Direct transformation of a clinical isolate of Candida parapsilosis using a dominant selection marker

Candida parapsilosis is a human pathogenic fungus with increasing importance, particularly in nosocomial infections. For detailed molecular genetic explorations of prototrophic clinical isolates of C. parapsilosis, we developed an efficient transformation system based on a dominant selectable marker. The gene encoding resistance to mycophenolic acid (MPA) was used for selection in yeast transformation. C. parapsilosis cells were transformed with a plasmid vector containing the Candida albicans inosine monophosphate dehydrogenase gene (IMH3) responsible for mycophenolic acid resistance. Transformation was carried out both by electroporation and by the lithium acetate (LiAc) method. The LiAc method resulted in very poor transformation efficiency, while the modified electroporation method yielded a high number of mitotically stable transformants exhibiting unambiguous MPA resistance. Two hundred transformants were analysed for the presence of the C. albicans IMH3(r) gene by polymerase chain reaction. Integration of single or multiple plasmid copies into the genomic DNA of C. parapsilosis was determined by Southern hybridization. To our knowledge, the present study is the first report about a method based on a dominant selectable marker for the transformation of a prototrophic, clinical isolate of C. parapsilosis. The described technique may prove to be an efficient tool for the examination of the biology and virulence of this pathogenic yeast.     

Elongation factor 3 (EF-3) from Candida albicans shows both structural and functional similarity to EF-3 from Saccharomyces cerevisiae

As with many other fungi, including the budding yeast Saccharomyces cerevisiae, the dimorphic fungus Candida albicans encodes the novel translation factor, elongation factor 3 (EF-3). Using a rapid affinity chromatography protocol, EF-3 was purified to homogeneity from C. albicans and shown to have an apparent molecular mass of 128 kDa. A polyclonal antibody raised against C. albicans EF-3 also showed cross-reactivity with EF-3 from S. cerevisiae. Similarly, the S. cerevisiae TEF3 gene (encoding EF-3) showed cross-hybridization with genomic DNA from C. albicans in Southern hybridization analysis, demonstrating the existence of a single gene closely related to TEF3 in the C. albicans genome. This gene was cloned by using a 0.7 kb polymerase chain reaction-amplified DNA fragment to screen to C. albicans gene library. DNA sequence analysis of 200 bp of the cloned fragment demonstrated an open reading frame showing 51% predicted amino acid identity between the putative C. albicans EF-3 gene and its S. cerevisiae counterpart over the encoded 65-amino-acid stretch. That the cloned C. albicans sequence did indeed encode EF-3 was confirmed by demonstrating its ability to rescue an otherwise non-viable S. cerevisiae tef3:HIS3 null mutant. Thus EF-3 from C. albicans shows both structural and functional similarity to EF-3 from S. cerevisiae.     

The Candida albicans ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) is essential for growth

The Candida albicans ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) was cloned by complementing a Saccharomyces cerevisiae erg26 mutant with a C. albicans genomic library. Sequence analysis showed a 70% identity between the C. albicans and S. cerevisiae ERG26 genes at the amino acid level. Sequential disruption of both copies of the ERG26 gene in the presence of an integrated rescue cassette containing a third copy of the ERG26 gene under the control of the inducible pMAL2 promoter, resulted in cells capable of growing only in the presence of the inducer. The results establish that the ERG26 gene is essential for growth and that inhibitors of the Erg26p may represent a new and highly effective class of antifungal agents.     

Effects of chlorhexidine diacetate on Candida albicans, C. glabrata and Saccharomyces cerevisiae.

The effects of chlorhexidine diacetate (CHA) on Candida albicans, C. glabrata and wild-type and mannan, and permeability mutants of Saccharomyces cerevisiae have been studied. A CHA concentration of 10 micrograms/ml had little lethal activity against the Candida strains, but was more effective against S. cerevisiae. Concentrations of 100 and especially 1000 micrograms/ml brought about a much more rapid death of cells. 2-Mercaptoethanol enhanced the activity of CHA to some extent. Some of the mutant strains of S. cerevisiae were rather more sensitive than the wild-type strain. The age of cultures of C. albicans and C. glabrata influenced their response to CHA.     

Effects of iprodione and fludioxonil on glycerol synthesis and hyphal development in Candida albicans

We investigated the effects of iprodione and fludioxonil on the pathogenic yeast Candida albicans. Growth of the wild-type IFO1385 strain of C. albicans was inhibited by both fungicides, while Saccharomyces cerevisiae was basically unaffected by them even at a concentration of 25 microg/ml. Both fungicides stimulated glycerol synthesis in C. albicans but not in S. cerevisiae. The antioxidant alpha-tocopherol acetate and the cytochrome P-450 inhibitor piperonyl butoxide antagonized the fungitoxicity of iprodione and fludioxonil in C. albicans. It is known that mutations within the histidine kinase NIK1/OS-1 gene confer resistance to iprodione and fludioxonil in Neurospora crassa, while the fungicide-insensitive S. cerevisiae has only one histidine kinase SLN1 gene in its genome. In contrast, C. albicans has three histidine kinase genes, namely CaSLN1, CaNIK1/COS1, and CaHK1, the null mutants of which were found to impair the hyphal formation. Iprodione and fludioxonil were found to suppress filamentation when the IFO1385 strain was incubated on a solid medium containing fetal bovine serum. These observations suggest that iprodione and fludioxonil interfere with the CaNIK1/COS1 signal transduction pathway, resulting in glycerol synthesis stimulation and the inhibition of hyphal formation.     

Cloning of the RHO1 gene from Candida albicans and its regulation of beta-1,3-glucan synthesis.

The Saccharomyces cerevisiae RHO1 gene encodes a low-molecular-weight GTPase. One of its recently identified functions is the regulation of beta-1,3-glucan synthase, which synthesizes the main component of the fungal cell wall (J. Drgonova et al., Science 272:277-279, 1996; T. Mazur and W. Baginsky, J. Biol. Chem. 271:14604-14609, 1996; and H. Qadota et al., Science 272:279-281, 1996). From the opportunistic pathogenic fungus Candida albicans, we cloned the RHO1 gene by the PCR and cross-hybridization methods. Sequence analysis revealed that the Candida RHO1 gene has a 597-nucleotide region which encodes a putative 22.0-kDa peptide. The deduced amino acid sequence predicts that Candida albicans Rho1p is 82.9% identical to Saccharomyces Rho1p and contains all the domains conserved among Rho-type GTPases from other organisms. The Candida albicans RHO1 gene could rescue a S. cerevisiae strain containing a rho1 deletion. Furthermore, recombinant Candida albicans Rho1p could reactivate the beta-1,3-glucan synthesis activities of both C. albicans and S. cerevisiae membranes in which endogenous Rho1p had been depleted by Tergitol NP-40-NaCl treatment. Candida albicans Rho1p was copurified with the beta-1,3-glucan synthase putative catalytic subunit, Candida albicans Gsc1p, by product entrapment. Candida albicans Rho1p was shown to interact directly with Candida albicans Gsc1p in a ligand overlay assay and a cross-linking study. These results indicate that Candida albicans Rho1p acts in the same manner as Saccharomyces cerevisiae Rho1p to regulate beta-1,3-glucan synthesis.     

Isolation of the gene for cytochrome P450L1A1 (lanosterol 14 alpha-demethylase) from Candida albicans

The Saccharomyces cerevisiae cytochrome P450 L1A1 (lanosterol 14 alpha-demethylase)-coding gene was used as a hybridization probe to isolate two HindIII fragments of 2.5 kb and 6.85 kb from a phage lambda library of Candida albicans nucleotide sequences. Restriction endonuclease mapping and Southern blot hybridization experiments indicated that these fragments represent two allelic forms of the same gene. This cloned sequence, when introduced into S. cerevisiae or C. albicans on a multiple copy vector, produced an increase in cytochrome P450 content and resistance to imidazole antifungal agents which are inhibitors of cytochrome P450 L1A1. In addition, the cloned sequence was able to complement a cytochrome P450 L1A1 gene disruption when introduced into S. cerevisiae. These data indicate that the cloned sequence codes for the lanosterol 14 alpha-demethylase cytochrome P450 L1A1 from C. albicans.     

A zinc finger protein from Candida albicans is involved in sucrose utilization.

A sucrose-inducible alpha-glucosidase activity that hydrolyzes sucrose in Candida albicans has been demonstrated previously. The enzyme is assayable in whole cells and was inhibited by both sucrose and maltose. A C. albicans gene (CASUC1) that affects sucrose utilization and alpha-glucosidase activity was cloned by expression in a Saccharomyces cerevisiae suc2 mutant (2102) devoid of invertase genes. CASUC1 enabled the S. cerevisiae mutant to utilize both sucrose and maltose. DNA sequence analysis revealed that CASUC1 encodes a putative zinc finger-containing protein with 28% identity to a maltose-regulatory gene (MAL63) of S. cerevisiae. The gene products of CASUC1 and MAL63 are approximately the same size (501 and 470 amino acids, respectively), and each contains a single zinc finger located at the N terminus. The zinc fingers of CASUC1 and MAL63 comprise six conserved cysteines (C6 zinc finger) and are of the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaavariable-Cys-Xaa2-Cys-+ ++Xaa6-Cys (where Xaan indicates a stretch of the indicated number of any amino acids). Both contain five amino acids in the variable region. CASUC1 also complemented the maltose utilization defect of an S. cerevisiae mutant (TCY-137) containing a defined mutation in a maltose-regulatory gene. The sucrose utilization defect of type II Candida stellatoidea, a sucrase-negative mutant of C. albicans, was corrected by CASUC1. Determinations of alpha-glucosidase activity in whole cells revealed that activity was restored in transformants cultivated on either sucrose or maltose. To our knowledge, this is the first zinc finger-encoding gene, as well as the first putative regulatory gene, to be identified in C. albicans.     

In-vitro activity of 5-fluorocytosine against 1,021 Spanish clinical isolates of Candida and other medically important yeasts

The aim of this study was to determine the prevalence of primary resistance to 5-fluorocytosine (5FC) among clinical isolates of yeasts in Spain where this drug is not currently available for therapy. We have tested the in vitro activity of 5FC against 1,021 recent yeast clinical isolates, including 522 Candida albicans, 140 Candida parapsilosis, 68 Candida glabrata, 41 Candida dubliniensis, 50 Candida guilliermondii, 34 Candida tropicalis, 28 Candida krusei, 20 Candida famata, 11 Cryptococcus neoformans, 5 Cryptococcus albidus, 43 Rhodotorula spp., 24 Trichosporon spp., 5 Saccharomyces cerevisiae, 9 Pichia spp., and 21 isolates from other 11 yeast species. The MICs were determined by the ATB Fungus agar microdilution test (bioMerieux, France) and the following interpretive breakpoints were used: susceptible, > 4 microg/ml; intermediate, 8 to 16 microg/ml; resistant, > 32 microg/ml. 5FC was very active against Candida spp. and other medically important yeasts as 852 (83.4%) of the studied isolates were susceptible (MIC < 4 microg/ml). The species most susceptible to 5FC were C. dubliniensis (100%of isolates; MIC90, 0.25 microg/ml), C. famata (100% of isolates; MIC90, 0.25 microg/ml), C. guilliermondii (98%of isolates; MIC90, 0.25 microg/ml), C. glabrata (95.5% of isolates; MIC90, 0.25 microg/ml), and C. neoformans (90.9% of isolates; MIC90, 2 microg/ml). Primary resistance to 5FC was very uncommon, and a MIC > 32 microg/ml, indicator of in vitro resistance, was observed in 106 isolates (10.4%): 77 C. albicans (16.5% of isolates; MIC90, > 128 microg/ml), 9 C. parapsilosis (6.4% of isolates; MIC90, 8 microg/ml), 4 C. albidus (80% of isolates, MIC50, > 128 microg/ml), 3 C. glabrata (4.4% of isolates; MIC90, 0.25 microg/ml), 3 C. tropicalis (8.8% of isolates; MIC90, 4 microg/ml), 2 C. krusei (7.1% of isolates; MIC90, 8 microg/ml), 2 Rhodotorula spp. (4.6% of isolates, MIC90, 1 microg/ml), 8 Trichosporon spp. (33.3% of isolates; MIC90, 64 microg/ml), and 1 C. lipolytica (50% of isolates). Interestingly, most C. albicans (67 out of 77 isolates) resistant to 5FC were serotype B isolates.     

Comparative study of the activity and kinetic properties of malate dehydrogenase and pyruvate decarboxylase from Candida albicans, Malassezia pachydermatis, and Saccharomyces cerevisiae

Candida albicans and Malassezia pachydermatis cause human and animal infections of the skin and internal organs. We compare the properties of two enzymes, pyruvate decarboxylase (PDC) and malate dehydrogenase (MDH), from these species and from Saccharomyces cerevisiae cultivated under aerobic and anaerobic conditions to find differences between the enzymes that adapt pathogens for virulence and help us in searching for new antifungal agents. Malassezia pachydermatis did not show any growth under anaerobic conditions, as opposed to C. albicans and S. cerevisiae. Under aerobic conditions, C. albicans showed the highest growth rate. Malassezia pachydermatis, contrary to the others, did not show any PDC activity, simultaneously showing the highest MDH activity under aerobic conditions and a Km value for oxaloacetate lower than S. cerevisiae. Candida albicans and S. cerevisiae showed a strong decrease in MDH activity under anaerobic conditions. Candida albicans shows four different isoforms of MDH, while M. pachydermatis and S. cerevisiae are characterized by two and three isoforms. Candida albicans shows about a twofold lower activity of PDC but, simultaneously, almost a threefold lower Km value for pyruvate in comparison with S. cerevisiae. The PDC apoform share under aerobic conditions in C. albicans was 47%, while in S. cerevisiae was only 26%; under anaerobic conditions, the PDC apoform decreased to 12% and 8%, respectively. The properties of enzymes from C. albicans show its high metabolic flexibility (contrary to M. pachydermatis) and cause easy switching between fermentative and oxidative metabolism. This feature allows C. albicans to cause both surface and deep infections. We take into consideration the use of thiamin antimetabolites as antifungal factors that can affect both oxidative and fermentative metabolism.     

Effects of chlorhexidine diacetate on Candida albicans, C. glabrata and Saccharomyces cerevisiae

S.J. HIOM, J.R. FURR, A.D. RUSSELL AND J.R. DICKINSON, 1992. The effects of chlorhexidine diacetate (CHA) on Candida albicans, C. glabrata and wild-type and mannan, and permeability mutants of Saccharomyces cerevisiae have been studied. A CHA concentration of 10 μg/ml had little lethal activity against the Candida strains, but was more effective against S. cerevisiae. Concentrations of 100 and especially 1000 μg/ml brought about a much more rapid death of cells. 2-Mercaptoethanol enhanced the activity of CHA to some extent. Some of the mutant strains of S. cerevisiae were rather more sensitive than the wild-type strain. The age of cultures of C. albicans and C. glabrata influenced their response to CHA.     

Lysine biosynthesis in selected pathogenic fungi: characterization of lysine auxotrophs and the cloned LYS1 gene of Candida albicans.

The alpha-aminoadipate pathway for the biosynthesis of lysine is present only in fungi and euglena. Until now, this unique metabolic pathway has never been investigated in the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. Five of the eight enzymes (homocitrate synthase, homoisocitrate dehydrogenase, alpha-aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase) of the alpha-aminoadipate pathway and glucose-6-phosphate dehydrogenase, a glycolytic enzyme used as a control, were demonstrated in wild-type cells of these organisms. All enzymes were present in Saccharomyces cerevisiae and the pathogenic organisms except C. neoformans 32608 serotype C, which exhibited no saccharopine reductase activity. The levels of enzyme activity varied considerably from strain to strain. Variation among organisms was also observed for the control enzyme. Among the pathogens, C. albicans exhibited much higher homocitrate synthase, homoisocitrate dehydrogenase, and alpha-aminoadipate reductase activities. Seven lysine auxotrophs of C. albicans and one of Candida tropicalis were characterized biochemically to determine the biochemical blocks and gene-enzyme relationships. Growth responses to alpha-aminoadipate- and lysine-supplemented media, accumulation of alpha-aminoadipate semialdehyde, and the lack of enzyme activity revealed that five of the mutants (WA104, WA153, WC7-1-3, WD1-31-2, and A5155) were blocked at the alpha-aminoadipate reductase step, two (STN57 and WD1-3-6) were blocked at the saccharopine dehydrogenase step, and the C. tropicalis mutant (X-16) was blocked at the saccharopine reductase step. The cloned LYS1 gene of C. albicans in the recombinant plasmid YpB1078 complemented saccharopine dehydrogenase (lys1) mutants of S. cerevisiae and C. albicans. The Lys1+ transformed strains exhibited significant saccharopine dehydrogenase activity in comparison with untransformed mutants. The cloned LYS1 gene has been localized on a 1.8-kb HindIII DNA insert of the recombinant plasmid YpB1041RG1. These results established the gene-enzyme relationship in the second half of the alpha-aminoadipate pathway. The presence of this unique pathway in the pathogenic fungi could be useful for their rapid detection and control.     

Prion-forming ability of Ure2 of yeasts is not evolutionarily conserved

[URE3] is a prion (infectious protein) of the Saccharomyces cerevisiae Ure2p, a regulator of nitrogen catabolism. We show that wild S. paradoxus can be infected with a [URE3] prion, supporting the use of S. cerevisiae as a prion test bed. We find that the Ure2p of Candida albicans and C. glabrata also regulate nitrogen catabolism. Conservation of amino acid sequence within the prion domain of Ure2p has been proposed as evidence that the [URE3] prion helps its host. We show that the C. albicans Ure2p, which does not conserve this sequence, can nonetheless form a [URE3] prion in S. cerevisiae, but the C. glabrata Ure2p, which does have the conserved sequence, cannot form [URE3] as judged by its performance in S. cerevisiae. These results suggest that the sequence is not conserved to preserve prion forming ability.     

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.