A new class of IMP dehydrogenase with a role in self-resistance of mycophenolic acid producing fungi
© Hansen et al; licensee BioMed Central Ltd. 2011
Received: 5 April 2011
Accepted: 16 September 2011
Published: 16 September 2011
Many secondary metabolites produced by filamentous fungi have potent biological activities, to which the producer organism must be resistant. An example of pharmaceutical interest is mycophenolic acid (MPA), an immunosuppressant molecule produced by several Penicillium species. The target of MPA is inosine-5'-monophosphate dehydrogenase (IMPDH), which catalyses the rate limiting step in the synthesis of guanine nucleotides. The recent discovery of the MPA biosynthetic gene cluster from Penicillium brevicompactum revealed an extra copy of the IMPDH-encoding gene (mpaF) embedded within the cluster. This finding suggests that the key component of MPA self resistance is likely based on the IMPDH encoded by mpaF.
In accordance with our hypothesis, heterologous expression of mpaF dramatically increased MPA resistance in a model fungus, Aspergillus nidulans, which does not produce MPA. The growth of an A. nidulans strain expressing mpaF was only marginally affected by MPA at concentrations as high as 200 μg/ml. To further substantiate the role of mpaF in MPA resistance, we searched for mpaF orthologs in six MPA producer/non-producer strains from Penicillium subgenus Penicillium. All six strains were found to hold two copies of IMPDH. A cladistic analysis based on the corresponding cDNA sequences revealed a novel group constituting mpaF homologs. Interestingly, a conserved tyrosine residue in the original class of IMPDHs is replaced by a phenylalanine residue in the new IMPDH class.
We identified a novel variant of the IMPDH-encoding gene in six different strains from Penicillium subgenus Penicillium. The novel IMPDH variant from MPA producer P. brevicompactum was shown to confer a high degree of MPA resistance when expressed in a non-producer fungus. Our study provides a basis for understanding the molecular mechanism of MPA resistance and has relevance for biotechnological and pharmaceutical applications.
The MPA biosynthetic gene cluster from Penicillium brevicompactum was identified only recently . Interestingly, it turned out that the MPA gene cluster, in addition to the MPA biosynthetic genes, contains a putative IMPDH-encoding gene (mpaF). The study also revealed an additional putative IMPDH-encoding gene by probing the P. brevicompactum genomic DNA . A BLAST search using mpaF as query resulted in only a single IMPDH encoding gene per organism for all fully sequenced non-Penicillium filamentous fungi (see the Results and Discussion section for details). Thus, the discovery of mpaF identifies P. brevicompactum as the first filamentous fungus known to feature two IMPDH encoding genes. In this study, we have identified additional species from the Penicillium subgenus Penicillium that contain two putative IMPDH encoding genes. Furthermore, we show that the two copies that are present in each fungus are dissimilar, and that one of them forms a new distinct group in a cladistic analysis. The IMPDH from the MPA cluster, mpaF, is the founding member of this novel group. The presence of mpaF within the biosynthesis cluster in P. brevicompactum hints at a role in MPA self-resistance. In this study, we examine this hypothesis and show that mpaF confers resistance to MPA when expressed in an otherwise highly sensitive non-producer fungus Aspergillus nidulans.
Results and discussion
Expression of mpaF in A. nidulansconfers resistance to MPA
A new class of IMPDHs found in the Penicillium subgenus Penicillium
The data above strongly suggest that mpaF encodes an IMPDH, which is resistant to MPA, hence strengthening the hypothesis that the IMPDH-encoding gene residing within the MPA gene cluster plays a distinctive role in MPA self-resistance. The results also lead to the next question - whether only MPA producers have two copies of IMPDH-encoding genes. We first performed a BLASTx search (default settings, August 2010, see Methods) by using the cDNA sequence of mpaF as a query. Two IMPDH-encoding genes from Penicillium chrysogenum, the only Penicillium species with a publicly available sequenced genome, produced the most significant hits (data not shown). As P. chrysogenum is not able to produce MPA, the presence of two IMPDH-encoding genes in this fungus is intriguing. Interestingly, the BLASTx search only revealed one IMPDH in the other filamentous fungi that have their genome sequence available in the public domain. Penicillium marneffei, another Penicillium species included in the search, was found to contain only one IMPDH-encoding gene in its genome. However, even though P. marneffei is named a Penicillium, it is only distantly related to Penicillium sensu stricto . Thus, the only two fungi known to have two IMPDH copies so far are the Penicillium species, P. brevicompactum and P. chrysogenum. An initial cladistic analysis showed that the P. brevicompactum IMPDH protein encoded by mpaF and one of the two IMPDHs from P. chrysogenum are phylogenetically highly distinct from the other IMPDHs from filamentous fungi. Furthermore, the IMPDH-encoding gene from P. brevicompactum that was not located within the MPA gene cluster and one of the two IMPDH-encoding genes from P. chrysogenum clustered together with the IMPDH-encoding genes from Aspergillus species (data not shown). Notably, this group was distinct from the group containing mpaF. Accordingly, we decided to name the original and mpaF types of IMPDH as IMPDH-A and IMPDH-B, respectively. The classification of IMPDHs was further substantiated with IMPDH sequences obtained from more Penicillium species as described in the following.
Strains and sequences
Other collection numbers
Sequences (Accession #)
Differences between IMPDH-A and IMPDH-B with a potential role in MPA resistance
IMPDH-B has possibly emerged through gene duplication
IMPDHs are highly conserved enzymes, which points to their important role in fitness. A high level of conservation was also observed for the sequences obtained from the six Penicillium strains investigated in our study. In a cladogram containing both fungal and prokaryotic IMPDHs, the fungal genes of type IMPDH-B clearly cluster with the fungal IMPDH-A type and not the prokaryotic IMPDH-encoding genes (data not shown). The identity of the IMPDH-B to prokaryotic IMPDHs is ~30-35% on amino acid level. This relatively low degree of conservancy, combined with the fact that IMPDH-A and IMPDH-B are ~80% identical in filamentous fungi, suggests that the genes coding for IMPDH-B arose by gene duplication in a filamentous fungus, rather than via a horizontal gene transfer from a prokaryotic organism. As both IMPDH-A and IMPDH-B classes are present in all Penicillium subgenus Penicillium strains tested, a gene duplication event in Penicillium is a plausible hypothesis. To gather further support for this hypothesis, β-tubulin sequences were obtained for all of the fungi tested in this study and a cladogram based on β-tubulin sequences was calculated (Figure 3). As β-tubulin is a highly conserved protein with a critical functional role in eukaryotes, it is often used to create an organismal cladogram [16, 17]. The cladogram based on IMPDH sequences is to a high degree in agreement with the β-tubulin cladogram, and both are in agreement with previously published β-tubulin cladograms . Based on the observations from the cladograms and the high level of identity (~80%) between IMPDH-B type and IMPDH-A type sequences, we postulate that the IMPDH-encoding gene duplication took place during the divergence of Penicillium subgenus Penicillium, or earlier. The large genome sequencing effort that is being carried out at the moment may shed further light on the evolutionary origin of IMPDH-B.
This is the first report elucidating the presence of a new class of IMP dehydrogenase, IMPDH-B, with a role in MPA resistance in filamentous fungi. The high level of resistance observed for IMPDH-B encoded by mpaF from P. brevicompactum is intriguing and stands as the strongest MPA tolerance reported for a eukaryotic IMPDH. Our study also provides insight into the possible molecular basis responsible for the high MPA resistance of IMPDH-B. In particular, we identified one amino acid residue, which is completely conserved in all previously identified IMPDHs, but which is different in the members of the group belonging to the type IMPDH-B. On the applied front, the identified genetic basis for self-resistance may help in efficient production of MPA in heterologous hosts and in understanding the MPA-related chemical ecology in filamentous fungi.
Strains and media
A.nidulans NID191 (argB2, pyrG89, veA1, nkuA-trS::AFpyrG, IS1::PgpdA-TtrpC::argB)  and NID495 (argB2, pyrG89, veA1, nkuA-trS::AFpyrG, ΔimdA::argB::mpaF) were grown on Minimal Medium (MM) containing 1% glucose, 10 mM NaNO3, 1 × salt solution , and 2% agar for solid media. MM was supplemented with 10 mM uridine, 10 mM uracil, and 4 mM L-arginine when necessary. P. brevicompactum IBT 23078 was grown on Czapek yeast autolysate (CYA) agar at 25°C. CYA: 5.0 g/l Yeast extract (Difco); 15 g/l agar; 35 g/l Czapek Dox broth (Difco); 10 mg/l ZnSO4·7H2O; 5 mg/l CuSO4·5H2O. The pH of the medium was adjusted to 6.5 by using NaOH/HCl.
Generating expression construct
List of primers
Sequence (5' → 3')
A. nidulansstrain construction
Protoplasting and gene-targeting procedures were performed as described previously [21, 22]. 5 μg pHC3 was digested with NotI to liberate the gene targeting substrate, which was used for transformation of NID3 . Transformants containing the desired gene targeting event were verified by PCR with primer-pairs BGHA98/BGHA256HC and BGHA255HC/BGHA225 using Taq-polymerase (Sigma-Aldrich) on genomic DNA obtained from streak purified transformants extracted using the FastDNA® SPIN for Soil Kit (MP Biomedicals, LLC).
MPA treatment of fungi
Spores from A. nidulans NID191 and A. nidulans NID495 were harvested. 10-fold dilution series was performed on freshly made MM-plates with 0, 5, 25, 100, 200 μg MPA/ml (Sigma). All plates contained 0.8% (v/v) methanol. Relative growth of the strains was assessed by visual inspection.
An alignment with the DNA sequence (including introns) of the genes encoding P. brevicompactum IMPDH-B, A. nidulans IMPDH-A, P. chrysogenum IMPDH-A, P. chrysogenum IMPDH-B and Aspergillus fumigatus IMPDH-A was created by using ClustalW . The ClustalW algorithm was accessed from the CLC DNA workbench 5 (CLC bio, http://www.clcbio.com/) with the following parameters: 'gap open cost = 20.0', 'gap extension cost = 1.0', and 'end gap cost = free'. The alignment was used to design degenerate primers to amplify either IMPDH-A like genes (BGHA236HC/BGHA246HC) or IMPDH-B like genes (BGHA240 HC/BGHA241 HC). The primer-set BGHA343/BGHA344 was used to amplify the β-tubulin sequence. Genomic DNA from P. brevicompactum IBT 23078 and four other fungi from Penicillium subgenus Penicillium were extracted using the FastDNA® SPIN for Soil Kit (MP Biomedicals, LLC). Touch-down PCR was carried out using Phusion polymerase (Finnzymes) and the following program. An initial denaturation cycle at 98°C for 2 min; followed by 35 cycles at 98°C for 30 s, an annealing step ranging from 61°C (first cycle) to 54°C (last cycle) for 30 s, and extension at 72°C for 45 s. PCR mixture was made according to the manufacture's instructions. PCR products generated by degenerate PCR were purified from agarose gels using illustra™ DNA and Gel band purification kit (GE Healthcare). Sequencing of purified PCR products was performed by StarSeq (Germany).
BLASTx search was performed with standard settings: 'blastp algorithm', 'expect threshold = 10', 'word size = 3', 'max matches in query range = 0', 'matrix = BLOSUM62', 'gap open cost = 11', 'gap extension cost = 1', and no filters were used. Alignment of DNA coding regions were performed with ClustalW  as implemented in the CLC DNA workbench 5 (CLC bio, http://www.clcbio.com/) and by using the following parameters: 'gap open cost = 20.0', 'gap extension cost = 1.0', and 'end gap cost = free'. A cladogram was constructed with the same software using the neighbour-joining method and 1000 bootstrap replicates . The DNA sequence of IMPDH and β-tubulin from selected fungi with sequenced genome were retrieved from NCBI. These included IMPDH sequence from A. nidulans [GenBank:ANIA_10476], Aspergillus terreus [GenBank:XM_001218149], Aspergillus niger [GenBank:XM_001391855], P. chrysogenum putative IMPDH-A coding gene, [GenBank:XM_002562313], putative IMPDH-B coding gene [GenBank:XM_002559146], P. marneffei [GenBank:XM_002151867]. β-tubulin sequences from A. nidulans [GenBank:XM_653694], A. terreus [GenBank:XM_001215409], A. niger [GenBank:XM_001392399], P. chrysogenum [GenBank:XM_002559715] and P. marneffei [GenBank:XM_002151381]. The MPA gene cluster sequence from P. brevicompactum, which contains the IMPDH-B sequence (mpaF) is available from GenBank under accession number [GenBank:HQ731031].
Amino acid sequences were aligned with ClustalW  as implemented in the CLC DNA workbench 5 (CLC bio, http://www.clcbio.com/) by using the following parameters: 'gap open cost = 20.0', 'gap extension cost = 1.0', and 'end gap cost = free'.
Nucleotide sequence accession numbers
All sequences obtained via degenerate PCR were submitted to GenBank (Table 1). Penicillium bialowiezense β-tubulin [GenBank:JF302653], putative IMPDH-A coding gene [GenBank:JF302658], putative IMPDH-B coding gene [GenBank:JF302662], P. brevicompactum β-tubulin [GenBank:JF302653], imdA [GenBank:JF302657], Penicillium carneum β-tubulin [GenBank:JF302650], putative IMPDH-A coding gene [GenBank:JF302656], putative IMPDH-B coding gene [GenBank:JF302660], Penicillium paneum β-tubulin [GenBank:JF302651], putative IMPDH-A coding gene [GenBank:JF302654], putative IMPDH-B coding gene [GenBank:JF302661], Penicillium roqueforti β-tubulin [GenBank:JF302649], putative IMPDH-A coding gene [GenBank:JF302655], putative IMPDH-B coding gene [GenBank:JF302659].
The work was supported by grant number 09-064967 and 09-064240 from the The Danish Council for Independent Research, Technology and Production Sciences to KRP and UHM. We thank Martin Engelhard Kornholt and Ellen Kirstine Lyhne for valuable technical assistance in the laboratory. We are grateful to Steven Sheridan for his comments on the manuscript.
- Hedstrom L: IMP dehydrogenase: structure, mechanism, and inhibition. Chemical reviews. 2009, 109: 2903-28. 10.1021/cr900021w.PubMedPubMed CentralView ArticleGoogle Scholar
- Weber G, Nakamura H, Natsumeda Y, Szekeres T, Nagai M: Regulation of GTP biosynthesis. Advances in enzyme regulation. 1992, 32: 57-69.PubMedView ArticleGoogle Scholar
- Frisvad JC, Smedsgaard JR, Larsen TO, Samson RA: Mycotoxins, drugs and other extrolites produced by species in Penicillium subgenus Penicillium. Stud Mycol. 2004, 49: 201-41.Google Scholar
- Demain AL: How do antibiotic-producing microorganisms avoid suicide?. Annals of the New York Academy of Sciences. 1974, 235: 601-12. 10.1111/j.1749-6632.1974.tb43294.x.PubMedView ArticleGoogle Scholar
- Hopwood DA: How do antibiotic-producing bacteria ensure their self-resistance before antibiotic biosynthesis incapacitates them?. Molecular microbiology. 2007, 63: 937-40. 10.1111/j.1365-2958.2006.05584.x.PubMedView ArticleGoogle Scholar
- Schrettl M, Carberry S, Kavanagh K, Haas H, Jones GW, O'Brien J, Nolan A, Stephens J, Fenelon O, Doyle S: Self-protection against gliotoxin--a component of the gliotoxin biosynthetic cluster, GliT, completely protects Aspergillus fumigatus against exogenous gliotoxin. PLoS pathogens. 2010, 6: e1000952-10.1371/journal.ppat.1000952.PubMedPubMed CentralView ArticleGoogle Scholar
- Scharf DH, Remme N, Heinekamp T, Hortschansky P, Brakhage A, Hertweck C: Transannular disulfide formation in gliotoxin biosynthesis and its role in self-resistance of the human pathogen Aspergillus fumigatus. Journal of the American Chemical Society. 2010, 132: 10136-41. 10.1021/ja103262m.PubMedView ArticleGoogle Scholar
- Gardiner DM, Jarvis RS, Howlett BJ: The ABC transporter gene in the sirodesmin biosynthetic gene cluster of Leptosphaeria maculans is not essential for sirodesmin production but facilitates self-protection. Fungal genetics and biology. 2005, 42: 257-63. 10.1016/j.fgb.2004.12.001.PubMedView ArticleGoogle Scholar
- Amnuaykanjanasin A, Daub ME: The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae. Fungal genetics and biology. 2009, 46: 146-58. 10.1016/j.fgb.2008.11.007.PubMedView ArticleGoogle Scholar
- Chanda A, Roze L, Kang S, Artymovich K, Hicks GR, Raikhel NV, Calvo AM, Linz JE: A key role for vesicles in fungal secondary metabolism. Proceedings of the National Academy of Sciences of the United States of America. 2009, 106: 19533-8. 10.1073/pnas.0907416106.PubMedPubMed CentralView ArticleGoogle Scholar
- Sirikantaramas S, Yamazaki M, Saito K: Mutations in topoisomerase I as a self-resistance mechanism coevolved with the production of the anticancer alkaloid camptothecin in plants. Proceedings of the National Academy of Sciences of the United States of America. 2008, 105: 6782-6. 10.1073/pnas.0801038105.PubMedPubMed CentralView ArticleGoogle Scholar
- Regueira TB, Kildegaard KR, Hansen BG, Mortensen UH, Hertweck C, Nielsen J: Discovery of the Mycophenolic Acid Biosynthesis genes of Penicillium brevicompactum. Appl Environ Microbiol. 77 (9): 3035-43.Google Scholar
- Riera TV, Wang W, Josephine HR, Hedstrom L: A kinetic alignment of orthologous inosine-5'-monophosphate dehydrogenases. Biochemistry. 2008, 47: 8689-96. 10.1021/bi800674a.PubMedPubMed CentralView ArticleGoogle Scholar
- Köhler GA, Gong X, Bentink S, Theiss S, Pagani GM, Agabian N, Hedstrom L: The functional basis of mycophenolic acid resistance in Candida albicans IMP dehydrogenase. The Journal of biological chemistry. 2005, 280: 11295-302. 10.1074/jbc.M409847200.PubMedView ArticleGoogle Scholar
- Berbee ML, Yoshimura A, Sugiyama J, Taylor JW: Is Penicillium Monophyletic? An Evaluation of Phylogeny in the Family Trichocomaceae from 18S, 5.8S and ITS ribosomal DNA sequence data. Mycologia. 1995, 87: 210-22. 10.2307/3760907.View ArticleGoogle Scholar
- Samson RA, Seifert KA, Kuijpers AFA, Houbraken JAMP, Frisvad JC: Phylogenetic analysis of Penicillium subgenus Penicillium using partial β-tubulin sequences. Studies in Mycology. 2004, 49: 175-200.Google Scholar
- Seifert KA, Samson RA, De Waard JR, Houbraken J, Lévesque CA, Moncalvo J-M, Louis-Seize G, Hebert PDN: Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case. Proc Nat Acad Sci. 2007, 104: 3901-6. 10.1073/pnas.0611691104.PubMedPubMed CentralView ArticleGoogle Scholar
- Hansen BG, Salomonsen B, Nielsen MT, Nielsen JB, Hansen NB, Nielsen KF, Regueira TB, Nielsen J, Patil KR, Mortensen UH: Versatile Enzyme Expression and Characterization System for Aspergillus nidulans, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case. Appl Environ Microbiol. 77 (9): 3044-51.Google Scholar
- Cove DJ: The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim Biophys Acta. 1966, 113: 51-6.PubMedView ArticleGoogle Scholar
- Nour-Eldin HH, Hansen BG, Nørholm MHH, Jensen JK, Halkier BA: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic acids research. 2006, 34: e122-10.1093/nar/gkl635.PubMedPubMed CentralView ArticleGoogle Scholar
- Johnstone IL, Hughes SG, Clutterbuck AJ: Cloning an Aspergillus nidulans developmental gene by transformation. The EMBO journal. 1985, 4: 1307-11.PubMedPubMed CentralGoogle Scholar
- Nielsen ML, Albertsen L, Lettier G, Nielsen JB, Mortensen UH: Efficient PCR-based gene targeting with a recyclable marker for Aspergillus nidulans. Fungal Genet Biol. 2006, 43: 54-64. 10.1016/j.fgb.2005.09.005.PubMedView ArticleGoogle Scholar
- Nielsen JB, Nielsen ML, Mortensen UH: Transient disruption of non-homologous end-joining facilitates targeted genome manipulations in the filamentous fungus Aspergillus nidulans. Fungal Genet Biol. 2008, 45: 165-70. 10.1016/j.fgb.2007.07.003.PubMedView ArticleGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic acids research. 1994, 22: 4673-80. 10.1093/nar/22.22.4673.PubMedPubMed CentralView ArticleGoogle Scholar
- Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular biology and evolution. 1987, 4: 406-25.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.