Discrimination of Human Pathogen Clostridium Species Especially of the Heterogeneous C. sporogenes and C. botulinum by MALDI-TOF Mass Spectrometry.

Clostridium species cause several local and systemic diseases. Conventional identification of these microorganisms is in part laborious, not always reliable, time consuming or does not always distinguish different species, i.e., C. botulinum and C. sporogenes. All in, there is a high interest to find out a reliable, powerful and rapid method to identify Clostridium spp. not only on genus but also on species level. The aim of the present study was to identify Clostridium spp. strains and also to find differences and metabolic groups of C. botulinum by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). A total of 123 strains of Clostridium spp. (C. botulinum, n = 40, C. difficile, n = 11, C. tetani, n = 11, C. sordellii, n = 20, C. sporogenes, n = 18, C. innocuum, n = 10, C. perfringens, n = 13) were analyzed by MALDI-TOF MS in combination with methods of multivariate statistical analysis. MALDI-TOF MS analysis in combination with methods of multivariate statistical analysis was able to discriminate between the different tested Clostridium spp., even between species which are closely related and difficult to differentiate by traditional methods, i.e., C. sporogenes and C. botulinum. Furthermore, the method was able to separate the different metabolic groups of C. botulinum. Especially, E gene-positive C. botulinum strains are clearly distinguishable from the other species but also from those producing other toxin types. Thus, MALDI-TOF MS represents a reliable and above all quick method for identification of cultivated Clostridium species.

Proteomic and functional consequences of hexokinase deficiency in glucose-repressible Kluyveromyces lactis.

The analysis of glucose signaling in the Crabtree-positive eukaryotic model organism Saccharomyces cerevisiae has disclosed a dual role of its hexokinase ScHxk2, which acts as a glycolytic enzyme and key signal transducer adapting central metabolism to glucose availability. In order to identify evolutionarily conserved characteristics of hexokinase structure and function, the cellular response of the Crabtree-negative yeast Kluyveromyces lactis to rag5 null mutation and concomitant deficiency of its unique hexokinase KlHxk1 was analyzed by means of difference gel electrophoresis. In total, 2,851 fluorescent spots containing different protein species were detected in the master gel representing all of the K. lactis proteins that were solubilized from glucose-grown KlHxk1 wild-type and mutant cells. Mass spectrometric peptide analysis identified 45 individual hexokinase-dependent proteins related to carbohydrate, short-chain fatty acid and tricarboxylic acid metabolism as well as to amino acid and protein turnover, but also to general stress response and chromatin remodeling, which occurred as a consequence of KlHxk1 deficiency at a minimum 3-fold enhanced or reduced level in the mutant proteome. In addition, three proteins exhibiting homology to 2-methylcitrate cycle enzymes of S. cerevisiae were detected at increased concentrations, suggesting a stimulation of pyruvate formation from amino acids and/or fatty acids. Experimental validation of the difference gel electrophoresis approach by post-lysis dimethyl labeling largely confirmed the abundance changes detected in the mutant proteome via the former method. Taking into consideration the high proportion of identified hexokinase-dependent proteins exhibiting increased proteomic levels, KlHxk1 is likely to have a repressive function in a multitude of metabolic pathways. The proteomic alterations detected in the mutant classify KlHxk1 as a multifunctional enzyme and support the view of evolutionary conservation of dual-role hexokinases even in organisms that are less specialized than S. cerevisiae in terms of glucose utilization.

A step towards the discrimination of beta-lactamase-producing clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa by MALDI-TOF mass spectrometry.

BACKGROUND: Matrix-Assisted Laser-Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has already proven to be a powerful tool for species identification in microbiological laboratories. As adequate and rapid screening methods for antibiotic resistance are crucially needed, the present study investigated the discrimination potential of MALDI-TOF MS among extended-spectrum-beta-lactamase (ESBL) or metallo-beta-lactamases- (MBL) producing and the nonproducing strains of Escherichia coli (n=19), Klebsiella pneumoniae (n=19), and Pseudomonas aeruginosa (n=38), respectively. MATERIAL/METHODS: We used a MALDI-TOF MS protocol, usually applied for species identification, in order to integrate a screening method for beta-lactamases into the routine species identification workflow. The acquired spectra were analyzed by visual inspection, statistical similarity analysis and support vector machine (SVM) classification algorithms. RESULTS: Neither visual inspection nor mathematical similarity analysis allowed discrimination between spectra of beta-lactamase-producing and the nonproducing strains, but classification within a species by SVM-based algorithms could achieve a correct classification rate of up to 70%. CONCLUSIONS: This shows that MALDI-TOF MS has definite potential to discriminate antibiotic-resistant strains due to ESBL and MBL production from nonproducing strains, but this performance is not yet sufficiently reliable for routine microbiological diagnostics.

Rapid identification of oral Actinomyces species cultivated from subgingival biofilm by MALDI-TOF-MS.

BACKGROUND: Actinomyces are a common part of the residential flora of the human intestinal tract, genitourinary system and skin. Isolation and identification of Actinomyces by conventional methods is often difficult and time consuming. In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has become a rapid and simple method to identify bacteria. OBJECTIVE: The present study evaluated a new in-house algorithm using MALDI-TOF-MS for rapid identification of different species of oral Actinomyces cultivated from subgingival biofilm. DESIGN: Eleven reference strains and 674 clinical strains were used in this study. All the strains were preliminarily identified using biochemical methods and then subjected to MALDI-TOF-MS analysis using both similarity-based analysis and classification methods (support vector machine [SVM]). The genotype of the reference strains and of 232 clinical strains was identified by sequence analysis of the 16S ribosomal RNA (rRNA). RESULTS: The sequence analysis of the 16S rRNA gene of all references strains confirmed their previous identification. The MALDI-TOF-MS spectra obtained from the reference strains and the other clinical strains undoubtedly identified as Actinomyces by 16S rRNA sequencing were used to create the mass spectra reference database. Already a visual inspection of the mass spectra of different species reveals both similarities and differences. However, the differences between them are not large enough to allow a reliable differentiation by similarity analysis. Therefore, classification methods were applied as an alternative approach for differentiation and identification of Actinomyces at the species level. A cross-validation of the reference database representing 14 Actinomyces species yielded correct results for all species which were represented by more than two strains in the database. CONCLUSIONS: Our results suggest that a combination of MALDI-TOF-MS with powerful classification algorithms, such as SVMs, provide a useful tool for the differentiation and identification of oral Actinomyces.

Oral brush biopsy analysis by MALDI-ToF Mass Spectrometry for early cancer diagnosis.

OBJECTIVES: Intact cell peptidome profiling (ICPP) with MALDI-ToF Mass Spectrometry holds promise as a non-invasive method to detect head and neck squamous cell carcinoma (HNSCC) objectively, which may significantly improve the early diagnosis of oral cancer. The present study was designed to discriminate between tumour samples and non-cancer controls (healthy mucosa and oral lesions) by analysing complete spectral patterns of intact cells using MALDI-ToF MS. MATERIALS AND METHODS: In the first step, a database consisting of 26 patients suffering from HNSCC was established by taking brush biopsy samples of the diseased area and of the healthy buccal mucosa of the respective contralateral area. After performing MALDI-ToF MS on these samples, classification analysis was used as the basis for further classification of an additional 26 blinded samples including HNSCC, oral lesions and healthy mucosa. RESULTS: By analysing spectral patterns of the blinded samples, all cancerous lesions were defined accurately. One incorrect evaluation (false positive) occurred in the lesion cohort, leading to a sensitivity of 100%, a specificity of 93% and an overall accuracy of 96.5%. CONCLUSION: ICPP using MALDI-ToF MS is able to distinguish between healthy and cancerous mucosa and between oral lesions and oral cancer with excellent sensitivity and specificity, which may lead to more accurate early diagnosis of HNSCC.

Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry.

Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has proven to be an effective identification tool in medical microbiology. Discrimination to subspecies or serovar level has been found to be challenging using commercially available identification software. By forming our own reference database and using alternative analysis methods, we could reliably identify all implemented Enterobacteriaceae and non-fermenting gram negative bacilli by MALDI-TOF MS and even succeeded to distinguish Shigella sonnei from Escherichia coli (E. coli) and Salmonella enterica spp. enterica serovar Enteritidis from Salmonella enterica spp. enterica serovar Typhimurium. Furthermore, the method showed the ability to separate Enterohemorrhagic E. coli (EHEC) and Enteropathogenic E. coli (EPEC) from non-enteropathogenic E. coli.

Oral brush biopsy analysis by matrix assisted laser desorption/ionisation-time of flight mass spectrometry profiling--a pilot study.

Oral squamous cell carcinomas (OSCCs) often present as advanced tumours requiring aggressive local and regional therapy and result in significant functional impairment. The objective is to develop pre-symptomatic screening detection of OSCC by a brush biopsy method which is less invasive than the conventional biopsy for histology. Given the molecular heterogeneity of oral cancer, it is unlikely that even a panel of tumour markers would provide accurate diagnosis. Therefore, approaches such as the matrix-assisted-laser-desorption/ionisation-time-of-flight-mass-spectrometry (MALDI-TOF-MS) allow several biomarkers or peptide profile patterns to be simultaneously assessed. Brush biopsies from 27 patients with histology-proven OSCCs plus 40 biopsies from 10 healthy controls were collected. MALDI-TOF-MS profiling was performed and additional statistical analysis of the data was used to classify the disease status according to the biological behaviour of the lesion. For classification a support vector machine algorithm was trained using spectra of brush biopsy samples to distinguish healthy control patients from patients with histology-proven OSCC. MALDI-TOF-MS was able to distinguish between healthy patients and OSCC patients with a sensitivity of 100% and specificity of 93%. In summary, MALDI-TOF-MS in combination with sophisticated bioinformatic methods can distinguish OSCC patients from non-cancer controls with excellent sensitivity and specificity. Further improvement and validation of this approach is necessary to determine its feasibility to assist the pre-symptomatic detection of head and neck cancer screening in routine daily practice.

Outcome prediction in pneumonia induced ALI/ARDS by clinical features and peptide patterns of BALF determined by mass spectrometry.

BACKGROUND: Peptide patterns of bronchoalveolar lavage fluid (BALF) were assumed to reflect the complex pathology of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) better than clinical and inflammatory parameters and may be superior for outcome prediction. METHODOLOGY/PRINCIPAL FINDINGS: A training group of patients suffering from ALI/ARDS was compiled from equal numbers of survivors and nonsurvivors. Clinical history, ventilation parameters, Murray's lung injury severity score (Murray's LISS) and interleukins in BALF were gathered. In addition, samples of bronchoalveolar lavage fluid were analyzed by means of hydrophobic chromatography and MALDI-ToF mass spectrometry (MALDI-ToF MS). Receiver operating characteristic (ROC) analysis for each clinical and cytokine parameter revealed interleukin-6>interleukin-8>diabetes mellitus>Murray's LISS as the best outcome predictors. Outcome predicted on the basis of BALF levels of interleukin-6 resulted in 79.4% accuracy, 82.7% sensitivity and 76.1% specificity (area under the ROC curve, AUC, 0.853). Both clinical parameters and cytokines as well as peptide patterns determined by MALDI-ToF MS were analyzed by classification and regression tree (CART) analysis and support vector machine (SVM) algorithms. CART analysis including Murray's LISS, interleukin-6 and interleukin-8 in combination was correct in 78.0%. MALDI-ToF MS of BALF peptides did not reveal a single identifiable biomarker for ARDS. However, classification of patients was successfully achieved based on the entire peptide pattern analyzed using SVM. This method resulted in 90% accuracy, 93.3% sensitivity and 86.7% specificity following a 10-fold cross validation (AUC = 0.953). Subsequent validation of the optimized SVM algorithm with a test group of patients with unknown prognosis yielded 87.5% accuracy, 83.3% sensitivity and 90.0% specificity. CONCLUSIONS/SIGNIFICANCE: MALDI-ToF MS peptide patterns of BALF, evaluated by appropriate mathematical methods can be of value in predicting outcome in pneumonia induced ALI/ARDS.

Identification and characterization of CaApe2--a neutral arginine/alanine/leucine-specific metallo-aminopeptidase from Candida albicans.

The proteolytic potential of the pathogenic fungus Candida albicans was evaluated by the identification and functional characterization of a peptidolytic enzyme isolated from the cell wall of the microorganism. Determination of basic structural and kinetic data identified a neutral arginine/alanine/leucine-specific metallo-aminopeptidase of unknown function termed CaApe2, which is encoded by ORF CaO19.5197 (GenBank RefSeq XM_705313). Mass spectrometric tryptic peptide analysis and N-terminal protein sequencing revealed serine-88 to represent the N-terminus of CaApe2. Taking into account the results of DNA and protein sequence analysis including inspection of the genomic region upstream of ORF CaO19.5197, the gene CaAPE2 is likely to consist of two exons linked by a phase-2 intron with exons 1 and 2 encoding a signal peptide and the amino acids 88-954 of ORF CaO19.5197, respectively. The isolated CaApe2 protein shares an equally high similarity with the gene products ScAap1 and ScApe2, suggesting duplication of a phylogenetically ancient precursor gene in Saccharomyces cerevisiae. The observed failure to cleave human type-I and type-IV collagen in vitro challenges a direct role that secreted CaApe2 might play in the degradation of extracellular matrix components during host colonization, but does not exclude per se a contribution of the aminopeptidase to the pathogenicity of C. albicans.

Ethyl pyruvate and ethyl lactate down-regulate the production of pro-inflammatory cytokines and modulate expression of immune receptors.

Esters of alpha-oxo-carbonic acids such as ethyl pyruvate (EP) have been demonstrated to exert inhibitory effects on the production of anti-inflammatory cytokines. So far, there is no information about effects, if any, of ethyl lactate (EL), an obviously inactive analogue of EP, on inflammatory immune responses. In the present study, we provide evidence that the anti-inflammatory action of alpha-oxo-carbonic acid esters is mediated by inhibition of glyoxalases (Glo), cytosolic enzymes that catalyse the conversion of alpha-oxo-aldehydes such as methylglyoxal (MGO) into the corresponding alpha-hydroxy acids using glutathione as a cofactor. In vitro enzyme activity measurements revealed the inhibition of human Glo1 by alpha-oxo-carbonic acid esters, whilst alpha-hydroxy-carbonic acid esters such as EL were not inhibitory. In contrast, both EP and EL were shown to suppress the Lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and IL-8 from human immunocompetent cells, and modulated the expression of the immune receptors HLA-DR, CD14 and CD91 on human monocytes. Here, we show a crossing link between glyoxalases and the immune system. The results described herein introduce glyoxalases as a possible target for therapeutic approaches of immune suppression.

Curcumin inhibits glyoxalase 1: a possible link to its anti-inflammatory and anti-tumor activity.

BACKGROUND: Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor. METHODOLOGY/PRINCIPAL FINDINGS: Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (K(i) = 5.1+/-1.4 microM). Applying a whole blood assay, IC(50) values of pro-inflammatory cytokine release (TNF-alpha, IL-6, IL-8, IL-1beta) were found to be positively correlated with the K(i)-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1. CONCLUSIONS/SIGNIFICANCE: The results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.

A novel form of 6-phosphofructokinase. Identification and functional relevance of a third type of subunit in Pichia pastoris.

Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of alpha subunits and alpha/beta subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, alpha, beta, and gamma. The sequence of the gamma subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the gamma subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the gamma subunit is not required for enzymatic activity, the gamma subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and gamma subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the gamma subunit. Therefore, we suggest that the gamma subunit contributes to adaptation of P. pastoris to energy resources.

6-phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme.

6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6-phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6-phosphate is significantly lower. Aqueous two-phase affinity partitioning with Cibacron Blue F3G-A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero-octameric 6-phosphofructokinases from other yeast species. The enzyme was found to be a hetero-oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS-PAGE), respectively, and a third non-covalently complexed protein component (34 kDa, SDS-PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N-terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI-TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein.