scholarly journals Nonribosomal Peptide Synthetase GenespesLandpes1Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

2012 ◽  
Vol 78 (9) ◽  
pp. 3166-3176 ◽  
Author(s):  
Karen A. O'Hanlon ◽  
Lorna Gallagher ◽  
Markus Schrettl ◽  
Christoph Jöchl ◽  
Kevin Kavanagh ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Opportunistic Pathogen ◽  
Mass Spectrometry Analysis ◽  
Infection Model ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Peptide Synthetases ◽  
Increased Sensitivity

ABSTRACTThe identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen,Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway inA. fumigatus. Deletion of eitherpesL(ΔpesL) orpes1(Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion ofpesLresulted in severely reduced virulence in an invertebrate infection model (P< 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster forA. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in bothA. fumigatuswild-type and ΔpesLstrains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesisin vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature.

scholarly journals Structure, Mechanism, and Inhibition of Aspergillus fumigatus Thioredoxin Reductase

2019 ◽  
Vol 63 (3) ◽  
Author(s):  
Andrew C. Marshall ◽  
Sarah E. Kidd ◽  
Stephanie J. Lamont-Friedrich ◽  
Georgia Arentz ◽  
Peter Hoffmann ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Enzyme Inhibitor ◽  
Redox Homeostasis ◽  
Thioredoxin Reductase ◽  
Antifungal Drugs ◽  
Mass Spectrometry Analysis ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Future Design

ABSTRACT Aspergillus fumigatus infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals. Thioredoxin reductase (TrxR) is essential for maintaining redox homeostasis and presents as a promising target for novel antifungals. We show that ebselen [2-phenyl-1,2-benzoselenazol-3(2H)-one] is an inhibitor of A. fumigatus TrxR (Ki = 0.22 μM) and inhibits growth of Aspergillus spp., with in vitro MIC values of 16 to 64 µg/ml. Mass spectrometry analysis demonstrates that ebselen interacts covalently with a catalytic cysteine of TrxR, Cys148. We also present the X-ray crystal structure of A. fumigatus TrxR and use in silico modeling of the enzyme-inhibitor complex to outline key molecular interactions. This provides a scaffold for future design of potent and selective antifungal drugs that target TrxR, improving the potency of ebselen toward inhbition of A. fumigatus growth.

scholarly journals Thiol Peroxidase Is an Important Component of Streptococcus pneumoniae in Oxygenated Environments

2012 ◽  
Vol 80 (12) ◽  
pp. 4333-4343 ◽  
Author(s):  
Barak Hajaj ◽  
Hasan Yesilkaya ◽  
Rachel Benisty ◽  
Maayan David ◽  
Peter W. Andrew ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mass Spectrometry Analysis ◽  
Activity Levels ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Cellular Processes ◽  
Thiol Peroxidase ◽  
Fine Tune

ABSTRACTStreptococcus pneumoniaeis an aerotolerant Gram-positive bacterium that causes an array of diseases, including pneumonia, otitis media, and meningitis. During aerobic growth,S. pneumoniaeproduces high levels of H2O2. SinceS. pneumoniaelacks catalase, the question of how it controls H2O2levels is of critical importance. Thepsalocus encodes an ABC Mn2+-permease complex (psaBCA) and a putative thiol peroxidase,tpxD. This study shows thattpxDencodes a functional thiol peroxidase involved in the adjustment of H2O2homeostasis in the cell. Kinetic experiments showed that recombinant TpxD removed H2O2efficiently. However,in vivoexperiments revealed that TpxD detoxifies only a fraction of the H2O2generated by the pneumococcus. Mass spectrometry analysis demonstrated that TpxD Cys58undergoes selective oxidationin vivo, under conditions where H2O2is formed, confirming the thiol peroxidase activity. Levels of TpxD expression and synthesisin vitrowere significantly increased in cells grown under aerobic versus anaerobic conditions. The challenge with D39 and TIGR4 with H2O2resulted intpxDupregulation, whilepsaBCAexpression was oppositely affected. However, the challenge of ΔtpxDmutants with H2O2did not affectpsaBCA, implying that TpxD is involved in the regulation of thepsaoperon, in addition to its scavenging activity. Virulence studies demonstrated a notable difference in the survival time of mice infected intranasally with D39 compared to that of mice infected intranasally with D39ΔtpxD. However, when bacteria were administered directly into the blood, this difference disappeared. The findings of this study suggest that TpxD constitutes a component of the organism's fundamental strategy to fine-tune cellular processes in response to H2O2.

scholarly journals An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases inMyxococcus xanthusDK1622

2018 ◽  
Vol 200 (21) ◽  
Author(s):  
Karla J. Esquilín-Lebrón ◽  
Tye O. Boynton ◽  
Lawrence J. Shimkets ◽  
Michael G. Thomas
Keyword(s):  
Myxococcus Xanthus ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases

ABSTRACTOne mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 fromMyxococcus xanthusDK1622 for characterization. TheM. xanthusDK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination ofin vivoandin vitroassays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping ofM. xanthusDK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a “universal” MLP for generating functional hybrid NRPSs.IMPORTANCEMbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP fromMyxococcus xanthusDK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a “universal” MLP during the construction of functional hybrid NRPSs.

scholarly journals A Highly Conserved Basidiomycete Peptide Synthetase Produces a Trimeric Hydroxamate Siderophore

2017 ◽  
Vol 83 (21) ◽  
Author(s):  
Eileen Brandenburger ◽  
Markus Gressler ◽  
Robin Leonhardt ◽  
Gerald Lackner ◽  
Andreas Habel ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Nonribosomal Peptide Synthetase ◽  
Full Length ◽  
Amide Bond ◽  
White Rot ◽  
Mass Spectrometry Analysis ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Peptide Synthetase

ABSTRACT The model white-rot basidiomycete, Ceriporiopsis (Gelatoporia) subvermispora B, encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet-uncharacterized fungal type VI siderophore synthetase family, which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module, and two thiolation/condensation (T-C) didomain partial modules which together constitute an AT1C1T2C2T3C3 domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as a polyhistidine fusion in Aspergillus niger as a soluble and active protein. N 5-acetyl-N 5-hydroxy-l-ornithine (l-AHO) and N 5-cis-anhydromevalonyl-N 5 -hydroxy-l-ornithine (l-AMHO) were accepted as the substrates, based on results of an in vitro substrate-dependent [32P]ATP-pyrophosphate radioisotope exchange assay. Full-length holo-CsNPS2 catalyzed amide bond formation between three l-AHO molecules to release the linear l-AHO trimer, called basidioferrin, as the product in vitro, which was verified by liquid chromatography–high-resolution electrospray ionization–mass spectrometry analysis. Phylogenetic analyses suggested that type VI family siderophore synthetases are widespread in mushrooms and evolved in a common ancestor of basidiomycetes. IMPORTANCE The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 support a further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the Aspergillus niger/SM-Xpress-based system as a suitable platform to heterologously express multimodular basidiomycete biosynthesis enzymes in the >250-kDa range in soluble and active form.

scholarly journals Quorum-Sensing Regulation of Constitutive Plantaricin by Lactobacillus plantarum Strains under a Model System for Vegetables and Fruits

2013 ◽  
Vol 80 (2) ◽  
pp. 777-787 ◽  
Author(s):  
Carlo G. Rizzello ◽  
Pasquale Filannino ◽  
Raffaella Di Cagno ◽  
Maria Calasso ◽  
Marco Gobbetti
Keyword(s):  
Quorum Sensing ◽  
Lactobacillus Plantarum ◽  
Antimicrobial Activities ◽  
Model System ◽  
Mass Spectrometry Analysis ◽  
Carrot Juice ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Vegetables And Fruits

ABSTRACTThis study aimed at investigating the regulatory system of bacteriocin synthesis byLactobacillus plantarumstrains in vegetables and fruits in a model system. Sterile and neutralized cell-free supernatant (CFS) fromL. plantarumstrains grown in MRS broth showedin vitroantimicrobial activities toward various indicator strains. The highest activity was that ofL. plantarumC2. The antimicrobial activity was further assayed on vegetable and fruit agar plates (solid conditions) and in juices (liquid conditions). A regulatory mechanism of bacteriocin synthesis via quorum sensing was hypothesized. The synthesis of antimicrobial compounds seemed to be constitutive under solid conditions of growth on vegetable and fruit agar plates. In contrast, it depended on the size of the inoculum whenL. plantarumC2 was grown in carrot juice. Only the inoculum of ca. 9.0 log CFU ml−1produced detectable activity. The genesplnA,plnEF,plnG, andplnHwere found in allL. plantarumstrains. The genesplnJKandplnNwere detected in only three or four strains. Reverse-phase high-performance liquid chromatography purification and mass spectrometry analysis revealed the presence of a mixture of eight peptides in the most active fraction of the CFS fromL. plantarumC2. Active peptides were encrypted into bacteriocin precursors, such as plantaricins PlnJ/K and PlnH and PlnG, which are involved in the ABC transport system. A real-time PCR assay showed an increase in the expression ofplnJKandplnGduring growth ofL. plantarumC2 in carrot juice.

scholarly journals Serum-Mediated Cleavage ofBacillus anthracisProtective Antigen Is a Two-Step Process That Involves a Serum Carboxypeptidase

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
David L. Goldman ◽  
Edward Nieves ◽  
Antonio Nakouzi ◽  
Johanna Rivera ◽  
Ei Ei Phyu ◽  
...  
Keyword(s):  
Host Response ◽  
Protective Antigen ◽  
Serum Proteins ◽  
Circulatory System ◽  
Mass Spectrometry Analysis ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Intimate Association ◽  
Carboxy Terminal

ABSTRACTMuch of our understanding of the activity of anthrax toxin is based onin vitrosystems, which delineate the interaction betweenBacillus anthracistoxins and the cell surface. However, these systems fail to account for the intimate association ofB. anthraciswith the circulatory system, including the contribution of serum proteins to the host response and processing of anthrax toxins. Using a variety of immunological techniques to inhibit serum processing ofB. anthracisprotective antigen (PA) along with mass spectrometry analysis, we demonstrate that serum digests PA via 2 distinct reactions. In the first reaction, serum cleaves PA83into 2 fragments to produce PA63and PA20fragments, similarly to that observed following furin digestion. This is followed by carboxypeptidase-mediated removal of the carboxy-terminal arginine and lysines from PA20.IMPORTANCEOur findings identify a serum-mediated modification of PA20that has not been previously described. These observations further imply that the processing of PA is more complex than currently thought. Additional study is needed to define the contribution of serum processing of PA to the host response and individual susceptibility to anthrax.

scholarly journals Targeted Disruption of Nonribosomal Peptide Synthetasepes3Augments the Virulence of Aspergillus fumigatus

2011 ◽  
Vol 79 (10) ◽  
pp. 3978-3992 ◽  
Author(s):  
Karen A. O'Hanlon ◽  
Timothy Cairns ◽  
Deirdre Stack ◽  
Markus Schrettl ◽  
Elaine M. Bignell ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Opportunistic Pathogen ◽  
Immune Recognition ◽  
Phenotypic Analysis ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Targeted Gene Deletion ◽  
Wild Type Phenotype ◽  
Murine Tissue ◽  
Triacetylfusarinine C

ABSTRACTNonribosomal peptide synthesis (NRPS) is a documented virulence factor for the opportunistic pathogenAspergillus fumigatusand other fungi. Secreted or intracellularly located NRP products include the toxic molecule gliotoxin and the iron-chelating siderophores triacetylfusarinine C and ferricrocin. No structural or immunologically relevant NRP products have been identified in the organism. We investigated the function of the largest gene inA. fumigatus, which encodes the NRP synthetase Pes3 (AFUA_5G12730), by targeted gene deletion and extensive phenotypic analysis. It was observed that in contrast to other NRP synthetases, deletion ofpes3significantly increases the virulence ofA. fumigatus, whereby thepes3deletion strain (A. fumigatusΔpes3) exhibited heightened virulence (increased killing) in invertebrate (P< 0.001) and increased fungal burden (P= 0.008) in a corticosteroid model of murine pulmonary aspergillosis. Complementation restored the wild-type phenotype in the invertebrate model. Deletion ofpes3also resulted in increased susceptibility to the antifungal, voriconazole (P< 0.01), shorter germlings, and significantly reduced surface β-glucan (P= 0.0325). Extensive metabolite profiling revealed that Pes3 does not produce a secreted or intracellularly stored NRP inA. fumigatus. Macrophage infections and histological analysis of infected murine tissue indicate thatΔpes3heightened virulence appears to be mediated by aberrant innate immune recognition of the fungus. Proteome alterations inA. fumigatusΔpes3strongly suggest impaired germination capacity. Uniquely, our data strongly indicate a structural role for the Pes3-encoded NRP, a finding that appears to be novel for an NRP synthetase.

scholarly journals Bordetella parapertussis PagP Mediates the Addition of Two Palmitates to the Lipopolysaccharide Lipid A

2014 ◽  
Vol 197 (3) ◽  
pp. 572-580 ◽  
Author(s):  
L. E. Hittle ◽  
J. W. Jones ◽  
A. M. Hajjar ◽  
R. K. Ernst ◽  
A. Preston
Keyword(s):  
Lipid A ◽  
Mutational Analysis ◽  
Mass Spectrometry Analysis ◽  
Toll Like Receptor 4 ◽  
Membrane Function ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Proinflammatory Responses ◽  
Acyl Chains ◽  
Increased Sensitivity

Bordetella bronchisepticaPagP (PagPBB) is a lipid A palmitoyl transferase that is required for resistance to antibody-dependent complement-mediated killing in a murine model of infection.B. parapertussiscontains a putativepagPhomolog (encodingB. parapertussisPagP [PagPBPa]), but its role in the biosynthesis of lipid A, the membrane anchor of lipopolysaccharide (LPS), has not been investigated. Mass spectrometry analysis revealed that wild-typeB. parapertussislipid A consists of a heterogeneous mixture of lipid A structures, with penta- and hexa-acylated structures containing one and two palmitates, respectively. Through mutational analysis, we demonstrate that PagPBPais required for the modification of lipid A with palmitate. While PagPBBtransfers a single palmitate to the lipid A C-3′ position, PagPBPatransfers palmitates to the lipid A C-2 and C-3′ positions. The addition of two palmitate acyl chains is unique toB. parapertussis. Mutation ofpagPBParesulted in a mutant strain with increased sensitivity to antimicrobial peptide killing and decreased endotoxicity, as evidenced by reduced proinflammatory responses via Toll-like receptor 4 (TLR4) to the hypoacylated LPS. Therefore, PagP-mediated modification of lipid A regulates outer membrane function and may be a means to modify interactions between the bacterium and its human host during infection.

scholarly journals MomL, a Novel Marine-DerivedN-Acyl Homoserine Lactonase from Muricauda olearia

2014 ◽  
Vol 81 (2) ◽  
pp. 774-782 ◽  
Author(s):  
Kaihao Tang ◽  
Ying Su ◽  
Gilles Brackman ◽  
Fangyuan Cui ◽  
Yunhui Zhang ◽  
...  
Keyword(s):  
Metal Binding ◽  
Catalytic Efficiency ◽  
Quorum Quenching ◽  
Site Directed Mutagenesis ◽  
Mass Spectrometry Analysis ◽  
Infection Model ◽  
Liquid Chromatography Mass Spectrometry ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Virulence Factor Production

ABSTRACTGram-negative bacteria useN-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed thatMuricauda oleariaTh120, belonging to the classFlavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution ofoxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C6-HSL is ∼2.9 × 105s−1M−1. Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the “HXHXDH” motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence ofPseudomonas aeruginosain aCaenorhabditis elegansinfection model, which suggests that MomL has the potential to be used as a therapeutic agent.

scholarly journals Evaluation of theIn VitroActivity of Voriconazole As Predictive ofIn VivoOutcome in a Murine Aspergillus fumigatus Infection Model

2013 ◽  
Vol 57 (3) ◽  
pp. 1404-1408 ◽  
Author(s):  
Valentina Salas ◽  
F. Javier Pastor ◽  
Enrique Calvo ◽  
Deanna A. Sutton ◽  
Annette W. Fothergill ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Infection Model ◽  
Broth Microdilution ◽  
Excellent Correlation ◽  
Cutoff Value ◽  
Content Type ◽  
Fungal Load ◽  
Strain Dependent

ABSTRACTWe have evaluated thein vitroactivity of voriconazole against 61 strains ofAspergillus fumigatusby using broth microdilution, disk diffusion, and minimal fungicidal concentration procedures. We observed an excellent correlation between the results obtained with the three methods. Five percent of the strains showed MICs greater than or equal to the epidemiological cutoff value (ECV; 1 μg/ml). To assess whether MICs were predictive ofin vivooutcome, we tested the efficacy of voriconazole at 25 mg/kg of body weight daily in an immunosuppressed murine model of disseminated infection using 10 strains representing various patterns of susceptibility to the drug as determined by thein vitrostudy. Voriconazole prolonged survival and reduced fungal load in the kidneys and brain in those mice infected with strains with MICs of ≤0.25 μg/ml, while in mice infected with strains with MICs of 0.5 to 2 μg/ml, the efficacy was varied and strain dependent and in mice infected with the strain with a MIC of 4 μg/ml, the antifungal did not show efficacy. Voriconazole reduced galactomannan antigenemia against practically all strains with a MIC of <4 μg/ml. Our results demonstrate that some relationship exists between voriconazole MICs andin vivoefficacy; however, further studies testing additional strains are needed to better ascertain which MIC values can predict clinical outcome.

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