scholarly journals Secretome of the Coprophilous Fungus Doratomyces stemonitis C8, Isolated from Koala Feces

2011 ◽  
Vol 77 (11) ◽  
pp. 3793-3801 ◽  
Author(s):  
Robyn Peterson ◽  
Jasmine Grinyer ◽  
Helena Nevalainen
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Plant Biomass ◽  
Glycosyl Hydrolase ◽  
Tandem Mass ◽  
Glycosyl Hydrolase Family ◽  
Coprophilous Fungi ◽  
Content Type ◽  
Coprophilous Fungus ◽  
Hydrolase Family

ABSTRACTCoprophilous fungi inhabit herbivore feces, secreting enzymes to degrade the most recalcitrant parts of plant biomass that have resisted the digestive process. Consequently, the secretomes of coprophilous fungi have high potential to contain novel and efficient plant cell wall degrading enzymes of biotechnological interest. We have used one-dimensional and two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization–time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), and quadrupole time-of-flight liquid chromatography–tandem mass spectrometry (Q-TOF LC-MS/MS) to identify proteins from the secretome of the coprophilous fungusDoratomyces stemonitisC8 (EU551185) isolated from koala feces. As the genome ofD. stemonitishas not been sequenced, cross-species identification,de novosequencing, and zymography formed an integral part of the analysis. A broad range of enzymes involved in the degradation of cellulose, hemicellulose, pectin, lignin, and protein were revealed, dominated by cellobiohydrolase of the glycosyl hydrolase family 7 and endo-1,4-β-xylanase of the glycosyl hydrolase family 10. A high degree of specialization for pectin degradation in theD. stemonitisC8 secretome distinguishes it from the secretomes of some other saprophytic fungi, such as the industrially exploitedT. reesei. In the first proteomic analysis of the secretome of a coprophilous fungus reported to date, the identified enzymes provide valuable insight into how coprophilous fungi subsist on herbivore feces, and these findings hold potential for increasing the efficiency of plant biomass degradation in industrial processes such as biofuel production in the future.

scholarly journals Identification of the OXA-48 Carbapenemase Family by Use of Tryptic Peptides and Liquid Chromatography-Tandem Mass Spectrometry

2019 ◽  
Vol 57 (5) ◽  
Author(s):  
Jeffrey R. Strich ◽  
Honghui Wang ◽  
Ousmane H. Cissé ◽  
Jung-Ho Youn ◽  
Steven K. Drake ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Accuracy Assessment ◽  
Multiple Reaction Monitoring ◽  
Carbapenem Resistance ◽  
Tandem Mass ◽  
Content Type ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

ABSTRACT Phenotypic detection of the OXA-48-type class D β-lactamases in Enterobacteriaceae is challenging. We describe a rapid (less than 90 min) assay for the identification of OXA-48 family carbapenemases in subcultured bacterial isolates based on a genoproteomic approach. Following in silico trypsin digestion to ascertain theoretical core peptides common to the OXA-48 family, liquid chromatography-tandem mass spectrometry (LC-MS/MS) data-dependent acquisition was used to identify candidate peptide markers. Two peptides were selected based on performance characteristics: ANQAFLPASTFK, a core peptide common to all 12 OXA-48 family β-lactamase members, and YSVVPVYQEFAR, a highly specific peptide common to 11 of 12 OXA-48 family proteins providing the basis for an LC-MS/MS multiple reaction monitoring assay. An accuracy assessment was performed that included 98 isolates, 26 of which were OXA-48 positive. Two additional specificity assessments were performed including a mixture of isolates positive for OXA-48, KPC, NDM, VIM, and IMP carbapenemases. A combination of expert rules and expert judgment was applied by blinded operators to identify positive isolates. All isolates containing an OXA-48 family carbapenemase across all three test sets were correctly identified with no false positives, demonstrating 100% sensitivity (95% confidence interval [CI], 91.2% to 100%) and 100% specificity (95% CI, 96.2% to 100%) for the assay. These findings provide a framework for an LC-MS/MS-based method for the direct detection of OXA-48 family carbapenemases from cultured isolates that may have utility in predicting carbapenem resistance and tracking hospital outbreaks of OXA-48-carrying organisms.

scholarly journals Simultaneous Identification of Multiple β-Lactamases in Acinetobacter baumannii in Relation to Carbapenem and Ceftazidime Resistance, Using Liquid Chromatography-Tandem Mass Spectrometry: TABLE 1

2015 ◽  
Vol 53 (6) ◽  
pp. 1927-1930 ◽  
Author(s):  
Hein Trip ◽  
Katrin Mende ◽  
Joanna A. Majchrzykiewicz-Koehorst ◽  
Norbert J. A. Sedee ◽  
Albert G. Hulst ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Acinetobacter Baumannii ◽  
Shotgun Proteomics ◽  
Added Value ◽  
Tandem Mass ◽  
Content Type ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Shotgun proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to detect β-lactamases in clinicalAcinetobacter baumanniiisolates. The correlation of the detection of β-lactamase proteins (rather than PCR detection of the corresponding genes) with the resistance phenotypes demonstrated an added value for LC-MS/MS in antimicrobial susceptibility testing.

scholarly journals Proteomic Analysis of Bacillus thuringiensis at Different Growth Phases by Using an Automated Online Two-Dimensional Liquid Chromatography-Tandem Mass Spectrometry Strategy

2012 ◽  
Vol 78 (15) ◽  
pp. 5270-5279 ◽  
Author(s):  
Shaoya Huang ◽  
Xuezhi Ding ◽  
Yunjun Sun ◽  
Qi Yang ◽  
Xiuqing Xiao ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Bacillus Thuringiensis ◽  
Tandem Mass Spectrometry ◽  
Identification System ◽  
Crystal Formation ◽  
Tandem Mass ◽  
Two Dimensional ◽  
Content Type ◽  
Three Phases

ABSTRACTThe proteome of a newBacillus thuringiensissubsp.kurstakistrain, 4.0718, from the middle vegetative (T1), early sporulation (T2), and late sporulation (T3) phases was analyzed using an integrated liquid chromatography (LC)-based protein identification system. The system comprised two-dimensional (2D) LC coupled with nanoscale electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. After deletion of redundant proteins from the different batches andB. thuringiensissubspecies, 918, 703, and 778 proteins were identified in the respective three phases. Their molecular masses ranged from 4.6 Da to 477.4 Da, and their isoelectric points ranged from 4.01 to 11.84. Function clustering revealed that most of the proteins in the three phases were functional metabolic proteins, followed by proteins participating in cell processes. Small molecular and macromolecular metabolic proteins were further classified according to the Kyoto Encyclopedia of Genes and Genome and BioCyc metabolic pathway database. Three protoxins (Cry2Aa, Cry1Aa, and Cry1Ac) as well as a series of potential intracellular active factors were detected. Many significant proteins related to spore and crystal formation, including sporulation proteins, help proteins, chaperones, and so on, were identified. The expression patterns of two identified proteins, CotJc and glutamine synthetase, were validated by Western blot analysis, which further confirmed the MS results. This study is the first to use shotgun technology to research the proteome ofB. thuringiensis. Valuable experimental data are provided regarding the methodology of analyzing theB. thuringiensisproteome (which can be used to produce insecticidal crystal proteins) and have been added to the related protein database.

scholarly journals Fluconazole Pharmacokinetics in Galleria mellonella Larvae and Performance Evaluation of a Bioassay Compared to Liquid Chromatography-Tandem Mass Spectrometry for Hemolymph Specimens

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Karen Marie Thyssen Astvad ◽  
Joseph Meletiadis ◽  
Sarah Whalley ◽  
Maiken Cavling Arendrup
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Human Exposure ◽  
Galleria Mellonella ◽  
Tandem Mass ◽  
Time Curve ◽  
Content Type ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

ABSTRACT The invertebrate model organism Galleria mellonella can be used to assess the efficacy of treatment of fungal infection. The fluconazole dose best mimicking human exposure during licensed dosing is unknown. We validated a bioassay for fluconazole detection in hemolymph and determined the fluconazole pharmacokinetics and pharmacodynamics in larval hemolymph in order to estimate a humanized dose for future experiments. A bioassay using 4-mm agar wells, 20 μl hemolymph, and the hypersusceptible Candida albicans DSY2621 was established and compared to a validated liquid chromatography-tandem mass spectrometry (LC–MS-MS) method. G. mellonella larvae were injected with fluconazole (5, 10, and 20 mg/kg of larval weight), and hemolymph was harvested for 24 h for pharmacokinetics calculations. The exposure was compared to the human exposure during standard licensed dosing. The bioassay had a linear standard curve between 1 and 20 mg/liter. Accuracy and coefficients of variation (percent) values were below 10%. The Spearman coefficient between assays was 0.94. Fluconazole larval pharmacokinetics followed one-compartment linear kinetics, with the 24-h area under the hemolymph concentration-time curve (AUC24 h) being 93, 173, and 406 mg · h/liter for the three doses compared to 400 mg · h/liter in humans under licensed treatment. In conclusion, a bioassay was validated for fluconazole determination in hemolymph. The pharmacokinetics was linear. An exposure comparable to the human exposure during standard licensed dosing was obtained with 20 mg/kg.

scholarly journals Anaerobic gut fungi are an untapped reservoir of natural products

2021 ◽  
Vol 118 (18) ◽  
pp. e2019855118
Author(s):  
Candice L. Swift ◽  
Katherine B. Louie ◽  
Benjamin P. Bowen ◽  
Heather M. Olson ◽  
Samuel O. Purvine ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Natural Products ◽  
Tandem Mass Spectrometry ◽  
Plant Biomass ◽  
Biosynthetic Enzymes ◽  
Tandem Mass ◽  
Anaerobic Fungi ◽  
Peptide Synthetases ◽  
Representative Species ◽  
Liquid Chromatography Tandem Mass

Anaerobic fungi (class Neocallimastigomycetes) thrive as low-abundance members of the herbivore digestive tract. The genomes of anaerobic gut fungi are poorly characterized and have not been extensively mined for the biosynthetic enzymes of natural products such as antibiotics. Here, we investigate the potential of anaerobic gut fungi to synthesize natural products that could regulate membership within the gut microbiome. Complementary 'omics' approaches were combined to catalog the natural products of anaerobic gut fungi from four different representative species: Anaeromyces robustus (A. robustus), Caecomyces churrovis (C. churrovis), Neocallimastix californiae (N. californiae), and Piromyces finnis (P. finnis). In total, 146 genes were identified that encode biosynthetic enzymes for diverse types of natural products, including nonribosomal peptide synthetases and polyketide synthases. In addition, N. californiae and C. churrovis genomes encoded seven putative bacteriocins, a class of antimicrobial peptides typically produced by bacteria. During standard laboratory growth on plant biomass or soluble substrates, 26% of total core biosynthetic genes in all four strains were transcribed. Across all four fungal strains, 30% of total biosynthetic gene products were detected via proteomics when grown on cellobiose. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of fungal supernatants detected 72 likely natural products from A. robustus alone. A compound produced by all four strains of anaerobic fungi was putatively identified as the polyketide-related styrylpyrone baumin. Molecular networking quantified similarities between tandem mass spectrometry (MS/MS) spectra among these fungi, enabling three groups of natural products to be identified that are unique to anaerobic fungi. Overall, these results support the finding that anaerobic gut fungi synthesize natural products, which could be harnessed as a source of antimicrobials, therapeutics, and other bioactive compounds.

scholarly journals Detection of Carbapenemase-Producing Bacteria by Using an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method

2013 ◽  
Vol 58 (2) ◽  
pp. 1231-1234 ◽  
Author(s):  
Anne Carricajo ◽  
Paul O. Verhoeven ◽  
Salim Guezzou ◽  
Nathalie Fonsale ◽  
Gerald Aubert
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Rapid Detection ◽  
Ultra Performance Liquid Chromatography ◽  
Tandem Mass ◽  
Spectrometry Method ◽  
Content Type ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

ABSTRACTThe emergence of carbapenemase-producing bacteria poses a new challenge in the management of antibiotic therapies for patients. This report describes a new method using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for rapid detection of carbapenemase activity in enterobacteria,Pseudomonas aeruginosa, andAcinetobacter baumannii. In a panel of 78 isolates, including 41 carbapenemase-producing strains, the ULPC-MS/MS assay showed 100% agreement with molecular characterization, whereas six carbapenemase-producing isolates were not detected by the modified Hodge test.

scholarly journals Prediction of Fluoroquinolone Susceptibility Directly from Whole-Genome Sequence Data by Using Liquid Chromatography-Tandem Mass Spectrometry To Identify Mutant Genotypes

2017 ◽  
Vol 62 (3) ◽  
Author(s):  
Wan Ahmad Kamil Wan Nur Ismah ◽  
Yuiko Takebayashi ◽  
Jacqueline Findlay ◽  
Kate J. Heesom ◽  
Juan-Carlos Jiménez-Castellanos ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Sequence Data ◽  
Whole Genome Sequence ◽  
Tandem Mass ◽  
Whole Genome ◽  
Content Type ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

ABSTRACTFluoroquinolone resistance in Gram-negative bacteria is multifactorial, involving target site mutations, reductions in fluoroquinolone entry due to reduced porin production, increased fluoroquinolone efflux, enzymes that modify fluoroquinolones, and Qnr, a DNA mimic that protects the drug target from fluoroquinolone binding. Here we report a comprehensive analysis, using transformation andin vitromutant selection, of the relative importance of each of these mechanisms for fluoroquinolone nonsusceptibility usingKlebsiella pneumoniaeas a model system. Our improved biological understanding was then used to generate 47 rules that can predict fluoroquinolone susceptibility inK. pneumoniaeclinical isolates. Key to the success of this predictive process was the use of liquid chromatography-tandem mass spectrometry to measure the abundance of proteins in extracts of cultured bacteria, identifying which sequence variants seen in the whole-genome sequence data were functionally important in the context of fluoroquinolone susceptibility.

scholarly journals Characterization of Two Distinct Glycosyl Hydrolase Family 78 α-l-Rhamnosidases from Pediococcus acidilactici

2011 ◽  
Vol 77 (18) ◽  
pp. 6524-6530 ◽  
Author(s):  
Herbert Michlmayr ◽  
Walter Brandes ◽  
Reinhard Eder ◽  
Christina Schümann ◽  
Andrés M. del Hierro ◽  
...  
Keyword(s):  
Glycosyl Hydrolase ◽  
Pediococcus Acidilactici ◽  
Glycosyl Hydrolase Family ◽  
Negative Effects ◽  
Content Type ◽  
Linalool Oxide ◽  
Hydrolysis Of ◽  
Flavanone Glycoside ◽  
Hydrolase Family

ABSTRACTα-l-Rhamnosidases play an important role in the hydrolysis of glycosylated aroma compounds (especially terpenes) from wine. Although several authors have demonstrated the enological importance of fungal rhamnosidases, the information on bacterial enzymes in this context is still limited. In order to fill this important gap, two putative rhamnosidase genes (ramandram2) fromPediococcus acidilacticiDSM 20284 were heterologously expressed, and the respective gene products were characterized. In combination with a bacterial β-glucosidase, both enzymes released the monoterpenes linalool andcis-linalool oxide from a muscat wine extract under ideal conditions. Additionally, Ram could release significant amounts of geraniol and citronellol/nerol. Nevertheless, the potential enological value of these enzymes is limited by the strong negative effects of acidity and ethanol on the activities of Ram and Ram2. Therefore, a direct application in winemaking seems unlikely. Although both enzymes are members of the same glycosyl hydrolase family (GH 78), our results clearly suggest the distinct functionalities of Ram and Ram2, probably representing two subclasses within GH 78: Ram could efficiently hydrolyze only the synthetic substratep-nitrophenyl-α-l-rhamnopyranoside (Vmax= 243 U mg−1). In contrast, Ram2 displayed considerable specificity toward hesperidin (Vmax= 34 U mg−1) and, especially, rutinose (Vmax= 1,200 U mg−1), a disaccharide composed of glucose and rhamnose. Both enzymes were unable to hydrolyze the flavanone glycoside naringin. Interestingly, both enzymes displayed indications of positive substrate cooperativity. This study presents detailed kinetic data on two novel rhamnosidases, which could be relevant for the further study of bacterial glycosidases.

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