Proteomic Profiling of the Outer Membrane Fraction of the Obligate Intracellular Bacterial Pathogen Ehrlichia ruminantium

The outer membrane proteins (OMPs) of Gram-negative bacteria play a crucial role in virulence and pathogenesis. Identification of these proteins represents an important goal for bacterial proteomics, because it aids in vaccine development. Here, we have developed such an approach for Ehrlichia ruminantium, the obligate intracellular bacterium that causes heartwater. A preliminary whole proteome analysis of elementary bodies, the extracellular infectious form of the bacterium, had been performed previously, but information is limited about OMPs in this organism and about their role in the protective immune response. Identification of OMPs is also essential for understanding Ehrlichia’s OM architecture, and how the bacterium interacts with the host cell environment. First, we developed an OMP extraction method using the ionic detergent sarkosyl, which enriched the OM fraction. Second, proteins were separated via one-dimensional electrophoresis, and digested peptides were analyzed via nano-liquid chromatographic separation coupled with mass spectrometry (LC-MALDI-TOF/TOF). Of 46 unique proteins identified in the OM fraction, 18 (39%) were OMPs, including 8 proteins involved in cell structure and biogenesis, 4 in transport/virulence, 1 porin, and 5 proteins of unknown function. These experimental data were compared to the predicted subcellular localization of the entire E. ruminantium proteome, using three different algorithms. This work represents the most complete proteome characterization of the OM fraction in Ehrlichia spp. The study indicates that suitable subcellular fractionation experiments combined with straightforward computational analysis approaches are powerful for determining the predominant subcellular localization of the experimentally observed proteins. We identified proteins potentially involved in E. ruminantium pathogenesis, which are good novel targets for candidate vaccines. Thus, combining bioinformatics and proteomics, we discovered new OMPs for E. ruminantium that are valuable data for those investigating new vaccines against this organism. In summary, we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.     

Global proteomic profiling of native outer membrane vesicles derived from Escherichia coli

Gram-negative bacteria constitutively secrete native outer membrane vesicles (OMVs) into the extracellular milieu. Although recent progress in this area has revealed that OMVs are essential for bacterial survival and pathogenesis, the mechanism of vesicle formation and the biological roles of OMVs have not been clearly defined. Using a proteomics approach, we identified 141 protein components of Escherichia coli-derived native OMVs with high confidence; two separate analyses yielded identifications of 104 and 117 proteins, respectively, with 80 proteins overlapping between the two trials. In the group of identified proteins, the outer membrane proteins were highly enriched, whereas inner membrane proteins were lacking, suggesting that a specific sorting mechanism for vesicular proteins exists. We also identified proteins involved in vesicle formation, the removal of toxic compounds and attacking phage, and the elimination of competing organisms, as well as those involved in facilitating the transfer of genetic material and protein to other bacteria, targeting host cells, and modulating host immune responses. This study provides a global view of native bacterial OMVs. This information will help us not only to elucidate the biogenesis and functions of OMV from nonpathogenic and pathogenic bacteria but also to develop vaccines and antibiotics effective against pathogenic strains.     

Killing of gram-negative bacteria by complement. Fractionation of cell membranes after complement C5b-9 deposition on to the surface of Salmonella minnesota Re595.

The effect of C5b-9 deposition on the envelope of target Gram-negative bacteria was studied. In order to understand the changes occurring after complement deposition on the bacterial surface, the preparation of Gram-negative bacterial membranes by different methods involving the osmotic lysis of spheroplasts was investigated. Subsequent fractionation of the outer membrane (OM) and cytoplasmic membrane (CM) by sucrose-density-gradient centrifugation showed differences in the membrane profiles obtained. The results indicate that optimum separation of OM and CM components requires effective digestion of DNA in the total membrane preparation before density-gradient fractionation. Salmonella minnesota Re595 carrying the intermediate complement complex C5b-7 (BC1-7) or C5b-8 (BC1-8) were efficiently killed upon incubation with purified C8 + C9 or C9 respectively. Human-alpha-thrombin-cleaved C9 (C9n), which is unable to form tubular poly(C9), was shown to be more effective at killing than native C9. By using an optimized system for the separation of OM and CM, it was found that, subsequent to lethal complement attack, the CM could not be recovered when C9 was used as the terminal complement component, but was recovered with reduced yield when C9n replaced C9. The results show that inability to recover the CM on sucrose density gradients after complement attack may not be a consequence of an essential membrane damage event required for complement-mediated killing of Gram-negative bacteria.     

Rapid screening of highly efficient vaccine candidates by immunoproteomics

Diseases caused by microorganisms can be controlled by vaccines, which require neutralizing antigens. Therefore, it is very important to identify highly efficient immunogens for immune prevention. By combining immunoproteomics and bacterial challenge after immunization, we developed a rapid method for screening protected antigens of pathogenic bacteria in aquaculture. Our approach may be divided into three consecutive steps. First, dominant immunogens of outer membrane proteins are screened by immunoproteomics. Second, proteins with the ability to induce production of neutralizing antibodies are identified from the immunogens by virulent bacterium challenge following vaccination. Third, vaccine candidates are determined by evaluation of neutralizing abilities. Information on the candidates has been obtained for further gene cloning by mass spectrometry. Our results indicate that highly efficient protected antigens were identified from the outer membrane proteome of Aeromonas hydrophila, in which an immunogen showed 71.4% protective ability with multivalent functions to A. hydrophila and Aeromonas sobria. In summary, we have developed a high-throughout, accurate, rapid and highly efficient method which will play an active role in immune prevention for microbiological diseases.     

亚细胞蛋白质组研究进展

运用蛋白质组学方法对亚细胞组分进行研究是目前的研究热点。传统的亚细胞分离技术结合质谱鉴定技术为亚细胞蛋白质组学的研究提供了技术平台。本文对快速发展的亚细胞蛋白质组研究进展及其所揭示的生物学意义进行了综述。     

大肠杆菌外膜蛋白的分离及其双向电泳图谱的建立

本文利用温度诱导的两相分离萃取技术选择性分离未经机械破碎的大肠杆菌细胞外膜蛋白,研究TritonX.114的浓度及处理时间对提取外膜蛋白的影响.实验结果表明,Triton X-114的使用浓度和作用时间均显著影响外膜蛋白的提取效率.SDS-PAGE结果表明不同Triton X-114的使用浓度和作用时间只是影响了外膜蛋白的提取效率而对外膜蛋白提取的种类没有影响.实验发现8%的Triton X-114处理3小时为最佳分离条件,分离得到的样品可用于双向电泳分析.通过对比实验发现样品裂解液中包含低浓度的Tris是外膜蛋白双向电泳成功的关键因素,CHAPS与ASB-14或NP-40结合使用可显著提高外膜蛋白的溶解能力,缩短聚焦时间,从而优化了大肠杆茵外膜蛋白双向电泳技术体系,建立了其双向电泳图谱.     

The bacterial outer membrane β-barrel assembly machinery

β-Barrel proteins found in the outer membrane of Gram-negative bacteria serve a variety of cellular functions. Proper folding and assembly of these proteins are essential for the viability of bacteria and can also play an important role in virulence. The β-barrel assembly machinery (BAM) complex, which is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, has been the focus of many recent studies. This review summarizes the significant progress that has been made toward understanding the structure and function of the bacterial BAM complex.     

Differential detergent fractionation for non-electrophoretic eukaryote cell proteomics

Differential detergent fractionation (DDF), which relies on detergents to sequentially extract proteins from eukaryotic cells, has been used to increase proteome coverage of 2D-PAGE. Here, we used DDF extraction in conjunction with the nonelectrophoretic proteomics method of liquid chromatography and electrospray ionization tandem mass spectrometry. We demonstrate that DDF can be used with 2D-LC ESI MS2 for comprehensive cellular proteomics, including a large proportion of membrane proteins. Compared to some published methods designed to isolate membrane proteins specifically, DDF extraction yields comprehensive proteomes which include twice as many membrane proteins. Two-thirds of these membrane proteins have more than one trans-membrane domain. Since DDF separates proteins based upon their physicochemistry and subcellular localization, this method also provides data useful for functional genome annotation. As more genome sequences are completed, methods which can aid in functional annotation will become increasingly important.     

Analysing the outer membrane subproteome of Methylococcus capsulatus (Bath) using proteomics and novel biocomputing tools

High-resolution two-dimensional gel electrophoresis and mass spectrometry has been used to identify the outer membrane (OM) subproteome of the Gram-negative bacterium Methylococcus capsulatus (Bath). Twenty-eight unique polypeptide sequences were identified from protein samples enriched in OMs. Only six of these polypeptides had previously been identified. The predictions from novel bioinformatic methods predicting β-barrel outer membrane proteins (OMPs) and OM lipoproteins were compared to proteins identified experimentally. BOMP () predicted 43 β-barrel OMPs (1.45%) from the 2,959 annotated open reading frames. This was a lower percentage than predicted from other Gram-negative proteomes (1.8–3%). More than half of the predicted BOMPs in M. capsulatus were annotated as (conserved) hypothetical proteins with significant similarity to very few sequences in Swiss-Prot or TrEMBL. The experimental data and the computer predictions indicated that the protein composition of the M. capsulatus OM subproteome was different from that of other Gram-negative bacteria studied in a similar manner. A new program, Lipo, was developed that can analyse entire predicted proteomes and give a list of recognised lipoproteins categorised according to their lipo-box similarity to known Gram-negative lipoproteins (). This report is the first using a proteomics and bioinformatics approach to identify the OM subproteome of an obligate methanotroph.     

A review on recent developments in mass spectrometry instrumentation and quantitative tools advancing bacterial proteomics

Proteomics has evolved substantially since its early days, some 20 years ago. In this mini-review, we aim to provide an overview of general methodologies and more recent developments in mass spectrometric approaches used for relative and absolute quantitation of proteins. Enhancement of sensitivity of the mass spectrometers as well as improved sample preparation and protein fractionation methods are resulting in a more comprehensive analysis of proteomes. We also document some upcoming trends for quantitative proteomics such as the use of label-free quantification methods. Hopefully, microbiologists will continue to explore proteomics as a tool in their research to understand the adaptation of microorganisms to their ever changing environment. We encourage them to incorporate some of the described new developments in mass spectrometry to facilitate their analyses and improve the general knowledge of the fascinating world of microorganisms.     

生物质谱技术在蛋白质组学研究中的应用

随着技术的进步,蛋白质组学的研究重心由最初旨在鉴定细胞或组织内基因组所表达的全部蛋白质转移到从整个蛋白质组水平上阐述包括蛋白翻译后修饰、生物大分子相互作用等反映蛋白质功能的层次。多种质谱离子化技术的突破使质谱技术成为蛋白质组学研究必不可少的手段。质谱技术联合蛋白质组学多角度、深层次探索生命系统分子本质成为现阶段生命科学研究领域的主旋律之一。本文简要综述了肽和蛋白质等生物大分子质谱分析的原理、方式和应用,并对其发展前景做出展望。     

MetaLocGramN: A meta-predictor of protein subcellular localization for Gram-negative bacteria

Subcellular localization is a key functional characteristic of proteins. It is determined by signals encoded in the protein sequence. The experimental determination of subcellular localization is laborious. Thus, a number of computational methods have been developed to predict the protein location from sequence. However predictions made by different methods often disagree with each other and it is not always clear which algorithm performs best for the given cellular compartment. We benchmarked primary subcellular localization predictors for proteins from Gram-negative bacteria, PSORTb3, PSLpred, CELLO, and SOSUI-GramN, on a common dataset that included 1056 proteins. We found that PSORTb3 performs best on the average, but is outperformed by other methods in predictions of extracellular proteins. This motivated us to develop a meta-predictor, which combines the primary methods by using the logistic regression models, to take advantage of their combined strengths, and to eliminate their individual weaknesses. MetaLocGramN runs the primary methods, and based on their output classifies protein sequences into one of five major localizations of the Gram-negative bacterial cell: cytoplasm, plasma membrane, periplasm, outer membrane, and extracellular space. MetaLocGramN achieves the average Matthews correlation coefficient of 0.806, i.e. 12% better than the best individual primary method. MetaLocGramN is a meta-predictor specialized in predicting subcellular localization for proteins from Gram-negative bacteria. According to our benchmark, it performs better than all other tools run independently. MetaLocGramN is a web and SOAP server available for free use by all academic users at the URL http://iimcb.genesilico.pl/MetaLocGramN. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction.     

Comparative proteomics analysis of sarcosine insoluble outer membrane proteins from clarithromycin resistant and sensitive strains of Helicobacter pylori

Helicobacter pylori causes disease manifestations in humans including chronic gastric and peptic ulcers, gastric cancer, and lymphoid tissue lymphoma. Increasing rates of H. pylori clarithromycin resistance has led to higher rates of disease development. Because antibiotic resistance involves modifications of outer membrane proteins (OMP) in other Gram-negative bacteria, this study focuses on identification of H. pylori OMP’s using comparative proteomic analyses of clarithromycin-susceptible and -resistant H. pylori strains. Comparative proteomics analyses of isolated sarcosine-insoluble OMP fractions from clarithromycin-susceptible and -resistant H. pylori strains were performed by 1) one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis protein separation and 2) in-gel digestion of the isolated proteins and mass spectrometry analysis by Matrix Assisted Laser Desorption Ionization-tandem mass spectrometry. Iron-regulated membrane protein, UreaseB, EF-Tu, and putative OMP were down-regulated; HopT (BabB) transmembrane protein, HofC, and OMP31 were up-regulated in clarithromycin-resistant H. pylori. Western blotting and real time PCR, respectively, validated UreaseB subunit and EF-Tu changes at the protein level, and mRNA expression of HofC and HopT. This limited proteomic study provides evidence that alteration of the outer membrane proteins’ profile may be a novel mechanism involved in clarithromycin resistance in H. pylori.     

Eukaryotic cell signaling and transcriptional activation induced by bacterial porins

The protein composition of the outer membrane of Gram-negative bacteria consists of about 20 immunochemically distinct proteins, termed outer membrane proteins (OMPs). Apart from their structural role, OMPs have been shown to have other functions, particularly with regard to transport, and have been classified as permeases and porins. Porins, during their interaction with the host, are immunogenic and also directly stimulate several cellular functions. Porins work both as molecules present on the bacterial surface and as molecules released by bacteria. Lipopolysaccharide and OMPs, the major structural macromolecular constituents of the outer membrane, carry out a fundamental role in the pathogenesis of Gram-negative infections. This brief review describes the multiple facets of the biological activities of porins both in vitro and in vivo, particularly focusing on their ability to induce the expression of cytokines and other factors that modulate cellular activities with either pathological or adaptive consequences. This brief discussion will focus on the signal transmission mechanisms induced by bacterial porins.     

Identification and characterization of the Sulfolobus solfataricus P2 proteome

Via combined separation approaches, a total of 1399 proteins were identified, representing 47% of the Sulfolobus solfataricus P2 theoretical proteome. This includes 1323 proteins from the soluble fraction, 44 from the insoluble fraction and 32 from the extra-cellular or secreted fraction. We used conventional 2-dimensional gel electrophoresis (2-DE) for the soluble fraction, and shotgun proteomics for all three cell fractions (soluble, insoluble, and secreted). Two gel-based fractionation methods were explored for shotgun proteomics, namely: (i) protein separation utilizing 1-dimensional gel electrophoresis (1-DE) followed by peptide fractionation by iso-electric focusing (IEF), and (ii) protein and peptide fractionation both employing IEF. Results indicate that a 1D-IEF fractionation workflow with three replicate mass spectrometric analyses gave the best overall result for soluble protein identification. A greater than 50% increment in protein identification was achieved with three injections using LC-ESI-MS/MS. Protein and peptide fractionation efficiency; together with the filtration criteria are also discussed.     

Classification and identification of bacteria using mass spectrometry-based proteomics

Timely classification and identification of bacteria is of vital importance in many areas of public health. Mass spectrometry-based methods provide an attractive alternative to well-established microbiologic procedures. Mass spectrometry methods can be characterized by the relatively high speed of acquiring taxonomically relevant information. Gel-free mass spectrometry proteomics techniques allow for rapid fingerprinting of bacterial proteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or, for high-throughput sequencing of peptides from protease-digested cellular proteins, using mass analysis of fragments from collision-induced dissociation of peptide ions. The latter technique uses database searching of product ion mass spectra. A database contains a comprehensive list of protein sequences translated from protein-encoding open reading frames found in bacterial genomes. The results of such searches allow the assignment of experimental peptide sequences to matching theoretical bacterial proteomes. Phylogenetic profiles of sequenced peptides are then used to create a matrix of sequence-to-bacterium assignments, which are analyzed using numerical taxonomy tools. The results thereof reveal the relatedness between bacteria, and allow the taxonomic position of an investigated strain to be inferred.     

Classification and identification of bacteria using mass spectrometry-based proteomics

Timely classification and identification of bacteria is of vital importance in many areas of public health. Mass spectrometry-based methods provide an attractive alternative to well-established microbiologic procedures. Mass spectrometry methods can be characterized by the relatively high speed of acquiring taxonomically relevant information. Gel-free mass spectrometry proteomics techniques allow for rapid fingerprinting of bacterial proteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or, for high-throughput sequencing of peptides from protease-digested cellular proteins, using mass analysis of fragments from collision-induced dissociation of peptide ions. The latter technique uses database searching of product ion mass spectra. A database contains a comprehensive list of protein sequences translated from protein-encoding open reading frames found in bacterial genomes. The results of such searches allow the assignment of experimental peptide sequences to matching theoretical bacterial proteomes. Phylogenetic profiles of sequenced peptides are then used to create a matrix of sequence-to-bacterium assignments, which are analyzed using numerical taxonomy tools. The results thereof reveal the relatedness between bacteria, and allow the taxonomic position of an investigated strain to be inferred.     

Subcellular tissue proteomics of hepatocellular carcinoma for molecular signature discovery

Hepatocellular carcinoma (HCC) is one of the leading causes of mortality from solid organ malignancy worldwide. Because of the complexity of proteins within liver cells and tissues, the discovery of therapeutic targets of HCC has been difficult. To investigate strategies for decreasing the complexity of tissue samples for detecting meaningful protein mediators of HCC, we employed subcellular fractionation combined with 1D-gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis. Moreover, we utilized a statistical method, namely, the Power Law Global Error Model (PLGEM), to distinguish differentially expressed proteins in a duplicate proteomic data set. Mass spectrometric analysis identified 3045 proteins in nontumor and HCC from cytosolic, membrane, nuclear, and cytoskeletal fractions. The final lists of highly differentiated proteins from the targeted fractions were searched for potentially translocated proteins in HCC from soluble compartments to the nuclear or cytoskeletal compartments. This analysis refined our targets of interest to include 21 potential targets of HCC from these fractions. Furthermore, we validated the potential molecular targets of HCC, MATR3, LETM1, ILF2, and IQGAP2 by Western blotting, immunohistochemisty, and immunofluorescent microscopy. Here we demonstrate an efficient strategy of subcellular tissue proteomics toward molecular target discovery of one of the most complicated human disease, HCC.     

An Outer Membrane Receptor of Neisseria meningitidis Involved in Zinc Acquisition with Vaccine Potential

Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract.     

Serum amyloid A protein binds to outer membrane protein A of gram-negative bacteria

Serum amyloid A (SAA) is the major acute phase protein in man and most mammals. We observed SAA binding to a surprisingly large number of Gram-negative bacteria, including Escherichia coli, Salmonella typhimurium, Shigella flexneri, Klebsiella pneumoniae, Vibrio cholerae, and Pseudomonas aeruginosa. The binding was found to be high affinity and rapid. Importantly, this binding was not inhibited by high density lipoprotein with which SAA is normally complexed in serum. Binding was also observed when bacteria were offered serum containing SAA. Ligand blots following SDS-PAGE or two-dimensional gels revealed two major ligands of 29 and 35 kDa that bound SAA when probing with radiolabeled SAA or SAA and monoclonal anti-SAA. Following fractionation the ligand was found in the outer membrane fraction of E. coli and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to be outer membrane protein A (OmpA). OmpA-deficient E. coli did not bind SAA, and following purification of OmpA the protein retained binding activity. The ligands on other bacteria were likely to be homologues of OmpA because wild type, but not OprF-deficient, P. aeruginosa bound SAA.