Evaluation of the genotoxic spectrum of cisplatin for Candida albicans.

The platinum co-ordination complex cisplatin [cis-diamminedichloroplatinum (II)] is a highly effective anticancer drug whose activity derives from its ability to form adducts crosslinking neighbouring purine bases in DNA. Under in vitro conditions, cisplatin induced cellular inactivation, forward and reverse mutations, reciprocal and nonreciprocal mitotic recombinations and phenotypic switching in the opportunistically pathogenic yeast Candida albicans. Mutant and recombinant yields were higher with post-treatment growth at 25 degrees C rather than 37 degrees C: the reverse was true for cell death or phenotypic switches. These responses comport with prior evidence that, generally, the higher recovery temperature discourages DNA repair processes in C. albicans. Thiosulphate, an agent used therapeutically to reduce nephrotoxic side effects of cisplatin in humans, greatly decreased both the lethality and recombinagenicity of cisplatin for C. albicans. Implications of these observations for possible genetic destabilization of C. albicans populations born naturally by cancer patients undergoing treatments with cisplatin are discussed.

Modification of responses of Candida albicans to cisplatin by membrane damaging antimycotic agents.

The genotoxic, antineoplastic platinum coordination complex, cisplatin (cis-diamminedichloroplatinum (II], exists as a positively charged aquated complex in water solution and as a neutral, nonaquated complex in saline solution. Candida albicans exhibited greater susceptibilities to cellular inactivation and induction of mitotic recombination when treated with the aquated rather than the nonaquated drug. The differential in responses was expressed by cells grown after treatment at 37 degrees C or at 25 degrees C, a temperature which promotes recovery from DNA damages by the yeast generally. Studies with protoplasts established that cell wall components do not influence cellular reactions to either form of the drug. However, membrane damaging antimycotic agents markedly affected responses. Pretreatments with fungistatic ketoconazole or with miconazole, under fungistatic or fungicidal conditions, enhanced cellular resistance to inactivation by aquated cisplatin: the effect was more pronounced with post-cisplatin growth at 25 degrees C than 37 degrees C. Fungicidal pretreatments with amphotericin B or miconazole greatly increased susceptibilities of surviving cells to the lethal and recombinagenic effects of nonaquated cisplatin with post-cisplatin recovery at 25 degrees C or 37 degrees C. Possible mechanisms underlying these responses and their implications for stability of C. albicans populations in cancer patients undergoing therapy with cisplatin are discussed.

Genetic destabilization of Candida albicans by hydroxyurea.

Candida albicans is a commensal component of the normal human microflora, but frequently causes infections in persons undergoing treatment for malignancies. The cytotoxicity and genotoxicity of the antineoplastic agent hydroxyurea (HU) for the yeast is demonstrated. Cultivation of the organism on defined complete medium in the presence of HU induced growth inhibition or cell death, gene mutations, segregations of heterozygous loci through reciprocal and nonreciprocal mitotic recombinations, and a special heritable system for high frequency switching between phenotypes reflecting cellular growth characteristics and susceptibilities to HU. Each of these responses is influenced differently by variations in drug concentration and temperature. The biochemical complexity underlying cytotoxic and genetic effects of HU, and the implications for the likelihood of HU induced changes occurring in indigenous yeast populations of persons undergoing therapy with the drug, are discussed.

Recombinagenicity of caffeine for Candida albicans.

Caffeine at concentrations of 0.5 x 10(-2) M or higher inhibited cell replication and induced gene segregations in Candida albicans cultured on defined complete medium. Both responses increased incrementally with increasing caffeine concentrations, and were more severe during incubation at 37 degrees C than 25 degrees C; at 37 degrees C, caffeine levels above 1.5 x 10(-2) M caused cellular inactivation. Caffeine effects occurred only under conditions permitting cell growth, and their magnitudes were greater for unbudded than budding cells, were influenced by cellular genetic backgrounds, and were unaffected by the presence of adenine in the medium. Evaluations of segregations for recessive auxotrophic markers of a four member linkage group carried heterozygously in a cis arrangement in treated cells established that induced segregants arise through either reciprocal or nonreciprocal recombinations. The frequency distributions of classes of reciprocal and nonreciprocal recombinants for these markers conformed with those previously obtained following induction by ultraviolet radiation, indicating that the probabilities of recombinational events within the chromosomal regions defined by the markers are not biased by the differences in kinds of initial DNA lesions caused by the two recombinagens. A panel of four protoplast fusion hybrids considered deficient for DNA repair because of enhanced susceptibilities to UV induced cellular inactivation and mitotic recombination exhibited corresponding increased sensitivities to caffeine, signifying that DNA damage induced by caffeine is subject to repair. Caffeine did not affect behavior of a variant strain exhibiting high frequency phenotypic switching between minute smooth and large rough colonial forms, and no evidence for mutagenicity of the drug was obtained with systems for detection of forward or reverse mutations. The mechanism of caffeine's recombinagenicity, and the implications of that property for genetic studies of C. albicans are discussed.

Anaerobically induced production of hybrid monokaryons by heterokaryons of Candida albicans.

During aerobic replication, balanced heterokaryons (hets) of Candida albicans produced by fusing protoplasts of complementing auxotrophic strains characteristically segregate low frequencies of prototrophic monokaryons bearing hybrid nuclei formed either through karyogamy or unidirectional internuclear genetic transfers within het cells. Anaerobic growth causes exponential inactivation of hets and induces their production of hybrid monokaryons. Both responses are functions of heterokaryosis as such and not the genetic backgrounds of hets. Evidence is presented that (i) the nuclei of anaerobically generated hybrids arise through induction in hets of karyogamy not internuclear genetic transfers and that (ii) the events underlying that induction are different from those responsible for inactivation of the cells.

Unidirectional internuclear transfer of linked genes in heterokaryons of Candida albicans.

Nutritionally balanced heterokaryons of the naturally diploid, asexual yeast, Candida albicans are produced by fusing protoplasts of complementing auxotrophic strains. Spontaneous unidirectional internuclear transfers of an intact gene linkage group in established heterokaryons is demonstrated. Evidence is presented that a transfer event (i) typically involves a single chromosome which is added to the resident homologues of a recipient nucleus, (ii) can occur equally well in either direction between complementing nuclei and (iii) may encourage gene conversion at transferred loci in the recipient nucleus. The bearing of these findings on application of protoplast fusion to parasexual genetic analysis of C. albicans is discussed.

Variations for susceptibilities to ultraviolet induced cellular inactivation and gene segregation among protoplast fusion hybrids of Candida albicans.

Hybrids of the naturally diploid, asexual and opportunistically pathogenic yeast, Candida albicans, can be obtained artificially by protoplast fusion. Evidence is presented that gene conversion and reciprocal recombination contribute to ultraviolet (UV)-induced segregations of heterozygous markers from both diploid and hybrid strains, and that hybrids also segregate through induced chromosome loss. Heterozygous diploid strains independently derived from the same wild-type diploid stock were alike in post-UV survival and segregational responses, and the organization of a four gene linkage group identified in diploids from the segregant products of reciprocal recombinations was transmitted intact to all hybrids from fusions between diploids of isogenic or nonisogenic backgrounds. However, hybrids arising independently from a given fusion cross differed significantly from each other in post-UV survival, absolute ability to segregate some parental markers, frequency of gene segregation, and proclivities for each of the three mechanisms of gene segregation. The bearings of the genetic backgrounds of parental strains and of growth temperatures during hybrid formation on each of these variables are described. The findings emphasize that awareness of the intrinsic heterogeneities of fusion hybrids is essential for reliable application of the protoplast fusion procedure to genetic analysis of C. albicans.

Selective inactivation of heterokaryons of Candida albicans by anaerobiosis.

Heterokaryons (hets), but not monokaryons of Candida albicans die when grown anaerobically on minimal medium. Their rates of inactivation increase with decreases in growth temperatures from 37 degrees C to 25 degrees C. At 10 degrees C, however, anaerobiosis is not lethal and suppresses the inactivation which normally occurs among hets cultured aerobically at that temperature. Killing of hets by anaerobiosis can be altered significantly by certain exogenously provided amino acids or intermediates of oxidative respiration. Aspartic acid alone promotes inactivation whereas alanine, glutamic acid or lysine individually have no effects. However, glutamate and lysine combined afford slight protection against inactivation while aspartate and glutamate combined, with or without lysine, are highly protective: the activity of the aspartate-glutamate combination is completely negated by the addition of alanine. Other common amino acids have no effects on het responses to anaerobiosis other than the ability, when combined, to relieve the antagonism of alanine for the aspartate-glutamate combination. Anaerobic survivals are also enhanced by oxalacetic acid or alpha-ketoglutaric acid, and even more so by a combination of these two intermediates. The resistances to inactivation elicited by the oxalacetate alpha-ketoglutarate or aspartate-glutamate combinations are not additive. These relationships are interpreted to signify that inactivation of hets by anaerobic growth is largely, if not exclusively, due to depletion of their oxalacetic acid and alpha-ketoglutaric acid contents for amino acid biosyntheses, and the unique inability of het cells to replenish those keto acids upon subsequent return to aerobic conditions. The observations are consistent with previous indications that mitochondria formed by hets are functionally abnormal.

Segregant-defective heterokaryons of Candida albicans.

Heterokaryons (hets) of the asexual, pathogenic yeast Candida albicans obtained by fusing protoplasts of complementing auxotrophic strains generate large numbers of parental-type auxotrophic monokaryons by random assortment of single nuclei into blastospores, and smaller numbers of monokaryons bearing hybrid nuclei formed through either karyogamy or the transfer of genetic material from one het nucleus to another. Het populations grown at 30 degrees C or 37 degrees C contain high frequencies (approx. 5%-10%) of two kinds of stable variants peculiar specifically for segregation of parental-type monokaryons: NS variants produce inviable auxotrophic monokaryons of one or both parental classes while AT variants yield parental-type monokaryons which grow very slowly. Variant frequencies are not affected by the wild-type strain background of hets, or the auxotrophies used to force heterokaryosis. However, both kinds of variants are induced by growth at 25 degrees C or by treatments with certain chemical or physical metabolic inhibitors. Evidence is presented that variant nuclei of independent origins carry different nutritionally irreparable recessive lethal (NS) or debilitating (AT) defects acquired in the course of actual or potential internuclear transfers of genetic material within het cells. The high incidence of variants, therefore, indicates considerable intrinsic genetic instability among het nuclei. Significances of these observations for parasexual genetic analyses of C. albicans and other yeasts through protoplast fusions are considered.

Nutritional enrichments do not affect growth or stability of heterokaryons of Candida albicans.

Complex nutritional supplements which stimulate growth of monokaryons of Candida albicans have no effect on growth or stability of heterokaryons. This evidence that heterokaryotic growth is characteristically limited by something other than nutritional circumstances is consistent with prior indications of naturally dysfunctional ribosomes in heterokaryons.

Transmission and expression of mutations to nalidixic acid resistance among products of protoplast fusion crosses of Candida albicans.

Earlier studies suggested that heritable resistance to nalidixic acid (Nal) induced in the asexual, pathogenic yeast Candida albicans by growth on Nal results from mitochondrial mutation. To determine conclusively whether mutations to Nal resistance are cytoplasmic or nuclear, several stable Nal-resistant (Nalr) mutants exhibiting distinctive differences in degrees of Nal resistance were obtained from each of two doubly auxotrophic strains (Ade-, Thr- and Arg-, His-), both derived from the same wild-type stock. Inheritance of Nal resistance was then assessed in a series of protoplast fusion crosses between complementing auxotrophs. The initial, intact cellular products of a fusion cross are prototrophic heterokaryons which frequently assort single parental nuclei into monokaryotic blastospores containing biparental cytoplasms. Occasional karyogamy within heterokaryons also yields prototrophic hybrid monokaryons which can undergo recombinations for chromosomal markers through spontaneous or induced mitotic crossing-over. Segregation and expression of Nal resistance among non-hybrid, parental-type monokaryons from Nalr X Nals heterokaryons showed that Nalr mutations are nuclear and that their expressions are not noticeably affected by admixture of cytoplasms of sensitive and resistant parental strains. Analyses of heterokaryons and hybrid monokaryons from Nalr X Nals and Nalr X Nalr crosses demonstrated that Nal resistance is recessive to sensitivity, and that independent Nalr mutations arise at one gene in the Ade-, Thr- strain and at a separate, complementing single gene in the Arg-, His- strain. Prior work demonstrated that induction of Nalr mutations in wild-type C. albicans depends profoundly on the (i) carbon and nitrogen, (ii) growth temperature, (iii) contact with particular metabolic inhibitors and (iv) division stage of cells during exposure to Nal. The present observations indicate that the character of cellular auxotrophies can determine the genetic loci at which Nalr mutations can be recovered.

Cellular inactivation and mitotic recombination induced by ultraviolet radiation in aneuploid and euploid strains of Candida albicans.

Prototrophic aneuploid and euploid derivatives of wild type Candida albicans strains 207 were produced by fusing protoplasts of complementing auxotrophs obtained from strain 207. Comparisons of cell survivals and incidences of mitotic recombinants occurring after ultraviolet irradiation (UV) of these strains indicate that (i) aneuploids are categorically less efficient than euploids for repair of pyrimidine dimers induced in DNA by UV and that (ii) such repair is enhanced by growing irradiated cells at 25 degrees C, on minimal medium or in the presence of ergosterol rather than at 37 degrees C, on amino acid enriched medium or medium unsupplemented with ergosterol. In addition, the comparisons establish than one cannot discriminate between strains of C. albicans which differ in cellular DNA contents or genomic constitutions on the basis of their UV survival curves.

Temperature-dependent internuclear transfer of genetic material in heterokaryons of Candida albicans.

Heterokaryons (hets) of Candida albicans are produced by fusing protoplasts of complementing auxotrophic strains and can be propagated continuously on minimal medium despite their tendency to assort nuclei into monokaryotic blastospores. Most mono-karyons have parental-type nuclei, but some are nuclear hybrids with DNA contents between one and two times that of their parental strains. Evidence is presented that hybrids arise by transfer of a portion of the genetic material of one bet nucleus to another, and that the amount of material conveyed during transfer increases with increasing het growth temperatures over the range 25°C to 41°C. This partial hybridization is a general property of hets and is not determined by the wild-type strain backgrounds of their parental components or by the kinds of auxotrophies forcing heterokaryosis. Frequencies of mitotic recombinants induced in partial hybrids by ultraviolet radiation indicate that nuclei of C. albicans are naturally diploid.

Effects of growth temperatures on plating efficiencies and stabilities of heterokaryons of Candida albicans.

Heterokaryons (hets) of Candida albicans constructed by fusing protoplasts of complementing auxotrophs produce heterogeneous clones on minimal medium consisting of (i) a minority of slow-growing hets, (ii) a preponderance of non-growing, parental-type auxotrophic monokaryons, and (iii) some prototrophic monokaryons bearing hybrid nuclei. Hets grown at a given temperature within the range 25 degrees C to 41 degrees C replate with higher efficiencies at any lower temperature and exhibit progressively declining plating efficiencies as plate temperatures increase beyond that at which they were initially grown. Neither auxotrophic nor prototrophic monokaryons show such responses. Growth of colonies produced by hets, wild-type strains or prototrophic hybrid monokaryons is stimulated by temperatures in the order, 37 degrees C greater than 30 degrees C greater than 41 degrees C greater than 25 degrees C. However, the proportion of hets to auxotrophic monokaryons within individual het clones increases directly from 25 degrees C to 41 degrees C. Though this pattern obtains whether colonies are compared at equivalent sizes or ages, het frequencies decline as colonies age at all temperatures. Appearance of hybrid monokaryons within het clones is unaffected by growth temperature. The relationships of temperatures to plating efficiencies and stabilities of hets are independent of the natures of their complementing auxotrophies or the wild-type backgrounds of their nuclear components and are, therefore, functions of heterokaryosis per se. Modifications of these relationships by selective metabolic antagonists or by growth of hets on different pre- and post-plating carbon sources indicate that they reflect temperature-dependent properties of mitochondria which are peculiar to hets.

Recombinogenic activity of nalidixic acid for artificial hybrids but not for natural strains of Candida albicans: evidence for the monoploidy of natural strains.

Nalidixic acid induces segregation of auxotrophs from prototrophic hybrids of Candida albicans artifically produced by fusing complementing auxotrophic protoplasts. The auxotrophies recovered are limited to those introduced through the fusion process, and patterns of segregations for linked auxotrophic markers demonstrate the segregants are products mitotic crossing-over. Nalidixic acid does not induce auxotrophies of any sort in clinical isolates of C. albicans. These findings are contrary to a current hypothesis that natural strains of C. albicans are diploid and heterozygous for a variety of auxotrophic mutations.

Cold-sensitive of heterokaryons of Candida albicans.

Heterokaryons (hets) of Candida albicans are formed by fusing protoplasts of complementing auxotrophs. Het clones typically contain two kinds of monokaryons--parental-type auxotrophs arising by segregation of constituent nuclei, and prototrophs resulting from segregation of hybred nuclei formed through infrequent karyogamy within het cells. Hets, but not their parental strains or monokaryotic derivatives, die in cultures held at 5 degrees C-15 degrees C. Death occurs at a high exponential rate during the first two days at low temperature and at a lesser exponential rate thereafter: death rates are highest at 10 degrees C. Inactivation is not influenced significantly by the kinds of auxotrophies forcing heterokaryosis or by difference or identity in the wild type backgrounds of the auxotrophs used to construct the hets. It is also unaffected by amino acid analogues, specific inhibitors of mitochondrial protein synthesis or the general inhibitor of DNA synthesis, hydroxyurea. Inactivation is promoted by purine or pyrimidine analogues and impeded by specific inhibitors of mitochondrial DNA synthesis and transcription or oxidative phosphorylation. As a rule, cold-sensitive mutants of eukaryotic microorganisms are defective for production and assembly of components of cytoplasmic or mitochondrial ribosomes at restrictive temperatures. Our observations suggest that comparable defects in biogenesis of mitochondrial ribosomes are normal properties of C. albicans hets.

Differentiation of Candida stellatoidea from C. albicans and C. tropicalis by temperature-dependent growth responses on defined media.

C. stellatoidea differs from both C. albicans and C. tropicalis in its i) much greater growth differential on minimal and amino acid enriched media and ii) unique inability to grow on minimal medium containing glycerol as carbon source at 37C. The relative responses to amino acid enrichment occur on media containing either fermentative or oxidative carbon sources, at 25C or 37C. Under any given conditions of carbon source and temperature, different assortments of individual amino acids are stimulatory for each of the three species. All assortments include one or more members of the glutamic acid family. However, sulfur amino acids stimulate only C. stellatoidea on all three carbon sources. On minimal-glycerol medium, wild type strains of C. stellatoidea grow prototrophically at 25C but are auxotrophic for amino acids at 37C; the particular auxotrophies expressed vary from strain to strain. Slow growing, mycelial mutants, prototrophic on glycerol at 37C arise spontaneously in wild type strains at frequencies indicating nuclear gene mutation. Such mutants can be induced by both transition and frame shift mutagens. The implications of these observations for the taxonomic relationships between the three Candida species and for identification of C. stellatoidea in particular are discussed.

Hybridization of Candida albicans through fusion of protoplasts.

Protoplasts of complementing auxotrophs of Candida albicans can fuse in the presence of polyethylene glycol and generate prototrophic cells. The yields of prototrophs from fusion mixtures depend greatly on the particular combinations of auxotrophies involved but not on other features of the strain backgrounds of protoplasts. The initial cellular products of fusions isolated on selective media are heterokaryons which replicate slowly but also segregate single parental nuclei into blastospores in high frequency. Karyogamy within heterokaryons produces hybrid nuclei which, on segregation, give rise to rapidly growing, uninucleate substrains. Analyses of the substrains show that hybrid nuclei either stabilize as diploid or undergo random loss of chromosomes to stabilize at various levels of aneuploidy prior to segregation. Chromosome losses and radiation induced mitotic crossing-over can effect recombination for parental auxotrophic markers in hybrids; patterns of recombination for ader and arg markers provide the first documented example of chromosomal linkage in C. albicans. Thus, protoplast fusions offer opportunities otherwise unavailable for applying the incisive tools of genetic recombination to analysis of this important, asexual yeast.

Production of heterokaryons of Candida albicans by protoplast fusions: Effects of differences in proportions and regenerative abilities of fusion partners.

Production of heterokaryons of Candida albicans by polyethylene glycol-induced fusion of complementing auxotrophic protoplasts was assayed in two series of fusion crosses designed to test the significances of the in-put ratios and regenerative abilities of parental protoplasts. The findings indicate that productive fusions require interaction of multiple protoplasts of each complementing type and can occur equally well with either regenerable or non-regenerable protoplasts.

Nalidixic acid inhibition of post-ultraviolet recovery by nalidixic acid sensitive and resistant strains of Candida albicans.

Nalidixic acid (Nal) can kill Candida albicans directly or suppress the organism's recovery from ultraviolet irradiation. Mutants selected for resistance to inactivation by Nal alone have generally enhanced DNA repair proficiencies evidenced by their coincident increased resistances to ultraviolet radiation, ethylmethane sulfonate, and nitrous acid. The effects of Nal, erythromycin, and inhibitors of oxidative phosphorylation on survivals of mutant and wild type strains following ultraviolet exposure indicate that different mechanisms underly the direct lethality of Nal and its ability to inhibit post-irradiation recovery.