Anaerobically induced production of hybrid monokaryons by heterokaryons of Candida albicans

Summary 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.     

Utilization of siderophores by Candida albicans

Candida albicans secretes both hydroxamate and phenolate-type siderophores when grown under iron-restricted conditions. The inhibition of candidal growth by iron limitation was reversed by the addition of supplemental hydroxamate on phenolate siderophores. Both siderophores produced equal stimulation of growth suggesting that C. albicans could utilize both siderophores with equal efficiency. Addition of heterologous siderophores from both bacteria and fungi also supported growth of the yeast in a deferrated medium. These results suggest that C. albicans has an iron-uptake mechanism which enables it to obtain iron by utilizing candidal and non-candidal siderophores.     

Cell-associated collagenolytic activity by Candida albicans

Cell associated collagenolytic activity of Candida albicans was quantified by measuring the degradation of synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which is a specific substrate for collagenase, by the freeze-thaw procedure method. This collagenolytic activity was enhanced by cells cultured in the presence of bovine serum albumin (BSA) in culture medium. However, this activity was inhibited in the presence of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), but not by the serine proteinase inhibitor p-amidinophenyl methanesulfonyl fluoride (APMSF), nor the aspartyl proteinase inhibitor pepstatin A. These results suggested the presence of a metalloenzyme on pericellular C. albicans. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection of Candida albicans in disseminated candidosis by immunofluorescence staining

An indirect immunofluorescence (IF) method using rabbit anti-Candida albicans was used to detect C. albicans in blood samples of 12 patients with systemic candidosis defined clinically, histologically and by blood cultures. Positive staining of C. albicans could be detected in all of the patients. The findings suggest that IF-method offers a more rapid method in the diagnosis of disseminated candidosis.     

Recombinagenicity of caffeine for Candida albicans

Caffeine at concentrations of 0.5 × 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 °C than 25 °C; at 37 °C, caffeine levels above 1.5 × 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.     

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 ade^r 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.     

Photodynamic inactivation for controlling Candida albicans infections

Antimicrobial photodynamic therapy (APDT) combines a non-toxic dye, termed photosensitizer, which is activated by visible light of appropriate wavelength which will produce reactive oxygen species (ROS). These ROS will react with cellular components inducing oxidative processes, leading to cell death. A wide range of microorganisms, have already showed susceptibility to APDT. Therefore, this treatment might consist in an alternative for the management of fungal infections that is mainly caused by biofilms, since they respond poorly to conventional antibiotics and may play a role in persistent infections. Biofilms are the leading cause of microbial infections in humans, thus representing a serious problem in health care. Candida albicans is the main type of fungi able to form biofilms, which cause superficial skin and mucous membrane infections as well as deep-seated mycoses, particularly in immunocompromised patients. In these patients, invasive infections are often associated with high morbidity and mortality. Furthermore, the increase in antifungal resistance has decreased the efficacy of conventional therapies. Treatments are time-consuming and thus demanding on health care budgets. Additionally, current antifungal drugs only have a limited spectrum of action and toxicity. The use of APDT as an antimicrobial topical agent against superficial and cutaneous diseases represents an effective method for eliminating microorganisms.     

Fungispecificity of fluconazole against Candida albicans

Candida albicans is an opportunistic pathogen of human mucosal surfaces. Colonization of oral and vaginal mucosa by this yeast is antagonized by the resident normal bacterial population. However, antibacterial therapy can alter the normal flora to allow fungal cells to attach, grow and invade host tissues. We studied the antimicrobic activity of fluconazole against clinical isolates of oral and vaginal bacteria and Candida albicans in vitro and in vivo by scanning and transmission electron microscopy; we also compared the bactericidal activity of fluconazole with clotrimazole in vitro by microbiologie assay. Fluconazole lysed fungi but did not change the ultrastructure of bacteria. Clotrimazole, but not fluconazole, was bactericidal against lactobacillus and streptococcus, the principal species of the oral and vaginal cavities. We conclude that Candida albicans, but not oral and vaginal bacteria, is susceptible to fluconazole. These observations help explain the antimycotic specificity of fluconazole and its efficacy against candidiasis in humans.     

Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans

Human cathelicidin LL37 and its fragments LL13–37 and LL17–32 exhibited similar potencies in inhibiting growth of the yeast Candida albicans. After treatment with 0.5 μM and 5 μM LL13–37, the hyphae changed from a uniformly thick to an increasingly slender appearance, with budding becoming less normal in appearance and cell death could be detected. Only the yeast form and no hyphal form could be observed following exposure to 50 μM LL13–37. LL13–37 at a concentration of 5 μM was able to permeabilize the membrane of yeast form as well as hyphal form of C. albicans since the nuclear stain SYTOX Green was localized in both forms. Mycelia treated with LL13–37 stained with SYTOX Green, but did not stain with MitoTracker deep red, indicating that the mitochondria were adversely affected by LL13–37. Bimane-labeled LL13–37 was able to enter some of the hyphae, but not all hyphae were affected, suggesting that LL37impaired membrane permeability characteristics in some of the hyphae. Reactive oxygen species was detectable in the yeast form of C. albicans cells after treatment with LL13–37 but not in the untreated cells. The results suggest that the increased membrane permeability caused by LL13–37 might not be the sole cause of cell death. It might lead to the uptake of the peptide, which might have some intracellular targets.     

Antibody formation in experimental immunizations with Candida albicans ribosomal fractions

Sera of mice immunized with ribosomal fractions of Candida albicans showed the presence of anti-C. albicans antibodies, detected by the gel-immunodiffusion, agglutination and immune adherence tests.Candida infections are among the most prevalent opportunistic yeast infections, attacking debilitated individuals, and against which there is no effective prophylactic treatment currently available (1, 2, 3, 4, 5). In view of the succes reported in experimental immunizations with ribosomal fractions from various bacteria and some fungi, as summarized by Youmans and Tewari (7, 8), a similar approach for immunization in experimental candidiasis appears reasonable. The present work describes preliminary results on circulating antibodies elicited in the course of immunizations with ribosomal fractions of Candida albicans.Ribosomal preparations were obtained from mechanically disrupted cell-pellets of C. albicans by differential centrifugation and purification in a 15% sucrose and 5% ammonium sulfate solution (in sodium-magnesium-Tris buffer), using a modification of the procedure described by Rubin (6). Concentration of ribosomal-RNA was determined by the absorbance at 260 nm; ribosomal-protein concentration by the Lowry reaction; and purity of the ribosomal preparation checked by the ratio of absorbance at 260 nm to 280, and at 260 to 235 nm. Mice (ICR strain) were immunized with these ribosomal preparations in amounts of 50–100 g ribosomalprotein/mouse, by 2–3 subcutaneous inoculations with Frend's adjuvant, with a 10–21 day interval between the inoculations.This work constitutes part of Ruth Levy's research study towards the Ph.D. degree.     

Cytochemical and ultrastructural studies of Candida albicans

Ultrastructural modifications of the cell wall coat of Candida albicans during adherence to host cells were investigated using various cytochemical techniques. Attachment of the fungus to buccal epithelial cells appeared to involve spatial rearrangement of their cell wall surface. In particular adhering yeast developed a fibrogranular surface layer visualized by the periodic acid — thiocarbohydrazide silver proteinate technique (a polysaccharide detectron technique); Concanavalin A binding sites detected on their cell wall coat were highly increased. Attachment of yeasts to epithelial cells appeared mediated by fibrillar structures or polysaccharidic granules distributed on the cell wall coat. But free extra-cell wall material containing mannoproteins released from the yeast surface suggested additional mechanisms.Abbreviations Con A Concanavalin A - Man-fer mannosyl ferritin - PATAg Periodic acid-thiocarbohydrazide-silver proteinate     

Development of amphotericin B liposomes bearing antibody specific to Candida albicans

Summary Liposomes expressing external antibody specific for Candida albicans and encapsulating amphotericin B were developed and characterized in this study. Antibody was first modified by the covalent attachment of palmitic acid residues. Liposomes were produced by reverse-phase evaporation and modified antibody was incorporated into these liposomes via the hydrophobic interaction between the palmitic acid and the phospholipids composing the liposomes. The liposomes were characterized as to the amount of amphotericin B by spectroscopy and for the presence of antibody by protein analysis and secondary immunolabeling by fluorescent and electron microscopic methods. Immunogold labeling showed that the antibody was being expressed externally on the liposomes in the electron microscopic studies and the specificity of these liposomes for C. albicans was observed by secondary immunofluorescence.     

One-step gene disruption by cotransformation to isolate double auxotrophs in Candida albicans

Summary The Candida albicans LEU2 gene was disrupted by substituting lambda DNA for a small deletion within the LEU2 gene. Cotransformation with a selectable URA3 ARS vector was used to introduce a linear fragment containing the disruption into the genome of a C. albicans ura3 deletion mutant. Cotransformants containing the lambda DNA were identified by colony hybridization and the URA3 plasmid was subsequently cured. Leu2 disrupted heterozygotes were detected by Southern hybridization and one disruptant was subsequently treated with UV irradiation. Only one leu2 ura3 mutant (SGY-484) was isolated out of 11,000 mutagenized cells. SGY-484 was transformed to Leu⁺ with either the C. albicans or Saccharomyces cerevisiae LEU2 gene. Southern hybridization analysis revealed that the mutant is not homozygous for the disruption; the leu2 mutation reverts and is most likely a point mutation. Unexpectedly, an ade2 ura3 mutant was isolated from the same mutagenesis.     

Re-expression by Candida albicans germ tubes of antigens lost during subculture of blastospores

The effect of germ tube induction on the antigenic variability in C. albicans was studied in strains from blood cultures (Group I) and superficial candidiasis (Group II). When compared by immunoblotting with a rabbit antiserum, antigenic extracts from Group I strains grown as blastospores showed a higher reactivity than that of Group II strains. Major bands in Group I strains (45–47, 33, 30 kDa) were continuously expressed through the subcultures in vitro but, with the exception of the 45 kDa band, the reactivity of all of them decreased or disappeared after the tenth subculture in Group II strains. The induction of the germ tubes produced the re-expression of the antigens lost during subculture in the yeast form, the effect being very clear in Group II strains. The re-expression by C. albicans germ tubes of antigens lost during subculture of blastospores in vitro and the higher reactivity shown by Group I strains grown in mycelial phase should be taken into consideration when a test to detect anti-C. albicans antibodies is to be developed.Abbreviations GYE glucose-yeast extract agar     

Development of phenotypic resistance to direct lethal miconazole action by Candida albicans entering stationary phase

In the late logarithmic or very early stationary phase of the growth cycle, yeast cells of Candida albicans undergo a shift from susceptibility to resistance to the direct lethal action of miconazole. Regulation of this phenotypic shift was examined. Experiments based on viable count determinations and the construction of time-kill curves showed that reestablishment of resistance is independent of both pH and the attainment of some critical viable cell density. However, it was found that development of resistance requires the continued availability of an appropriate energy source toward the end of exponential growth.     

A new minimal synthetic medium for germ-tube production in Candida albicans

A new minimal synthetic medium, with low amount of glucose, without aminoacids, vitamins and neutral pH, which induces germ-tubes production in Candida albicans, is reported in this work. The results indicate a perfect agreement between the germ-tube test performed with the standard method in human or animal serum and this test performed in minimal synthetic medium. In this medium the germ-tube test for the presumptive identification of Candida albicans can be performed with the same formality, time and reproducibility as those in human or animal serum. This constitutes an interesting finding because it is easy to prepare, to store and is highly reproducible.     

Selective Advantages of a Parasexual Cycle for the Yeast Candida albicans

The yeast Candida albicans can mate. However, in the natural environment mating may generate progeny (fusants) fitter than clonal lineages too rarely to render mating biologically significant: C. albicans has never been observed to mate in its natural environment, the human host, and the population structure of the species is largely clonal. It seems incapable of meiosis, and most isolates are diploid and carry both mating-type-like (MTL) locus alleles, preventing mating. Only chromosome loss or localized loss of heterozygosity can generate mating-competent cells, and recombination of parental alleles is limited. To determine if mating is a biologically significant process, we investigated if mating is under selection. The ratio of nonsynonymous to synonymous mutations in mating genes and the frequency of mutations abolishing mating indicated that mating is under selection. The MTL locus is located on chromosome 5, and when we induced chromosome 5 loss in 10 clinical isolates, most of the resulting MTL-homozygotes could mate with each other, producing fusants. In laboratory culture, a novel environment favoring novel genotypes, some fusants grew faster than their parents, in which loss of heterozygosity had reduced growth rates, and also faster than their MTL-heterozygous ancestors—albeit often only after serial propagation. In a small number of experiments in which co-inoculation of an oral colonization model with MTL-homozygotes yielded small numbers of fusants, their numbers declined over time relative to those of the parents. Overall, our results indicate that mating generates genotypes superior to existing MTL-heterozygotes often enough to be under selection.     

Non-glucan Attached Proteins of Candida albicans Biofilm Formed on Various Surfaces

Non-glucan attached proteins of the cell surface and extracellular matrix of Candida albicans biofilms formed on two catheter surfaces and denture acrylic were examined. The SDS-PAGE protein profiles of these proteins compared with that obtained from planktonic yeast cells and germ tubes were generally similar. This observation suggested that this class of biofilm surface proteins is not composed of a unique set of extracellular proteins or that one or a few proteins dominate the non-glucan attached proteins of biofilm. However, differences were observed in the proteins obtained from biofilm formed on one catheter surface and two proteins, Grp2p and ORF19.822p, identified by mass spectrometry following two-dimensional separation. These proteins have previously been associated with drug resistance and their presence or abundance appeared to be influenced by the surface on which the biofilm was formed.     

Effect of Candida albicans dsDNA in Gastrointestinal Candida Infection

Neonates are highly sensitive to infections because they are biased to develop Th2 immune responses. When exposed to certain agents, such as DNA vaccines or CpG DNA motifs, neonates are capable to mount adult-like Th1 protective responses. This study investigates the capacity of Candida albicans (C. albicans) dsDNA to induce host resistance in newborn mice against gastrointestinal C. albicans infection. The protective properties of dsDNA are related to an increased number of spleen CD4+ T cells secreting IFN-γ. In infected DNA-treated mice, an enhanced production of IFN-γ by Peyer’s patch cells was observed together with reduced colonization and histopathological changes in the stomach. Our results indicated that C. albicans dsDNA administration in neonates elicited the protective immune response against gastrointestinal Candida infection.     

Oxidative stress by antimicrobial peptide pleurocidin triggers apoptosis in Candida albicans

Pleurocidin (GWGSFFKKAAHVGKHVGKAALTHYL-NH₂), found in skin mucous secretions of the winter flounder Pleuronectes americanus, is known to possess a high potency and broad-spectrum antimicrobial peptide without cytotoxicity. In this study, to investigate the impact of pleurocidin on apoptotic progress, we observed morphological and physiological changes in Candida albicans. In cells exposed to pleurocidin, intracellular reactive oxygen species (ROS) which is a major cause of apoptosis were increased, and hydroxyl radicals were especially a large part of ROS. The increase of ROS induced oxidative stress and mitochondrial membrane depolarization which causes release of pro-apoptotic factors. Using FITC-VAD-FMK staining, we confirmed activation of yeast metacaspases which lead to apoptosis and phosphatidylserine externalization at early stage apoptosis was observed using annexin V FITC. In addition, pleurocidin induced-apoptotic cells underwent apoptotic morphological changes, showing the reduced cell size (low FSC) and enhanced intracellular density (high SSC) in flow cytometry dot plots. Under the influence of oxidative stress, DNA and nuclei were fragmented and condensed in cells, and they were visualized by 4′,6-diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. These apoptotic phenomena represent that oxidative stress by inducing pleurocidin must be an important factor of the apoptotic process in C. albicans.