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-.     

Proteome map of normal rat retina and comparison with the proteome of diabetic rat retina: new insight in the pathogenesis of diabetic retinopathy

We have employed proteomics to establish a proteome map of the normal rat retina. This baseline map was then used for comparison with the early diabetic rat retinal proteome. Diabetic rat retinae were obtained from Dark Agouti rats after 10 wk of streptozotocin-induced hyperglycaemia. Extracted proteins from normal and diabetic rat retinae were separated and compared using 2-DE. A total of 145 protein spots were identified in the normal rat retina using MALDI-MS and database matching. LC-coupled ESI-MS increased the repertoire of identified proteins by 23 from 145 to 168. Comparison with early diabetic rat retinae revealed 24 proteins unique to the diabetic gels, and 37 proteins absent from diabetic gels. Uniquely expressed proteins identified included the HSPs 70.1A and 8, and platelet activating factor. There were eight spots with increased expression and 27 with decreased expression on diabetic gels. Beta catenin, phosducin and aldehyde reductase were increased in expression in diabetes whilst succinyl coA ligase and dihydropyrimidase-related protein were decreased. Identification of such changes in protein expression has given new insights and a more comprehensive understanding of the pathogenesis of diabetic retinopathy, widening the scope of potential avenues for new therapies for this common cause of blindness.     

ProteomeWeb: a web-based interface for the display and interrogation of proteomes

The analysis of proteomes, i.e., the proteins expressed by biological organisms under a given set of conditions at a given time, requires separating complex protein mixtures into discrete protein components, measuring their relative abundances, and identifying the individual protein components. Many types of data are generated during the course of proteome analysis, including graphic images of the protein profiles, flat files containing numeric data, spreadsheets for assimilating numeric data, and relational database tables for integrating data from multiple experiments. As part of a project to describe the proteomes of microbes of interest to the U.S. Department of Energy, a World-Wide Web-based interface has been developed for the display of protein profiles generated by two-dimensional gel electrophoresis. The web interface is capable of obtaining protein identifications on the fly, interrogating the quantitative data in the context of available genome sequence information, and relating the proteome data to existing metabolic pathway databases. Analysis of protein expression profiles is expedited, providing the capability to efficiently determine the gene locations for proteins modulated in abundance in response to different growth conditions and to locate the positions of the proteins within specific metabolic pathways. The proteome of the archaeon Methanococcus jannaschii, a microbe for which the complete genome sequence is available, is used to demonstrate the capabilities of this evolving web interface (http://proteomeweb.anl.gov).     

Flexibility of the metabolism of Corynebacterium glutamicum 2262, a glutamic acid-producing bacterium,in response to temperature upshocks

In order to test the temperature sensitivity of glutamate production metabolism, several temperature shifts, from 33 to 37, 38, 39, 40 or 41°C, were applied to the temperature-sensitive strain, Corynebacterium glutamicum 2262, cultivated in a 24-h fed-batch process. Whereas glucose was entirely dedicated to biomass synthesis when cells were grown at 33°C, applying temperature upshocks, whatever their range, triggered a redistribution of the carbon utilisation between glutamate, biomass and lactate production. Although increasing the culture temperature from 33 to 37, 38, 39 or 40°C resulted in final glutamate titers superior to 80 g/l, temperatures resulting in the best chanelling of the carbon flow towards glutamic acid synthesis were 39 and 40°C. Moreover, this study showed that the higher the temperature, the slower the growth rate and the higher the lactate accumulation. Journal of Industrial Microbiology & Biotechnology (2002) 28, 333–337 DOI: 10.1038/sj/jim/7000251 Received 26 September 2001/ Accepted in revised form 23 February 2002     

A comparative proteomic approach to understand the adaptations of an H+ -ATPase-defective mutant of Corynebacterium glutamicum ATCC14067 to energy deficiencies

F172-8, an H(+)-ATPase-defective mutant of the glutamic acid-producing bacterium Corynebacterium glutamicum ATCC 14067, exhibits enhanced rates of glucose consumption and respiration compared to the parental strain when cultured in a biotin-rich medium with glucose as the carbon source. We conducted a comparative proteomic analysis to clarify the mechanism by which the enhanced glucose metabolism in this mutant is established using a proteome reference map for strain ATCC 14067. A comparison of the proteomes of the two strains revealed the up-regulated expression of the several important enzymes such as pyruvate kinase (Pyk), malate:quinone oxidoreductase (Mqo), and malate dehydrogenase (Mdh) in the mutant. Because Pyk activates glycolysis in response to cellular energy shortages in this bacterium, its increased expression may contribute to the enhanced glucose metabolism of the mutant. A unique reoxidation system has been suggested for NADH in C. glutamicum consisting of coupled reactions between Mqo and Mdh, together with the respiratory chain; therefore, the enhanced expression of both enzymes might contribute to the reoxidation of NADH during increased respiration. The proteomic analysis allowed the identification of unique physiological changes associated with the H(+)-ATPase defect in F172-8 and contributed to the understanding of the adaptations of C. glutamicum to energy deficiencies.     

Proteome analysis of glandular parotid and submandibular-sublingual saliva in comparison to whole human saliva by two-dimensional gel electrophoresis

The secretions of the salivary parotid and submandibular-sublingual (SMSL) glands constitute the main part of whole human saliva (WS) in which proline-rich proteins (PRPs) and mucins represent dominant groups. Although proteome analysis had been performed on WS, no identification of PRPs or mucins by 2-DE and MS was achieved in WS and no comprehensive analysis of both glandular secretions is available so far. The aim of this study was to compare the protein map of WS to parotid and SMSL secretions for the display of PRPs and mucins. WS and glandular secretions were subjected to 2-DE and spots were analyzed by MALDI-MS. New components identified in WS were cyclophilin-B and prolyl-4-hydroxylase. Also acidic and basic PRPs as well as the proline-rich glycoprotein (PRG) could now be mapped in WS. Acidic PRPs were found equally in parotid and SMSL secretions, whereas basic PRPs and PRG were found primarily in parotid secretion. Salivary mucin MUC7 was identified in SMSL secretion. Thus, the more abundant proteins of WS can be explained mainly by mixed contributions of parotid and SMSL secretions with only few components remaining that may be derived from local sources in the oral cavity.     

Susceptibility to oxidative stress: proteomic analysis of bronchoalveolar lavage from ozone-sensitive and ozone-resistant strains of mice

Previous studies have shown that the pulmonary response to ozone (O(3)) varies greatly among strains of mice, but the factor(s) and the mechanism(s) that are responsible for this differential susceptibility have not yet been clearly identified. The present study explores the molecular bases for this differential O(3) susceptibility by studying the expression of proteins associated to the epithelial lining fluid (ELF) from two strains of mice, C57BL/6J and the C3H/HeJ, respectively described as O(3)-sensitive and O(3)-resistant. The ELF proteins of these two strains were displayed by two-dimensional gel electrophoresis (2-DE) of bronchoalveolar lavage fluids (BALFs) and the protein patterns obtained with BALF samples of both strains were compared. Two major differences were observed between the BALF 2-DE protein maps obtained from C57BL/6J and C3H/HeJ strains. First, two isoforms of the antioxidant protein 2 (AOP2) were detected in a strain-dependent manner: C3J/HeJ possesses only AOP2a (isoelectric point 5.7) and C57BL/6J exhibits only AOP2b (isoelectric point 6.0). Second, the levels of anti-inflammatory and immunosuppressive Clara cell protein-16 (CC16) were 1.3 times higher in the BALF from resistant C3H/HeJ than from sensitive C57BL/6 mice. Moreover, two 6 kDa isoforms of CC16 with isoelectric points of 4.9 (CC16a) and 5.2 (CC16b) are detected in both strains. Interestingly, the C57BL/6J strain had a twice decreased level of the acidic isoform of CC16 compared to C3H/HeJ. Our results suggest that AOP2 and CC16 might participate in the protection of the pulmonary tract to O(3)-induced lung injury. The possible differential contribution of specific protein isoforms in the differential susceptibility to oxidative stress is discussed.     

The first two-dimensional reference map of the fission yeast, Schizosaccharomyces pombe proteins

Cytosolic proteins of Schizosaccharomyces pombe were separated by two-dimensional (2-D) gel electrophoresis, to construct the first 2-D reference map. In the pI range 4-7, more than 500 spots were detected by silver staining, and 70 different proteins corresponding to 111 spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry, where necessary. In the pI range 6-9, approximately 330 spots were detected, and 31 proteins corresponding to 38 spots were identified by mass spectrometry. More than 50% of the identified proteins were involved in amino acid, carbohydrate or nucleotide metabolism, and energy production. A second large group of identified proteins comprises heat shock and other stress related proteins and chaperones.     

Functional subgenomics of Clostridium thermocellum cellulosomal genes: identification of the major catalytic components in the extracellular complex and detection of three new enzymes

Clostridium thermocellum produces the most efficient enzyme-complex for the degradation of polysaccharides in biomass, the large extracellular cellulosome. The draft complete genomic sequence of Clostridium thermocellum was screened for open reading frames (ORF) containing cellulosomal dockerin sequences. Seventy-one putative cellulosomal genes were detected. One third of these ORFs may be involved in cellulose hydrolysis. Most of the others showed homology to hemicellulases, pectinases, chitinases, glycosidases or esterases potentially involved in the unwrapping of cellulose fibers. To identify the predominant catalytic components, cellulosomes were purified and the components were separated by an adapted two-dimensional gel electrophoresis technique. The apparent major spots were identified by MALDI-TOF/TOF. Ten of the components were previously known: the structural protein CipA, the endo-glucanases Cel8A, Cel5G, Cel9N, the cellobiohydrolases Cbh9A, Cel9K, Cel48S, the xylanases Xyn10C, Xyn10Z, and the chitinase Chi18A. In addition, three hitherto unknown major components were detected, Cel9R, Xyn10D and Xgh74A. These major components in the cellulosomal particles most probably constitute the essential enzymes for crystalline cellulose hydrolysis.     

Proteomic analysis of the extremely thermoacidophilic archaeon Picrophilus torridus at pH and temperature values close to its growth limit

The thermoacidophilic archaeon Picrophilus torridus belongs to the Thermoplasmatales order and is the most acidophilic organism known to date, growing under extremely acidic conditions around pH 0 (pH(opt) 1) and simultaneously at high temperatures up to 65°C. Some genome features that may be responsible for survival under these harsh conditions have been concluded from the analysis of its 1.55 megabase genome sequence. A proteomic map was generated for P. torridus cells grown to the mid-exponential phase. The soluble fraction of the cells was separated by isoelectric focusing in the pH ranges 4-7 and 3-10, followed by a two dimension (2D) on SDS-PAGE gels. A total of 717 Coomassie collodial-stained protein spots from both pH ranges (pH 4-7 and 3-10) were excised and subjected to LC-MS/MS, leading to the identification of 665 soluble protein spots. Most of the enzymes of the central carbon metabolism were identified on the 2D gels, corroborating biochemically the metabolic pathways predicted from the P. torridus genome sequence. The 2D master gels elaborated in this study represent useful tools for physiological studies of this thermoacidophilic organism. Based on quantitative 2D gel electrophoresis, a proteome study was performed to find pH- or temperature-dependent differences in the proteome composition under changing growth conditions. The proteome expression patterns at two different temperatures (50 and 70°C) and two different pH conditions (pH 0.5 and 1.8) were compared. Several proteins were up-regulated under most stress stimuli tested, pointing to general roles in coping with stress.     

Protein expression profiling of glutathione S-transferase pi null mice as a strategy to identify potential markers of resistance to paracetamol-induced toxicity in the liver

GST pi (GSTP) is a member of the glutathione S-transferase (EC 2.5.1.18; GST) family of enzymes that catalyse the conjugation of electrophilic species with reduced glutathione and thus play an important role in the detoxification of electrophilic metabolites. Deletion of GSTP in mice has previously been shown to lead to enhanced susceptibility to chemical-induced skin carcinoma, consistent with its known metabolic functions. A decreased susceptibility to paracetamol hepatotoxicity has also been observed, which has not been fully explained. One possibility is that deletion of the GSTP gene locus results in compensatory changes in other proteins involved in defence against chemical stress. We have therefore used complementary protein expression profiling techniques to perform a systematic comparison of the protein expression profiles of livers from GSTP null and wild-type mice. Analysis of liver proteins by two-dimensional electrophoresis confirmed the absence of GSTP in null mice whereas GSTP represented 3-5% of soluble protein in livers from wild-type animals. There was a high degree of quantitative and qualitative similarity in other liver proteins between GSTP null and wild-type mice. There was no evidence that the absence of GSTP in null animals resulted in enhanced expression of other GST isoforms in the null mice (GST alpha, 1.48%, GST mu, 1.68% of resolved proteins) compared with the wild-type animals (GST alpha, 1.50%, GST mu, 1.40%). In contrast, some members of the thiol specific antioxidant family of proteins, notably antioxidant protein 2 and thioredoxin peroxidases, were expressed at a higher level in the GSTP null mouse livers. These changes presumably reflect the recently described role of GSTP in cell signalling and may underlie the protection against paracetamol toxicity seen in these animals.     

The Corynebacterium glutamicum gene pmt encoding a glycosyltransferase related to eukaryotic protein-O-mannosyltransferases is essential for glycosylation of the resuscitation promoting factor (Rpf2) and other secreted proteins

Two-dimensional gel electrophoresis and immunoassays revealed several proteins of the secretory subproteome of Corynebacterium glutamicum to be glycosylated. By genome-wide searches for genes involved in glycosylation, the C. glutamicum gene cg1014 was found to exhibit significant similarity to eukaryotic protein-O-mannosyltransferases (PMTs) and to a recently identified orthologue of Mycobacterium tuberculosis, Rv1002c, which is responsible for protein-O-mannosylation. The putative membrane protein Cg1014 showed the same predicted transmembrane topology as Saccharomyces cerevisiae PMT1 and M. tuberculosis Rv1002c along with conserved amino acid residues responsible for catalytic activity. Deletion of the C. glutamicum pmt gene (cg1014) caused a complete loss of glycosylation of secreted proteins including the resuscitation promoting factor 2 (Rpf2), which is involved in intercellular communication and growth stimulation of C. glutamicum. Because the gene pmt as well as rpf genes are present in the genomes of all actinobacteria sequenced so far, this work provides new insights into bacterial protein glycosylation and new opportunities to elucidate the molecular mechanisms of Rpf activity in pathogenic growth and infection.     

Exposure to ultraviolet radiation causes proteomic changes in embryos of the purple sea urchin, Strongylocentrotus purpuratus

We performed experiments to determine how environmentally relevant ultraviolet radiation (UVR) affects protein expression during early development in the sea urchin, Strongylocentrotus purpuratus. To model the protein-mediated cell cycle response to UV-irradiation, six batches of embryos were exposed to UVR, monitored for both delays in the first mitotic division and changes in the proteome at two specific developmental time points. Embryos were exposed to or protected from artificial UVR (11.5 W/m²) for 25 or 60 min. These levels of UVR are within the range we have measured in coastal waters between 0.5 and 2 m. Embryos treated with UVR for 60 min cleaved an average of 23.2 min (± 1.92 s.e.m.) after UV-protected embryos. Differential protein spot migration between UV-protected and UV-treated embryos was examined at 30 and 90 min post-fertilization using two-dimensional SDS-PAGE (2D GE). A total of 1306 protein spots were detected in all gels, including differences in 171 protein spots (13% of the detected proteome) in UV-treated embryos at 30 min post-fertilization and 187 spots (14%) at 90 min post-fertilization (2-way ANOVA, P = 0.03, n = 6). The majority of the proteins affected by UVR were subsequently identified using matrix assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS). Our results indicate UVR affects proteins from multiple cellular pathways and indicate that the mechanisms involved in UV-stress and UV-induced developmental delay in sea urchin embryos are integrated among multiple pathways for cellular stress, protein turnover and translation, signal transduction, cytoskeletal dynamics, and general metabolism.     

Proteomic analysis identified N-cadherin, clusterin, and HSP27 as mediators of SPARC (secreted protein, acidic and rich in cysteines) activity in melanoma cells

Secreted protein, acidic and rich in cysteines (SPARC) is a secreted protein associated with increased aggressiveness of different human cancer types. In order to identify downstream mediators of SPARC activity, we performed a 2-DE proteomic analysis of human melanoma cells following antisense-mediated downregulation of SPARC expression. We found 23/504 differential spots, 15 of which were identified by peptide fingerprinting analysis. Three of the differential proteins (N-cadherin (N-CAD), clusterin (CLU), and HSP27) were validated by immunoblotting, confirming decreased levels of N-CAD and CLU and increased amounts of HSP27 in conditioned media of cells with diminished SPARC expression. Furthermore, transient knock down of SPARC expression in melanoma cells following adenoviral-mediated transfer of antisense RNA confirmed these changes. We next developed two different RNAs against SPARC that were able to inhibit in vivo melanoma cell growth. Immunoblotting of the secreted fraction of RNAi-transfected melanoma cells confirmed that downregulation of SPARC expression promoted decreased levels of N-CAD and CLU and increased secretion of HSP27. Transient re-expression of SPARC in SPARC-downregulated cells reverted extracellular N-CAD, CLU, and HSP27 to levels similar to those in the control. These results constitute the first evidence that SPARC, N-CAD, CLU, and HSP27 converge in a unique molecular network in melanoma cells.     

Drug induced proteome changes in Candida albicans: comparison of the effect of beta(1,3) glucan synthase inhibitors and two triazoles,fluconazole and itraconazole

The dimorphic fungus Candida albicans is an opportunistic human pathogen. Candidiasis is usually treated with azole antifungal agents. However clinical treatments may fail due to the appearance of resistance to this class of antifungal agents in Candida. Echinocandin derivatives are an alternative for the treatment of these fungal infections and are active against azole resistant isolates of C. albicans. Azoles inhibit the lanosterol 14 alpha demethylase which is a key enzyme in the synthesis of ergosterol. In contrast, the echinocandin class of antibiotics inhibit noncompetitively beta-(1,3)-D-glucan synthesis in vitro. We have investigated the impact of mulundocandin on the proteome of C. albicans and compared it to those of a mulundocandin derivative, as well as to two azoles of different structure, fluconazole and itraconazole. The changes in gene expression underlying the antifungal responses were analyzed by comparative 2-D PAGE. Dose dependant responses were kinetically studied on C. albicans grown at 25 degrees C (yeast form) in synthetic dextrose medium. This study shows that antifungals with a common mechanism of action lead to comparable effects at the proteome level and that a proteomics approach can be used to distinguish different antifungals, with the promise to become a useful tool to study drugs of unknown mechanism of action.     

Proteomic analysis of the role of S-nitrosoglutathione reductase in lipopolysaccharide-challenged mice

S-Nitrosoglutathione reductase (GSNOR) is a key regulator of protein S-nitrosylation, the covalent modification of cysteine residues by nitric oxide that can affect activities of many proteins. We recently discovered that excessive S-nitrosylation from GSNOR deficiency in mice under inflammation inactivates the key DNA repair protein O(6) -alkylguanine-DNA alkyltransferase and promotes both spontaneous and carcinogen-induced hepatocellular carcinoma. To explore further the mechanism of tumorigenesis due to GSNOR deficiency, we compared the protein expression profiles in the livers of wild-type and GSNOR-deficient (GSNOR(-/-) ) mice that were challenged with lipopolysaccharide to induce inflammation and expression of inducible nitric oxide synthase (iNOS). Two-dimensional difference gel electrophoresis analysis identified 38 protein spots of significantly increased intensity and 31 protein spots of significantly decreased intensity in the GSNOR(-/-) mice compared to those in the wild-type mice. We subsequently identified 19 upregulated and 19 downregulated proteins in GSNOR(-/-) mice using mass spectrometry. Immunoblot analysis confirmed in GSNOR(-/-) mice a large increase in the expression of the pro-inflammatory mediator S100A9, a protein previously implicated in human liver carcinogenesis. We also found a decrease in the expression of multiple members of the protein disulfide-isomerase (PDI) family and an alteration in the expression pattern of the endoplasmic reticulum (ER) chaperones in GSNOR(-/-) mice. Furthermore, altered expression of these proteins from GSNOR deficiency was prevented in mice lacking both GSNOR and iNOS. In addition, we detected S-nitrosylation of two members of the PDI protein family. These results suggest that S-nitrosylation resulting from GSNOR deficiency may promote carcinogenesis under inflammatory conditions in part through the disruption of inflammatory and ER stress responses.     

The proteomic to biology inference,a frequently overlooked concern in the interpretation of proteomic data: A plea for functional validation

Proteomics will celebrate its 20th year in 2014. In this relatively short period of time, it has invaded most areas of biology and its use will probably continue to spread in the future. These two decades have seen a considerable increase in the speed and sensitivity of protein identification and characterization, even from complex samples. Indeed, what was a challenge twenty years ago is now little more than a daily routine. Although not completely over, the technological challenge now makes room to another challenge, which is the best possible appraisal and exploitation of proteomic data to draw the best possible conclusions from a biological point of view. The point developed in this paper is that proteomic data are almost always fragmentary. This means in turn that although better than an mRNA level, a protein level is often insufficient to draw a valid conclusion from a biological point of view, especially in a world where PTMs play such an important role. This means in turn that transformation of proteomic data into biological data requires an important intermediate layer of functional validation, i.e. not merely the confirmation of protein abundance changes by other methods, but a functional appraisal of the biological consequences of the protein level changes highlighted by the proteomic screens.