The parasexual cycle in Candida albicans provides an alternative pathway to meiosis for the formation of recombinant strains

Candida albicans has an elaborate, yet efficient, mating system that promotes conjugation between diploid a and α strains. The product of mating is a tetraploid a/α cell that must undergo a reductional division to return to the diploid state. Despite the presence of several “meiosis-specific” genes in the C. albicans genome, a meiotic program has not been observed. Instead, tetraploid products of mating can be induced to undergo efficient, random chromosome loss, often producing strains that are diploid, or close to diploid, in ploidy. Using SNP and comparative genome hybridization arrays we have now analyzed the genotypes of products from the C. albicans parasexual cycle. We show that the parasexual cycle generates progeny strains with shuffled combinations of the eight C. albicans chromosomes. In addition, several isolates had undergone extensive genetic recombination between homologous chromosomes, including multiple gene conversion events. Progeny strains exhibited altered colony morphologies on laboratory media, demonstrating that the parasexual cycle generates phenotypic variants of C. albicans. In several fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, the conserved Spo11 protein is integral to meiotic recombination, where it is required for the formation of DNA double-strand breaks. We show that deletion of SPO11 prevented genetic recombination between homologous chromosomes during the C. albicans parasexual cycle. These findings suggest that at least one meiosis-specific gene has been re-programmed to mediate genetic recombination during the alternative parasexual life cycle of C. albicans. We discuss, in light of the long association of C. albicans with warm-blooded animals, the potential advantages of a parasexual cycle over a conventional sexual cycle.     

The Parasexual Cycle in Candida albicans Provides an Alternative Pathway to Meiosis for the Formation of Recombinant Strains

Candida albicans has an elaborate, yet efficient, mating system that promotes conjugation between diploid a and α strains. The product of mating is a tetraploid a/α cell that must undergo a reductional division to return to the diploid state. Despite the presence of several “meiosis-specific” genes in the C. albicans genome, a meiotic program has not been observed. Instead, tetraploid products of mating can be induced to undergo efficient, random chromosome loss, often producing strains that are diploid, or close to diploid, in ploidy. Using SNP and comparative genome hybridization arrays we have now analyzed the genotypes of products from the C. albicans parasexual cycle. We show that the parasexual cycle generates progeny strains with shuffled combinations of the eight C. albicans chromosomes. In addition, several isolates had undergone extensive genetic recombination between homologous chromosomes, including multiple gene conversion events. Progeny strains exhibited altered colony morphologies on laboratory media, demonstrating that the parasexual cycle generates phenotypic variants of C. albicans. In several fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, the conserved Spo11 protein is integral to meiotic recombination, where it is required for the formation of DNA double-strand breaks. We show that deletion of SPO11 prevented genetic recombination between homologous chromosomes during the C. albicans parasexual cycle. These findings suggest that at least one meiosis-specific gene has been re-programmed to mediate genetic recombination during the alternative parasexual life cycle of C. albicans. We discuss, in light of the long association of C. albicans with warm-blooded animals, the potential advantages of a parasexual cycle over a conventional sexual cycle.     

Parasexual Ploidy Reduction Drives Population Heterogeneity Through Random and Transient Aneuploidy in Candida albicans

The opportunistic pathogen Candida albicans has a large repertoire of mechanisms to generate genetic and phenotypic diversity despite the lack of meiosis in its life cycle. Its parasexual cycle enables shifts in ploidy, which in turn facilitate recombination, aneuploidy, and homozygosis of whole chromosomes to fuel rapid adaptation. Here we show that the tetraploid state potentiates ploidy variation and drives population heterogeneity. In tetraploids, the rate of losing a single heterozygous marker [loss of heterozygosity (LOH)] is elevated ∼30-fold higher than the rate in diploid cells. Furthermore, isolates recovered after selection for LOH of one, two, or three markers were highly aneuploid, with a broad range of karyotypes including strains with a combination of di-, tri-, and tetrasomic chromosomes. We followed the ploidy trajectories for these tetraploid- and aneuploid-derived isolates, using a combination of flow cytometry and double-digestion restriction-site-associated DNA analyzed with next-generation sequencing. Isolates derived from either tetraploid or aneuploid isolates predominately resolved to a stable euploid state. The majority of isolates reduced to the conventional diploid state; however, stable triploid and tetraploid states were observed in ∼30% of the isolates. Notably, aneuploid isolates were more transient than tetraploid isolates, resolving to a euploid state within a few passages. Furthermore, the likelihood that a particular isolate will resolve to the same ploidy state in replicate evolution experiments is only ∼50%, supporting the idea that the chromosome loss process of the parasexual cycle is random and does not follow trajectories involving specific combinations of chromosomes. Together, our results indicate that tetraploid progenitors can produce populations of progeny cells with a high degree of genomic diversity, from altered ploidy to homozygosis, providing an excellent source of genetic variation upon which selection can act.     

Completion of a parasexual cycle in Candida albicans by induced chromosome loss in tetraploid strains

The human pathogenic fungus Candida albicans has traditionally been classified as a diploid, asexual organism. However, mating-competent forms of the organism were recently described that produced tetraploid mating products. In principle, the C.albicans life cycle could be completed via a sexual process, via a parasexual mechanism, or by both mechanisms. Here we describe conditions in which growth of a tetraploid strain of C.albicans on Saccharomyces cerevisiae 'pre-sporulation' medium induced efficient, random chromosome loss in the tetraploid. The products of chromosome loss were often strains that were diploid, or very close to diploid, in DNA content. If they inherited the appropriate MTL (mating-type like) loci, these diploid products were themselves mating competent. Thus, an efficient parasexual cycle can be performed in C.albicans, one that leads to the reassortment of genetic material in this organism. We show that this parasexual cycle-consisting of mating followed by chromosome loss-can be used in the laboratory for simple genetic manipulations in C.albicans.     

Voice Prosthetic Biofilm Formation and Candida Morphogenic Conversions in Absence and Presence of Different Bacterial Strains and Species on Silicone-Rubber

Morphogenic conversion of Candida from a yeast to hyphal morphology plays a pivotal role in the pathogenicity of Candida species. Both Candida albicans and Candida tropicalis, in combination with a variety of different bacterial strains and species, appear in biofilms on silicone-rubber voice prostheses used in laryngectomized patients. Here we study biofilm formation on silicone-rubber by C. albicans or C. tropicalis in combination with different commensal bacterial strains and lactobacillus strains. In addition, hyphal formation in C. albicans and C. tropicalis, as stimulated by Rothia dentocariosa and lactobacilli was evaluated, as clinical studies outlined that these bacterial strains have opposite results on the clinical life-time of silicone-rubber voice prostheses. Biofilms were grown during eight days in a silicone-rubber tube, while passing the biofilms through episodes of nutritional feast and famine. Biofilms consisting of combinations of C. albicans and a bacterial strain comprised significantly less viable organisms than combinations comprising C. tropicalis. High percentages of Candida were found in biofilms grown in combination with lactobacilli. Interestingly, L. casei, with demonstrated favorable effects on the clinical life-time of voice prostheses, reduced the percentage hyphal formation in Candida biofilms as compared with Candida biofilms grown in absence of bacteria or grown in combination with R. dentocariosa, a bacterial strain whose presence is associated with short clinical life-times of voice prostheses.     

Hemolytic Factor Production by Clinical Isolates of Candida Species

Most cases of fungal bloodstream infections (BIs) are attributed to Candida albicans; however, non-Candida albicans Candida species have recently been identified as common pathogens. Although hemolytic factor is known to be putative virulence factor contributing to pathogenicity in Candida species, its production is poorly evaluated. The present study was undertaken to analyze the production of hemolytic factor by C. albicans (10), C. tropicalis (13), and C. parapsilosis (8) isolates associated with BIs. Data of hemolysis zones on plate assay revealed that the majority of C. albicans isolates produced mild hemolytic activity whereas the majority of C. tropicalis produced strong activity. None of the tested C. parapsilosis isolates exhibited hemolysis on plate assay. We also evaluated the hemolytic activity in the cell-free broth. There were no significant differences (P > 0.05) in the secreted hemolytic activity among intra-species isolates. Different levels of secreted hemolytic factor were observed for Candida species, where C. tropicalis exhibited the highest production of hemolytic factor (P < 0.05) followed by C. albicans and C. parapsilosis. Inhibition of hemolysis (up to 89.12 %) from culture supernatant, following incubation with the lectin Concanavalin A (Con A), was observed for all three Candida species. This finding suggests that the secreted hemolytic factor of C. tropicalis and C. parapsilosis may be a mannoprotein, similar to that described for C. albicans.     

The Influence of Chemical Composition of Commercial Lemon Essential Oils on the Growth of Candida Strains

Candida yeasts are saprophytes naturally present in the environment and forming colonies on human mucous membranes and skin. They are opportunistic fungi that cause severe and even fatal infections in immunocompromised individuals. Several essential oils, including eucalyptus, pine, cinnamon and lemon, have been shown to be effective against Candida strains. This study addresses the chemical composition of some commercial lemon essential oils and their antifungal potential against selected Candida yeast strains. Antifungal potential and minimum inhibitory concentrations were determined for six commercial lemon essential oils against five Candida yeast strains (Candida albicans 31, Candida tropicalis 32, Candida glabrata 33, Candida glabrata 35 and Candida glabrata 38). On the basis of the GCMS analysis, it was found that the tested lemon essential oils had different chemical compositions, but mostly, they contained almost exclusively terpenes and oxygenated terpenes. The tests show that antifungal potential of lemon essential oils against Candida yeast strains was related to the high content of monoterpenoids and the type of Candida strains. From six tested commercial oils, only four (ETJA, Vera-Nord, Avicenna-Oil and Aromatic Art) shows antifungal potential against three Candida species (C. albicans, C. tropicalis and C. glabrata). Vera-Nord and Avicenna-Oil show the best activity and effectively inhibit the growth of the C. albicans strain across the full range of the concentrations used. Our study characterises lemon essential oils, which could be used as very effective natural remedies against candidiasis caused by C. albicans.     

Does stress induce (para)sex? Implications for Candida albicans evolution

Theory predicts that stress is a key factor in explaining the evolutionary role of sex in facultatively sexual organisms, including microorganisms. Organisms capable of reproducing both sexually and asexually are expected to mate more frequently when stressed, and such stress-induced mating is predicted to facilitate adaptation. Here, we propose that stress has an analogous effect on the parasexual cycle in Candida albicans, which involves alternation of generations between diploid and tetraploid cells. The parasexual cycle can generate high levels of diversity, including aneuploidy, yet it apparently occurs only rarely in nature. We review the evidence that stress facilitates four major steps in the parasexual cycle and suggest that parasex occurs much more frequently under stress conditions. This may explain both the evolutionary significance of parasex and its apparent rarity.     

Oral Candida carriage of patients attending a dental clinic in Braga,Portugal

BackgroundThe ability of the Candida species to colonize surfaces can be considered as a risk factor for oral infection.AimsTo establish oral Candida carriage in patients attending a dental clinic in Braga, Portugal.MethodsA total of 97 patients were analysed. Swab samples were collected and directly cultured onto CHROMagar Candida. Representative yeasts were identified by polymerase chain reaction.ResultsFrom the samples analysed 54.6% (n=53) were Candida positive, and Candida albicans was the most frequently isolated species, accounting for 79% of all the species identified. Non-C. albicans Candida (NCAC) species recovered included Candida parapsilosis, Candida glabrata, Candida tropicalis, and Candida guilliermondii. There was a lack of association between the presence of C. albicans or NCAC species, and age, gender, or prostheses wearing in this population. In 17% of the cases (n=9), polymicrobial cultures, with two different Candida species, were identified.ConclusionsThis study shows a high Candida carriage rate among this population, thus pointing to the relevance of an accurate diagnostic approach in Candida species identification.     

A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

Candida albicans, the most prevalent human fungal pathogen, is generally diploid. However, 50% of isolates that are resistant to fluconazole (FLC), the most widely used antifungal, are aneuploid and some aneuploidies can confer FLC resistance. To ask if FLC exposure causes or only selects for aneuploidy, we analyzed diploid strains during exposure to FLC using flow cytometry and epifluorescence microscopy. FLC exposure caused a consistent deviation from normal cell cycle regulation: nuclear and spindle cycles initiated prior to bud emergence, leading to “trimeras,” three connected cells composed of a mother, daughter, and granddaughter bud. Initially binucleate, trimeras underwent coordinated nuclear division yielding four daughter nuclei, two of which underwent mitotic collapse to form a tetraploid cell with extra spindle components. In subsequent cell cycles, the abnormal number of spindles resulted in unequal DNA segregation and viable aneuploid progeny. The process of aneuploid formation in C. albicans is highly reminiscent of early stages in human tumorigenesis in that aneuploidy arises through a tetraploid intermediate and subsequent unequal DNA segregation driven by multiple spindles coupled with a subsequent selective advantage conferred by at least some aneuploidies during growth under stress. Finally, trimera formation was detected in response to other azole antifungals, in related Candida species, and in an in vivo model for Candida infection, suggesting that aneuploids arise due to azole treatment of several pathogenic yeasts and that this can occur during the infection process.     

The new mutation L321F in Candida albicans ERG11 gene may be associated with fluconazole resistance

BackgroundFor many years fluconazole has been commonly used to treat Candida infections. However, the indiscriminate use of this antimycotic therapy has favored the emergence of resistant isolates. Mutations in the ERG11 gene have been described as one of the primary mechanisms of resistance in Candida species.AimsIn this study we investigated missense mutations in ERG11 genes of Candida albicans, Candida glabrata and Candida tropicalis isolates previously evaluated by susceptibility testing to fluconazole.MethodsScreening for these mutations was performed on 19 Candida clinical isolates (eight C. albicans, five C. glabrata and six C. tropicalis) resistant and susceptible to fluconazole. The ERG11 gene was amplified by PCR with specific primers for each Candida species and analyzed by automated sequencing.ResultsWe identified 14 different missense mutations, five of which had not been described previously. Among them, a new mutation L321F was identified in a fluconazole resistant C. albicans isolate and it was analyzed by a theoretical three-dimensional structure of the ERG11p.ConclusionThe L321F mutation in C. albicans ERG11 gene may be associated with fluconazole resistance.     

Species Distribution and Virulence Factors of Candida spp. Isolated from the Oral Cavity of Kidney Transplant Recipients in Brazil

Although yeasts belonging to the genus Candida are frequently seen as commensals in the oral cavity, they possess virulence attributes that contribute for pathogenicity. The aims of the present study were to study the prevalence of Candida spp. isolated from the oral cavity of renal transplant recipients and to analyze strains virulence factors. We isolated a total of 70 Candida strains from 111 transplant recipients, and Candida albicans was the most prevalent species (82.86 %). Oral candidiasis was diagnosed in 14.4 % kidney transplant patients, while 11 isolates (15.7 %) corresponded to non-Candida albicans Candida (NCAC) species. C. albicans adhered to a higher extension than NCAC strains. Some isolates of Candida tropicalis were markedly adherent to human buccal epithelial cells and highly biofilm-forming strains. Regarding proteinase activity, Candida orthopsilosis was more proteolytic than Candida metapsilosis. Candida glabrata and Candida dubliniensis showed very low ability to form biofilm on polystyrene microtiter plates. We have demonstrated here diverse peculiarities of different Candida species regarding the ability to express virulence factors. This study will contribute for the understanding of the natural history and pathogenesis of yeasts belonging to the genus Candida in the oral cavity of patients who were submitted to kidney transplant and are under immunosuppressive therapies.     

Etiología de la candidiasis en pediatría: análisis comparativo con el paciente adulto

BackgroundCandida spp. represents a group of commensal yeasts that can act as pathogens and cause candidiasis in different anatomical locations.AimsThe aim of this study was to perform an epidemiological and comparative analysis between the isolates of Candida spp. in clinical specimens during a three year-period (2010-2012) from children (0-14 years) and adults (15-99 years) in the Valencian Community (RedMIVA).MethodsThe microbiological surveillance network of Valencian Community was used as the information source.Results and conclusionsCandida was isolated in 52,436 patients (1,604 [3.1%] children and 50,832 [96.9%] adults). Candida albicans was significantly (p < 0.05) the predominant species in both age groups, and in almost every type of clinical specimen. The distribution of other species varied depending on the sample type and age group. In blood specimens, Candida parapsilosis followed by C. albicans, Candida famata and Candida lusitaniae were the main species found in children, whereas C. albicans followed by C. parapsilosis, Candida glabrata and Candida tropicalis were the predominant species in adults. In sterile fluids, urine and lower respiratory tract samples, C. parapsilosis was the second most prevalent species in the children group, while C. glabrata and C. tropicalis were the main second species in adults.     

Use of CHROMagar in the Differentiation of Common Species of <Emphasis Type="Italic">Candida</Emphasis>

CHROMagar has been reported to be useful for the rapid and accurate identification of Candida species. We tested 135 isolates of Candida species isolated from oropharyngeal candidiasis in HIV patients and found that it was useful in the presumptive identification of Candida albicans and Candida krusei. Occasional strains of C. tropicalis produced colonies with a greenish tinge making it difficult to differentiate from C. albicans.     

Virulence phenotypes result from interactions between pathogen ploidy and genetic background

Studying fungal virulence is often challenging and frequently depends on many contexts, including host immune status and pathogen genetic background. However, the role of ploidy has often been overlooked when studying virulence in eukaryotic pathogens. Since fungal pathogens, including the human opportunistic pathogen Candida albicans, can display extensive ploidy variation, assessing how ploidy impacts virulence has important clinical relevance. As an opportunistic pathogen, C. albicans causes nonlethal, superficial infections in healthy individuals, but life‐threatening bloodstream infections in individuals with compromised immune function. Here, we determined how both ploidy and genetic background of C. albicans impacts virulence phenotypes in healthy and immunocompromised nematode hosts by characterizing virulence phenotypes in four near‐isogenic diploid and tetraploid pairs of strains, which included both laboratory and clinical genetic backgrounds. We found that C. albicans infections decreased host survival and negatively impacted host reproduction, and we leveraged these two measures to survey both lethal and nonlethal virulence phenotypes across the multiple C. albicans strains. In this study, we found that regardless of pathogen ploidy or genetic background, immunocompromised hosts were susceptible to fungal infection compared to healthy hosts. Furthermore, for each host context, we found a significant interaction between C. albicans genetic background and ploidy on virulence phenotypes, but no global differences between diploid and tetraploid pathogens were observed.     

Lack of relationship between genotype and virulence in Candida species

BackgroundThe virulence of isolates among different Candida species causing candidemia may play a role in the prognosis of the patients. Furthermore, the potential relationship between genotype and virulence is still unclear and need to be further studied.AimsWe aim to assess the relationship between genotype and virulence in Candida species using a Galleria mellonella larvae infection model.MethodsOne hundred and ninety-four isolates from 68 clusters (Candida albicans, 114/41; Candida parapsilosis, 74/24; Candida tropicalis, 6/3) were compared against the same number of each species singleton genotypes in terms of survival of G. mellonella larvae.ResultsThe median of survival and the IQR ranges of clusters and singleton were as follows: C. albicans (2 days, IQR 1.5–2 vs. 2 days, IQR 1–2.25), C. parapsilosis (2 days, IQR 1.5–2.6 vs. 2 days, IQR 2–3.3), and C. tropicalis (1 day, IQR 1–3.5 vs. 2 days, IQR 2–3.5; p < 0.05). High intra-cluster variability in terms of median of survival was found regardless the species.ConclusionsNo relationship between genotype and virulence in Candida was observed with the G. mellonella model.     

Zygote formation and recombination between like mating types in the yeast Saccharomycopsis lipolytica by protoplast fusion

Summary Protoplasts from auxotrophic strains of the alkane yeast, Saccharomycopsis (Candida) lipolytica, will hybridize despite identity in mating type. Fusion products following regeneration and selection form stable prototrophic diploids, and recombinant progeny can be obtained either through the parasexual or the sexual cycle. These results confirm that mating type alleles of this yeast control only the initial steps in the mating sequence, cell recognition and agglutination, but not karyogamy and meiosis.     

Flocculation phenomenon of Candida albicans by lysozyme

Young colonies of Sabouraud's glucose agar room temperature culture ofCandida species from human isolation were suspended in distilled water. The suspension was mixed with a solution of lysozyme and incubated in a 37° C water bath. Within 3–5 hours, various species ofCandida cells showed flocculation to varying degrees which occurred at varying periods of onset. Among sevenCandida species,Candida albicans andCandida stellatoidea showed the strongest flocculation, earliest onset and most solution clarity than did any other species.Candida stellatoidea was indistinguishable fromCandida albicans in its degree of flocculation, and in the clarity of solution.Candida species may be arranged in the following order according to their decreasing positivity in flocculation:

1. Candida albicans
2. Candida stellatoidea
3. Candida tropicalis
4. Candida krusei
5. Candida pseudotropicalis
6. Candida parapsilosis
7. Candida guilliermondii
8. Saccharomyces species may be placed afterCandida guilliermondii.

It seems possible to separate theCandida species into 3 groups by the rate of flocculation, and clarity of solution. Group I.Candida albicans andCandida stellatoidea. Group II.Candida tropicalis, C. krusei andCandida pseudotropicalis. Group III.Candida parapsilosis andCandida guilliermondii. Saccharomyces specimens (S. cerevisiae and others) were placed after group III.