[Show abstract][Hide abstract] ABSTRACT: The bark beetle-associated fungus Grosmannia clavigera participates in the large-scale destruction
of pine forests. In the tree, it must tolerate saturating levels of toxic conifer defense
chemicals (e.g. monoterpenes). The fungus can metabolize some of these compounds
through the ß-oxidation pathway and use them as a source of carbon. It also uses carbon
from pine triglycerides, where oleic acid is the most common fatty acid. High levels of free
fatty acids, however, are toxic and can cause additional stress during host colonization.
Fatty acids induce expression of neighboring genes encoding a cytochrome P450
(CYP630B18) and its redox partner, cytochrome P450 reductase (CPR2). The aim of this
work was to study the function of this novel P450 system. Using LC/MS, we biochemically
characterized CYP630 as a highly specific oleic acid ω-hydroxylase. We explain oleic acid
specificity using protein interaction modeling. Our results underscore the importance of ω-
oxidation when the main ß-oxidation pathway may be overwhelmed by other substrates
such as host terpenoid compounds. Because this CYP-CPR gene cluster is evolutionarily
conserved, our work has implications for metabolism studies in other fungi.
PLoS ONE 03/2015; 10(3). DOI:10.1371/journal.pone.0120119 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To successfully colonize and eventually kill pine trees, Grosmannia clavigera (Gs), the main fungal pathogen associated with the mountain-pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes (especially (+)-limonene). We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography mass spectrometry (GC/MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone, p-mentha-2,8-dienol, perillyl alcohol and isopiperitenol. It can also degrade (+)-limonene through the C1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. RNA-seq data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation, then valine and TCA metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knock-outs indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl-CoA hydratase. Our work provides information on enzyme-mediated limonene utilization or modification, and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals.
[Show abstract][Hide abstract] ABSTRACT: Among the most devastating pests of Norway spruce (Picea abies) are the European spruce bark beetle (Ips typographus) and the associated pathogenic blue-stain fungus Ceratocystis polonica. Following attack and colonization, the beetle and the fungus must cope with induced host chemical defenses, such as monoterpenes that are generally thought to be toxic to both symbionts. The goal of this study was to better understand the response of Norway spruce following C. polonica inoculation at low density that does not overwhelm the tree and to identify monoterpenes mobilized toward the fungus. We inoculated healthy mature trees and monitored monoterpene profiles 2, 3, and 5 months post-inoculation. We also exposed three different C. polonica strains to the most abundant or significantly up-regulated monoterpenes to determine differences in monoterpene toxicity and resistance among strains. Total monoterpene levels, including limonene, were increased at 2 and 3 months after inoculation and had dropped after 5 months. In in vitro assays, all monoterpenes were inhibitory to C. polonica. Limonene and β-pinene were the most potent inhibitors of fungal growth. The extent of inhibition varied between the three strains tested. These results showed a defense response of Norway spruce to C. polonica, in which limonene may play a critical role in inhibiting the spread of the fungus. We also showed that differences between strains of C. polonica must be taken into account when assessing the role of the fungus in this bark beetle–symbiont system.
European Journal of Forest Research 12/2013; 133(4). DOI:10.1007/s10342-013-0772-4 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aims: CYP53A15, from the sorghum pathogen Cochliobolus lunatus, is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi. Because this enzyme is unique to fungi, it is a promising drug target in fungal pathogens of other eukaryotes.
Methods and Results: In our work, we showed high antifungal activity of seven cinnamic acid derivatives against C. lunatus and two other fungi, Aspergillus niger and Pleurotus ostreatus. To elucidate the mechanism of action of cinnamic acid derivatives with the most potent antifungal properties, we studied the interactions between these compounds and the active site of C. lunatus cytochrome P450, CYP53A15.
Conclusion: We demonstrated that cinnamic acid and at least four of the 42 tested derivatives inhibit CYP53A15 enzymatic activity.
Significance and Impact of the Study: By identifying selected derivatives of cinnamic acid as possible antifungal drugs, and CYP53 family enzymes as their targets, we revealed a potential inhibitor-target system for antifungal drug development.
Journal of Applied Microbiology 12/2013; [Epub ahead of print](xx):1-12.. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CYP53A15, from the sorghum pathogen Cochliobolus lunatus is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi. Because this enzyme is unique to fungi, it is a promising drug target in fungal pathogens of other eukaryotes.
In our work we showed high antifungal activity of seven cinnamic acid derivatives against C. lunatus, and two other fungi, Aspergillus niger and Pleurotus ostreatus. In order to elucidate the mechanism of action of cinnamic acid derivatives with the most potent antifungal properties, we studied the interactions between these compounds and the active site of C. lunatus cytochrome P450, CYP53A15.
We demonstrated that cinnamic acid and at least four of the 42 tested derivatives inhibit CYP53A15 enzymatic activity.
By identifying selected derivatives of cinnamic acid as possible antifungal drugs, and CYP53 family enzymes as their targets, we revealed a potential inhibitor-target system for antifungal drug development. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Grosmannia clavigera is a fungal associate of the mountain pine beetle (Dendroctonus ponderosae) and a pathogen of lodgepole pine (Pinus contorta) that must overcome terpenoid oleoresin and phenolic defenses of host trees. G. clavigera responds to monoterpene influx with complementary mechanisms that include export and the use of these compounds as a carbon source. Cytochromes P450 (CYPs) may also be involved in the metabolism of host defense compounds. We have identified and phylogenetically classified G. clavigera CYPs (CYPome). We show that although the G. clavigera CYPome has contracted in evolution, certain CYP families have expanded by duplication. We analyzed RNA-seq data for CYP expression following treatment with terpenes and pine phloem extracts to identify CYPs potentially involved in detoxification of these pine defense compounds. We also used transcriptome analysis of G. clavigera grown on monoterpenes, triglycerides or oleic acid as a carbon source to identify up-regulated CYPs that may be involved in the utilization of these compounds to support fungal growth. Finally, we identify secondary metabolite biosynthetic gene clusters that contain CYPs, and CYPs in clusters that may be involved in conversion of host chemicals.
[Show abstract][Hide abstract] ABSTRACT: Fungal CYP53 enzymes are highly conserved proteins, involved in phenolic detoxification and have no homologues in higher eukaryotes, rendering them favorable drug targets. Aiming to discover novel CYP53 inhibitors, we employed two parallel virtual screening protocols and evaluated highest scoring hit compounds by analyzing the spectral binding interactions, by surveying the antifungal activity, and assessing the inhibition of catalytic activity. Based on combined results, we selected 3-methyl-4-(1H-pyrrol-1-yl)benzoic acid (compound 2) as the best candidate for hit-to-lead follow-up in the antifungal drug discovery process.
Journal of Chemical Information and Modeling 10/2012; 52(11):3053-3063. DOI:10.1021/ci3004418 · 3.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Results: Big Ideas:
In July 2012, we asked young scientists to describe the one big idea in their field that they wish every non-scientist understood. Why?
We heard from nearly 200 readers. A sample of the best responses can be found below.
In the 5 October 2012 issue, we ran excerpts from 17 of the many interesting responses we received. Below, you will find the full versions of those 17 essays (in the order they were printed) as well as the best (in alphabetical order) of the other submissions we received.
To read the complete versions, as well as many more, go to http://scim.ag/NextGen4Results.
[Show abstract][Hide abstract] ABSTRACT: Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.
[Show abstract][Hide abstract] ABSTRACT: Aegerolysins, discovered in fungi, bacteria and plants, are highly similar proteins with interesting biological properties. Certain aegerolysins possess antitumoral, antiproliferative, and antibacterial activities. Further possible medicinal applications include their use in the prevention of atherosclerosis, or as vaccines. Additional biotechnological value of fungal aegerolysins lies in their involvement in development, which could improve cultivation of commercially important edible mushrooms. Besides, new insights on microheterogeneity of raft-like membrane domains could be gained by using aegerolysins as specific markers in cell and molecular biology. Although the exact function of aegerolysins in their producing organisms remains to be explained, they are biochemically well characterized all-beta structured proteins sharing the following common features: low isoelectric points, similar molecular weights (15-17 kDa), and stability in a wide pH range.
Protein Science 05/2009; 18(4):694-706. DOI:10.1002/pro.85 · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A large number of proteins involved in calcium and intracellular pH signaling and homeostasis have previously been discovered and characterized in Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana,but relatively few have been identified in Aspergillus species. The aim of this study was to identify proteins regulating the intracellular concentration of calcium ions and protons in Aspergillus spp. and compare these with other fungi. For Aspergillus spp. we identified 46, 97 and 105 putative Ca2+-permeable channels, cation/proton transporters and P-ATPases, respectively, the majority of them previously unknown. The subunits composing V-type H+ ATPase and F0F1 ATP synthase (F-type ATPase) from Aspergillus spp. were identified. The greater redundancy of Ca2+-permeable channels, cation/proton exchangers and P-ATPases in filamentous fungi (between 28 putative proteins from A. clavatus and 34 from A. oryzae)compared to that of S. cerevisiae (19 proteins) reflects a more complex cellular organization and filamentous growth form. On the other hand the complexity of V-type H+ ATPase and F0F1 ATP synthase in filamentous fungi is comparable to that in ascomycetous yeast species indicating that both ATPase complexes are a basic universal requirement of the fungal cell.
[Show abstract][Hide abstract] ABSTRACT: A novel cytochrome P450, CYP53A15, was identified in the pathogenic filamentous ascomycete Cochliobolus lunatus. The protein, classified into the CYP53 family, was capable of para hydroxylation of benzoate. Benzoate is a key intermediate in the metabolism of aromatic compounds in fungi and yet basically toxic to the organism. To guide functional analyses, protein structure was predicted by homology modeling. Since many naturally occurring antifungal phenolic compounds are structurally similar to CYP53A15 substrates, we tested their putative binding into the active site of CYP53A15. Some of these compounds inhibited CYP53A15. Increased antifungal activity was observed when tested in the presence of benzoate. Some results suggest that CYP53A15 O-demethylation activity is important in detoxification of other antifungal substances. With the design of potent inhibitors, CYP53 enzymes could serve as alternative antifungal drug targets.
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 reductase (CPR) is the redox partner of P450 monooxygenases, involved in primary and secondary metabolism of eukaryotes. Two novel CPR genes, sharing 34% amino acid identity, were found in the filamentous ascomycete Cochliobolus lunatus. Fungal genomes were searched for putative CPR enzymes. Phylogenetic analysis suggests that multiple independent CPR duplication events occurred in fungi, whereas P450-CPR fusion occurred before the diversification of Dikarya and Zygomycota. Additionally, losses of methionine synthase reductase were found in certain fungal taxa; a truncated form of this enzyme was conserved in Pezizomycotina. In fungi, high numbers of cytochrome P450 enzymes, multiple CPRs, and P450-CPR fusion proteins were associated with filamentous growth. Evolution of multiple CPR-like oxidoreductases in filamentous fungi might have been driven by the complexity of biochemical functions necessitated by their growth form, as opposed to yeast. __________________________________________________________________________________________LINK: http://www.sciencedirect.com/science/article/pii/S1087184507001831
[Show abstract][Hide abstract] ABSTRACT: Suppression subtractive hybridization (SSH) was employed to study differential gene expression upon progesterone treatment of the filamentous fungus Cochliobolus lunatus, a plant and opportunistic human pathogen. The transcription profile of progesterone-induced vs. non-induced C. lunatus revealed changes in the number of genes involved in facilitated and vesicle mediated transport, amino acid and derivative metabolism, protein biosynthesis, cell wall biogenesis, lipid metabolism, carbohydrate metabolism, and generation of precursor metabolites and energy. These results suggest that progesterone induces a global adaptive stress response in the organism. Such a response is not surprising, as the steroidal ring structure is similar to certain antifungal plant defense compounds. In C. lunatus, the conversion of such molecules to hydroxylated and less-toxic substances is mediated by enzymes of the cytochrome P450 superfamily, however little is known of the genes encoding them. We identified several putative cytochrome P450 cDNA sequences and quantitatively analyzed their relative mRNA levels upon progesterone induction using Real-time RT-PCR. None of the selected cytochromes P450 showed significant up-regulation (more than 2 fold induction). As an additional inevitable consequence of the large-scale sequencing of cDNA clones, valuable insight into the genome of this non-model organism was obtained.