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1.  Description of the interaction between Candida albicans and macrophages by mixed and quantitative proteome analysis without isolation 
AMB Express  2015;5:41.
Candida albicans is an opportunistic pathogen that causes fatal diseases in immunocompromised hosts. Host resistance against C. albicans relies on ingestion of the pathogen by macrophages. Analysis of the escaping behavior of C. albicans from macrophages is required to understand the onset of systemic candidiasis. In this study, native interactions of C. albicans with macrophages were investigated by proteome analysis using high efficiency of long monolithic silica capillary column. Using this system, we developed a method of “mixed and quantitative proteome analysis” in which C. albicans and macrophages were simultaneously analyzed by nanoLC–MS/MS without the need to isolate the two individual living cells. Two hundred twenty-seven proteins from C. albicans and five proteins from macrophages were identified as candidate interaction-specific molecules. C. albicans seemed to produce glucose through a β-oxidation pathway, a glyoxylate cycle, and gluconeogenesis for escape from macrophages. Up-regulation of stress-related and candidate pathogenic proteins in C. albicans indicated how C. albicans endured the harsh environment inside the macrophages. Down-regulation of apoptosis-associated protein NOA1- and chaperone HSPA1A-syntheses in macrophage indicated that C. albicans was able to escape from macrophages in part by suppressing the production of these macrophage proteins.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-015-0127-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4503712  PMID: 26179440
Candida albicans; Macrophage; Mixed proteome analysis; Quantitative proteome analysis; Apoptosis; Chaperone
2.  Elucidation of the recognition mechanisms for hemicellulose and pectin in Clostridium cellulovorans using intracellular quantitative proteome analysis 
AMB Express  2015;5:29.
Clostridium cellulovorans is an anaerobic, cellulolytic bacterium, capable of effectively degrading and metabolizing various types of substrates, including cellulose, hemicellulose (xylan and galactomannan), and pectin. Among Clostridia, this ability to degrade and metabolize a wide range of hemicellulose and pectin substrates is a unique feature; however, the mechanisms are currently unknown. To clarify the mechanisms of hemicelluloses and pectin recognition and metabolism, we carried out a quantitative proteome analysis of C. cellulovorans cultured with these substrates. C. cellulovorans was cultured in the medium of glucose (control), xylan, galactomannan (Locus bean gum, LBG), or pectin for 36 h. Xylan and galactomannan were used to search for the common recognition mechanisms of hemicellulose, and pectin was used to search for unique recognition systems in C. cellulovorans. Using an isobaric tag method and liquid chromatograph/mass spectrometer equipped with a long monolithic silica capillary column, we identified 734 intracellular proteins from all substrates. We performed KEGG analyses and cluster analyses of the resulting proteins. In the KEGG analyses, we found common degradation mechanisms for hemicellulose and pectin. In the cluster analysis corresponding to the genome analysis, we detected substrate-specific clusters that include genes involved in substrate recognition, substrate degradation, and metabolism. Combining the results of the KEGG analyses and cluster analyses, we propose the mechanisms involved in the recognition and metabolism of hemicellulose and pectin in C. cellulovorans.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-015-0115-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4441647  PMID: 26020016
Clostridium cellulovorans; Proteome analysis; Monolithic column; Substrate recognition; Hemicellulose; Pectin; Metabolism
3.  Exoproteome analysis of Clostridium cellulovorans in natural soft-biomass degradation 
AMB Express  2015;5(1):2.
Clostridium cellulovorans is an anaerobic, cellulolytic bacterium, capable of effectively degrading various types of soft biomass. Its excellent capacity for degradation results from optimization of the composition of the protein complex (cellulosome) and production of non-cellulosomal proteins according to the type of substrates. In this study, we performed a quantitative proteome analysis to determine changes in the extracellular proteins produced by C. cellulovorans for degradation of several types of natural soft biomass. C. cellulovorans was cultured in media containing bagasse, corn germ, rice straw (natural soft biomass), or cellobiose (control). Using an isobaric tag method and a liquid chromatograph equipped with a long monolithic silica capillary column/mass spectrometer, we identified 372 proteins in the culture supernatant. Of these, we focused on 77 saccharification-related proteins of both cellulosomal and non-cellulosomal origins. Statistical analysis showed that 18 of the proteins were specifically produced during degradation of types of natural soft biomass. Interestingly, the protein Clocel_3197 was found and commonly involved in the degradation of every natural soft biomass studied. This protein may perform functions, in addition to its known metabolic functions, that contribute to effective degradation of natural soft biomass.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-014-0089-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4305082  PMID: 25642399
Clostridium cellulovorans; Cellulosome; Soft-biomass degradation; Proteome analysis; Monolithic column
4.  Insect-Induced Daidzein, Formononetin and Their Conjugates in Soybean Leaves 
Metabolites  2014;4(3):532-546.
In response to attack by bacterial pathogens, soybean (Gylcine max) leaves accumulate isoflavone aglucones, isoflavone glucosides, and glyceollins. In contrast to pathogens, the dynamics of related insect-inducible metabolites in soybean leaves remain poorly understood. In this study, we analyzed the biochemical responses of soybean leaves to Spodoptera litura (Lepidoptera: Noctuidae) herbivory and also S. litura gut contents, which contain oral secretion elicitors. Following S. litura herbivory, soybean leaves displayed an induced accumulation of the flavone and isoflavone aglycones 4’,7-dihyroxyflavone, daidzein, and formononetin, and also the isoflavone glucoside daidzin. Interestingly, foliar application of S. litura oral secretions also elicited the accumulation of isoflavone aglycones (daidzein and formononetin), isoflavone 7-O-glucosides (daidzin, ononin), and isoflavone 7-O-(6’-O-malonyl-β-glucosides) (malonyldaidzin, malonylononin). Consistent with the up-regulation of the isoflavonoid biosynthetic pathway, folair phenylalanine levels also increased following oral secretion treatment. To establish that these metabolitic changes were the result of de novo biosynthesis, we demonstrated that labeled (13C9) phenylalanine was incorporated into the isoflavone aglucones. These results are consistent with the presence of soybean defense elicitors in S. litura oral secretions. We demonstrate that isoflavone aglycones and isoflavone conjugates are induced in soybean leaves, not only by pathogens as previously demonstrated, but also by foliar insect herbivory.
PMCID: PMC4192678  PMID: 25000357
plant-insect interaction; plant induced resistance; insect-produced elicitors; isoflavones; insect herbivory; secondary metabolites; liquid chromatography; mass spectrometry
5.  Exoproteome Profiles of Clostridium cellulovorans Grown on Various Carbon Sources 
Applied and Environmental Microbiology  2013;79(21):6576-6584.
The cellulosome is a complex of cellulosomal proteins bound to scaffolding proteins. This complex is considered the most efficient system for cellulose degradation. Clostridium cellulovorans, which is known to produce cellulosomes, changes the composition of its cellulosomes depending on the growth substrates. However, studies have investigated only cellulosomal proteins; profile changes in noncellulosomal proteins have rarely been examined. In this study, we performed a quantitative proteome analysis of the whole exoproteome of C. cellulovorans, including cellulosomal and noncellulosomal proteins, to illustrate how various substrates are efficiently degraded. C. cellulovorans was cultured with cellobiose, xylan, pectin, or phosphoric acid-swollen cellulose (PASC) as the sole carbon source. PASC was used as a cellulose substrate for more accurate quantitative analysis. Using an isobaric tag method and a liquid chromatography mass spectrometer equipped with a long monolithic silica capillary column, 639 proteins were identified and quantified in all 4 samples. Among these, 79 proteins were involved in saccharification, including 35 cellulosomal and 44 noncellulosomal proteins. We compared protein abundance by spectral count and found that cellulosomal proteins were more abundant than noncellulosomal proteins. Next, we focused on the fold change of the proteins depending on the growth substrates. Drastic changes were observed mainly among the noncellulosomal proteins. These results indicate that cellulosomal proteins were primarily produced to efficiently degrade any substrate and that noncellulosomal proteins were specifically produced to optimize the degradation of a particular substrate. This study highlights the importance of noncellulosomal proteins as well as cellulosomes for the efficient degradation of various substrates.
PMCID: PMC3811513  PMID: 23956399
6.  Spatial Reorganization of Saccharomyces cerevisiae Enolase To Alter Carbon Metabolism under Hypoxia 
Eukaryotic Cell  2013;12(8):1106-1119.
Hypoxia has critical effects on the physiology of organisms. In the yeast Saccharomyces cerevisiae, glycolytic enzymes, including enolase (Eno2p), formed cellular foci under hypoxia. Here, we investigated the regulation and biological functions of these foci. Focus formation by Eno2p was inhibited temperature independently by the addition of cycloheximide or rapamycin or by the single substitution of alanine for the Val22 residue. Using mitochondrial inhibitors and an antioxidant, mitochondrial reactive oxygen species (ROS) production was shown to participate in focus formation. Focus formation was also inhibited temperature dependently by an SNF1 knockout mutation. Interestingly, the foci were observed in the cell even after reoxygenation. The metabolic turnover analysis revealed that [U-13C]glucose conversion to pyruvate and oxaloacetate was accelerated in focus-forming cells. These results suggest that under hypoxia, S. cerevisiae cells sense mitochondrial ROS and, by the involvement of SNF1/AMPK, spatially reorganize metabolic enzymes in the cytosol via de novo protein synthesis, which subsequently increases carbon metabolism. The mechanism may be important for yeast cells under hypoxia, to quickly provide both energy and substrates for the biosynthesis of lipids and proteins independently of the tricarboxylic acid (TCA) cycle and also to fit changing environments.
PMCID: PMC3754543  PMID: 23748432
7.  Fixation of CO2 in Clostridium cellulovorans analyzed by 13C-isotopomer-based target metabolomics 
AMB Express  2013;3:61.
Clostridium cellulovorans has been one of promising microorganisms to use biomass efficiently; however the basic metabolic pathways have not been completely known. We carried out 13C-isotopomer-based target metabolome analysis, or carbohydrate conversion process analysis, for more profound understanding of metabolic pathways of the bacterium. Our findings that pyruvate + oxaloacetate, fumarate, and malate inside and outside cells exhibited 13C incorporation suggest that C. cellulovorans exactly fixed CO2 and partly operated the TCA cycle in a reductive manner. Accompanied with CO2 fixation, the microorganism was also found to produce and secrete lactate. Overall, our study demonstrates that a part of C. cellulovorans metabolic pathways related to glycolysis and the TCA cycle are involved in CO2 fixation.
PMCID: PMC4124662  PMID: 24103325
CO2 fixation; Clostridium cellulovorans; Target metabolomics
8.  Disclosure of the differences of Mesorhizobium loti under the free-living and symbiotic conditions by comparative proteome analysis without bacteroid isolation 
BMC Microbiology  2013;13:180.
Rhizobia are symbiotic nitrogen-fixing soil bacteria that show a symbiotic relationship with their host legume. Rhizobia have 2 different physiological conditions: a free-living condition in soil, and a symbiotic nitrogen-fixing condition in the nodule. The lifestyle of rhizobia remains largely unknown, although genome and transcriptome analyses have been carried out. To clarify the lifestyle of bacteria, proteome analysis is necessary because the protein profile directly reflects in vivo reactions of the organisms. In proteome analysis, high separation performance is required to analyze complex biological samples. Therefore, we used a liquid chromatography-tandem mass spectrometry system, equipped with a long monolithic silica capillary column, which is superior to conventional columns. In this study, we compared the protein profile of Mesorhizobium loti MAFF303099 under free-living condition to that of symbiotic conditions by using small amounts of crude extracts.
We identified 1,533 and 847 proteins for M. loti under free-living and symbiotic conditions, respectively. Pathway analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that many of the enzymes involved in the central carbon metabolic pathway were commonly detected under both conditions. The proteins encoded in the symbiosis island, the transmissible chromosomal region that includes the genes that are highly upregulated under the symbiotic condition, were uniquely detected under the symbiotic condition. The features of the symbiotic condition that have been reported by transcriptome analysis were confirmed at the protein level by proteome analysis. In addition, the genes of the proteins involved in cell surface structure were repressed under the symbiotic nitrogen-fixing condition. Furthermore, farnesyl pyrophosphate (FPP) was found to be biosynthesized only in rhizobia under the symbiotic condition.
The obtained protein profile appeared to reflect the difference in phenotypes under the free-living and symbiotic conditions. In addition, KEGG pathway analysis revealed that the cell surface structure of rhizobia was largely different under each condition, and surprisingly, rhizobia might provided FPP to the host as a source of secondary metabolism. M. loti changed its metabolism and cell surface structure in accordance with the surrounding conditions.
PMCID: PMC3750425  PMID: 23898917
Mesorhizobium loti; Lotus japonicus; Symbiosis; Proteome analysis; Plant-microbe interaction; Monolithic column; Nitrogen fixation; Rhizobase; KEGG
9.  Secretome Analysis of the Pine Wood Nematode Bursaphelenchus xylophilus Reveals the Tangled Roots of Parasitism and Its Potential for Molecular Mimicry 
PLoS ONE  2013;8(6):e67377.
Since it was first introduced into Asia from North America in the early 20th century, the pine wood nematode Bursaphelenchus xylophilus has caused the devastating forest disease called pine wilt. The emerging pathogen spread to parts of Europe and has since been found as the causal agent of pine wilt disease in Portugal and Spain. In 2011, the entire genome sequence of B. xylophilus was determined, and it allowed us to perform a more detailed analysis of B. xylophilus parasitism. Here, we identified 1,515 proteins secreted by B. xylophilus using a highly sensitive proteomics method combined with the available genomic sequence. The catalogue of secreted proteins contained proteins involved in nutrient uptake, migration, and evasion from host defenses. A comparative functional analysis of the secretome profiles among parasitic nematodes revealed a marked expansion of secreted peptidases and peptidase inhibitors in B. xylophilus via gene duplication and horizontal gene transfer from fungi and bacteria. Furthermore, we showed that B. xylophilus secreted the potential host mimicry proteins that closely resemble the host pine’s proteins. These proteins could have been acquired by host–parasite co-evolution and might mimic the host defense systems in susceptible pine trees during infection. This study contributes to an understanding of their unique parasitism and its tangled roots, and provides new perspectives on the evolution of plant parasitism among nematodes.
PMCID: PMC3689755  PMID: 23805310
10.  Tracing Putative Trafficking of the Glycolytic Enzyme Enolase via SNARE-Driven Unconventional Secretion 
Eukaryotic Cell  2012;11(8):1075-1082.
Glycolytic enzymes are cytosolic proteins, but they also play important extracellular roles in cell-cell communication and infection. We used Saccharomyces cerevisiae to analyze the secretory pathway of some of these enzymes, including enolase, phosphoglucose isomerase, triose phosphate isomerase, and fructose 1,6-bisphosphate aldolase. Enolase, phosphoglucose isomerase, and an N-terminal 28-amino-acid-long fragment of enolase were secreted in a sec23-independent manner. The enhanced green fluorescent protein (EGFP)-conjugated enolase fragment formed cellular foci, some of which were found at the cell periphery. Therefore, we speculated that an overview of the secretory pathway could be gained by investigating the colocalization of the enolase fragment with intracellular proteins. The DsRed-conjugated enolase fragment colocalized with membrane proteins at the cis-Golgi complex, nucleus, endosome, and plasma membrane, but not the mitochondria. In addition, the secretion of full-length enolase was inhibited in a knockout mutant of the intracellular SNARE protein-coding gene TLG2. Our results suggest that enolase is secreted via a SNARE-dependent secretory pathway in S. cerevisiae.
PMCID: PMC3416056  PMID: 22753847
11.  Profile of native cellulosomal proteins of Clostridium cellulovorans adapted to various carbon sources 
AMB Express  2012;2:37.
We performed a focused proteome analysis of cellulosomal proteins predicted by a genome analysis of Clostridium cellulovorans [Tamaru, Y., et al.. 2010. J. Bacteriol. 192:901–902]. Our system employed a long monolithic column (300 cm), which provides better performance and higher resolution than conventional systems. Twenty-three cellulosomal proteins were, without purification, identified by direct analysis of the culture medium. Proteome analysis of the C. cellulovorans cellulosome after culture in various carbon sources demonstrated the production of carbon source-adapted cellulosome components.
PMCID: PMC3444338  PMID: 22839966
Clostridium cellulovorans; Cellulosome; Focused proteome analysis; Monolithic column
12.  Putative Role of Cellulosomal Protease Inhibitors in Clostridium cellulovorans Based on Gene Expression and Measurement of Activities▿ 
Journal of Bacteriology  2011;193(19):5527-5530.
This study is the first to demonstrate the activity of putative cellulosomal protease/peptidase inhibitors (named cyspins) of Clostridium cellulovorans, using the Saccharomyces cerevisiae display system. Cyspins exhibited inhibitory activities against several representative plant proteases. This suggests that these inhibitors protect their microbe and cellulosome from external attack by plant proteases.
PMCID: PMC3187468  PMID: 21784939
13.  Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease 
PLoS ONE  2012;7(2):e32513.
Candida albicans, a commensal organism, is a part of the normal flora of healthy individuals. However, once the host immunity is compromised, C. albicans opportunistically causes recurrent superficial or fatal systemic candidiasis. Secreted aspartic proteases (Sap), encoded by 10 types of SAP genes, have been suggested to contribute to various virulence processes. Thus, it is important to elucidate their biochemical properties for better understanding of the molecular mechanisms that how Sap isozymes damage host tissues.
Methodology/Principal Findings
The SAP7 gene was cloned from C. albicans SC5314 and heterogeneously produced by Pichia pastoris. Measurement of Sap7 proteolytic activity using the FRETS-25Ala library showed that Sap7 was a pepstatin A-insensitive protease. To understand why Sap7 was insensitive to pepstatin A, alanine substitution mutants of Sap7 were constructed. We found that M242A and T467A mutants had normal proteolytic activity and sensitivity to pepstatin A. M242 and T467 were located in close proximity to the entrance to an active site, and alanine substitution at these positions widened the entrance. Our results suggest that this alteration might allow increased accessibility of pepstatin A to the active site. This inference was supported by the observation that the T467A mutant has stronger proteolytic activity than the wild type.
We found that Sap7 was a pepstatin A-insensitive protease, and that M242 and T467 restricted the accessibility of pepstatin A to the active site. This finding will lead to the development of a novel protease inhibitor beyond pepstatin A. Such a novel inhibitor will be an important research tool as well as pharmaceutical agent for patients suffering from candidiasis.
PMCID: PMC3287985  PMID: 22384266

Results 1-13 (13)