OmpW and OmpV are required for NaCl regulation in Photobacterium damsela

Photobacterium damsela is a marine pathogen to both fish and human beings. The bacterium can shift between the ambient seawater and hosts, suggesting the existence of proteins rapidly responding to salt concentration. In the current study, proteomic methodologies were applied to screen the outer membrane proteins (OMPs) related to salt stress. OmpW and OmpV were determined in the response in this bacterium as OmpC and OmpF did in E. coli. Furthermore, the two genes were overexpressed in E. coli Top10F and complemented in V. paraheamolyticus mutants. The ability in salt-tolerance was elevated in the E. coli overexpressed OmpW and reduced in the cells overexpressed OmpV. These V. paraheamolyticus mutants could recover their response to environmental salt concentration when they were complemented by P. damsela OmpW and OmpV. These findings indicate that OmpW and OmpV are required for environmental salt regulation in P. damsela, in which OmpW and OmpV, respectively, elevate and reduce the ability in salinity-tolerance.     

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.     

Proteomic analysis reveals responsive proteins of Vibrio parahaemolyticus on exposure to cationic antimicrobial peptides

Aims: To investigate whether Vibrio parahaemolyticus can sense and directly respond to the presence of cationic antimicrobial peptides (AMPs). Methods and Results: We performed proteomic methodologies to investigate the responsive proteins of V. parahaemolyticus on exposure to AMP Q6. Differential expression patterns of the outer membrane, inner membrane and cytoplasmic proteins (OMPs, IMPs and CPs) from the bacteria with or without Q6 treatment were obtained using two‐dimensional gel electrophoresis (2‐DE). Three OMPs (maltoporin, flagellin and OmpV), two IMPs (ATP synthase F1, alpha subunit; and OmpV) and three CPs (pyruvate dehydrogenase subunit E1, glyceraldehyde‐3‐phosphate dehydrogenase and inositol‐5‐monophosphate dehydrogenase) were identified using LC‐ESI‐Q‐TOF MS/MS and Mascot program. Real‐time quantitative polymerase chain reaction was also performed to determine the mRNA expression level of the target genes. Conclusions: Our results suggested that V. parahaemolyticus may directly respond to AMPs through the upregulation of the efflux channel, increased yield of energy, effective repair of damaged membranes and downregulation of carbohydrate and nucleotide metabolism for energy preservation. Significance and Impact of the Study: AMP‐responsive proteins identified in the current study could serve as attractive targets for developing more effective antimicrobial agents.     

Cloning, expression and immunogenicty analysis of five outer membrane proteins of Vibrio parahaemolyticus zj2003

Genes of five outer membrane proteins of Vibrio parahaemolyticus zj2003, including OmpW, OmpV, OmpK, OmpU and TolC, were cloned and expressed as N-terminal His(6)-tagged proteins in Escherichia coli. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of these proteins, large yellow croaker (Pseudosciaena crocea) were immunized by intraperitoneal injection. Antibody response was assessed by method of enzyme-linked immunosorbent assay. Titres to all five recombinant proteins increased during 4 to 8 weeks post immunization, within the range of log 2 values of 5.75 to 10.8. Recorded relative survival percent (RPS) of the vaccinated groups varied from 80% to 90%, while 10 fish in control group all died. Western blot tests were undertaken with the serum of survival fish after experimental infection. Except for recombinant TolC, the other four recombinant proteins were recognized by the serum. It is indicated that four outer membrane proteins of V. parahaemolyticus zj2003, including OmpW, OmpV, OmpU and OmpK, are immunogenic during in vivo infection, which would be of some significance in developing efficient vaccine in aquaculture. This is the first report of successful vaccination against V. parahaemolyticus with purified recombinant outer membrane proteins.     

The outer membrane protein OmpW forms an eight-stranded beta-barrel with a hydrophobic channel

Escherichia coli OmpW belongs to a family of small outer membrane proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. To gain insight into the function of these proteins A we have determined the crystal structure of E. coli OmpW to 2.7-A resolution. The structure shows that OmpW forms an 8-stranded beta-barrel with a long and narrow hydrophobic channel that contains a bound n-dodecyl-N,N-dimethylamine-N-oxide detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of n-dodecyl-N,N-dimethylamine-N-oxide. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial outer membrane.     

Systematic identification of the subproteome of Escherichia coli cell envelope reveals the interaction network of membrane proteins and membrane-associated peripheral proteins

Membrane proteins of Gram-negative bacteria are key molecules that interface the cells with the environment. Despite recent proteomic identification of numerous oligomer proteins in the Escherichia coli cell envelope, the protein complex of E. coli membrane proteins and their peripherally associated proteins remain ill-defined. In the current study, we systematically analyze the subproteome of E. coli cell envelope enriched in sarcosine-insoluble fraction (SIF) and sarcosine-soluble fraction (SSF) by using proteomic methodologies. One hundred and four proteins out of 184 spots on 2D electrophoresis gels are identified, which includes 31 outer membrane proteins (OMPs). Importantly, our further proteomic studies reveal a number of previously unrecognized membrane-interacting protein complexes, such as the complex consisting of OmpW and fumarate reductase. This established complete proteomic profile of E. coli envelope also sheds new insight into the function(s) of E. coli outer envelope.     

Serological properties and processing in Escherichia coli K12 of OmpV fusion proteins of Vibrio cholerae

Summary Fusion proteins comprising the amino-terminal 99 amino acids of the bacteriophage MS2 replicase and various portions of OmpV a major outer membrane protein of Vibrio cholerae were expressed in Escherichia coli K12. These fusions were expressed under the control of the P_L promoter of bacteriophage , and expression was controlled using a cI_ts repressor. Fusions occurring within the secretory signal sequence of OmpV gave rise to the production of mature OmpV. The efficiency, however, decreased with progressive deletion of the signal sequence within the fusions. The reactivity of various OmpV fusions with antisera raised against purified OmpV and whole bacteria demonstrated the existence of two antigenic domains: one present in the denatured form and another in the membrane-associated form of OmpV. These domains correspond to markedly hydrophilic regions of the protein as would be predicted for surface-exposed epitopes.     

Discrimination of outer membrane proteins using support vector machines

MOTIVATION: Discriminating outer membrane proteins from other folding types of globular and membrane proteins is an important task both for dissecting outer membrane proteins (OMPs) from genomic sequences and for the successful prediction of their secondary and tertiary structures. RESULTS: We have developed a method based on support vector machines using amino acid composition and residue pair information. Our approach with amino acid composition has correctly predicted the OMPs with a cross-validated accuracy of 94% in a set of 208 proteins. Further, this method has successfully excluded 633 of 673 globular proteins and 191 of 206 alpha-helical membrane proteins. We obtained an overall accuracy of 92% for correctly picking up the OMPs from a dataset of 1087 proteins belonging to all different types of globular and membrane proteins. Furthermore, residue pair information improved the accuracy from 92 to 94%. This accuracy of discriminating OMPs is higher than that of other methods in the literature, which could be used for dissecting OMPs from genomic sequences. AVAILABILITY: Discrimination results are available at http://tmbeta-svm.cbrc.jp.     

Characterization of Francisella tularensis outer membrane proteins

Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs.     

Proteomic studies highlight outer‐membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer‐membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer‐membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB‐dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT‐PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.     

大黄鱼三种病原弧菌外膜蛋白交叉保护性抗原筛选

弧菌是海水养殖环境中常见的条件性致病菌,弧菌病的暴发给水产养殖业造成了严重损失。鉴于水生动物尤其是鱼类弧菌病的发生常常是多种（血清型或亚种）弧菌的混合感染,筛选具有潜在的交叉保护性蛋白抗原,作为制备多价疫苗或联合疫苗的侯选成分具有重要意义。文中从患病大黄鱼中分离到8株弧菌,经生理生化和分子生物学鉴定分别为6株哈维氏弧菌Vibrio harveyi,1株溶藻弧菌Vibrio alginolyticus和1株副溶血弧菌Vibrio parahaemolyticus。选择典型的不同种的弧菌为代表,提取其外膜蛋白,经SDS-PAGE和Westernblotting分析,确定它们大约在45 kDa、35 kDa、22 kDa处出现了3条共同的免疫印迹条带,表明它们很有可能含有共同的能够彼此交叉保护的抗原。利用双向电泳和免疫印迹相结合的方法,借助于MALDI-TOF-MS质谱分析技术,发现溶藻弧菌V.alginolyticus的一种功能未知的孔蛋白（Porin,GenBank Accession No.ZP₀1260407）和副溶血弧菌V.parahaemolyticus的一种麦芽糖孔蛋白的前体蛋白（Maltoporin precursor,GenBank AccessionNo.NP₈01154）能够和哈维氏弧菌V.harveyi全菌多抗产生免疫反应,表明这两种蛋白可以作为3种弧菌的交叉保护性抗原,以此制备的疫苗可望对3种弧菌的感染产生交叉保护作用。     

使用多特征联合变量的支持向量机方法预测外膜蛋白

外膜蛋白(Outer Membrane Proteins, OMPs)是一类具有重要生物功能的蛋白质, 通过生物信息学方法来预测OMPs能够为预测OMPs的二级和三级结构以及在基因组发现新的OMPs提供帮助。文中提出计算蛋白质序列的氨基酸含量特征、二肽含量特征和加权多阶氨基酸残基指数相关系数特征, 将三类特征组合, 采用支持向量机(Support Vector Machine, SVM)算法来识别OMPs。计算了包括四种残基指数的多种组合特征的识别结果, 并且讨论了相关系数的阶次和权值对预测性能的影响。在数据集上的十倍交叉验证测试和独立性测试结果显示, 组合特征识别方法对OMPs和非OMPs的识别精度最高分别达到96.96%和97.33%, 优于现有的多种方法。在五种细菌基因组内识别OMPs的结果显示, 组合特征方法具有很高的特异性, 并且对PDB数据库中已知结构的OMPs识别准确度超过99%。表明该方法能够作为基因组内筛选OMPs的有效工具。     

Beta-barrel proteins that reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro

Little is known about the dynamic process of membrane protein folding, and few models exist to explore it. In this study we doubled the number of Escherichia coli outer membrane proteins (OMPs) for which folding into lipid bilayers has been systematically investigated. We cloned, expressed, and folded nine OMPs: outer membrane protein X (OmpX), OmpW, OmpA, the crcA gene product (PagP), OmpT, outer membrane phospholipase A (OmpLa), the fadl gene product (FadL), the yaet gene product (Omp85), and OmpF. These proteins fold into the same bilayer in vivo and share a transmembrane beta-barrel motif but vary in sequence and barrel size. We quantified the ability of these OMPs to fold into a matrix of bilayer environments. Several trends emerged from these experiments: higher pH values, thinner bilayers, and increased bilayer curvature promote folding of all OMPs. Increasing the incubation temperature promoted folding of several OMPs but inhibited folding of others. We discovered that OMPs do not have the same ability to fold into any single bilayer environment. This suggests that although environmental factors influence folding, OMPs also have intrinsic qualities that profoundly modulate their folding. To rationalize the differences in folding efficiency, we performed kinetic and thermal denaturation experiments, the results of which demonstrated that OMPs employ different strategies to achieve the observed folding efficiency.     

Expression, characterization and immunogenicity of a major outer membrane protein from Vibrio alginolyticus

Vibrio alginolyticus is one of the Vibrio pathogens common to humans and marine animals.During infection and induction of the host immune response,outer membrane proteins of bacteria play animportant role.In this study,an outer membrane protein gene(ompW)was cloned from V.alginolyticus andexpressed in Escherichia coli.The 645 bp open reading frame(ORF)encodes a protein of 214 amino acidresidues with a predicted molecular weight of 23.3 kDa.The amino acid sequence showed a high identitywith that of Photobacterium damselae(96.2%)and Vibrio parahaemolyticus(94.4%).The alignment analy-sis indicated that OmpW was highly conserved.Sodium dodecyl sulfate-polyacrylamide gel electrophoresisshowed that the gene was over-expressed in E.coli BL21(DE3).Western blot analysis revealed that theexpressed protein had immunoreactivity.The recombinant protein was purified by affinity chromatographyon Ni-NTA Superflow resin.Large yellow croaker vaccinated with the purified OmpW showed significantlyincreased antibody to OmpW,which could resist the infection by V.alginolyticus.A specific antibody wasdetected by enzyme-linked immunosorbent assay.This study suggested that the conserved OmpW could bean effective vaccine candidate against infection by V.alginolyticus.     

使用多特征联合变量的支持向量机方法预测外膜蛋白

外膜蛋白(Outer Membrane Proteins, OMPs)是一类具有重要生物功能的蛋白质, 通过生物信息学方法来预测OMPs能够为预测OMPs的二级和三级结构以及在基因组发现新的OMPs提供帮助。文中提出计算蛋白质序列的氨基酸含量特征、二肽含量特征和加权多阶氨基酸残基指数相关系数特征, 将三类特征组合, 采用支持向量机(Support Vector Machine, SVM)算法来识别OMPs。计算了包括四种残基指数的多种组合特征的识别结果, 并且讨论了相关系数的阶次和权值对预测性能的影响。在数据集上的十倍交叉验证测试和独立性测试结果显示, 组合特征识别方法对OMPs和非OMPs的识别精度最高分别达到96.96%和97.33%, 优于现有的多种方法。在五种细菌基因组内识别OMPs的结果显示, 组合特征方法具有很高的特异性, 并且对PDB数据库中已知结构的OMPs识别准确度超过99%。表明该方法能够作为基因组内筛选OMPs的有效工具。     

Identification of Outer Membrane Proteins Altered in Response to UVC-Radiation in Vibrio parahaemolyticus and Vibrio alginolyticus

Vibrio parahaemolyticus and V. alginolyticus, marine foodborne pathogens, were treated with UVC-radiation (240 J/m²) to evaluate alterations in their outer membrane protein profiles. Outer membrane protein patterns of UVC-irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Altered proteins were identified by mass spectrometry (MS and MS/MS) and analysis revealed that OmpW, OmpA, Long-chain fatty acid transport protein, Outer membrane receptor protein, Putative uncharacterized protein VP0167, Maltoporin (lamB), Polar flagellin B/D, Agglutination protein Peptidoglycan-associated lipoprotein and MltA-interacting protein MipA were appeared, thereby they can be considered as UVC-stress proteins in some vibrios. In addition, expression of OmpK decreased to non-detectable level. Furthermore, we observed a decrease or an increase in the expression level of other outer membrane proteins.     

Discrimination of outer membrane proteins using machine learning algorithms

Discriminating outer membrane proteins (OMPs) from other folding types of globular and membrane proteins is an important task both for identifying OMPs from genomic sequences and for the successful prediction of their secondary and tertiary structures. In this work, we have analyzed the performance of different methods, based on Bayes rules, logistic functions, neural networks, support vector machines, decision trees, etc. for discriminating OMPs. We found that most of the machine learning techniques discriminate OMPs with similar accuracy. The neural network-based method could discriminate the OMPs from other proteins [globular/transmembrane helical (TMH)] at the fivefold cross-validation accuracy of 91.0% in a dataset of 1,088 proteins. The accuracy of discriminating globular proteins is 88.8% and that of TMH proteins is 93.7%. Further, the neural network method is tested with globular proteins belonging to 30 different folding types and it could successfully exclude 95% of the considered proteins. The proteins with SAM domain such as knottins, rubredoxin, and thioredoxin folds are eliminated with 100% accuracy. These accuracy levels are comparable to or better than other methods in the literature. We suggest that this method could be effectively used to discriminate OMPs and for detecting OMPs in genomic sequences.     

Downregulation of Tsx and OmpW and upregulation of OmpX are required for iron homeostasis in Escherichia coli

Upregulation of outer membrane (OM) proteins was systematically investigated in response to poor iron availability in the host and natural environments, but downregulation of OM proteins was ill-defined in this response. We utilized proteomic methodologies to characterize altered OM proteins in the sarcosine-insoluble fraction of Escherichia coli K12 cultured in LB medium with iron limitation. Notably, three novel proteins, Tsx, OmpW, and OmpX, related to iron homeostasis were identified; Tsx and OmpW were downregulated, and OmpX was upregulated. These alterations were functionally validated with the use of gene overexpression and deletion methods. Of the two downregulated proteins, Tsx was more sensitive to an iron-deficient environment than OmpW. In addition, the significantly negative correlation between Tsx with OmpW was achieved when overexpressed strains were used. These findings strongly indicate that the downregulation of Tsx and OmpW and the upregulation of OmpX are required for iron homeostasis in E. coli.     

Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor

Heat shock proteins (Hsps) are known to be induced in response to short-term stress. The present study aimed to evaluate the potential of Hsp70 as a biomarker of stress produced by increased temperature, osmotic pressure, and exposure to cadmium and sodium chloride in marine macroalgae and fresh water plant species. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed with a working range of 0.025-10 μg ml^-1 using a monoclonal antibody raised against purified Hsp70 of Phaseolus aureus (mung bean). Fucus serratus (toothed wrack), Chondrus crispus (Stackhouse or Carrageen moss), Ulva lactuca (sea lettuce) and Lemna minor (common duckweed) sample extracts were stressed for up to 24 h and then tested in the IC-ELISA. The presence of Hsp70 and cross-reactivity of the monoclonal antibody was confirmed by Western blot. The heat shock response was confirmed in each species using a 2-h 42°C treatment. Following heat shock, Hsp70 concentrations increased to a peak at 2 h (F. serratus) or 4 h (L. minor), after which concentrations decreased. Osmotic and cadmium stresses also resulted in elevated Hsp70 concentrations in samples of F. serratus and L. minor when compared with unstressed controls. In both, osmotic and metal stress, the production of Hsp70 increased to a maximum and subsequently decreased as the stressor levels increased. Results suggest that Hsp70 IC-ELISA could potentially be applied to the detection of stress in these aquatic species, although it would probably be most effective when used in conjunction with other measurements to provide a stressor-specific biomarker profile or fingerprint.     

A proteomics approach for identifying osmotic-stress-related proteins in rice

Osmotic stress can endanger the survival of plants. To investigate the mechanisms of how plants respond to osmotic stress, rice protein profiles from mannitol-treated plants, were monitored using a proteomics approach. Two-week-old rice seedlings were treated with 400mM mannitol for 48h. After separation of proteins from the basal part of leaf sheaths by two-dimensional polyacrylamide gel electrophoresis, 327 proteins were detected. The levels of 12 proteins increased and the levels of three proteins decreased with increasing concentration or duration, of mannitol treatment. Levels of a heat shock protein and a dnaK-type molecular chaperone were reduced under osmotic, cold, salt and drought stresses, and ABA treatment, whereas a 26S proteasome regulatory subunit was found to be responsive only to osmotic stress. Furthermore, proteins whose accumulation was sensitive to osmotic stress are present in an osmotic-tolerant cultivar. These results indicate that specific proteins expressed in the basal part of rice leaf sheaths show a coordinated response to cope with osmotic stress.