Projects / Programmes
The role of cytochromes P450 in mycotoxin biosynthesis and virulence of the pathogenic fungus Aspergillus fumigatus
| Code |
Science |
Field |
Subfield |
| 3.01.00 |
Medical sciences |
Microbiology and immunology |
|
| Code |
Science |
Field |
| B230 |
Biomedical sciences |
Microbiology, bacteriology, virology, mycology |
| Code |
Science |
Field |
| 3.01 |
Medical and Health Sciences |
Basic medicine |
Cytochrome P450 monoxygenases, Aspergillus fumigatus, Virulence, Mycotoxins
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
21507 |
PhD Branka Korošec |
Systems and cybernetics |
Head |
2010 - 2013 |
61 |
Organisations (1)
| no. |
Code |
Research organisation |
City |
Registration number |
No. of publicationsNo. of publications |
| 1. |
0104 |
National Institute of Chemistry |
Ljubljana |
5051592000 |
20,968 |
Abstract
Scientific background: The pathogenic filamentous ascomycete fungus Aspergillus fumigatus causes a wide spectrum of serious diseases including allergic reactions, mycotoxicosis and invasive aspergillosis (IA). There appears to be more than one, essential virulence determinant in the pathogenicity of this fungus acting in an additive and/or synergistic manner against the host organism. They either function as fungal defence mechanisms, or directly damage the host organism. Several A. fumigatus secondary metabolites (e.g., conidial melanins and mycotoxins) have been implicated in virulence.
Recently, it has been shown that a single transcriptional regulator, LaeA, positively controls the expression of up to 40% of major classes of secondary metabolite biosynthesis genes, mostly arranged into contiguous clusters within the genome. Fifteen of the 23 cytochrome P450 monoxygenase genes regulated by LaeA are found in secondary metabolite gene clusters. These include clusters dedicated to the production of mycotoxins, such as fumitremorgens, ergot alkaloids (e.g., fumigaclavines), ETP (epipolythiodioxopiperazine) toxin and gliotoxin.
Problem presentation: Cytochrome P450 enzymes, which are particularly abundant in filamentous fungi, act in endogenous and xenobiotic metabolism. Sterol 14?-demethylase (CYP51), involved in biosynthesis of ergosterol, is cellular target of azole anti-fungals which are essential to the therapy of aspergillosis, despite many toxic side-effects. Since CYP51 is present in all eukaryotes, it is difficult to develop drugs with sufficient selectivity. A major problem is also the increasing frequency of azole drug resistance observed in A. fumigatus.
Project aims: Most cytochrome P450 genes discovered in recently sequenced fungal genomes, including those found in secondary metabolite gene clusters, are »orphans« in the sense that their regulation and function are still largely unknown. The purpose of our project is to identify and functionally characterize selected P450 gene products, regulated by LaeA, that are possibly involved in fungal virulence.
Methods: »Orphan« cytochromes P450 will be chosen on the basis of microarray studies and/or bioinformatic data. Selected cytochromes P450 will be expressed, purified and tested in reconstituted P450 monooxyenase systems with different cytochrome P450 reductases (CPRs). To determine catalytic activities of these enzymes two different medium-throughput screening assays will be used. With these techniques potential P450 substrates, as well as inhibitors of orphan P450 available in chemical libraries will be screened. Specific P450s can be further used in molecular modelling studies with the aim of designing complementary small compounds that would specifically inhibit P450 activities. Inhibitors of identified P450s may serve as compounds for the design of novel antifungal drugs.
Originality, relevance and potential influence: As sufficient knowledge of the molecules involved in mycotoxin biosynthetic pathways is a prerequisite for the development of diagnostic and therapeutic tools, characterisation and functional determination of the putative cytochrome P450 genes and their products is crucial. Because of the increasing number of aspergillus infections and the development of azole drug resistance in pathogenic fungi identified P450s could represent interesting targets for the development of new, more specific and efficient anti-fungal drugs.
Organisation and feasibility of the project: This project fits into the framework of the inter-institutional research program P1-0104 »Functional genomics and biotechnology for health«, and will be carried out at the National Institute of Chemistry in collaboration with the Chair of Pharmaceutical Chemistry at the Faculty of Pharmacy (University of Ljubljana) and University Clinic of Respiratory and Allergic Diseases Golnik, where we have expertise, competence and equipment to successfully execute our research.
Significance for science
Fungal genome sequencing projects have revealed high numbers of cytochrome P450 monooxygenases in species with filamentous growth form, but great majority of these cytochromes P450, including those found in secondary metabolite gene clusters, are „orphans“ in the sense that their regulation and function are still largely unknown. Thus, the relatively slow progress of assigning function to specific P450s poses a challenge. Fungal P450, often part of secondary metabolite gene clusters, are involved in the synthesis of different natural products, many of which are involved in fungal virulence. As sufficient knowledge of the molecules involved in mycotoxin biosynthetic pathways is a prerequisite for the development of diagnostic and therapeutic tools, characterisation and functional determination of the putative cytochrome P450 genes and their products is crucial. By characterization of two novel cytochromes, which potentially contribute to the virulence of the fungus Aspergillus fumigatus, we enhance knowledge of the involvement of cytochromes in the virulence of the fungus Aspergillus fumigatus.Specific P450s can be further used in molecular modeling studies with the aim of designing complementary small compounds that would specifically inhibit P450 activities. Inhibitors of identified P450s may serve as compounds for the design of novel antifungal drugs
Significance for the country
Despite current diagnostic and therapeutic options, mortality associated with invasive aspergillosis caused by A. fumigatus remains high. A major problem is also the increasing frequency of azole drug resistance observed in A. fumigatus. Thus the development of new rapid diagnostic methods with higher sensitivity and more efficacious antifungal drugs with novel mechanisms of action are needed. We suspect that many of the functionally yet uncharacterized cytochromes P450 of pathogenic fungi, involved in biosynthesis of secondary metabolites putatively contribute to fungal virulence and as such could represent interesting targets for the development of new more specific and efficient anti-fungal drugs. In our study, we functionally characterized two cytochrome P450s and showed their potential involvement in the pathogenicity of the fungus Aspergillus fumigatus. By defining the role of the new cytochrome P450 were exposed those which do not have homologues in higher eukaryotes and thus may represent a potential antifungal targets, which could enable the development of more selective and potent inhibitors against pathogenic fungi, with less adverse side effects. Our results represent progress in development of novel antifungal compounds, based on natural defence molecules, which could prove useful in combating infectious and toxin-producing fungi in medicine as well as in agriculture, since fungal infections cause economically significant crop losses annually.
Most important scientific results
Annual report
2012,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Final report,
complete report on dLib.si
