Comparison of responses of DNA topoisomerase I from Candida albicans and human cells to four new agents which stimulate topoisomerase-dependent DNA nicking

Abstract DNA topoisomerase I is a potential target for therapeutic antifungal agents predicted to have a fungicidal mode of action. This report describes four agents with varying degrees of selectivity for the fungal topoisomerase I compared to the human enzyme: 5-hydroxy-1H-indole-3-acetic acid (5-HIAA), quinizarin, dibenzo- p -dioxin-2-carboxylic acid and 7-amino-4-hydroxy-2-naphthalenesulfonic acid. Taken together with the response of topoisomerase to camptothecin and aminocatechol, these data suggest that there are sufficient structural differences between the topoisomerase I from Candida albicans and human cells to allow selective targeting of the fungal topoisomerase I over its human counterpart.     

The essential role of yeast topoisomerase III in meiosis depends on recombination

Yeast cells mutant for TOP3, the gene encoding the evolutionary conserved type I-5' topoisomerase, display a wide range of phenotypes including altered cell cycle, hyper-recombination, abnormal gene expression, poor mating, chromosome instability and absence of sporulation. In this report, an analysis of the role of TOP3 in the meiotic process indicates that top3Delta mutants enter meiosis and complete the initial steps of recombination. However, reductional division does not occur. Deletion of the SPO11 gene, which prevents recombination between homologous chromosomes in meiosis I division, allows top3Delta mutants to form viable spores, indicating that Top3 is required to complete recombination successfully. A topoisomerase activity is involved in this process, since expression of bacterial TopA in yeast top3Delta mutants permits sporulation. The meiotic block is also partially suppressed by a deletion of SGS1, a gene encoding a helicase that interacts with Top3. We propose an essential role for Top3 in the processing of molecules generated during meiotic recombination.     

Inactivation of homologous recombination suppresses defects in topoisomerase III-deficient mutants

The Saccharomyces cerevisiae TOP3 gene encodes the type IA topoisomerase (Top3p) that is highly conserved in evolution. Deletion of TOP3 leads to a reduction in cell viability, hyper-recombination between repetitive DNA sequences, and abnormalities in both cell cycle progression and responses to DNA damaging agents. Deletion of SGS1, encoding the sole RecQ family helicase in S. cerevisiae, strongly suppresses the phenotypic effects of loss of TOP3 function. Here, we show that many of the adverse phenotypic effects of TOP3 deletion can also be partially alleviated by disruption of homologous recombination (HR) functions. This genetic interaction is seen both in strains deleted for TOP3 and in wild-type strains over-expressing a dominant-negative Top3p mutant form that confers a top3-like phenotype. Moreover, we show that this genetic interaction is conserved in the distantly-related fission yeast, Schizosaccharomyces pombe. Our results implicate topoisomerase III enzymes in recombination repair events required for cellular protection against DNA damaging agents and DNA replication inhibitors.     

Molecular analysis of the Cryptococcus neoformans ADE2 gene, a selectable marker for transformation and gene disruption.

Cryptococcus neoformans is an important fungal pathogen of man. The incidence of cryptococcal disease has increased dramatically in patients immunocompromised because of HIV infection, organ transplantation, or treatment with cytotoxic chemotherapy or corticosteroids. This organism is an excellent model for molecular dissection of fungal pathogenesis and virulence factors. Here we report the nucleotide sequence of the C. neoformans serotype D genomic ADE2 gene, which encodes a phosphoribosylaminoimidazole carboxylase required for purine biosynthesis. Importantly, this version of the ADE2 gene has been used as the selectable marker for virtually all gene disruptions by transformation and homologous recombination in C. neoformans. We compare the nucleotide and amino acid sequences of the ADE2 gene and product to other highly related adenine biosynthetic genes and enzymes from other yeasts and fungi. We also describe a series of convenient ADE2 cassettes for gene disruption construct preparation. Finally, we have identified the ade2 mutations in strains M001 and M049, adenine auxotrophic mutants derived from the serotype A strain H99. These mutant strains have served as recipients for targeted gene disruptions using the ADE2 gene. These studies should facilitate transformation and gene disruption approaches using the ADE2 selectable marker in this important human fungal pathogen.     

Mitotic recombination in the rDNA of S. cerevisiae is suppressed by the combined action of DNA topoisomerases I and II

We have found that mitotic recombination within the S. cerevisiae rDNA cluster (200 tandemly repeated 9.1 kb units) is strongly suppressed and that this suppression requires the combined action of DNA topoisomerases I and II. Strains with a null mutation in the TOP1 gene (encoding topoisomerase I) or a ts mutation in the TOP2 gene (encoding topoisomerase II) grown at a semipermissive temperature show 50- to 200-fold higher frequencies of mitotic recombination in rDNA relative to TOP+ controls. Suppression of recombination is specific to the rDNA because the recombination frequency at another tandem array, the CUP1 locus, at a simple HIS4 duplication, or among dispersed repeats (MAT and HML or HMR) is not elevated in top1 or top2 mutants. The high frequency of mitotic recombination within the rDNA cluster in topoisomerase mutants shows that both TOP1 and TOP2 are required for suppression of recombination in this region of the genome.     

Rapamycin Antifungal Action Is Mediated via Conserved Complexes with FKBP12 and TOR Kinase Homologs in Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen that causes meningitis in patients immunocompromised by AIDS, chemotherapy, organ transplantation, or high-dose steroids. Current antifungal drug therapies are limited and suffer from toxic side effects and drug resistance. Here, we defined the targets and mechanisms of antifungal action of the immunosuppressant rapamycin in C. neoformans. In the yeast Saccharomyces cerevisiae and in T cells, rapamycin forms complexes with the FKBP12 prolyl isomerase that block cell cycle progression by inhibiting the TOR kinases. We identified the gene encoding a C. neoformans TOR1 homolog. Using a novel two-hybrid screen for rapamycin-dependent TOR-binding proteins, we identified the C. neoformans FKBP12 homolog, encoded by the FRR1 gene. Disruption of the FKBP12 gene conferred rapamycin and FK506 resistance but had no effect on growth, differentiation, or virulence of C. neoformans. Two spontaneous mutations that confer rapamycin resistance alter conserved residues on TOR1 or FKBP12 that are required for FKBP12-rapamycin-TOR1 interactions or FKBP12 stability. Two other spontaneous mutations result from insertion of novel DNA sequences into the FKBP12 gene. Our observations reveal that the antifungal activities of rapamycin and FK506 are mediated via FKBP12 and TOR homologs and that a high proportion of spontaneous mutants in C. neoformans result from insertion of novel DNA sequences, and they suggest that nonimmunosuppressive rapamycin analogs have potential as antifungal agents.     

Overexpression of type I topoisomerases sensitizes yeast cells to DNA damage

DNA topoisomerases play essential roles in many DNA metabolic processes. It has been suggested that topoisomerases play an essential role in DNA repair. Topoisomerases can introduce DNA damage upon exposure to drugs that stabilize the covalent protein-DNA intermediate of the topoisomerase reaction. Lesions in DNA are also able to trap topoisomerase-DNA intermediates, suggesting that topoisomerases have the potential to either assist in DNA repair by locating sites of damage or exacerbating DNA damage by generation of additional damage at the site of a lesion. We have shown that overexpression of yeast topoisomerase I (TOP1) conferred hypersensitivity to methyl methanesulfonate and other DNA-damaging agents, whereas expression of a catalytically inactive enzyme did not. Overexpression of topoisomerase II did not change the sensitivity of cells to these DNA-damaging agents. Yeast cells lacking TOP1 were not more resistant to DNA damage than cells expressing wild type levels of the enzyme. Yeast topoisomerase I covalent complexes can be trapped efficiently on UV-damaged DNA. We suggest that TOP1 does not participate in the repair of DNA damage in yeast cells. However, the enzyme has the potential of exacerbating DNA damage by forming covalent DNA-protein complexes at sites of DNA damage.     

Structures of Cryptococcus neoformans protein farnesyltransferase reveal strategies for developing inhibitors that target fungal pathogens

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.     

Calcineurin is required for virulence of Cryptococcus neoformans.

Cyclosporin A (CsA) and FK506 are antimicrobial, immunosuppressive natural products that inhibit signal transduction. In T cells and Saccharomyces cerevisiae, CsA and FK506 bind to the immunophilins cyclophilin A and FKBP12 and the resulting complexes inhibit the Ca2+-regulated protein phosphatase calcineurin. We find that growth of the opportunistic fungal pathogen Cryptococcus neoformans is sensitive to CsA and FK506 at 37 degrees C but not at 24 degrees C, suggesting that CsA and FK506 inhibit a protein required for C. neoformans growth at elevated temperature. Genetic evidence supports a model in which immunophilin-drug complexes inhibit calcineurin to prevent growth at 37 degrees C. The gene encoding the C. neoformans calcineurin A catalytic subunit was cloned and disrupted by homologous recombination. Calcineurin mutant strains are viable but do not survive in vitro conditions that mimic the host environment (elevated temperature, 5% CO2 or alkaline pH) and are no longer pathogenic in an animal model of cryptococcal meningitis. Introduction of the wild-type calcineurin A gene complemented these growth defects and restored virulence. Our findings demonstrate that calcineurin is required for C. neoformans virulence and may define signal transduction elements required for fungal pathogenesis that could be targets for therapeutic intervention.     

A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase

A hyper-recombination mutation was isolated that causes an increase in recombination between short repeated delta sequences surrounding the SUP4-omicron gene in S. cerevisiae. The wild-type copy of this gene was cloned by complementation of one of its pleiotropic phenotypes, slow growth. DNA sequence of the clone revealed a 656 amino acid open reading frame capable of encoding a protein homologous to the bacterial type I topoisomerase. No homology was detected with previously identified eukaryotic topoisomerases. Construction of double mutants with either of the two known yeast topoisomerase genes revealed synergistic effects on growth suggesting overlapping functions. Expression of bacterial topoisomerase I in yeast can fully complement the slow growth defect of a null mutation. We have named this locus TOP3 and suggest that it defines a novel eukaryotic topoisomerase gene.     

Cloning and Characterization of an Arabidopsis thaliana Topoisomerase I Gene

cDNA and genomic clones encoding DNA topoisomerase I were isolated from Arabidopsis thaliana λgt11 and λFix libraries by low stringency hybridization with a Saccharomyces cerevisiae TOP1 probe. The cDNA clones include a 2748-base pair open reading frame predicting an amino acid sequence that is highly homologous to sequences encoded by TOP1 from yeast and human sources. The sequence of the upstream genomic region reveals two putative TATA-like elements and a purine-rich region, but no other obvious controlling elements. Southern blot analysis shows that the gene is present as a single copy in the Arabidopsis genome. When expressed in a S. cerevisiae top1 mutant under the control of the GAL1 promoter, the gene complements the phenotype caused by loss of topoisomerase activity and directs the expression of a protein that cross-reacts with a human anti-topoisomerase I antibody.     

Cloning and characterization of the lanosterol 14alpha-demethylase (ERG11) gene in Cryptococcus neoformans

The ergosterol pathway in fungal pathogens is an attractive antimicrobial target because it is unique from the major sterol (cholesterol) producing pathway in humans. Lanosterol 14alpha-demethylase is the target for a major class of antifungals, the azoles. In this study we have isolated the gene for this enzyme from Cryptococcus neoformans. The gene, ERG11, was recovered using degenerate PCR with primers designed with a novel algorithm called CODEHOP. Sequence analysis of Erg11p identified a highly conserved region typical of the cytochrome P450 class of mono-oxygenases. The gene was present in single copy in the genome and mapped to one end of the largest chromosome. Comparison of the protein sequence to a number of major human fungal pathogen Erg11p homologs revealed that the C. neoformans protein was highly conserved, and most closely related to the Erg11p homologs from other basidiomycetes. Functional studies demonstrated that the gene could complement a Saccharomyces cerevisiae erg11 mutant, which confirmed the identity of the C. neoformans gene.     

Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans

Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans. Fungal Genetics and Biology 29, 38-48. Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle and well-developed genetic and molecular approaches. Two different transformation systems have been developed, and a number of genes have been disrupted by homologous recombination. However, the frequency of homologous recombination achieved by these approaches has differed dramatically between strains of the A and D serotypes. Transformation by electroporation in serotype D strains results in homologous recombination at frequencies of 1/1000 to 1/100,000, whereas transformation by the biolistic method has resulted in gene disruption at frequencies between 2 and 50% in serotype A strains. We find that gene disruption by homologous recombination can be achieved in the congenic serotype D strain series by biolistic transformation with frequencies of approximately 1 to 4%. By this approach, we have readily disrupted the genes encoding a MAPK homolog (CPK1), the calcineurin A catalytic subunit (CNA1), and a G protein alpha subunit (GPA1). By physical and genetic methods, we show that these mutations result from targeted recombination events without ectopic integrations. Because genetic approaches can be applied in the congenic serotype D strains, our observations represent a significant advance in molecular approaches to understand the physiology and virulence of this important human pathogen.     

Genome rearrangement in top3 mutants of Saccharomyces cerevisiae requires a functional RAD1 excision repair gene.

Saccharomyces cerevisiae cells that are mutated at TOP3, a gene that encodes a protein homologous to bacterial type I topoisomerases, have a variety of defects, including reduced growth rate, altered gene expression, blocked sporulation, and elevated rates of mitotic recombination at several loci. The rate of ectopic recombination between two unlinked, homologous loci, SAM1 and SAM2, is sixfold higher in cells containing a top3 null mutation than in wild-type cells. Mutations in either of the two other known topoisomerase genes in S. cerevisiae, TOP1 and TOP2, do not affect the rate of recombination between the SAM genes. The top3 mutation also changes the distribution of recombination events between the SAM genes, leading to the appearance of novel deletion-insertion events in which conversion tracts extend beyond the coding sequence, replacing the DNA flanking the 3' end of one SAM gene with nonhomologous DNA flanking the 3' end of the other. The effects of the top3 null mutation on recombination are dependent on the presence of an intact RAD1 excision repair gene, because both the rate of SAM ectopic gene conversion and the conversion tract length were reduced in rad1 top3 mutant cells compared with top3 mutants. These results suggest that a RAD1-dependent function is involved in the processing of damaged DNA that results from the loss of Top3 activity, targeting such DNA for repair by recombination.     

Chitosan, the deacetylated form of chitin, is necessary for cell wall integrity in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. The fungal cell wall is an excellent target for antifungal therapies as it is an essential organelle that provides cell structure and integrity, it is needed for the localization or attachment of known virulence factors, including the polysaccharide capsule, melanin, and phospholipase, and it is critical for host-pathogen interactions. In C. neoformans, chitosan produced by the enzymatic removal of acetyl groups from nascent chitin polymers has been implicated as an important component of the vegetative cell wall. In this study, we identify four putative chitin/polysaccharide deacetylases in C. neoformans. We have demonstrated that three of these deacetylases, Cda1, Cda2, and Cda3, can account for all of the chitosan produced during vegetative growth in culture, but the function for one, Fpd1, remains undetermined. The data suggest a model for chitosan production in vegetatively growing C. neoformans where the three chitin deacetylases convert chitin generated by the chitin synthase Chs3 into chitosan. Utilizing a collection of chitin/polysaccharide deacetylase deletion strains, we determined that during vegetative growth, chitosan helps to maintain cell integrity and aids in bud separation. Additionally, chitosan is necessary for maintaining normal capsule width and the lack of chitosan results in a "leaky melanin" phenotype. Our analysis indicates that chitin deacetylases and the chitosan made by them may prove to be excellent antifungal targets.     

Molecular cloning and genetic mapping of the DNA topoisomerase II gene of Saccharomyces cerevisiae

The structural gene for DNA topoisomerase II from the yeast Saccharomyces cerevisiae has been cloned. The clones were selected from a YEp13 plasmid bank of yeast DNA by complementing a temperature-sensitive mutation (top2-1) in the topoisomerase II gene, TOP2. Chromosomal integrants of the clone were derived by homologous recombination in strains lacking the 2 mu circle plasmid. Genetic analysis of these integrants indicates that we have cloned the TOP2 gene and not an extragenic suppressor. A YEp13-TOP2 hybrid plasmid integrant was used to localize the TOP2 gene to the left arm of chromosome XIV by the 2 mu circle-directed marker loss method. Results from standard meiotic mapping experiments indicate that TOP2 is about 16 centi-Morgans to the centromere proximal side of MET4. Northern blot analysis of TOP2 RNA isolated from a wild-type strain and from an rna2 mutant shows the RNA to be 4.5 kb long in both cases, thus indicating that the TOP2 gene has no large introns.     

Identification of the gene encoding DNA topoisomerase I from Candida albicans

Abstract A gene encoding a type I topoisomerase (TOP1) was isolated from Candida albicans , sequenced, and expressed in Saccharomyces cerevisiae . The TOP1 gene was identified from a C. albicans genomic library by hybridization with the product of a polymerase chain reaction with degenerate primer sets encoding regions conserved in other TOP1 genes. A clone containing an open reading frame of 2463 bp and predicted to encode a protein of 778 amino acids with sequence similarity to eukaryotic type I topoisomerases was identified. The C. albicans TOP1 gene restored camptothecin sensitivity and increased the topoisomerase activity in S. cerevisiae , indicating that the DNA fragment encodes a functional C. albicans topoisomerase I.     

Influence of Iron Regulation on the Metabolome of Cryptococcus neoformans

Iron is an essential nutrient for virtually all organisms and acts as a cofactor for many key enzymes of major metabolic pathways. Furthermore, iron plays a critical role in pathogen-host interactions. In this study, we analyzed metabolomic changes associated with iron availability and the iron regulatory protein Cir1 in a human fungal pathogen Cryptococcus neoformans. Our metabolite analysis revealed that Cir1 influences the glycolytic pathway, ergosterol biosynthesis and inositol metabolism, which require numerous iron-dependent enzymes and play important roles in pathogenesis and antifungal sensitivity of the fungus. Moreover, we demonstrated that increased cellular iron content and altered gene expression in the cir1 mutant contributed to metabolite changes. Our study provides a new insight into iron regulation and the role of Cir1 in metabolome of C. neoformans.