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1.
Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G4 总被引：1，自引：0，他引：1
In order to identify genes encoding the outer membrane proteins (OMPs) of the myxobacter Flavobacterium columnare G(4), the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. Two genes encoding a membrane-associated zinc metalloprotease and prolyl oligopeptidase are reported in this paper. The membrane-associated zinc metalloprotease gene (map) is 1800 bp in length, coding for 449 amino acids (aa). Despite the presence of a conserved motif HEXXH for all metalloproteases, the special HEXXH approximately 32 aa approximately E motif of the F. columnare G(4) Map and its low level of identity with other reported zinc-containing metalloproteases may imply that the membrane-associated zinc metalloprotease of F. columnare G(4) represents a new family of zincins. The gene encoding prolyl oligopeptidase (Pop), a serine proteinase, is 2352 bp in length, coding for 649 aa. Sequence homology analysis revealed that the Pop is also novel as it has < 50% identity with other reported prolyl oligopeptidase family proteins. The present study represents the first to employ anti-fish bacterial OMP sera to screen genes of membrane-associated proteases of fish pathogenic bacteria, and to provide necessary information for the examination of the role of the two genes in the infection and pathogenesis of F. columnare. 相似文献
2.
鱼肠道弧菌外膜蛋白抗原性分析 总被引：3，自引：0，他引：3
采用十二烷基肌氨酸钠法和苯甲基磺酰氟法提取鱼肠道弧菌外膜蛋白.SDS-PAGE图谱显示,PMSF提取的鱼肠道弧菌有19条带,Sarkosyl法提取的鱼肠道弧菌有14条带,其中111、105、87、78、61、58、53、48、45、40、36、33、32、31 kD蛋白带为2种方法共同条带,101、55、42、27、25 kD为PMSF法特有条带.此外,以制备的兔抗鱼肠道弧菌全菌血清为第一抗体,应用Western-blotting技术分析了鱼肠道弧菌外膜蛋白的抗原性,结果显示分子量为106、87、61、58、55、42、36、32 kD的8条蛋白带发生了免疫反应. 相似文献
3.
采用十二烷基肌氨酸钠(Sarkosyl)和苯甲基磺酰氟(PMSF)2种方法提取秦皇岛弧菌HQ010712-1(Vibrio qinhuangdaora sp.nov.)外膜蛋白,结果显示 Sarkosyl法提取效果较好,且所提取的主要外膜蛋白分子量为102kD、45 kD、39 kD、36 kD、30 kD、28 kD、24 kD、22 kD;为比较该菌株与弧菌属其他细菌外膜蛋白组分及抗原性异同,以鳗弧菌(Vibrio anguillarum)、副溶血弧菌(Vibrio parahaemolyticus)、溶藻胶弧菌(Vibrio alginolyticus)为对照,电泳图谱显示4种弧菌外膜蛋白的分子量主要集中在22～48 kD之间;利用抗秦皇岛弧菌HQ010712-1血清的免疫印迹表明菌株HQ010712-1外膜蛋白中分子量为45 kD、36 kD的蛋白条带呈现阳性反应,其他3种弧菌外膜蛋白中均有与该抗血清反应的条带,且分子量为36 kD的反应带为菌株HQ010712-1、副溶血弧菌、溶藻胶弧菌共有.本研究旨在为进一步筛选和研究致病性弧菌的共同保护性抗原提供参考. 相似文献
4.
柱状黄杆菌(Flavobacterium columnare)是一种世界范围的水产动物致病菌,是中国重要养殖鱼类草鱼(Ctenopharyngodon idellus)、鳜(Siniperca chuatsi)等烂鳃病的病原。本研究以1972年从患"烂鳃病"草鱼上分离的两株冻干柱状黄杆菌G4和G18菌株为研究对象,并将G4株再次分离纯化得纯化菌株,命名为G4R3。对草鱼鱼苗浸泡攻毒结果显示,G4R3的LD50至少比G18的高3个数量级,因此G4R3为"强毒株",G18为"弱毒株"。利用蛋白质组学方法分析柱状黄杆菌强毒株G4R3和弱毒株G18的胞外蛋白,经过双向电泳并结合图像分析,共发现了34个点是差异表达的蛋白。胶内酶解、肽质量指纹图谱和串联质谱分析后,鉴定出其中的7个蛋白点,代表滑动蛋白K、腺酐甲硫氨酸合成酶和一种可能的膜蛋白等3种蛋白,它们可能是柱状黄杆菌的毒力因子。 相似文献
5.
R. L. DAVIES 《Journal of fish diseases》1991,14(5):563-570
Abstract. The expression of iron-regulated outer membrane proteins (OMPs) was examined in 36 isolates of Yersinia ruckeri selected to represent a range of biotypes, serotypes and OMP-types. Iron-restricted growth was achieved in tryptone soya broth by chelation with 200 μm α,α'-dipyridyl, although similar results could be achieved with 25 μm transferrin or 280 μM ethylenediaminedihydroxyphenylacetic acid (EDDA). During growth under iron-restricted conditions, all 36 isolates produced four additional outer membrane proteins of molecular weights 72, 69·5, 68 and 66 kDa. Expression of these proteins was repressed by the addition of 100 μm FeCl3 to the growth medium, confirming that the proteins were iron-regulated. The facts that all of the isolates examined possessed the same four iron-regulated OMPs, and that the plasmid content of different serotypes is known to vary indicated that production of these proteins is probably chromosomally mediated and not plasmid-mediated. Production of siderophores was not detected in low iron media and suggested a direct iron-uptake mechanism for this organism. 相似文献
6.
摘要：对已分离的1株致病性鳗弧菌W1外膜蛋白图谱进行SDS-PAGE分析，并与8株不同血清型的鳗弧菌外膜蛋白进行比较。结果表明，鳗弧菌W1主要外膜蛋白分别为24kD，38kD，42kD和47kD，主要外膜蛋白图谱与鳗弧菌O1血清型标准菌株VIB1相似。抗生素药敏试验表明该菌对氨苄青霉素、强力霉素、磺胺嘧啶等14种常用药物产生了抗性，只对新生霉素、呋喃妥因、利福平、新霉素等9种药物敏感。研究不同浓度的氨苄青霉素、强力霉素、磺胺嘧啶和庆大霉素对鳗弧菌外膜蛋白表达的影响。结果表明，氨苄青霉素、强力霉素和磺胺嘧啶明显地抑制鳗弧菌42kD的主要外膜蛋白的表达，随着抗菌素浓度的增加，该外膜蛋白的表达量逐渐减少甚至消失，而庆大霉素浓度的变化对其表达没有明显影响。对该42kD主要外膜蛋白进行N末端分析表明，其N末端序列为EAPTAINS，与已发表的细菌其他外膜蛋白序列没有同源性。 相似文献
7.
8.
The effect of iron limitation growth conditions on the cell and extracellular components of the fish pathogen Pasteurella piscicida 总被引：1，自引：0，他引：1
The effect of iron limitation, using the iron-chelating agent 2,2 dipyridyl, on the electrophoretic profiles of outer membrane proteins (OMPs) and extracellular products (ECPs) from 21 Pasteurella piscicida strains isolated from Europe and Japan was investigated. In addition, the effect of iron-limited and iron-surplus growth conditions on caseinase activity in culture supernatants of the pathogen was examined. The majority of P. piscicida strains, from Greece, Italy and France, cultured under iron-limited conditions, produced four novel OMPs (63 and three at and above 200 kDa). In contrast, iron-regulated outer membrane proteins were not induced in Japanese strains. Electrophoretic analysis of the ECPs from the pathogen grown under iron surplus and iron limitation revealed a large range of products and additional high molecular mass (MM) bands were evident under iron-limited conditions. When culture supernatants were analysed for their activity, most of the bacteria tested showed elevated activities under iron limited conditions. Finally, neither hydroxamate nor phenolate type siderophores could be detected with any of the chemical assays used. 相似文献
9.
Jang HB Kim YR Cha IS Noh SW Park SB Ohtani M Hikima J Aoki T Jung TS 《Journal of fish diseases》2011,34(7):555-562
Although the major capsid proteins (MCPs) of lymphocystis disease virus (LCDV) have been characterized, little is known about the host-derived immune response to MCPs and other LCDV antigenic proteins. To identify antigenic proteins of LCDV that could be used as vaccine candidates in olive flounder, Paralichthys olivaceus, we analysed the viral proteins responsible for its virulence by applying immuno-proteomics. LCDV proteins were separated by one-dimensional gel electrophoresis, transferred to polyvinylidene difluoride membrane, and probed with homogeneous P. olivaceus antisera elicited by LCDV natural infection and vaccination with formalin-killed LCDV. Four immune-reactive proteins were obtained at 68-, 51-, 41- and 21 kDa using antisera collected from natural infection while two proteins at 51- and 21 kDa exhibited response to antisera from vaccinated fish, indicating that the latter two proteins have vaccine potential. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray MS/MS, the 51 and 21 kDa proteins were identified as MCP and an unknown protein, respectively. 相似文献
10.
Edwardsiella tarda is a causative agent of edwardsiellosis in freshwater and marine fish. Extracellular enzymic, haemolytic, hydrophobic and serum resistance activities, haemagglutination, autoagglutination and siderophores of high‐ and low‐ virulent E. tarda strains were examined. The results revealed different haemagglutination, autoagglutination, haemolytic, hydrophobic and serum resistance activities in different strains. Analysis of extracellular proteins (ECPs) and outer membrane proteins (OMPs) demonstrated several major, low molecular weight, virulent‐strain‐specific proteins, which could be virulence‐related. Based on the database search with MALDI‐TOF MS data, the closest homologies of the three protein bands Ed1, Ed2 and Ed3 were phosphotransferase enzyme family protein, nitrite reductase [NAD(P)H], large subunit and ATP‐dependent Lon protease, respectively. A comparison of pathogenicity of purified lipopolysaccharide (LPS) and lipid A from virulent and avirulent strains demonstrated that LPS was one of the virulence factors of the E. tarda isolates, and lipid A was a biologically active determinant of LPS. 相似文献
11.
用十二烷基肌氨酸钠(Sarkosyl)抽提结合超速离心的方法提取了一株大菱鲆致病性溶藻弧菌(Vibrio alginolyticus)SR1和其他7株弧菌的外膜蛋白。通过SDS-PAGE图谱分析比较了这8株弧菌外膜蛋白的组成,结果表明,8株弧菌的外膜蛋白电泳一般可得到6-12条条带,其分子量多集中在65-106 kD和28-48 kD,其中36 kD的蛋白带为8株弧菌所共有。用兔抗SR1全菌血清进行Western-blot印迹显示,菌株SR1的外膜蛋白条带中有6条发生了阳性反应,其分子量分别为73 kD、48 kD4、5 kD3、9 kD、36 kD和32 kD。而其他7株弧菌的外膜蛋白与兔抗SR1血清也发生程度不等的阳性反应,这些阳性反应条带的分子量集中在65-73 kD、45-48 kD和36-41 kD之间,其中36 kD的外膜蛋白在8株弧菌中均出现明显的阳性反应,说明36 kD的外膜蛋白是这8株弧菌共有的特异性抗原。 相似文献
12.
Abdullateef Ajadi M. Y. Sabri A. B. Dauda M. Y. Ina-Salwany A. H. Hasliza 《Aquaculture International》2018,26(2):673-684
The use of antibiotics to curtail vibriosis, which is a major infectious disease, plaguing shrimp and prawn is rather becoming less effective and the need for a better alternative is expedient. The outer membrane proteins (OMPs) of V. alginolyticus were extracted, mixed with powdered commercial feed and fed to the prawns to evaluate its effect on growth performance and protective potential. Sixty prawns were divided into groups A, B and C of 10 prawns each, with two replicates in six (150 L) glass aquaria. Groups A, B and C were fed with OMPs mixed diet, with OMPs-Freund’s incomplete adjuvant mixed diet and OMPs or adjuvant free diet (control diet) respectively. All the prawns were weighed weekly, and haemolymph was collected to determine the total haemocyte count (THC) and phenoloxidase (PO) activity. At the end of the feeding trial, prawns were intramuscularly challenged with 50 μL of 107 CFU V. alginolyticus. The treated groups were significantly higher in growth performance and THC than the control group, but no significant difference between the groups in terms of PO activity and mortality rate. The study, however, submitted that oral administration of OMPs with or without adjuvant is a good growth promoter and has the potential for protection against vibriosis in giant freshwater prawn (Macrobrachium rosenbergii). 相似文献
13.
Subcellular components of the probiotics Aeromonas sobria GC2 and Bacillus subtilis JB-1, when administered to rainbow trout, Oncorhynchus mykiss , conferred protection against a new biogroup of Yersinia ruckeri . Thus, intraperitoneal or intramuscular injection of rainbow trout with cell wall proteins (CWPs), outer membrane proteins (OMPs), lipopolysaccharides (LPS), whole cell proteins (WCPs) and live cells followed by challenge on day 8 with Y. ruckeri led to 80–100% survival compared with 10% survival in the controls. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of WCPs and OMPs from GC2 had 10 and 5 variable protein bands in comparison to 11 and 5 bands in the WCPs and CWPs from JB-1. Proteomic analyses were employed following SDS-PAGE to categorize one dominant protein of 104.7 kDa from the CWPs of JB-1 and equated it with ' Bacillus spp. endoglucanase' with a Mascot score >69. These results point to the potential of using cellular components of probiotics for protection of fish against bacterial diseases. 相似文献
14.
Flavobacterium columnare is the causative agent of columnaris disease. The presence of lesions on the gills, skin and fins of diseased fish suggests that F. columnare is able to utilize fish skin mucus as a substrate for growth and that exposure to this material would alter the expression of genes involved in the colonization of the outer surfaces of the fish. Growth, biofilm formation, extracellular protease production and changes in protein expression of F. columnare strain C#2 cultured in media supplemented with juvenile Atlantic salmon skin mucus were compared with the same media without mucus. C#2 was able to grow by using mucus as the sole nutrient source. Growth in mucus-containing media induced cells to grow as a biofilm and extracellular protease activity increased in mucus-containing cultures. SDS-PAGE protein profiles showed that expression of six extracellular proteins increased in mucus-containing media. These results demonstrate that salmon surface mucus promotes the growth of F. columnare and that exposure to mucus alters the growth characteristics of this bacterium with regard to protease production and biofilm formation. Further characterization of mucus-induced physiological changes will increase our understanding of the basis of virulence of this economically important fish pathogen. 相似文献
15.
Swati Sucharita Dash Basanta Kumar Das Phalguni Pattnaik Surya Kanta Samal Swagatika Sahu Subrato Ghosh 《Journal of the World Aquaculture Society》2009,40(2):236-247
Flavobacterium columnare is an important pathogen of freshwater fishes often causing high mortality. Seven strains of F. columnare have been isolated from gill necrosis, skin lesions and internal organs of Catla catla, Labeo rohita, Cirrhinus mrigala, Carassius auratus, Anabas testudineus and Clarias batrachus and characterized by biochemical reactions and serological tests viz indirect enzyme‐linked immunosorbent assay (ELISA), Dot‐ELISA, agglutination test. All the strains showed binding to Congo red dye as well as hemolysis. All the seven strains were susceptible to amikacin, gentamycin and ofloxacin. In vivo LD50 dose of virulent strain MS2 was found to be 6 × 104 CFU/mL after experimental infection to L. rohita. Serologically all the seven strains showed a positive result to agglutination, Dot‐ELISA and indirect ELISA. The agglutination titer was found to be in the range of 256‐131,072. The lysozyme activity of hyperimmune sera was found to be 39.37 ± 0.80 units/mL. This is the first extensive study that report about the various strains of F. columnare from freshwater fishes of Eastern India and their differentiation by biochemical and serological methods. 相似文献
16.
The early entry of the fish pathogen Flavobacterium columnare and enhancement by abrasion was studied in channel catfish, Ictalurus punctatus (Rafinesque), using the polymerase chain reaction and a species-specific primer set for a bacterial 16S rRNA gene product. Evaluations were conducted following an abrasion bath immersion challenge with F. columnare. Abrasion, a practice which has historically been used prior to bacterial challenge, had significant effects on the early entry of the pathogen and on cumulative percent survival (CPS). The FvpF1-FvpR1 primer set was useful in detecting the early entry of F. columnare in mucus, skin, gill, blood, liver and trunk kidney tissues in both abraded and unabraded fish following immersion challenge at 29 +/- 2 degrees C. Bacteria were detected earlier in all tissues in abraded fish, except in the trunk kidney. These differences were not significant, except in the case of blood. Mucus, skin and gill tissues were positive for F. columnare earliest regardless of treatment (after 5 min in abraded fish and after 15 min in unabraded fish). CPS following challenge with F. columnare was significantly affected by abrasion, which supports the use of abrasion for the F. columnare challenge model for channel catfish. 相似文献
17.
Lipopolysaccharide (LPS) and total protein profiles from four Flavobacterium columnare isolates were compar. These strains belonged to genetically different groups and/or presented distinct virulence properties. Flavobacterium columnare isolates ALG-00-530 and ARS-1 are highly virulent strains that belong to different genomovars while F. columnare FC-RR is an attenuated mutant used as a live vaccine against F. columnare. Strain ALG-03-063 is included in the same genomovar group as FC-RR and presents a similar genomic fingerprint. Electrophoresis of LPS showed qualitative differences among the four strains. Further analysis of LPS by immunoblotting revealed that the avirulent mutant lacks the higher molecular bands in the LPS. Total protein analysis displayed by immunoblotting showed differences between the strains analysed although common bands were present in all the isolates. FC-RR lacked two distinct common bands (34 and 33 kDa) shared by the other three isolates. Based on the difference of LPS and total protein profiles, it is possible to discriminate the attenuated mutant FC-RR from other F. columnare strains. 相似文献
18.
Figueiredo HC Klesius PH Arias CR Evans J Shoemaker CA Pereira DJ Peixoto MT 《Journal of fish diseases》2005,28(4):199-204
Flavobacterium columnare is an important pathogen of freshwater fish, implicated in skin and gill disease, often causing high mortality. An outbreak of skin disease in fingerling and adult Nile tilapia, Oreochromis niloticus (L.), cultivated in a recirculation system, was investigated. Four strains were isolated and characterized by biochemical reactions, enzyme production, fatty acid profile and analysis of the 16S-23S rDNA intergenic spacer region. All strains were identified as F. columnare. Experimental infection assays with one of these strains (BZ-5-02) were conducted and pathogenicity (by intramuscular route) was demonstrated in Nile tilapia and channel catfish, Ictalurus punctatus (Rafinesque). This is the first report of characterization of Brazilian strains of F. columnare. 相似文献
19.
A trial was performed to evaluate the efficacy of diquat (6,7-dihydrodipyrido[1,2-a:2',1'-c]pyrazinediium dibromide) against an acute experimental infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus . Diquat is an Environmental Protection Agency-approved herbicide and has the potential to be legally and practically used against columnaris. Channel catfish were challenged, by cutaneous abrasion, and waterborne exposure to F. columnare and treated once at 22-h post-challenge with 2.5, 5.0, 10.0 and 15 mg L−1 of diquat active ingredient for 6 h. At the conclusion of the trial, 21-day post-challenge, diquat at 5.0, 10.0 and 15 mg L−1 significantly ( P < 0.05) reduced the mortality of infected fish from 95% in the challenged non-treated fish to 68%, 59% and 49%, respectively. In vitro , the minimum inhibitory concentration (MIC) of 23 isolates of F. columnare was assayed. The majority of the isolates had an MIC value of 5 μg mL−1 (15 of the 23 isolates). Infected fish exhibited acute clinical signs similar to a natural infection. The skin had severe ulcerative necrotizing dermatitis and the muscles had severe necrotizing myositis. The gills had severe multifocal necrotizing branchitis. The results demonstrate that diquat would reduce mortalities caused by an acute columnaris infection. 相似文献