Proteomic analysis of the outer membrane of Protochlamydia amoebophila elementary bodies

Chlamydiae are obligate intracellular bacteria, comprising some of the most important bacterial pathogens of animals and humans. During their unique developmental cycle they have to attach to and enter their eukaryotic host cells, a process mediated by proteins in the chlamydial outer membrane. So far the only experimental data for chlamydial outer membrane proteins are available from members of the Chlamydiaceae, a family comprising exclusively human and animal pathogens. To get further insights into the evolution of the protein composition of the chlamydial outer membrane and into host-dependent differences, we performed an extensive experimental analysis of outer membrane fractions of Protochlamydia amoebophila elementary bodies, which constitute the infectious form of this non-pathogenic member of the Chlamydiae that thrives as a symbiont in Acanthamoeba spp. We used 1-D and 2-DE in combination with MALDI-TOF, MALDI-TOF/TOF and nanoLC-ESI-MS/MS, and compared our experimental results with a previously published in silico analysis of chlamydial outer membrane proteins. This resulted in the identification of 38 proteins supported by both studies and therefore very likely to be located in the P. amoebophila outer membrane. The obtained experimental data provide the first comprehensive overview of outer membrane proteins of a chlamydial organism outside the Chlamydiaceae. They reveal both fundamental differences and convergent evolution between pathogenic and symbiotic chlamydiae.     

Tapping the nucleotide pool of the host: novel nucleotide carrier proteins of Protochlamydia amoebophila

Protochlamydia amoebophila UWE25 is related to the Chlamydiaceae comprising major pathogens of humans, but thrives as obligate intracellular symbiont in the protozoan host Acanthamoeba sp. The genome of P. amoebophila encodes five paralogous carrier proteins belonging to the nucleotide transporter (NTT) family. Here we report on three P. amoebophila NTT isoforms, PamNTT2, PamNTT3 and PamNTT5, which possess several conserved amino acid residues known to be critical for nucleotide transport. We demonstrated that these carrier proteins are able to transport nucleotides, although substrate specificities and mode of transport differ in an unexpected manner and are unique among known NTTs. PamNTT2 is a counter exchange transporter exhibiting submillimolar apparent affinities for all four RNA nucleotides, PamNTT3 catalyses an unidirectional proton-coupled transport confined to UTP, whereas PamNTT5 mediates a proton-energized GTP and ATP import. All NTT genes of P. amoebophila are transcribed during intracellular multiplication in acanthamoebae. The biochemical characterization of all five NTT proteins from P. amoebophila in this and previous studies uncovered that these metabolically impaired bacteria are intimately connected with their host cell's metabolism in a surprisingly complex manner.     

Building the invisible wall: updating the chlamydial peptidoglycan anomaly

The existence of peptidoglycan (PG) in chlamydiae has long been debated. Genome sequencing of members of the Chlamydiaceae family and Protochlamydia amoebophila has uncovered a nearly complete pathway for PG synthesis in these organisms. The recent use of microarray and proteomic analysis methods has revealed that PG synthesis genes are expressed primarily during reticulate body development and division. Furthermore, key genes in the chlamydial PG synthesis pathway encode functional PG synthesis enzymes, some of which provide the basis for the susceptibility of chlamydiae to PG inhibitors. Recent studies shed light on how the construction of a cell wall in chlamydiae is taking shape and why the wall is being built.     

线粒体蛋白质组学研究进展

线粒体是真核生物中重要的细胞器,其包含的全部蛋白质称为线粒体蛋白质组。人类线粒体大约包含1500多种蛋白质,由核基因和线粒体基因共同编码。线粒体是细胞能量合成和物质代谢的中心,其功能障碍将直接或问接引起许多疾病。目前线粒体蛋白质组学正是系统性地研究线粒体在生理、病理过程中的功能变化以及研究疾病发生机制的重要方法。将线粒体蛋白质组的研究方法、研究进展、线粒体蛋白质组的性质及其在相关疾病研究中的作用进行综述,并对线粒体蛋白质组学在疾病发生机制和诊断治疗中的发展前景进行展望。     

Characterization of Chlamydia MurC-Ddl, a fusion protein exhibiting D-alanyl-D-alanine ligase activity involved in peptidoglycan synthesis and D-cycloserine sensitivity

Recent characterization of chlamydial genes encoding functional peptidoglycan (PG)-synthesis proteins suggests that the Chlamydiaceae possess the ability to synthesize PG yet biochemical evidence for the synthesis of PG has yet to be demonstrated. The presence of D-amino acids in PG is a hallmark of bacteria. Chlamydiaceae do not appear to encode amino acid racemases however, a D-alanyl-D-alanine (D-Ala-D-Ala) ligase homologue (Ddl) is encoded in the genome. Thus, we undertook a genetics-based approach to demonstrate and characterize the D-Ala-D-Ala ligase activity of chlamydial Ddl, a protein encoded as a fusion with MurC. The full-length murC-ddl fusion gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible ara promoter and transformed into a D-Ala-D-Ala ligase auxotroph of Escherichia coli possessing deletions of both the ddlA and ddlB genes. Viability of the E. coliDeltaddlADeltaddlB mutant in the absence of exogenous D-Ala-D-Ala dipeptide became dependent on the expression of the chlamydial murC-ddl thus demonstrating functional ligase activity. Domain mapping of the full-length fusion protein and site-directed mutagenesis of the MurC domain revealed that the structure of the full fusion protein but not MurC enzymatic activity was required for ligase activity in vivo. Recombinant MurC-Ddl exhibited substrate specificity for D-Ala. Chlamydia growth is inhibited by D-cycloserine (DCS) and in vitro analysis provided evidence for the chlamydial MurC-Ddl as the target for DCS sensitivity. In vivo sensitivity to DCS could be reversed by addition of exogenous D-Ala and D-Ala-D-Ala. Together, these findings further support our hypothesis that PG is synthesized by members of the Chlamydiaceae family and suggest that D-amino acids, specifically D-Ala, are present in chlamydial PG.     

Application of proteomics technology for analyzing the interactions between host cells and intracellular infectious agents

Host-pathogen interactions involve protein expression changes within both the host and the pathogen. An understanding of the nature of these interactions provides insight into metabolic processes and critical regulatory events of the host cell as well as into the mechanisms of pathogenesis by infectious microorganisms. Pathogen exposure induces changes in host proteins at many functional levels including cell signaling pathways, protein degradation, cytokines and growth factor production, phagocytosis, apoptosis, and cytoskeletal rearrangement. Since proteins are responsible for the cell biological functions, pathogens have evolved to manipulate the host cell proteome to achieve optimal replication. Intracellular pathogens can also change their proteome to adapt to the host cell and escape from immune surveillance, or can incorporate cellular proteins to invade other cells. Given that the interactions of intracellular infectious agents with host cells are mainly at the protein level, proteomics is the most suitable tool for investigating these interactions. Proteomics is the systematic analysis of proteins, particularly their interactions, modifications, localization and functions, that permits the study of the association between pathogens with their host cells as well as complex interactions such as the host-vector-pathogen interplay. A review on the most relevant proteomic applications used in the study of host-pathogen interactions is presented.     

Detection of sequence variation in PCR-amplified fragments of omp2 gene from three species of the family Chlamydiaceae using agarose gel electrophoresis containing bisbenzimide-PEG

A simple technique providing a means for rapid genetic differentiation of chlamydial strains is described. The technique is based on a single-step sequence-specific separation of PCR-amplified DNA fragments by electrophoresis in an agarose gel containing a DNA ligand - bisbenzimide-PEG. A hypervariable region at the 5' end of the omp2 gene of Chlamydiaceae species encoding the 60-kDa cysteine-rich outer membrane protein was selected as a target for PCR. The appropriate fragments were amplified from strains of Chlamydia trachomatis, Chlamydophila pneumoniae, and Chlamydophila psittaci, and the PCR products originating from different species were electrophoretically separated in the presence of the DNA ligand. We therefore demonstrated that PCR with a single pair of primers followed by simple agarose gel electrophoresis with bisbenzimide-PEG can be applied to the differentiation of three members of the family Chlamydiaceae which are commonly recognized as human pathogens.     

Determination and comparison of the baseline proteomes of the versatile microbe Rhodopseudomonas palustris under its major metabolic states

Rhodopseudomonas palustris is a purple nonsulfur anoxygenic phototrophic bacterium that is ubiquitous in soil and water. R. palustris is metabolically versatile with respect to energy generation and carbon and nitrogen metabolism. We have characterized and compared the baseline proteome of a R. palustris wild-type strain grown under six metabolic conditions. The methodology for proteome analysis involved protein fractionation by centrifugation, subsequent digestion with trypsin, and analysis of peptides by liquid chromatography coupled with tandem mass spectrometry. Using these methods, we identified 1664 proteins out of 4836 predicted proteins with conservative filtering constraints. A total of 107 novel hypothetical proteins and 218 conserved hypothetical proteins were detected. Qualitative analyses revealed over 311 proteins exhibiting marked differences between conditions, many of these being hypothetical or conserved hypothetical proteins showing strong correlations with different metabolic modes. For example, five proteins encoded by genes from a novel operon appeared only after anaerobic growth with no evidence of these proteins in extracts of aerobically grown cells. Proteins known to be associated with specialized growth states such as nitrogen fixation, photoautotrophic, or growth on benzoate, were observed to be up-regulated under those states.     

Proteomics analysis of the Francisella tularensis LVS response to iron restriction: induction of the F. tularensis pathogenicity island proteins IglABC

Francisella tularensis is a highly virulent, facultative intracellular pathogen that causes tularemia in humans and animals. Although it is one of the most infectious bacterial pathogens, little is known about its virulence mechanisms. In this study, the response of F. tularensis live vaccine strain to iron depletion, which simulates the environment within the host, was investigated. In order to detect alterations in protein synthesis, metabolic labeling, followed by 2D-PAGE analysis was used. Globally, 141 protein spots were detected whose levels were significantly altered in the iron-restricted medium. About 65% of the spots were successfully identified using mass spectrometric approaches. Importantly, among the proteins produced at an increased level during iron-limited growth, three proteins were found encoded by the igl operon, located in the F. tularensis pathogenicity island I (FPI). Of these, the IglC and IglA proteins were previously reported to be necessary for full virulence of F. tularensis. These results, obtained at the proteome level, support and confirm recently published data showing that the igl operon genes are transcribed in response to iron limitation.     

Insights into the gene expression profile of uncultivable hemotrophic Mycoplasma suis during acute infection, obtained using proteome analysis

Hemotrophic mycoplasmas, bacteria without cell walls whose niche is the erythrocytes of their hosts, have never been cultivated in vitro. Therefore, knowledge of their pathogenesis is fundamental. Mycoplasma suis infects pigs, causing either acute fatal hemolytic anemia or chronic low-grade anemia, growth retardation, and immune suppression. Recently, the complete genomes of two hemotrophic mycoplasma species, M. suis and M. haemofelis, were sequenced, offering new strategies for the analysis of their pathogenesis. In this study we implemented a proteomic approach to identify M. suis proteins during acute infection by using tandem mass spectrometry. Twenty-two percent of the predicted proteins encoded in M. suis strain KI_3806 were identified. These included nearly all encoded proteins of glycolysis and nucleotide metabolism. The proteins for lipid metabolism, however, were underrepresented. A high proportion of the detected proteins are involved in information storage and processing (72.6%). In addition, several proteins of different functionalities, i.e., posttranslational modification, membrane genesis, signal transduction, intracellular trafficking, inorganic ion transport, and defense mechanisms, were identified. In its reduced genome, M. suis harbors 65.3% (strain Illinois) and 65.9% (strain KI_3806) of the genes encode hypothetical proteins. Of these, only 6.3% were identified at the proteome level. All proteins identified in this study are present in both M. suis strains and are encoded in more highly conserved regions of the genome sequence. In conclusion, our proteome approach is a further step toward the elucidation of the pathogenesis and life cycle of M. suis as well as the establishment of an in vitro cultivation system.     

Unity in variety--the pan-genome of the Chlamydiae

Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.     

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).     

Relative quantification of proteins across the species boundary through the use of shared peptides

We show that shared peptides of proteins that are encoded in different species are suitable for cross-species relative protein quantification. A 14N-containing proteome from the thermoacidophilic archaeon Sulfolobus tokodaii was mixed with a 15N-labeled proteome from Sulfolobus solfataricus. Using three shared peptides per protein, the relative abundance of six orthologous proteins was calculated. Observed standard deviations were approximately 10%, indicating that the trypsin accessibility to cleavage sites was not altered in the orthologs. The abundance ratios of the and subunits of the Thermosome were 0.64 and 1.24 in Sulfolobus tokodaii compared to Sulfolobus solfataricus, suggesting a different stoichiometry of the complex in both species. In addition, an in silico study was performed on the occurrence of shared peptides. Inter- and intra-species peptide redundancy was investigated in the model organisms Homo sapiens, Mus musculus, Escherichia coli K12, Escherichia coli O157:H7, S. solfataricus, and S. tokodaii. M. musculus and H. sapiens share 30-50% of all peptides (6-15 residues). Moreover, approximately one-third of all proteins shared > or = 40% of their peptides with at least one other protein in the related species, thus offering strong potential for cross-species relative protein quantification. Conversely, approximately 40% of all peptides (6-15 residues) encoded in H. sapiens are encoded multiple times and therefore complicate identification and quantification.     

Mapping and comprehensive analysis of the extracellular and cell surface proteome of the human pathogen Corynebacterium diphtheriae

Secreted proteins of the human pathogen Corynebacterium diphtheriae might be involved in important pathogen-host cell interactions. Here, we present the first systematic reference map of the extracellular and cell surface proteome fractions of the type strain C. diphtheriae C7s(-)tox-. The analysis window of 2-DE covered the pI range from 3 to 10 along with a MW range from 8 to 150 kDa. Computational analysis of the 2-D gels detected almost 150 protein spots in the extracellular proteome fraction and about 80 protein spots of the cell surface proteome. MALDI-TOF-MS and PMF with trypsin unambiguously identified 107 extracellular protein spots and 53 protein spots of the cell surface, representing in total 85 different proteins of C. diphtheriae C7s(-)tox-. Several of the identified proteins are encoded by pathogenicity islands and might represent virulence factors of C. diphtheriae. Additionally, four solute-binding proteins (HmuT, Irp6A, CiuA, and FrgD) of different iron ABC transporters were identified, with the hitherto uncharacterized FrgD protein being the most abundant one of the cell surface proteome of C. diphtheriae C7s(-)tox-.     

Shotgun proteomic analysis of Chlamydia trachomatis

Chlamydiae are widespread bacterial pathogens responsible for a broad range of diseases, including sexually transmitted infections, pneumonia and trachoma. To validate the existence of hitherto hypothetical proteins predicted from recent chlamydial genome sequencing projects and to examine the patterns of expression of key components at the protein level, we have surveyed the expressed proteome of Chlamydia trachomatis strain L2. A combination of two-dimensional gel analysis, multi-dimensional protein identification (MudPIT) and nanocapillary liquid chromatography-tandem mass spectrometry allowed a total of 328 chlamydial proteins to be unambiguously assigned. Proteins identified as being expressed in the metabolically inert form, elementary body, of Chlamydia include the entire set of predicted glycolytic enzymes, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. An enzyme central to cell wall biosynthesis was also detected in the intracellular form, reticulate body, of Chlamydia, suggesting that the peptidoglycan is produced during growth within host cells. Other sets of proteins identified include 17 outer membrane-associated proteins of potential significance in vaccine studies and 67 proteins previously annotated as hypothetical or conserved hypothetical. Taken together, >/=35% of the predicted proteome for C. trachomatis has been experimentally verified, representing the most extensive survey of any chlamydial proteome to date.     

Lipoproteins of Mycobacterium tuberculosis: an abundant and functionally diverse class of cell envelope components

Mycobacterium tuberculosis remains the predominant bacterial scourge of mankind. Understanding of its biology and pathogenicity has been greatly advanced by the determination of whole genome sequences for this organism. Bacterial lipoproteins are a functionally diverse class of membrane-anchored proteins. The signal peptides of these proteins direct their export and post-translational lipid modification. These signal peptides are amenable to bioinformatic analysis, allowing the lipoproteins encoded in whole genomes to be catalogued. This review applies bioinformatic methods to the identification and functional characterisation of the lipoproteins encoded in the M. tuberculosis genomes. Ninety nine putative lipoproteins were identified and so this family of proteins represents ca. 2.5% of the M. tuberculosis predicted proteome. Thus, lipoproteins represent an important class of cell envelope proteins that may contribute to the virulence of this major pathogen.     

A soluble 3D LC/MS/MS proteome of the filamentous cyanobacterium Nostoc punctiforme

Nostoc punctiforme is an oxygenic photoautotrophic cyanobacterium with multiple developmental states, which can form nitrogen-fixing symbioses with a variety of terrestrial plants. 3D LC/MS/MS shotgun peptide sequencing was used to analyze the proteome when N. punctiforme is grown in continuous moderate light with ammonia as the nitrogen source. The soluble proteome includes 1575 proteins, 50% of which can be assigned to core metabolic and transport functions. Another 39% are assigned to proteins with no known function, a substantially higher fraction than in the Escherichia coli proteome. Many expressed proteins protect against oxidative and light stress. Seventy-one sensor histidine kinases, response regulators, and serine/threonine kinases, individually and as hybrid, multidomain proteins, were identified, reflecting a substantial capacity to sense and respond to environmental change. Proteins encoded by each of the five N. punctiforme plasmids were identified, as were 10 transposases, reflecting the plasticity of the N. punctiforme genome. This core proteome sets the stage for comparison with that of other developmental states.     

Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs

Chlamydiae sp. are obligate intracellular pathogens that cause a variety of diseases in humans. Adhesion of the infectious elementary body to the eukaryotic host cell is a pivotal step in chlamydial pathogenesis. Here we describe the characterization of members of the polymorphic membrane protein family (Pmp), the largest protein family (with up to 21 members) unique to Chlamydiaceae. We show that yeast cells displaying Pmp6, Pmp20 or Pmp21 on their surfaces, or beads coated with the recombinant proteins, adhere to human epithelial cells. A hallmark of the Pmp protein family is the presence of multiple repeats of the tetrapeptide motifs FxxN and GGA(I, L, V) and deletion analysis shows that at least two copies of these motifs are needed for adhesion. Importantly, pre-treatment of human cells with recombinant Pmp6, Pmp20 or Pmp21 protein reduces infectivity upon subsequent challenge with Chlamydia pneumoniae and correlates with diminished attachment of Chlamydiae to target cells. Antibodies specific for Pmp21 can neutralize infection in vitro. Finally, a combination of two different Pmp proteins in infection blockage experiments shows additive effects, possibly suggesting similar functions. Our findings imply that Pmp6, Pmp20 and Pmp21 act as adhesins, are vital during infection and thus represent promising vaccine candidates.     

Reconstructing the evolution of the mitochondrial ribosomal proteome

For production of proteins that are encoded by the mitochondrial genome, mitochondria rely on their own mitochondrial translation system, with the mitoribosome as its central component. Using extensive homology searches, we have reconstructed the evolutionary history of the mitoribosomal proteome that is encoded by a diverse subset of eukaryotic genomes, revealing an ancestral ribosome of alpha-proteobacterial descent that more than doubled its protein content in most eukaryotic lineages. We observe large variations in the protein content of mitoribosomes between different eukaryotes, with mammalian mitoribosomes sharing only 74 and 43% of its proteins with yeast and Leishmania mitoribosomes, respectively. We detected many previously unidentified mitochondrial ribosomal proteins (MRPs) and found that several have increased in size compared to their bacterial ancestral counterparts by addition of functional domains. Several new MRPs have originated via duplication of existing MRPs as well as by recruitment from outside of the mitoribosomal proteome. Using sensitive profile–profile homology searches, we found hitherto undetected homology between bacterial and eukaryotic ribosomal proteins, as well as between fungal and mammalian ribosomal proteins, detecting two novel human MRPs. These newly detected MRPs constitute, along with evolutionary conserved MRPs, excellent new screening targets for human patients with unresolved mitochondrial oxidative phosphorylation disorders.