Rosemarie Kelly

Specific Substitutions in the Echinocandin Target Fks1p Account for Reduced Susceptibility of Rare Laboratory and Clinical Candida sp. Isolates

ABSTRACT An association between reduced susceptibility to echinocandins and changes in the 1,3-β-d-glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Cafks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei.

An improved protocol for the preparation of yeast cells for transformation by electroporation

Pretreatment of yeast cells with lithium acetate (LiAc) and dithiothreitol (DTT) enhances the frequency of transformation by electroporation. The method shows improvements of 6–67‐fold in wild‐type strains derived from commonly used Saccharomyces cerevisiae genetic backgrounds. In addition, 15–300‐fold improvement in transformation frequency was achieved with several mutant strains of S. cerevisiae that transformed poorly by conventional procedures. Both DTT and lithium acetate were necessary for maximal transformation frequencies. Pretreatment with lithium and DTT also resulted in an ∼3·5‐fold increase in the electroporation transformation frequency of the pathogenic fungus Candida albicans.

Transformation of Candida albicans by electroporation

There is disclosed a procedure for DNA-mediated transformation of Candida albicans by electroporation utilizing lithium acetate and dithiothreitol to weaken the cell wall structure and optimize the yield of transformants.In contrast to a variety of other yeasts, Candida albicans has proved difficult to transform with high efficiency. Lithium acetate transformation is fast and simple but provides a very low efficiency of DNA transfer (50–100 transformants/µg DNA), while spheroplast transformation, although more efficient (∼300 transformants/µg integrative DNA and 103–104 transformants/µg replicative DNA), is complicated and time‐consuming. In this study we applied various yeast transformation techniques to C. albicans and selected an electroporation procedure for further optimization. Transformation efficiencies of up to 300 transformants/µg were obtained for an integrative plasmid and up to 4500 transformants/µg for a CARS‐carrying plasmid. This reasonably high transformation efficiency, combined with the ease and speed of electroporation in comparison to alternative techniques, make it the preferred method for transformation of C. albicans. Copyright

Purification of geranylgeranyltransferase I from Candida albicans and cloning of the CaRAM2 and CaCDC43 genes encoding its subunits.

All previously characterized protein geranylgeranyltransferases I (GGTase I) are heterodimeric zinc metalloenzymes which catalyse geranylgeranylation of a cysteine residue in proteins containing a C-terminal CaaL motif (C, Cys; a, aliphatic amino acid; L, Leu). The alpha and beta subunits of GGTase I of Saccharomyces cerevisiae are encoded by RAM2 and CDC43, respectively, and are essential for yeast viability. The authors are therefore investigating the role of geranylgeranylation in the related pathogenic yeast, Candida albicans, which is the most prevalent human fungal pathogen. GGTase I was purified to near homogeneity and also found to be a heterodimeric magnesium-dependent, zinc metalloenzyme displaying selectivity for CaaL-containing protein substrates. GGTase I peptide sequences were obtained from the purified protein and used to clone the genes encoding both subunits. CaRAM2 and CaCDC43 encode proteins that are 42 and 34% identical to their corresponding S. cerevisiae homologues, respectively, and 30% identical to their human homologues. Despite the limited overall homology, key zinc- and substrate-binding residues of the beta subunit (Cdc43p) are conserved. A unique feature of CaCdc43p is a tract of polyasparagine whose length varies from 6 to 17 residues among C. albicans strains and between alleles. Coexpression of both CaCDC43 and CaRAM2 under their native promoters complemented the ts defect of a S. cerevisiae cdc43 mutant but expression of the beta-subunit alone did not correct the growth defect, suggesting that hybrid GGTase I heterodimers are nonfunctional.

New fungal metabolite geranylgeranyltransferase inhibitors with antifungal activity

Geranylgeranyltransferase I (GGTase I) catalyzes the post-translational transfer of lyophilic diterpenoid geranylgeranyl to the cysteine residue of proteins terminating with a CaaX motif such as Rho1p and Cdc42p. It has been shown that GGTase I activity is essential for viability of Saccharomyces cerevisiae and hence its inhibition is a potential antifungal target. From natural product screening, a number of azaphilones including one novel analog were isolated as broad-spectrum inhibitors of GGTase I. Isolation, structure elucidation, GGTase I inhibitory activities and antifungal activities of these compounds are described.

Heterologous transformation of Zalerion arboricola

A heterologous DNA-mediated transformation system was developed for the pneumocandin-producing fungus Z. arboricola that was based on either conferral of hygromycin B resistance or complementation of a nitrate reductase mutant. Hygromycin-resistant transformants were selected with plasmid pCSN43 which contains the E. coli hygromycin B phosphotransferase gene under the control of Aspergillus nidulans trpC transcription signals. Transformation frequencies were about four transformants per μg of circular DNA and could be improved four- to six-fold by linearizing the transforming DNA. The transformants differed from one another with respect to the copy number of the integrated plasmid and the site of integration. Adding an autonomously-replicating sequence (AMA1) from A. nidulans to pCSN43 enhanced transformation three-fold and produced, in addition, numerous abortive transformants. However, it is unlikely that the AMA1 sequence promoted plasmid replication in Z. arboricola. Nitrate reductase mutants of Z. arboricola were isolated by positive selection on chlorate-containing medium, and one mutant was subsequently transformed with pSTA700 which contains the nitrate reductase gene (niaD) from Cephalosporium acremonium. Introduction of the niaD gene restored sensitivity to chlorate in the mutant; therefore, using the niaD gene as a selectable marker provides a system for both positive and negative selection. To our knowledge, this is the first report describing transformation of a member of the genus Zalerion.

Isolation and sequence analysis of the cDNA encoding Δ1-pyrroline-5-carboxylate reductase from Zalerion arboricola

A cDNA encoding delta 1-pyrroline-5-carboxylate reductase (P5CR) was isolated from the pneumocandin (Pmo)-producing fungus, Zalerion arboricola (Za), by complementation of a P5CR-deficient mutant (pro3) of Saccharomyces cerevisiae (Sc). The cloned cDNA was placed under control of the Sc galactokinase (GAL1) promoter and restored P5CR activity to the pro3 mutant. Sequence analysis revealed that the Za P5CR-encoding cDNA encodes an approx. 35 kDa protein with substantial amino acid (aa) identity to P5CR from another filamentous fungus, Neurospora crassa (Nc). Za P5CR exhibits a moderate degree of aa identity to P5CR from plants, bacteria, human and Sc. This is the first gene to be isolated from Za.

Discovery, Isolation, and Structure Elucidation of Dretamycin

The Candida albicans fitness test is a whole cell screening platform that utilizes a mixed-pool of C. albicans mutants, each of which carries a heterozygous deletion of a particular gene. In the presence of an antifungal inhibitor, a subset of these mutants exhibits a growth phenotype of hypersensitivity or hyposensitivity. Collectively these mutants reflect aspects of the mechanism of action of the compound in question. In the course of screening natural products a culture of Streptomyces sp. MS-1-4 was discovered to produce a compound, dretamycin, which yielded a fitness profile exhibiting significant hypersensitivity of the DRE2 heterozygote and hyposensitivity of the DIP5 heterozygote. Herein we report the production, isolation, and structure elucidation of dretamycin.