Proteomic variation and diversity in clinical Streptococcus pneumoniae isolates from invasive and non-invasive sites.

Streptococcus pneumoniae is responsible for a variety of invasive and non-invasive human infections. There are over 90 serotypes of S. pneumoniae differing in their ability to adapt to the different niches within the host. Two-dimensional gel electrophoresis was used to discriminate clinical S. pneumoniae isolates recovered from either blood cultures (invasive site isolates) or other sites, including sputum, tracheal aspirate, ear, eye and skin swabs (non-invasive site isolates). Global protein expression profiles for five invasive site and six non-invasive site isolates representing five different serotypes (serotypes 4, 6, 9, 14 and 23) were obtained for each isolate and combined into a single data set using Progenesis SameSpots™ software. One-hundred and eighty six protein spots (39% of the protein spots in the dataset) differed significantly (ANOVA, p<0.05) in abundance between the invasive site (101 upregulated protein spots) and non-invasive site (85 upregulated protein spots) isolates. Correlations between the bacterial proteomes and their sites of isolation were determined by Principal Component Analysis (PCA) using the significantly different protein spots. Out of the 186 variable protein spots, 105 exhibited a serotype-associated pattern of variability. The expression of the remaining 81 protein spots was concluded to be uniquely linked to the site of bacterial isolation. Mass spectrometry was used to identify selected protein spots that showed either constant or differential abundance levels. The identified proteins had a diverse range of functions including, capsule biogenesis, DNA repair, protein deglycation, translation, stress response and virulence as well as amino acid, carbohydrate, lipid and nucleotide metabolism. These findings provide insight on the proteins that contribute towards the adaptation of the bacteria to different sites within the host.

Streptococcus pneumoniae proteomics: determinants of pathogenesis and vaccine development.

Streptococcus pneumoniae is a major pathogen that is responsible for a variety of invasive diseases. The bacteria gain entry initially by establishing a carriage state in the nasopharynx from where they migrate to other sites in the body. The worldwide distribution of the bacteria and the severity of the diseases have led to a significant level of interest in the development of vaccines against the bacteria. Current vaccines, based on the bacterial polysaccharide, have a number of limitations including poor immunogenicity and limited effectiveness against all pneumococcal serotypes. There are many challenges in developing vaccines that will be effective against the diverse range of isolates and serotypes for this highly variable bacterial pathogen. This review considers how proteomic technologies have extended our understanding of the pathogenic mechanisms of nasopharyngeal colonization and disease development as well as the critical areas in developing protein-based vaccines.

Proteomic changes in the brain of the western painted turtle (Chrysemys picta bellii) during exposure to anoxia.

During anoxia, overall protein synthesis is almost undetectable in the brain of the western painted turtle. The aim of this investigation was to address the question of whether there are alterations to specific proteins by comparing the normoxic and anoxic brain proteomes. Reductions in creatine kinase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase reflected the reduced production of adenosine triphosphate (ATP) during anoxia while the reduction in transitional endoplasmic reticulum ATPase reflected the conservation of ATP or possibly a decrease in intracellular Ca(2+). In terms of neural protection programed cell death 6 interacting protein (PDCD6IP; a protein associated with apoptosis), dihydropyrimidinase-like protein, t-complex protein, and guanine nucleotide protein G(o) subunit alpha (Go alpha; proteins associated with neural degradation and impaired cognitive function) also declined. A decline in actin, gelsolin, and PDCD6IP, together with an increase in tubulin, also provided evidence for the induction of a neurological repair response. Although these proteomic alterations show some similarities with the crucian carp (another anoxia-tolerant species), there are species-specific responses, which supports the theory of no single strategy for anoxia tolerance. These findings also suggest the anoxic turtle brain could be an etiological model for investigating mammalian hypoxic damage and clinical neurological disorders.

Proteomic changes in the crucian carp brain during exposure to anoxia.

During exposure to anoxia, the crucian carp brain is able to maintain normal overall protein synthesis rates. However, it is not known if there are alterations in the synthesis or expression of specific proteins. This investigation addresses this issue by comparing the normoxic and anoxic brain proteome. Nine proteins were found to be reduced by anoxia. Reductions in the glycolytic pathway proteins creatine kinase, fructose biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and lactate dehydrogenase reflect the reduced production and requirement for adenosine tri-phosphate during anoxia. In terms of neural protection, voltage-dependent anion channel, a protein associated with neuronal apoptosis, was reduced, along with gefiltin, a protein associated with the subsequent need for neuronal repair. Additionally the expression of proteins associated with neural degeneration and impaired cognitive function also declined; dihydropyrimidinase-like protein-3 and vesicle amine transport protein-1. One protein was found to be increased by anoxia; pre-proependymin, the precursor to ependymin. Ependymin fulfils multiple roles in neural plasticity, memory formation and learning, neuron growth and regeneration, and is able to reverse the possibility of apoptosis, thus further protecting the anoxic brain.

A proteomic and targeted metabolomic approach to investigate change in Lolium perenne roots when challenged with glycine.

A combined proteomic and isotope tracer approach was used to investigate the impact of supplying N as glycine to roots of Lolium perenne. Initially, ammonium nitrate was supplied to all plants, after which half received glycine as their sole N source, while the remainder continued to receive ammonium nitrate. Plants supplied with glycine acquired less N than those receiving the mineral source, resulting in reduced root nitrate concentrations. The amino acid complement of roots was also strongly affected by the form of N supplied, and 15N labelling indicated that the biochemical fate of acquired N in roots was dependent on the form of N available for uptake. Proteomic analysis of Lolium roots indicated that 6% of 627 root proteins resolved on 2D gels changed in abundance in response to the form of N applied. Multivariate analysis of protein abundance clearly discriminated the proteomes of L. perenne roots as a function of treatment applied. Seven affected proteins were identified (mostly by protein homology with sequenced species), including methionine adenosyltransferase, an enzyme involved in glycine metabolism. Although some changes in root amino acid and protein complements were due to responses to reduced N supply, both the distinct fate of 15N tracers and the abundances of identified proteins could be attributed specifically to the form of N available to roots. The results demonstrate the potential of targeted proteomic approaches to identify functioning of plants where more traditional methods cannot resolve multiple, co-incident biological interactions and element fluxes.

Growth-induced changes in the proteome of Helicobacter pylori.

Helicobacter pylori is a major human pathogen that is responsible for a number of gastrointestinal infections. We have used 2-DE to characterise protein synthesis in bacteria grown either on solid agar-based media or in each of two broth culture media (Brucella and brain heart infusion (BHI) broth). Significant differences were observed in the proteomes of bacteria grown either on agar-based or in broth media. Major changes in protein abundance were identified using principal component analysis (PCA), which delineated the profiles derived for the three key growth conditions (i.e. agar plates, Brucella and BHI broth). Proteins detected across the gel series were identified by peptide mass mapping and Edman sequencing. A number of proteins associated with protein synthesis in general as well as specific amino acid synthesis were depressed in broth-grown bacteria compared to plate-grown bacteria. A similar reduction was also observed in the abundance of proteins involved in detoxification. Two of the most abundant spots, identified as UreB and GroEL, in plate-grown bacteria showed a >140-fold drop in abundance in bacteria grown in Brucella broth compared to bacteria grown on agar plates. Two protein spots induced in bacteria grown in broth culture were both identified as glyceraldehyde 3-phosphate dehydrogenase based on their N-terminal amino acid sequences derived by Edman degradation. The underlying causes of the changes in the proteins abundance were not clear, but it was likely that a significant proportion of the changes were due to the alkaline pH of the broth culture media.

Proteomic analysis of the entomopathogenic nematode Steinernema feltiae IS-6 IJs under evaporative and osmotic stresses.

In order to improve the storage capability under desiccation of the widely sold biological insecticides based on entomopathogenic nematodes (EPNs), we need to understand how these organisms respond to desiccation stress. As part of our studies to achieve this, we studied survival and protein expression in infective juveniles of the EPN Steinernema feltiae IS-6 when exposed to evaporative (exposure to 97% relative humidity (RH) for 3 days, followed by a 1-day exposure to 85% RH) and osmotic (exposure to 24% glycerol for 8h) stresses. More than 400 protein spots that were detected by proteomic analysis showed reproducible abundance within replications. Of these, 10 spots and 7 spots showed detectable changes in abundance under evaporative and osmotic stress, respectively, compared to fully hydrated nematodes. Three spots exhibited a differential response pattern between evaporative and osmotic desiccation (one was down regulated and two were novel in evaporative desiccation). Peptide mass mapping with MALDI-TOF mass spectrometry (MS) identified 10 desiccation-response proteins, among which several are known to be stress responsive including heat shock protein 60, coenzyme q biosynthesis protein, inositol monophosphatase and fumarate lyase that were found in both stresses. Other identified proteins are known to be involved in the cell cycle regulation, regulation of gene transcription, organization of macromolecular structure and some currently have no known functions. Our results suggest that it is unlikely that improvement of desiccation tolerance in EPNs can be achieved through genetic transformation and addition of single genes and that selective breeding could be the best approach to generate desiccation resistant worms.

Apolipoprotein AI could be a significant determinant of epithelial integrity in rainbow trout gill cell cultures: a study in functional proteomics.

The freshwater fish gill forms a barrier against an external hypotonic environment. By culturing rainbow trout gill cells on permeable supports, as intact epithelia, this study investigates barrier property mechanisms. Under symmetrical conditions the apical and basolateral epithelial surfaces contact cell culture media. Replacing apical media with water, to generate asymmetrical conditions (i.e. the situation encountered by the freshwater gill), rapidly increases transepithelial resistance (TER). Proteomic analysis revealed that this is associated with enhanced expression of pre-apolipoprotein AI (pre-apoAI). To test the physiological relevance, gill cells were treated with a dose of 50 microg ml(-1) human apolipoprotein (apoAI). This was found to elevate TER in those epithelia which displayed a lower TER prior to apoAI treatment. These results demonstrate the action of apoAI and provide evidence that the rainbow trout gill may be a site of apoAI synthesis. TER does not differentiate between the trans-cellular (via the cell membrane) and para-cellular (via intercellular tight junctions) pathways. However, despite the apoAI-induced changes in TER, para-cellular permeability (measured by polyethylene glycol efflux) remained unaltered suggesting apoAI specifically reduces trans-cellular permeability. This investigation combines proteomics with functional measurements to show how a proteome change may be associated with freshwater gill function.

Proteomic analysis of urine in patients with intestinal segments transposed into the urinary tract.

Intestinal segments are used to replace or reconstruct the urinary bladder when it has become dysfunctional or develops life-threatening disease such as cancer. The quality of life in patients with intestinal segments used to either enlarge or completely replace the native bladder is adversely affected by recurrent urinary tract infections, excessive mucus production and the occasional development of malignancy. At present, there is no reliable method of predicting or noninvasively monitoring these patients for the development of these complications. The characterisation of proteins secreted into urine from the transposed intestinal segments could serve as important indicators of these clinical complications. Urine is an ideal source of material in which to search for biomarkers, since it bathes the affected tissues and can be obtained relatively easily by noninvasive methods. The urinary proteome of patients with intestinal segments transposed into the urinary tract is unknown and we present the first global description of the urinary protein profile in these patients. Sample preparation is a critical step in achieving accurate and reliable data. We describe a method to prepare urinary proteins that was compatible with their subsequent analysis using two-dimensional polyacrylamide gel electrophoresis. This method helped to overcome some of the technical problems encountered in analysing urine from this patient cohort. The method was used to analyse urinary proteins recovered from five healthy controls and ten patients with intestinal segments transposed into the urinary tract. Four low molecular weight proteins were found to be present in nine out of ten for the patient group but for none of the healthy controls. The four proteins were identified as lithostathine-1 alpha precursor, pancreatitis associated protein-1 precursor, liver fatty acid binding protein and testis expressed protein-12. The role of these proteins as potential biomarkers of intestinal cell activity within the reconstructed bladder is discussed.

A systematic approach to modeling, capturing, and disseminating proteomics experimental data.

Both the generation and the analysis of proteome data are becoming increasingly widespread, and the field of proteomics is moving incrementally toward high-throughput approaches. Techniques are also increasing in complexity as the relevant technologies evolve. A standard representation of both the methods used and the data generated in proteomics experiments, analogous to that of the MIAME (minimum information about a microarray experiment) guidelines for transcriptomics, and the associated MAGE (microarray gene expression) object model and XML (extensible markup language) implementation, has yet to emerge. This hinders the handling, exchange, and dissemination of proteomics data. Here, we present a UML (unified modeling language) approach to proteomics experimental data, describe XML and SQL (structured query language) implementations of that model, and discuss capture, storage, and dissemination strategies. These make explicit what data might be most usefully captured about proteomics experiments and provide complementary routes toward the implementation of a proteome repository.

Proteomics: the protein revolution.

Proteomics, the latest scientific buzzword and research field, represents a milestone in biological research, as proteins return to centre stage. But what has led to this protein renaissance and why has proteomics become a key research tool in the post-genomic era? The answers to these questions lie in the huge progress that has been made in the area of genomics.