Single-copy IMH3 allele is sufficient to confer resistance to mycophenolic acid in Candida albicans and to mediate transformation of clinical Candida species

Abstract

Parasexual genetic analysis of Candida albicans utilized the dominant selectable marker that conferred resistance to mycophenolic acid. We cloned and sequenced the IMH3(r) gene from C. albicans strain 1006, which was previously identified as resistant to mycophenolic acid (MPA) (A. K. Goshorn and S. Scherer, Genetics 123:213-218, 1989). MPA is an inhibitor of IMP dehydrogenase, an enzyme necessary for the de novo biosynthesis of GMP. G. A. Kohler et al. (J. Bacteriol. 179:2331-2338, 1997) have shown that the wild-type IMH3 gene, when expressed in high copy number, will confer resistance to this antibiotic. We demonstrate that the IMH3(r) gene from strain 1006 has three amino acid changes, two of which are nonconservative, and demonstrate that at least two of the three mutations are required to confer resistance to MPA. We used this gene as a dominant selectable marker in clinical isolates of C. albicans and Candida tropicalis. We also identified the presence of autonomously replicating sequence elements that permit autonomous replication in the promoter region of this gene. Finally, we found the excision of a phi-type long terminal repeat element outside the IMH3 open reading frame of the gene in some strains. We used the IMH3(r) allele to disrupt one allele of ARG4 in two clinical isolates, WO-1 and FC18, thus demonstrating that a single ectopic integration of this dominant selectable marker is sufficient to confer resistance to MPA.

FIG. 1

Typical selection for Mpa^r after transformation with p3408. WO-1 was transformed with uncut p3408 and plated onto medium containing MPA (1 μg/ml). The plates were incubated at 30°C for 3 days. Transformants were picked and replated onto MPA at 1, 10, and 20 μg/ml. Almost all colonies grew under all conditions tested.

FIG. 2

Plasmid p3408 is sufficient to confer Mpa^r in clinical isolates of C. tropicalis. Primers used to clone the IMH3^r allele were used to detect the presence of the IMH3^r allele in C. tropicalis strain 678 transformed with p3408 (Lanes 1 to 6, transformants). Sequence similarity between the IMH3 allele of C. tropicalis and the primers used was not sufficient to detect the 2.7-kb IMH3 homolog in untransformed C. tropicalis (lane 7). Lane 8, 1.kb Plus DNA ladder (Gibco-BRL, Grand Island, N.Y.).

FIG. 3

The IMH3^r allele possesses an ARS. FC18 and WO-1 were transformed with uncut p3408 and selected on MPA (1 μg/ml). Mpa^r transformants were replated onto MPA at 1, 5, 10, and 20 μg/ml. Colonies were picked from the second set of plates and grown overnight in YEPD. DNA was extracted, digested with HindIII, subjected to gel electrophoresis, and blotted. Blots were hybridized with a radiolabeled IMH3 probe. The bands at 11.0 kb are the genomic copies. The 6.7-kb bands correspond to the size of the linearized p3408 plasmid.

FIG. 4

Structure of the IMH3^r allele and location of primer pairs. The IMH3 gene consists of two exons, of 1.1 and 1.6 bp, respectively, and an intron of 248 bp. The mutations which distinguish the IMH3^r allele from the wild-type gene are shown in their approximate locations. The primers which were used to amplify the exons separately are shown as arrows. Amplification of the gene was carried out with the following primers: for exon 1, B15106(W) and B17359(Y); for exon 2, C15107(Z) and B17360(X).

FIG. 5

Disruption of one allele of ARG4 with IMH3. (Top) A 1.6-kb fragment of the ARG4 ORF in plasmid p1129 was replaced with the IMH3^r allele to create p3394. SnaBI-linearized p3394 was used to transform both FC18 and WO-1. Transformants were selected on the basis of Mpa^r. (Bottom) DNA from p3394-transformed WO-1 was extracted and digested with BamHI and NsiI, electrophoresed, and blotted. Blots were hybridized with a radiolabeled probe of p1129 (ARG4) (A) or IMH3 (B). These blots demonstrate that the IMH3^r allele integrated ectopically at the ARG4 locus. They further demonstrate that one copy of the IMH3^r allele is sufficient to confer Mpa^r.