Salt-responsive outer membrane proteins of Vibrio anguillarum serotype O1 as revealed by comparative proteome analysis

Aims:  Vibrio anguillarum is a universal marine pathogen causing vibriosis. Vibrio anguillarum encounters different osmolarity conditions between seawater and hosts, and its outer membrane proteins (OMPs) play a crucial role in the adaptation to changes of the surroundings. In the present study, proteomic approaches were applied to investigate the salt-responsive OMPs of V. anguillarum .
Methods and Results:  Lower salinity (0·85% NaCl) is more suitable for growth, survival and swimming motility of the bacterium. Comparative two-dimensional electrophoresis (2-DE) analysis reveals six differentially expressed protein spots among three different salinities, which were successfully identified as OmpU, maltoporin, flagellin B, Omp26La, Omp26La and OmpW respectively.
Conclusions:  OmpW and OmpU were highly expressed at 3·5% salinity, suggesting their role in the efficient efflux of NaCl. Maltoporin was downregulated in higher salinity, indicating that higher osmolarity inhibits carbohydrate transport and bacterial growth. Omp26La, the homologue of OmpV, functions as a salt-responsive protein in lower salinity.
Significance and Impact of the Study:  To the best of our knowledge, this is the first report describing salt stress-responsive proteins of V. anguillarum using proteomic approaches. Our results provide a useful strategy for delineating the osmoregulatory mechanism of the marine pathogens.     

A ToxR homolog from Vibrio anguillarum serotype O1 regulates its own production, bile resistance, and biofilm formation

ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.     

Variation of virulence and other properties among Sendai virus strains

The virulence of five Sendai virus strains (MN, Z, KN, Mol, and Hm) isolated from laboratory rodents was compared, using 3-week-old female Jcl-ICR mice. The virulence of the strains was Mol, MN, KN, Z, and Hm in decreasing order. The 50% lethal dose and 50% lung consolidation inducing dose of the highest virulent strain differed by the order of more than 10(3) and 10(6), respectively, from those of the lowest virulent one. Other properties such as the growth rate in LLC-MK2 cells, neuraminidase activities, and molecular weights of structural proteins also differed among the virus strains. These results indicate that Sendai virus prevailing in laboratory rodents is not homogenous with respect to virulence and some other properties.     

利用抑制性消减杂交（SSH）技术研究不同毒力的鳗弧菌菌株的基因多样性

[目的和方法]鳗弧菌是一种嗜盐的革兰氏阴性细菌,也是鱼类弧菌病的主要病原.对斑马鱼的半数致死量研究结果表明,鳗弧菌菌株VIB72具有较高的毒力,而菌株CW1的毒力较低.本文利用抑制性消减杂交(SSH)技术对这两个菌株的遗传差异进行了研究.[结果]通过对差减文库筛选,分离到59个对菌株VIB72的阳性克隆,并对这些克隆的DNA序列进行了测定.17个基因片断与其它细菌的已知功能的基因有较高的同源性,其中包括可溶性溶胞壁质转糖基酶、转移蛋白MobA和MobC、转座子IS66、抑制相关蛋白(金属β-内酰胺酶和乙酰转移酶家族)、毒素蛋白(DT-201和alveicin A免疫蛋白)、与OLD家族相似的ATP依赖性核酸内切酶以及SocE和GTP结合蛋白HflX(有高频率的溶原化).这些基因片断有可能是鳗弧菌毒力岛的一部分.其他的基因片断与其它的已知基因没有明显的相关性.[结论]这些结果表明,SSH技术成功地鉴定了不同致病性的鳗弧菌菌株的基因差异及潜在的毒力基因.     

Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium Vibrio anguillarum

Surface display of heterologous peptides and proteins such as receptors, antigens, and enzymes on live bacterial cells is of considerable value for various biotechnological and industrial applications. In this study, a series of novel cell surface display systems were examined by using Vibrio anguillarum outer membrane protein and outer membrane lipoprotein as anchoring motifs. These display systems consist of (i) the signal sequence and first 11 N-terminal amino acids of V. anguillarum outer membrane lipoprotein Wza, or the signal sequence and first 9 N-terminal amino acids of the mature major Escherichia coli lipoprotein Lpp, and (ii) transmembrane domains of V. anguillarum outer membrane proteins Omporf1, OmpU, or Omp26La. In order to assay the translocation efficiency of constructed display systems in bacteria, green fluorescent protein (GFP) was inserted to the systems and the results of GFP surface localization confirmed that four of the six surface display systems could successfully display GFP on the E. coli surface. For assaying its potential application in live bacteria carrier vaccines, an excellent display system Wza-Omporf1 was fused with the major capsid protein (MCP) of large yellow croaker iridovirus and introduced into attenuated V. anguillarum strain MVAV6203, and subsequent analysis of MCP surface localization proved that the novel display system Wza-Omporf1 could function as a strong tool in V. anguillarum carrier vaccine development.     

Role of the <Emphasis Type="Italic">toxR</Emphasis> Gene from Fish Pathogen <Emphasis Type="Italic">Vibiro alginolyticus</Emphasis> in the Physiology and Virulence

A mutant strain of Vibiro alginolyticus with an in-frame deletion of the toxR gene was constructed to reveal the role of ToxR in the physiology and virulence of V. alginolyticus. The statistical analysis showed no significant difference in the growth ability, swarming motility, activity of extracellular protease and the virulence by injection (the value of LD₅₀) between the wild-type and the toxR mutant. However, the deletion of toxR could decrease the level of biofilm formation. The comparative proteomic analysis demonstrated the deletion mutation of toxR could up-regulate the expression of glutamine synthetase and levansucrase, and down-regulate the expression of 10 proteins such as OmpU, DnaK, etc. These results suggest that ToxR may be involved in the early stages of infection by influencing colonization of the bacteria on the surface of the intestine through enhancing the biofilm information of V. alginolyticus via modulating the expression of glutamine synthetize, levansucrase and OmpU.     

Structural and functional importance of outer membrane proteins in Vibrio cholerae flagellum

Vibrio cholerae has a sheath-covered monotrichous flagellum that is known to contribute to virulence. Although the structural organization of the V. cholerae flagellum has been extensively studied, the involvement of outer membrane proteins as integral components in the flagellum still remains elusive. Here we show that flagella produced by V. cholerae O1 El Tor strain C6706 were two times thicker than those from two other Gram-negative bacteria. A C6706 mutant strain (SSY11) devoid of two outer membrane proteins (OMPs), OmpU and OmpT, produced thinner flagella. SSY11 showed significant defects in the flagella-mediated motility as compared to its parental strain. Moreover, increased shedding of the flagella-associated proteins was observed in the culture supernatant of SSY11. This finding was also supported by the observation that culture supernatants of the SSY11 strain induced the production of a significantly higher level of IL-8 in human colon carcinoma HT29 and alveolar epithelial A549 cells than those of the wild-type C6706 strain. These results further suggest a definite role of these two OMPs in providing the structural integrity of the V. cholerae flagellum as part of the surrounding sheath.     

Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and a non-virulent strain of the fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS). To identify antigens associated with virulence or host immunity, we compared total and immunogenic proteins of cellular and extracellular products (ECP) between a virulent (CSF-259-93) and non-virulent (ATCC 49418) strain of F. psychrophilum. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis of total cellular proteins revealed only minor differences between the strains; however, separation of ECP showed that proteins were differentially expressed. Western blot analysis using rainbow trout (Oncorhynchus mykiss) anti-CSF-259-93 sera showed greater reactivity to proteins of the virulent strain, including many > 50 kDa. Further analysis by 2-dimensional electrophoresis (2DE) identified numerous differences between the strains. Western blot analysis combined with 2DE identified several immunogenic proteins that reacted with the antisera and were shared between the 2 strains. However, at least 15 immunogenic proteins appeared to be unique to the virulent strain, while 4 such proteins were identified in the non-virulent strain; 8 proteins unique to the virulent strain and 6 shared proteins were further analyzed for identification by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Of these, 3 immunogenic proteins (heat shock proteins HSP 60 and HSP 70) and 2 other proteins (ATP synthase and thermolysin) were conclusively identified. The 2 highly immunogenic heat shock proteins were shown to share extensive homology with heat shock proteins of related bacteria. This approach for antigen identification may provide a basis for targeted vaccine development against CWD and RTFS.     

Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids,outer membrane protein profiles and siderophore production

The virulence of 18 strains of Vibrio anguillarum serogroup O1 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only strains that carried the 67 kbp virulence plasmid or derivatives of it produced the outer membrane protein, OM2. All virulent strains harboured the 67 kbp plasmid or derivatives of it, indicating its importance for virulence. However, some strains carrying the virulence plasmid or a derivative of it, produced siderophores as well as OM2 but were non-pathogenic to fish. Likewise, among the virulent strains, considerable variation in LD50 values was recorded. Plasmid profiling and restriction analysis showed that the virulence plasmid existed in various molecular weights from 26 to 80 kbp, with 65-67 kbp being the most common, and that this plasmid displayed various restriction profiles. The presence of other plasmids did not seem to affect the pathogenic properties.     

Comparative proteome analysis of fractions enriched for membrane-associated proteins from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica strains

The facultative intracellular pathogen Francisella tularensis is the causative agent of the serious infectious disease tularemia. Despite intensive research, the virulence factors and pathogenetic mechanisms remain largely unknown. To identify novel putative virulence factors, we carried out a comparative proteome analysis of fractions enriched for membrane-associated proteins isolated from the highly virulent subspecies tularensis strain SCHU S4 and three representatives of subspecies holarctica of different virulence including the live vaccine strain. We identified six proteins uniquely expressed and four proteins expressed at significantly higher levels by SCHU S4 compared to the ssp. holarctica strains. Four other protein spots represented mass and charge variants and seven spots were charge variants of proteins occurring in the ssp. holarctica strains. The genes encoding proteins of particular interest were examined by sequencing in order to confirm and explain the findings of the proteome analysis. Our studies suggest that the subspecies tularensis-specific proteins represent novel potential virulence factors.     

Evidence for the role of a siderophore in promoting Vibrio anguillarum infections

Vibrio anguillarum strain 775 harbouring the virulence plasmid pJM1 produces a plasmid-mediated siderophore that can crossfeed siderophore-deficient, receptor-proficient mutants of V. anguillarum in vitro. Experimental infections of salmonid fishes with mixtures consisting of the wild-type strain and a siderophore-deficient, receptor-proficient mutant resulted in recovery of both the wild-type and the mutant strain, while in infections with mixtures consisting of the wild-type strain and a siderophore-deficient, receptor-deficient mutant only the wild-type strain could be recovered. These results suggest that the V. anguillarum plasmid-mediated siderophore is produced in vivo in a diffusible form and that it is an important factor of virulence.     

Porins of Vibrio cholerae: purification and characterization of OmpU.

Three outer membrane proteins with molecular masses of 40, 38, and 27 kDa of the hypertoxinogenic strain 569B of Vibrio cholerae have been purified to homogeneity. The synthesis of all the three proteins is regulated by the osmolarity of the growth medium. The pore-forming ability of the 40-kDa protein, OmpT, and the 38-kDa protein, OmpU, has been demonstrated by using liposomes, in which these proteins were embedded. The 27-kDa protein, OmpX, though osmoregulated, is not a porin. OmpU constitutes 30% of the total outer membrane protein when grown in the presence of 1.0% NaCl in the growth medium and 60% in the absence of NaCl. OmpU is an acidic protein and is a homotrimer of 38-kDa monomeric units. Its secondary structure contains predominantly a beta-sheet, and three to four Ca2+ ions are associated with each monomeric unit. Removal of Ca2+ irreversibly disrupts the structure and pore-forming ability of the protein. The pore size of OmpU is 1.6 nm, and the specific activity of the OmpU channel is two- to threefold higher than that of Escherichia coli porin OmpF, synthesis of which resembles that of OmpU with respect to the osmolarity of the growth medium. The pore size of OmpT, which is analogous to OmpC of E. coli, is smaller than that of OmpU. Southern blot hybridization of V. cholerae genomic DNA digested with several restriction endonucleases with nick-translated E. coli ompF as the probe revealed no nucleotide sequence homology between the ompU and ompF genes. OmpU is also not antigenically related to OmpF. Anti-OmpF antiserum, however, cross-reacted with the 45-kDa V. cholerae outer membrane protein, OmpS, the synthesis of which is regulated by the presence of maltose in the growth medium. OmpU hemagglutinated with rabbit and human blood. This toxR-regulated protein is one of the possible virulence determinants in V. cholerae (V. L. Miller and J. J. Mekalanos, J. Bacteriol. 170:2575-2583, 1988).     

Secretory delivery of heterologous proteins in attenuated <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> for potential use in vaccine design

To synthesize and secrete heterologous proteins in an attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different antigen-delivery systems based on bacterial-originated secretion signal peptides (SPs) were designed and identified in this work. Four SPs were derived from hemolysin of Escherichia coli, RTX protein of V. cholerae, hemolysin of V. anguillarum, zinc-metalloprotease of V. anguillarum, respectively, and their abilities to support secretion of green fluorescent protein (GFP) in an attenuated V. anguillarum strain MVAV6203 were assayed. Immunodetection of GFP showed that the capability of the tested signal leaders to direct secretion of GFP varied greatly. Although all the four signal peptide-fused GFPs could be expressed correctly and trapped intracellularly in recombinant strains, only the EmpA signal peptide could confer efficient secretion to GFP. For the investigation of its potential application in live bacteria carrier vaccines, a heterologous protein EseB of Edwardsiella tarda was fused to the SP(empA) antigen-delivery system and introduced into the strain MVAV6203. Further analysis of EseB demonstrated that the constructed SP(empA) antigen-delivery system could be used to secrete foreign protein in attenuated V. anguillarum and be available for carrier vaccines development.     

Metal content and biochemical analyses of a recombinant collagenase PrtV from Vibrio parahaemolyticus

PrtV is an extracellular metalloprotease of Vibrio parahaemolyticus and regarded as a collagenase. Inductively coupled plasma-optical emission spectrometry analysis indicated that the recombinant PrtV contains 1 mol of zinc per mol of the native enzyme. On the basis of a kinetic study using 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA, the specific substrate for bacterial collagenase) as a substrate, it was suggested that metal ions may play a significant role in the binding and catalytic steps of the substrate. PrtV hydrolyzed type I, II, III, and IV collagens; however, it did not hydrolyze type V. In addition, the hydrolysis of native proteins and synthetic substrates revealed that PrtV possesses higher activity toward collagen and collagen-like sequences. The result of the thermal stability study indicated that PrtV was thermostable up to 40 C; at 50 C, stability gradually decreased. In addition, PrtV showed higher storage stability at -20 and 4 C, respectively, than at 25 C. Compared with collagenases from Clostridium histolyticum and Vibrio alginolyticus, PrtV was immunologically different and had no significant effect on the growth of CHO, HeLa, and Vero cells. Taken together, the results of the studies described in this paper advance our knowledge concerning the metal content and biochemical properties of PrtV.     

A ferret model of canine distemper virus virulence and immunosuppression

Canine distemper virus (CDV) infects many carnivores, including ferrets and dogs, and is the member of the Morbillivirus genus most easily amenable to experimentation in a homologous small-animal system. To gain insights into the determinants of CDV pathogenesis, we isolated a strain highly virulent for ferrets by repeated passaging in these animals. Sequence comparison of the genome of this strain with that of its highly attenuated precursor revealed 19 mutations distributed almost evenly in the six genes. We then recovered a virus from a cDNA copy of the virulent CDV strain's consensus sequence by using a modified reverse genetics system based on B cells. We infected ferrets with this virus and showed that it fully retained virulence as measured by the timing of rash appearance, disease onset, and death. Body temperature, leukocyte number, lymphocyte proliferation activity, and cell-associated viremia also had similar kinetics. We then addressed the question of the relative importance of the envelope and other viral constituents for virulence. Viruses in which the envelope genes (matrix, fusion, and hemagglutinin) of the virulent strain were combined with the other genes of the attenuated strain caused severe rash and fever even if the disease onset was delayed. Viruses in which the nucleocapsid, polymerase, and phosphoprotein genes (coding also for the V and C proteins) of the virulent strain were combined with the envelope genes of the attenuated strain caused milder signs of disease. Thus, virulence-inducing mutations have accumulated throughout the genome.     

弧菌外膜蛋白OmpU的免疫交叉反应性和交叉保护性北大核心CSCD

【目的】通过对弧菌外膜蛋白Omp U的克隆、表达以及免疫学特性分析,明确外膜蛋白Omp U是否为弧菌的共同抗原,并具有免疫交叉反应性和交叉保护性。【方法】对弧菌外膜蛋白omp U基因进行克隆和生物信息学分析。分别制备副溶血弧菌、溶藻弧菌、创伤弧菌、拟态弧菌和霍乱弧菌的Omp U重组蛋白抗血清,对Omp U的免疫交叉反应特性以及抗原表位定位情况进行比较分析。以霍乱弧菌的Omp U重组蛋白免疫小鼠后,再以多种弧菌进行攻毒,分析其交叉免疫保护作用。【结果】外膜蛋白Omp U在弧菌种内和种间相似性分别为73.0%–100%和58.6%–89.0%,并至少存在9个保守的B细胞抗原表位。Omp U重组蛋白抗血清在弧菌种内和种间均产生显著的免疫交叉反应,识别弧菌中分子量35–40 k Da的同源蛋白。副溶血弧菌ATCC17802、创伤弧菌ATCC27562和拟态弧菌ATCC33653来源的Omp U重组蛋白抗体能识别供试菌株,提示这些菌株的Omp U抗原表位定位于细胞表面。Omp U重组蛋白对免疫后的小鼠具有交叉免疫保护作用,攻毒实验后小鼠相对存活率(RPS)为43.0%–100%。【结论】上述结果表明,外膜蛋白Omp U是弧菌中一种保守的共同抗原,具有免疫交叉保护性,可以作为弧菌广谱疫苗的候选抗原。