A new class of IMP dehydrogenase with a role in self-resistance of mycophenolic acid producing fungi

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Role of IMPDH and MPA in GMP biosynthesis. MPA inhibits IMPDH. MPA: Mycophenolic acid. R: ribose 5'-monophosphate. IMP: inosine-5'-monophosphate, XMP: xanthosine-5'-monophosphate, guanosine-5'-monophosphate. GMP: Guanosine monophosphate. IMPDH: IMP dehydrogenase.

Figure 2

MpaFp confers resistance towards MPA. A) Replacing native IMPDH-A coding gene (AN10476, A. nidulans imdA) with mpaF by homologous recombination. The gene targeting substrate contains four parts: mpaF (IMPDH from MPA gene cluster), argB (selection marker) and finally TSI and TSII (targeting sequence I, 2197 bp; and II, 2244 bp flanking AN10476 (A. nidulans IMPDH)). B) Spot assay to determine sensitivity towards MPA. Ten-fold serial dilutions of spores from the two strains NID191 (reference strain with native A. nidulans imdA) and NID495 (A. nidulans imdA replaced with mpaF) were spotted on minimal medium plates with 0, 5, 25, 100 and 200 μg MPA/ml. Each row is composed of spots containing plated spores ranging from ~10⁶ (to the left) to ~10 (to the right) as indicated in the figure.

Figure 3

Identification and cladistic analysis of IMPDH-A and IMPDH-B coding genes from different fungi. A) Gene organization of imdA from A. nidulans and mpaF (coding for IMPDH-B in P. brevicompactum). The sequence region used for creating the cladograms in B is marked by a square. Introns are marked by a thin open line. B) and C): Rooted cladograms based on, B) IMPDH cDNA sequences (651-654 bp); and C) β-tubulin cDNA sequences (981 bp) from species from Penicillium subgenus Penicillium and from five fungi with sequenced genomes including the outgroup. P.: Penicillium and A.: Aspergillus. Bootstrap values (expressed as percentage of 1000 replications) are shown at the branch points. MPA production is indicated by "+" or "-". The clades with Penicillium subgenus Penicillium genes are boxed; red, IMPDH-A; blue, IMPDH-B; green, β-tubulin. Coccidioides immitis has been used as outgroup in both analyses B and C. Scale bars correspond to 0.130 and 0.060 nucleotide changes per site in cladograms B) and C) respectively.

Figure 4

Multiple sequence alignment of selected fungal IMPDHs. The region including the amino acid residue at position 415 and part of the flap-region (flap-region being spanned by residues 412 - 467) is presented in the figure. The position 415 is tyrosine in all IMPDHs identified prior to this work [1]. Note that the flap region is very variable, with only residue 415Y and key catalytic residues 441R and 442Y completely conserved in all IMPDHs identified prior to this work [1]. Residues conserved among all nine sequences are highlighted in grey. P. brevicompactum IMPDH-B (encoded by mpaF) is used as a reference while referring to position numbers. P, Penicillium; A, Aspergillus.