosts.