Mitochondrial carnitine palmitoyltransferase 2 is involved in Nε-(carboxymethyl)-lysine-mediated diabetic nephropathy.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease in the world. Advanced glycation end products (AGEs) are thought to be involved in the pathogenesis of DN via multifactorial mechanisms including the generation of oxidative stress and overproduction of various growth factors and cytokines. AGEs are heterogeneous cross-linked sugar-derived proteins, and Nε-(carboxymethyl)-lysine (CML)-conjugated BSA is a major component of AGEs. However, the proteins involved in DN induction by CML have never been reported. Herein, we investigated specific protein regulators of AGE-mediated DN via proteomic analysis of streptozotocin (STZ)-induced diabetic mice kidneys. We identified 937, 976, and 870 proteins in control, STZ, and STZ + CML-BSA samples, respectively. Bioinformatics analysis identified several CML-mediated proteins potentially involved in kidney damage, activation of fatty acid oxidation (FAO), and mitochondrial dysfunction. Furthermore, we identified the CML-specific differential protein carnitine palmitoyltransferase 2 (CPT2), related to FAO. To confirm the effect of CPT2 and the CML-mediated mechanism, human renal tubular HK-2 cells were treated with CML-BSA and cpt2 siRNA, and examined for FAO-mediated fibrosis and mitochondrial dysfunction. CML-BSA and CPT2 knockdown induced fibrosis-related gene expression and damage to mitochondrial membrane potential. Moreover, CPT2 overexpression recovered CML-induced fibrosis-related gene expression. Based on these results, a decrease in CML-induced CPT2 expression causes mitochondrial FAO damage, leading to renal fibrosis and DN.

Proteomic Analysis of Urothelium of Rats with Detrusor Overactivity Induced by Bladder Outlet Obstruction.

Overactive bladder (OAB) syndrome is a condition that has four symptoms: urgency, urinary frequency, nocturia, and urge incontinence and negatively affects a patient's life. Recently, it is considered that the urinary bladder urothelium is closely linked to pathogenesis of OAB. However, the mechanisms of pathogenesis of OAB at the molecular level remain poorly understood, mainly because of lack of modern molecular analysis. The goal of this study is to identify a potential target protein that could act as a predictive factor for effective diagnosis and aid in the development of therapeutic strategies for the treatment of OAB syndrome. We produced OAB in a rat model and performed the first proteomic analysis on the mucosal layer (urothelium) of the bladders of sham control and OAB rats. The resulting data revealed the differential expression of 355 proteins in the bladder urothelium of OAB rats compared with sham subjects. Signaling pathway analysis revealed that the differentially expressed proteins were mainly involved in the inflammatory response and apoptosis. Our findings suggest a new target for accurate diagnosis of OAB that can provide essential information for the development of drug treatment strategies as well as establish criteria for screening patients in the clinical environment.

Anti-Osteoarthritic Mechanisms of Chrysanthemum zawadskii var. latilobum in MIA-Induced Osteoarthritic Rats and Interleukin-1ß-Induced SW1353 Human Chondrocytes.

Background and objectives: Chrysanthemum zawadskii var. latilobum (CZ), which has traditionally been used as a oriental tea in Asia, is known to have anti-inflammatory effects in osteoarthritis (OA). But the mechanism of these effects has not been made clear and it needs to be elucidated specifically for the clinical use of CZE in OA. Materials and Methods: To reveal this mechanism, we first identified which biomarkers were expressed in the joints of rats in which OA had been induced with monosodium iodoacetate and determined whether CZ extract (CZE) could normalize these biomarkers in the progression of OA. The anti-osteoarthritis effect of CZE was evaluated for its capability to inhibit levels of extracellular matrix (ECM)-degrading enzymes and enhance ECM synthesis. We also sought to identify whether the marker compound of CZE, linarin, has anti-osteoarthritic effects in the human chondrosarcoma cell line SW1353. Results: The changes in matrix metalloproteinases (MMPs) were remarkable: among them, MMP-1, MMP-3, MMP-9 and MMP-13 were most strongly induced, whereas their expressions were inhibited by CZE dose dependently. The expressions of the ECM synthetic genes, COL2A1 and ACAN, and the transcription factor SOX9 of these genes were reduced by OA induction and significantly normalized by CZE dose dependently. SOX9 is also a repressor of ECM-degrading aggrecanases, ADAMTS-4 and ADAMTS-5, and CZE significantly reduced the levels of these enzymes dose dependently. Similar results were obtained using the human chondrosarcoma cell line SW1353 with linarin, the biologically active compound of CZE. Conclusions: These anti-osteoarthritic effects suggest that CZE has mechanisms for activating ECM synthesis with SOX9 as well as inhibiting articular ECM-degrading enzymes.

Clinical proteomic analysis of scrub typhus infection.

BACKGROUND: Scrub typhus is an acute and febrile infectious disease caused by the Gram-negative α-proteobacterium Orientia tsutsugamushi from the family Rickettsiaceae that is widely distributed in Northern, Southern and Eastern Asia. In the present study, we analysed the serum proteome of scrub typhus patients to investigate specific clinical protein patterns in an attempt to explain pathophysiology and discover potential biomarkers of infection. METHODS: Serum samples were collected from three patients (before and after treatment with antibiotics) and three healthy subjects. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry was performed to identify differentially abundant proteins using quantitative proteomic approaches. Bioinformatic analysis was then performed using Ingenuity Pathway Analysis. RESULTS: Proteomic analysis identified 236 serum proteins, of which 32 were differentially expressed in normal subjects, naive scrub typhus patients and patients treated with antibiotics. Comparative bioinformatic analysis of the identified proteins revealed up-regulation of proteins involved in immune responses, especially complement system, following infection with O. tsutsugamushi, and normal expression was largely rescued by antibiotic treatment. CONCLUSIONS: This is the first proteomic study of clinical serum samples from scrub typhus patients. Proteomic analysis identified changes in protein expression upon infection with O. tsutsugamushi and following antibiotic treatment. Our results provide valuable information for further investigation of scrub typhus therapy and diagnosis.

Antibiotic treatment modulates protein components of cytotoxic outer membrane vesicles of multidrug-resistant clinical strain, Acinetobacter baumannii DU202.

BACKGROUND: Outer membrane vesicles (OMVs) of Acinetobacter baumannii are cytotoxic and elicit a potent innate immune response. OMVs were first identified in A. baumannii DU202, an extensively drug-resistant clinical strain. Herein, we investigated protein components of A. baumannii DU202 OMVs following antibiotic treatment by proteogenomic analysis. METHODS: Purified OMVs from A. baumannii DU202 grown in different antibiotic culture conditions were screened for pathogenic and immunogenic effects, and subjected to quantitative proteomic analysis by one-dimensional electrophoresis and liquid chromatography combined with tandem mass spectrometry (1DE-LC-MS/MS). Protein components modulated by imipenem were identified and discussed. RESULTS: OMV secretion was increased > twofold following imipenem treatment, and cytotoxicity toward A549 human lung carcinoma cells was elevated. A total of 277 proteins were identified as components of OMVs by imipenem treatment, among which ß-lactamase OXA-23, various proteases, outer membrane proteins, ß-barrel assembly machine proteins, peptidyl-prolyl cis-trans isomerases and inherent prophage head subunit proteins were significantly upregulated. CONCLUSION: In vitro stress such as antibiotic treatment can modulate proteome components in A. baumannii OMVs and thereby influence pathogenicity.

Salt stress affects global protein expression profiles of extracellular membrane-derived vesicles of Listeria monocytogenes.

Our previous study has suggested that Listeria monocytogenes produces extracellular membrane vesicles (MVs) and its general stress transcription factor sigma B (σB) affects the production of MVs under energy stress. The objective of this study was to evaluate the production of MVs and perform global protein profiling for MVs with or without salt stress to understand the function of MVs in the pathogenesis of L. monocytogenes. When cells of L. monocytogenes were grown under 0.5 M salt stress, protein concentrations of MVs derived from wild-type strain and its isogenic ΔsigB mutant were approximately doubled compared to those of MVs derived from cells without salt stress. Proteomic analyses showed that the number of MV proteins expressed in wild-type strain was similar to that in ΔsigB mutant under salt stress. However, global protein expression profiles were dramatically changed under salt stress compared to those without salt stress. Fifteen σB dependent proteins were expressed in MVs of wild-type strain under salt stress, including osmolyte transporter OpuCABCD. In addition, MVs produced by salt stressed wild-type and ΔsigB mutant inhibited biofilm formation abilities of both strains. Taken together, our results suggest that salt stress can promote the production of MVs involved in carnitine transporter proteins, with σB playing a pivotal role in biological event.

Clostridium difficile-derived membrane vesicles induce the expression of pro-inflammatory cytokine genes and cytotoxicity in colonic epithelial cells in vitro.

Clostridium difficile is the most common etiological agent of antibiotic-associated diarrhea in hospitalized and non-hospitalized patients. This study investigated the secretion of membrane vesicles (MVs) from C. difficile and determined the expression of pro-inflammatory cytokine genes and cytotoxicity of C. difficile MVs in epithelial cells in vitro. C. difficile ATCC 43255 and two clinical isolates secreted spherical MVs during in vitro culture. Proteomic analysis revealed that MVs of C. difficile ATCC 43255 contained a total of 262 proteins. Translation-associated proteins were the most commonly identified in C. difficile MVs, whereas TcdA and TcdB toxins were not detected. C. difficile ATCC 43255-derived MVs stimulated the expression of pro-inflammatory cytokine genes, including interleukin (IL)-1ß, IL-6, IL-8, and monocyte chemoattractant protein-1 in human colorectal epithelial Caco-2 cells. Moreover, these extracellular vesicles induced cytotoxicity in Caco-2 cells. In conclusion, C. difficile MVs are important nanocomplexes that elicit a pro-inflammatory response and induce cytotoxicity in colonic epithelial cells, which may contribute, along with toxins, to intestinal mucosal injury during C. difficile infection.

Characterization of Site-Specific N-Glycopeptide Isoforms of α-1-Acid Glycoprotein from an Interlaboratory Study Using LC-MS/MS.

Glycoprotein conformations are complex and heterogeneous. Currently, site-specific characterization of glycopeptides is a challenge. We sought to establish an efficient method of N-glycoprotein characterization using mass spectrometry (MS). Using alpha-1-acid glycoprotein (AGP) as a model N-glycoprotein, we identified its tryptic N-glycopeptides and examined the data reproducibility in seven laboratories running different LC-MS/MS platforms. We used three test samples and one blind sample to evaluate instrument performance with entire sample preparation workflow. 165 site-specific N-glycopeptides representative of all N-glycosylation sites were identified from AGP 1 and AGP 2 isoforms. The glycopeptide fragmentations by collision-induced dissociation or higher-energy collisional dissociation (HCD) varied based on the MS analyzer. Orbitrap Elite identified the greatest number of AGP N-glycopeptides, followed by Triple TOF and Q-Exactive Plus. Reproducible generation of oxonium ions, glycan-cleaved glycopeptide fragment ions, and peptide backbone fragment ions was essential for successful identification. Laboratory proficiency affected the number of identified N-glycopeptides. The relative quantities of the 10 major N-glycopeptide isoforms of AGP detected in four laboratories were compared to assess reproducibility. Quantitative analysis showed that the coefficient of variation was <25% for all test samples. Our analytical protocol yielded identification and quantification of site-specific N-glycopeptide isoforms of AGP from control and disease plasma sample.

Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells.

Staphylococcus aureus secretes membrane-derived vesicles (MVs), which can deliver virulence factors to host cells and induce cytopathology. However, the cytopathology of host cells induced by MVs derived from different S. aureus strains has not yet been characterized. In the present study, the cytotoxic activity of MVs from different S. aureus isolates on host cells was compared and the proteomes of S. aureus MVs were analyzed. The MVs purified from S. aureus M060 isolated from a patient with staphylococcal scalded skin syndrome showed higher cytotoxic activity toward host cells than that shown by MVs from three other clinical S. aureus isolates. S. aureus M060 MVs induced HEp-2 cell apoptosis in a dose-dependent manner, but the cytotoxic activity of MVs was completely abolished by treatment with proteinase K. In a proteomic analysis, the MVs from three S. aureus isolates not only carry 25 common proteins, but also carry ≥60 strain-specific proteins. All S. aureus MVs contained δ-hemolysin (Hld), γ-hemolysin, leukocidin D, and exfoliative toxin C, but exfoliative toxin A (ETA) was specifically identified in S. aureus M060 MVs. ETA was delivered to HEp-2 cells via S. aureus MVs. Both rETA and rHld induced cytotoxicity in HEp-2 cells. In conclusion, MVs from clinical S. aureus isolates differ with respect to cytotoxic activity in host cells, and these differences may result from differences in the MV proteomes. Further proteogenomic analysis or mutagenesis of specific genes is necessary to identify cytotoxic factors in S. aureus MVs.

Acinetobacter nosocomialis secretes outer membrane vesicles that induce epithelial cell death and host inflammatory responses.

Acinetobacter nosocomialis is an important nosocomial pathogen that causes a variety of opportunistic infections; however, pathogenesis of this microorganism has not yet been characterized. The aim of this study was to investigate the secretion of outer membrane vesicles (OMVs) from A. nosocomialis and to determine their cytotoxic effects and their ability to induce inflammatory responses both in vitro and in vivo by using human epithelial HEp-2 cells and a mouse model, respectively. A. nosocomialis ATCC 17903(T) secreted spherical OMVs when cultured in vitro. Proteomic analysis revealed that 147 different proteins were associated with A. nosocomialis OMVs and virulence-associated proteins, such as outer membrane protein A (OmpA), CsuA, CsuC, CsuD, PilW, hemolysin, and serine protease, were identified. A. nosocomialis OMVs were cytotoxic to HEp-2 cells. These vesicles also induced the expression of pro-inflammatory cytokine genes in the HEp-2 cells. Early inflammatory responses, such as congestion and focal neutrophilic infiltration, were observed in the lungs of mice injected with A. nosocomialis OMVs. In conclusion, A. nosocomialis OMVs are important secretory nanocomplexes that induce cytotoxicity of epithelial cells and host inflammatory responses, which may contribute to the pathogenesis of A. nosocomialis.

Proteomic characterization of the outer membrane vesicle of Pseudomonas putida KT2440.

Outer membrane vesicles (OMVs) are produced by various pathogenic Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. In this study, we isolated OMVs from a representative soil bacterium, Pseudomonas putida KT2440, which has a biodegradative activity toward various aromatic compounds. Proteomic analysis identified the outer membrane proteins (OMPs) OprC, OprD, OprE, OprF, OprH, OprG, and OprW as major components of the OMV of P. putida KT2440. The production of OMVs was dependent on the nutrient availability in the culture media, and the up- or down-regulation of specific OMPs was observed according to the culture conditions. In particular, porins (e.g., benzoate-specific porin, BenF-like porin) and enzymes (e.g., catechol 1,2-dioxygenase, benzoate dioxygenase) for benzoate degradation were uniquely found in OMVs prepared from P. putida KT2440 that were cultured in media containing benzoate as the energy source. OMVs of P. putida KT2440 showed low pathological activity toward cultured cells that originated from human lung cells, which suggests their potential as adjuvants or OMV vaccine carriers. Our results suggest that the protein composition of the OMVs of P. putida KT2440 reflects the characteristics of the total proteome of P. putida KT2440.

Proteogenomic characterization of antimicrobial resistance in extensively drug-resistant Acinetobacter baumannii DU202.

OBJECTIVES: To determine the genomic sequence of extensively drug-resistant Acinetobacter baumannii DU202 and to perform proteomic characterization of antibiotic resistance in this strain using genome data. METHODS: The genome sequence of A. baumannii DU202 was determined using the Hi-Seq 2000 system and comparative analysis was performed to determine the unique characteristics of A. baumannii DU202. Previous proteomic results from the cell wall membrane fraction by one-dimensional electrophoresis and liquid chromatography combined with mass spectrometry analysis (1DE-LC-MS/MS), using the A. baumannii ATCC 17978 genome as a reference, were reanalysed to elucidate the resistance mechanisms of A. baumannii DU202 using strain-specific genome data. Additional proteomic data from the cytosolic fraction were also analysed. RESULTS: The genome of A. baumannii DU202 consists of 3660 genes and is most closely related to the Korean A. baumannii 1656-2 strain. More than 144 resistance genes were annotated in the A. baumannii DU202 genome, of which 72 that encoded proteins associated with antibiotic resistance were identified in the proteomic analysis of A. baumannii DU202 cultured in tetracycline, imipenem and Luria-Bertani broth (control) medium. Strong induction of ß-lactamases, a multidrug resistance efflux pump and resistance-nodulation-cell division (RND) multidrug efflux proteins was found to be important in the antibiotic resistance responses of A. baumannii DU202. CONCLUSIONS: Combining genomic and proteomic methods provided comprehensive information about the unique antibiotic resistance responses of A. baumannii DU202.

A global proteome study of Mycobacterium gilvum PYR-GCK grown on pyrene and glucose reveals the activation of glyoxylate, shikimate and gluconeogenetic pathways through the central carbon metabolism highway.

Various hydrocarbons have been released into the environment as a result of industrialization. An effective way of removing these materials without further environmental contamination is microbial bioremediation. Mycobacterium gilvum PYR-GCK, a bacteria isolated from a PAH polluted estuary, was studied using comparative shotgun proteomics to gain insight on its molecular activity while using pyrene and glucose as sole carbon and energy sources. Based on annotated genomic information, a confirmation analysis was first performed to confirm its pyrene degradation activity, using gas chromatography-mass spectrometry technology. One dimensional gel electrophoresis and liquid chromatography-mass spectrometry technologies employed in the proteomics analysis revealed the expression of pyrene degrading gene products along with upregulated expression of proteins functioning in the glyoxylate and shikimate pathways, in the pyrene-induced cells. The study also revealed the pathway of pyrene degraded intermediates, via partial gluconeogenesis, into the pentose phosphate pathway to produce precursors for nucleotides and amino acids biosynthesis.

Proteomic characterization of influenza H5N1 virus-like particles and their protective immunogenicity.

Recombinant virus-like particles (VLPs) have been shown to induce protective immunity. Despite their potential significance as promising vaccine candidates, the protein composition of VLPs produced in insect cells has not been well characterized. Here we report a proteomic analysis of influenza VLPs containing hemagglutinin (HA) and matrix M1 proteins from a human isolate of avian influenza H5N1 virus (H5 VLPs) produced in insect cells using the recombinant baculovirus expression system. Comprehensive proteomic analysis of purified H5 VLPs identified viral proteins and 37 additional host-derived proteins, many of which are known to be present in other enveloped viruses. Proteins involved in different cellular structures and functions were found to be present in H5 VLPs including those from the cytoskeleton, translation, chaperone, and metabolism. Immunization with purified H5 VLPs induced protective immunity, which was comparable to the inactivated whole virus containing all viral components. Unpurified H5 VLPs containing excess amounts of noninfluenza soluble proteins also conferred 100% protection against lethal challenge although lower immune responses were induced. These results provide important implications consistent with the idea that VLP production in insect cells may involve similar cellular machinery as other RNA enveloped viruses during synthesis, assembly, trafficking, and budding processes.

Quantitative proteomic analysis of cell wall and plasma membrane fractions from multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative, nonmotile aerobic bacterium that has emerged as an important nosocomial pathogen. Multidrug-resistant (MDR) A. baumannii is difficult to treat with antibiotics, and treatment failure in infected patients is of great concern in clinical settings. To investigate proteome regulation in A. baumannii under antibiotic stress conditions, quantitative membrane proteomic analyses of a clinical MDR A. baumannii strain cultured in subminimal inhibitory concentrations of tetracycline and imipenem were performed using a combination of label-free (one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry) and label (isobaric tag for relative and absolute quantitation) approaches. In total, 484 proteins were identified, and 302 were classified as outer membrane, periplasmic, or plasma membrane proteins. The clinical A. baumannii strain DU202 responded specifically and induced different cell wall and membrane protein sets that provided resistance to the antibiotics. The induction of resistance-nodulation-cell division transporters and protein kinases, and the repression of outer membrane proteins were common responses in the presence of tetracycline and imipenem. Induction of a tetracycline resistant pump, ribosomal proteins, and iron-uptake transporters appeared to be dependent on tetracycline conditions, whereas ß-lactamase and penicillin-binding proteins appeared to be dependent on imipenem conditions. These results suggest that combined liquid chromatography-based proteomic approaches can be used to identify cell wall and membrane proteins involved in the antibiotic resistance of A. baumannii.

Enrichment and proteome analysis of a hyperthermostable protein set of archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that can be used for the screening of thermophilic enzymes. Previously, we characterized the metabolic enzymes of the cytosolic proteome by two-dimensional electrophoresis/tandem mass spectrometry (2-DE/MS-MS). In this study, we identified a subset of hyperthermostable proteins in the cytosolic proteome using enrichment by in vitro heat treatment and protein identification. After heat treatment at 100°C for 2 h, 13 and 149 proteins were identified from the soluble proteome subset by 2-DE/MS-MS and 1-DE/MS-MS analysis, respectively. Representative proteins included intracellular protease I, thioredoxin reductase, triosephosphate isomerase, putative hydroperoxide reductase, proteasome, and translation initiation factors. Intracellular protease, deblocking aminopeptidases, and fructose-1,6-bisphosphatase were overexpressed in Escherichia coli and biological activity above 85°C was confirmed. The folding transition temperature (Tm) of identified proteins was analyzed using the in silico prediction program TargetStar. The proteins enriched with the heat treatment have higher Tm than the homologous proteins from mesophilic strains. These results suggested that the heat-stable protein set of hyperthermophilic T. onnurineus NA1 can be effectively fractionated and enriched by in vitro heat treatment.

Streptococcus equi-derived extracellular vesicles as a vaccine candidate against Streptococcus equi infection.

Streptococcus equi subspecies equi is a pathogenic bacterium that causes strangles, a highly contagious respiratory infection in horses and other equines. The limitations of current vaccines against S. equi infection warrants the development of an affordable, safe, and effective vaccine. Because gram-positive extracellular vesicles (EVs) transport various immunogenic antigens, they are attractive vaccine candidates. Here, we purified the EVs of S. equi ATCC 39506 and evaluated them as a vaccine candidate against S. equi infection in mice. As an initial step, comparative proteomic analysis was performed to characterize the functional features of the EVs. Reverse vaccinology and knowledge-based annotations were then used to screen potential vaccine candidates (PVCs) for S. equi ATCC 39506. Finally, 32 PVCs were found to be enriched in the EV fraction, suggesting the usefulness of this fraction as a vaccine. Importantly, a significantly higher survival rate after S. equi infection was detected in mice immunized with S. equi-derived EVs via the intraperitoneal route than in mice immunized with heat-killed bacteria. Of note, immunoprecipitation-mass spectrometry results validated various immunogenic antigens within the EV proteome. In conclusion, our results suggest that S. equi-derived EVs can serve as a vaccine candidate against S. equi infection.

Dataset of single nucleotide polymorphisms and comprehensive proteomic analysis of Streptococcus equi subsp. equi ATCC 39506.

Streptococcus equi subspecies equi (S. equi) is an opportunistic pathogen and a major causative agent of equine strangles, a contagious respiratory infection in horses and other equines. In this study, we provide the dataset associated with our research publication "Streptococcus equi-derived extracellular vesicles as a vaccine candidate against Streptococcus equi infections" [1]. We describe the genomic differences between S. equi 4047 and S. equi ATCC 39506 and outline the comprehensive proteome information of various fractions, including the whole cell lysate, membrane proteome, secretory proteome, and extracellular vesicle proteome. In addition, we included a dataset of highly immunoreactive proteins identified through immunoprecipitation. The specifications table provides a detailed summary of the gene annotation and quantitative information obtained for each proteome. The proteomics data were analyzed using shotgun proteomics with LTQ Velos and Q Exactive mass spectrometry in the data-dependent acquisition mode. We have deposited the acquired data, including the mass spectrometry raw files and exported MASCOT search results, in the PRIDE public repository under the accession numbers PXD025152 and PXD025527.

National surveillance of antifungal susceptibility of Candida species in South Korean hospitals.

We investigated the species distribution and antifungal susceptibility of Candida isolates from tertiary and non-tertiary hospitals in South Korea from 2002-2004. Of the 612 Candida isolates that were collected, Candida albicans, C. parapsilosis, C. tropicalis, and C. glabrata occurred most frequently, accounting for 97.3% and 96.8% of the isolates in tertiary and non-tertiary hospitals, respectively. C. albicans was the most common isolate, but the incidence of non-C. albicansCandida species was higher than that of C. albicans in tertiary hospitals. The Candida species had much lower MIC(90) to voriconazole (tertiary hospitals: 0.5 microg/ml, non-tertiary hospitals: 0.25 microg/ml) than to fluconazole (tertiary hospitals: 8 microg/ml, non-tertiary hospitals: 4 microg/ml). The MIC(90) of Candida isolates to 5-flucytosine in non-tertiary hospitals was two times higher than that observed in tertiary facilities. The C. glabrata isolates showed a tendency toward strong resistance to fluconazole, but C. parapsilosis isolates were susceptible to all of the evaluated antifungal agents. Voriconazole showed strong in vitro activity against Candida species, especially C. krusei, which is resistant to fluconazole and 5-flucytosine. To our knowledge, this is the first report of Candida antifungal susceptibility that includes non-tertiary hospitals in South Korea.

Application of free-flow electrophoresis/2-dimentional gel electrophoresis for fractionation and characterization of native proteome of Pseudomonas putida KT2440.

Free Flow Electrophoresis (FFE) is a liquid-based isoelectric focusing method. Unlike conventional in-gel fractionation of proteins, FFE can resolve proteins in their native forms and fractionation of subcellular compartments of the cell is also possible. To test the efficacy of the FFE method, the native cytosol proteome of a bacterium, Pseudomonas putida KT2440 was fractionated by FFE and the spectrum of protein elutes was characterized in association with 2-dimentional gel electrophoresis (2-DE). Major native proteins of P. putida KT2440 were eluted in the range of pH 4.8-6.0 in FFE, whereas the denatured proteome of P. putida KT2440 was widely distributed in the rage of pH 4 approximately 10 in the 2-DE analysis. In addition, one of the three FFE major fractions, which was eluted at pH 5.0, was further analyzed using 2-DE/MS-MS. Then, the pH range of identified proteins eluted in 2-DE/MS-MS was 4.72-5.89, indicating that observed pi values of native cytosolic proteomes in FFE were narrower than those of denatured cytosolic proteome. These results suggest that FFE fractionation and 2-DE/MS analysis may be useful tools for characterization of native proteomes of P. putida KT2440 and comparative analysis between denatured and native proteomes.