Selection of Candida albicans trisomy during oropharyngeal infection results in a commensal-like phenotype.

When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC. We also analyzed the expression of virulence-associated traits in vitro. All three trisomic strains exhibited characteristics of a commensal during OPC in mice. They achieved the same oral fungal burden as the diploid progenitor strain but caused significantly less weight loss and elicited a significantly lower inflammatory host response. In vitro, all three trisomic strains had reduced capacity to adhere to and invade oral epithelial cells and increased susceptibility to neutrophil killing. Whole genome sequencing of pre- and post-infection isolates found that the trisomies were usually maintained. Most post-infection isolates also contained de novo point mutations, but these were not conserved. While in vitro growth assays did not reveal phenotypes specific to de novo point mutations, they did reveal novel phenotypes specific to each lineage. These data reveal that during OPC, clones that are trisomic for Chr5 or Chr6 are selected and they facilitate a commensal-like phenotype.

The 'obligate diploid' Candida albicans forms mating-competent haploids.

Candida albicans, the most prevalent human fungal pathogen, is considered to be an obligate diploid that carries recessive lethal mutations throughout the genome. Here we demonstrate that C. albicans has a viable haploid state that can be derived from diploid cells under in vitro and in vivo conditions, and that seems to arise through a concerted chromosome loss mechanism. Haploids undergo morphogenetic changes like those of diploids, including the yeast-hyphal transition, chlamydospore formation and a white-opaque switch that facilitates mating. Haploid opaque cells of opposite mating type mate efficiently to regenerate the diploid form, restoring heterozygosity and fitness. Homozygous diploids arise spontaneously by auto-diploidization, and both haploids and auto-diploids show a similar reduction in fitness, in vitro and in vivo, relative to heterozygous diploids, indicating that homozygous cell types are transient in mixed populations. Finally, we constructed stable haploid strains with multiple auxotrophies that will facilitate molecular and genetic analyses of this important pathogen.

Microevolution of Candida albicans in macrophages restores filamentation in a nonfilamentous mutant.

Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1Δ/efg1Δ mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1Δ/efg1Δ strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

Evolution of pathogenicity and sexual reproduction in eight Candida genomes.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

Analysis of protein function in clinical C. albicans isolates.

Clinical isolates are prototrophic and hence are not amenable to genetic manipulation using nutritional markers. Here we describe a new set of plasmids carrying the NAT1 (nourseothricin) drug resistance marker (Shen et al., ), which can be used both in clinical isolates and in laboratory strains. We constructed novel plasmids containing HA-NAT1 or MYC-NAT1 cassettes to facilitate PCR-mediated construction of strains with C-terminal epitope-tagged proteins and a NAT1-pMet3-GFP plasmid to enable conditional expression of proteins with or without the green fluorescent protein fused at the N-terminus. Furthermore, for proteins that require both the endogenous N- and C-termini for function, we have constructed a GF-NAT1-FP cassette carrying truncated alleles that facilitate insertion of an intact, single copy of GFP internal to the coding sequence. In addition, GFP-NAT1, RFP-NAT1 and M-Cherry-NAT1 plasmids were constructed, expressing two differently labelled gene products for the study of protein co-expression and co-localization in vivo. Together, these vectors provide a useful set of genetic tools for studying diverse aspects of gene function in both clinical and laboratory strains of C. albicans.

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 alpha strains. The product of mating is a tetraploid a/alpha 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.

Genomic plasticity of the human fungal pathogen Candida albicans.

The genomic plasticity of Candida albicans, a commensal and common opportunistic fungal pathogen, continues to reveal unexpected surprises. Once thought to be asexual, we now know that the organism can generate genetic diversity through several mechanisms, including mating between cells of the opposite or of the same mating type and by a parasexual reduction in chromosome number that can be accompanied by recombination events (2, 12, 14, 53, 77, 115). In addition, dramatic genome changes can appear quite rapidly in mitotic cells propagated in vitro as well as in vivo. The detection of aneuploidy in other fungal pathogens isolated directly from patients (145) and from environmental samples (71) suggests that variations in chromosome organization and copy number are a common mechanism used by pathogenic fungi to rapidly generate diversity in response to stressful growth conditions, including, but not limited to, antifungal drug exposure. Since cancer cells often become polyploid and/or aneuploid, some of the lessons learned from studies of genome plasticity in C. albicans may provide important insights into how these processes occur in higher-eukaryotic cells exposed to stresses such as anticancer drugs.

Haplotype mapping of a diploid non-meiotic organism using existing and induced aneuploidies.

Haplotype maps (HapMaps) reveal underlying sequence variation and facilitate the study of recombination and genetic diversity. In general, HapMaps are produced by analysis of Single-Nucleotide Polymorphism (SNP) segregation in large numbers of meiotic progeny. Candida albicans, the most common human fungal pathogen, is an obligate diploid that does not appear to undergo meiosis. Thus, standard methods for haplotype mapping cannot be used. We exploited naturally occurring aneuploid strains to determine the haplotypes of the eight chromosome pairs in the C. albicans laboratory strain SC5314 and in a clinical isolate. Comparison of the maps revealed that the clinical strain had undergone a significant amount of genome rearrangement, consisting primarily of crossover or gene conversion recombination events. SNP map haplotyping revealed that insertion and activation of the UAU1 cassette in essential and non-essential genes can result in whole chromosome aneuploidy. UAU1 is often used to construct homozygous deletions of targeted genes in C. albicans; the exact mechanism (trisomy followed by chromosome loss versus gene conversion) has not been determined. UAU1 insertion into the essential ORC1 gene resulted in a large proportion of trisomic strains, while gene conversion events predominated when UAU1 was inserted into the non-essential LRO1 gene. Therefore, induced aneuploidies can be used to generate HapMaps, which are essential for analyzing genome alterations and mitotic recombination events in this clonal organism.

Parasexuality of Candida Species.

While most fungi have the ability to reproduce sexually, multiple independent lineages have lost meiosis and developed parasexual cycles in its place. Emergence of parasexual cycles is particularly prominent in medically relevant fungi from the CUG paraphyletic group of Candida species. Since the discovery of parasex in C. albicans roughly two decades ago, it has served as the model for Candida species. Importantly, parasex in C. albicans retains hallmarks of meiosis including genetic recombination and chromosome segregation, making it a potential driver of genetic diversity. Furthermore, key meiotic genes play similar roles in C. albicans parasex and highlights parallels between these processes. Yet, the evolutionary role of parasex in Candida adaptation and the extent of resulting genotypic and phenotypic diversity remain as key knowledge gaps in this facultative reproductive program. Here, we present our current understanding of parasex, the mechanisms governing its regulation, and its relevance to Candida biology.

Evolution in Candida albicans populations during a single passage through a mouse host.

The mechanisms and rates by which genotypic and phenotypic variation is generated in opportunistic, eukaryotic pathogens during growth in hosts are not well understood. We evaluated genomewide genetic and phenotypic evolution in Candida albicans, an opportunistic fungal pathogen of humans, during passage through a mouse host (in vivo) and during propagation in liquid culture (in vitro). We found slower population growth and higher rates of chromosome-level genetic variation in populations passaged in vivo relative to those grown in vitro. Interestingly, the distribution of long-range loss of heterozygosity (LOH) and chromosome rearrangement events across the genome differed for the two growth environments, while rates of short-range LOH were comparable for in vivo and in vitro populations. Further, for the in vivo populations, there was a positive correlation of cells demonstrating genetic alterations and variation in colony growth and morphology. For in vitro populations, no variation in growth phenotypes was detected. Together, our results demonstrate that passage through a living host leads to slower growth and higher rates of genomic and phenotypic variation compared to in vitro populations. Results suggest that the dynamics of population growth and genomewide rearrangement contribute to the maintenance of a commensal and opportunistic life history of C. albicans.

Aneuploid chromosomes are highly unstable during DNA transformation of Candida albicans.

Candida albicans strains tolerate aneuploidy, historically detected as karyotype alterations by pulsed-field gel electrophoresis and more recently revealed by array comparative genome hybridization, which provides a comprehensive and detailed description of gene copy number. Here, we first retrospectively analyzed 411 expression array experiments to predict the frequency of aneuploidy in different strains. As expected, significant levels of aneuploidy were seen in strains exposed to stress conditions, including UV light and/or sorbose treatment, as well as in strains that are resistant to antifungal drugs. More surprisingly, strains that underwent transformation with DNA displayed the highest frequency of chromosome copy number changes, with strains that were initially aneuploid exhibiting approximately 3-fold more copy number changes than strains that were initially diploid. We then prospectively analyzed the effect of lithium acetate (LiOAc) transformation protocols on the stability of trisomic chromosomes. Consistent with the retrospective analysis, the proportion of karyotype changes was highly elevated in strains carrying aneuploid chromosomes. We then tested the hypothesis that stresses conferred by heat and/or LiOAc exposure promote chromosome number changes during DNA transformation procedures. Indeed, a short pulse of very high temperature caused frequent gains and losses of multiple chromosomes or chromosome segments. Furthermore, milder heat exposure over longer periods caused increased levels of loss of heterozygosity. Nonetheless, aneuploid chromosomes were also unstable when strains were transformed by electroporation, which does not include a heat shock step. Thus, aneuploid strains are particularly prone to undergo changes in chromosome number during the stresses of DNA transformation protocols.

An isochromosome confers drug resistance in vivo by amplification of two genes, ERG11 and TAC1.

Acquired azole resistance is a serious clinical problem that is often associated with the appearance of aneuploidy and, in particular, with the formation of an isochromosome [i(5L)] in the fungal opportunist Candida albicans. Here we exploited a series of isolates from an individual patient during the rapid acquisition of fluconazole resistance (Flu(R)). Comparative genome hybridization arrays revealed that the presence of two extra copies of Chr5L, on the isochromosome, conferred increased Flu(R) and that partial truncation of Chr5L reduced Flu(R). In vitro analysis of the strains by telomere-mediated truncations and by gene deletion assessed the contribution of all Chr5L genes and of four specific genes. Importantly, ERG11 (encoding the drug target) and a hyperactive allele of TAC1 (encoding a transcriptional regulator of drug efflux pumps) made independent, additive contributions to Flu(R) in a gene copy number-dependent manner that was not different from the contributions of the entire Chr5L arm. Thus, the major mechanism by which i(5L) formation causes increased azole resistance is by amplifying two genes: ERG11 and TAC1.

Efficient and rapid identification of Candida albicans allelic status using SNP-RFLP.

Candida albicans is the most prevalent opportunistic fungal pathogen in the clinical setting, causing a wide spectrum of diseases ranging from superficial mucosal lesions to life-threatening deep-tissue infections. Recent studies provide strong evidence that C. albicans possesses an arsenal of genetic mechanisms promoting genome plasticity and that it uses these mechanisms under conditions of nutritional or antifungal drug stress. Two microarray-based methods, single nucleotide polymorphism (SNP) and comparative genome hybridization arrays, have been developed to study genome changes in C. albicans. However, array technologies can be relatively expensive and are not available to every laboratory. In addition, they often generate more data than needed to analyze specific genomic loci or regions. Here, we have developed a set of SNP-restriction fragment length polymorphism (RFLP) (or PCR-RFLP) markers, two per chromosome arm, for C. albicans. These markers can be used to rapidly and accurately detect large-scale changes in the C. albicans genome including loss of heterozygosity (LOH) at single loci, across chromosome arms or across whole chromosomes. Furthermore, skewed SNP-RFLP allelic ratios are indicative of trisomy at heterozygous loci. While less comprehensive than array-based approaches, we propose SNP-RFLP as an inexpensive, rapid, and reliable method to screen strains of interest for possible genome changes.

Genome plasticity in Candida albicans is driven by long repeat sequences.

Genome rearrangements resulting in copy number variation (CNV) and loss of heterozygosity (LOH) are frequently observed during the somatic evolution of cancer and promote rapid adaptation of fungi to novel environments. In the human fungal pathogen Candida albicans, CNV and LOH confer increased virulence and antifungal drug resistance, yet the mechanisms driving these rearrangements are not completely understood. Here, we unveil an extensive array of long repeat sequences (65-6499 bp) that are associated with CNV, LOH, and chromosomal inversions. Many of these long repeat sequences are uncharacterized and encompass one or more coding sequences that are actively transcribed. Repeats associated with genome rearrangements are predominantly inverted and separated by up to ~1.6 Mb, an extraordinary distance for homology-based DNA repair/recombination in yeast. These repeat sequences are a significant source of genome plasticity across diverse strain backgrounds including clinical, environmental, and experimentally evolved isolates, and represent previously uncharacterized variation in the reference genome.

Partner Choice in Spontaneous Mitotic Recombination in Wild Type and Homologous Recombination Mutants of Candida albicans.

Candida albicans, the most common fungal pathogen, is a diploid with a genome that is rich in repeats and has high levels of heterozygosity. To study the role of different recombination pathways on direct-repeat recombination, we replaced either allele of the RAD52 gene (Chr6) with the URA-blaster cassette (hisG-URA3-hisG), measured rates of URA3 loss as resistance to 5-fluoroorotic acid (5FOAR) and used CHEF Southern hybridization and SNP-RFLP analysis to identify recombination mechanisms and their frequency in wildtype and recombination mutants. FOAR rates varied little across different strain backgrounds. In contrast, the type and frequency of mechanisms underlying direct repeat recombination varied greatly. For example, wildtype, rad59 and lig4 strains all displayed a bias for URA3 loss via pop-out/deletion vs. inter-homolog recombination and this bias was reduced in rad51 mutants. In addition, in rad51-derived 5FOAR strains direct repeat recombination was associated with ectopic translocation (5%), chromosome loss/truncation (14%) and inter-homolog recombination (6%). In the absence of RAD52, URA3 loss was mostly due to chromosome loss and truncation (80-90%), and the bias of retained allele frequency points to the presence of a recessive lethal allele on Chr6B. However, a few single-strand annealing (SSA)-like events were identified and these were independent of either Rad59 or Lig4. Finally, the specific sizes of Chr6 truncations suggest that the inserted URA-blaster could represent a fragile site.

Rapid Phenotypic and Genotypic Diversification After Exposure to the Oral Host Niche in Candida albicans.

In vitro studies suggest that stress may generate random standing variation and that different cellular and ploidy states may evolve more rapidly under stress. Yet this idea has not been tested with pathogenic fungi growing within their host niche in vivo Here, we analyzed the generation of both genotypic and phenotypic diversity during exposure of Candida albicans to the mouse oral cavity. Ploidy, aneuploidy, loss of heterozygosity (LOH), and recombination were determined using flow cytometry and double digest restriction site-associated DNA sequencing. Colony phenotypic changes in size and filamentous growth were evident without selection and were enriched among colonies selected for LOH of the GAL1 marker. Aneuploidy and LOH occurred on all chromosomes (Chrs), with aneuploidy more frequent for smaller Chrs and whole Chr LOH more frequent for larger Chrs. Large genome shifts in ploidy to haploidy often maintained one or more heterozygous disomic Chrs, consistent with random Chr missegregation events. Most isolates displayed several different types of genomic changes, suggesting that the oral environment rapidly generates diversity de novo In sharp contrast, following in vitro propagation, isolates were not enriched for multiple LOH events, except in those that underwent haploidization and/or had high levels of Chr loss. The frequency of events was overall 100 times higher for C. albicans populations following in vivo passage compared with in vitro These hyper-diverse in vivo isolates likely provide C. albicans with the ability to adapt rapidly to the diversity of stress environments it encounters inside the host.

A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans.

TAC1, a Candida albicans transcription factor situated near the mating-type locus on chromosome 5, is necessary for the upregulation of the ABC-transporter genes CDR1 and CDR2, which mediate azole resistance. We showed previously the existence of both wild-type and hyperactive TAC1 alleles. Wild-type alleles mediate upregulation of CDR1 and CDR2 upon exposure to inducers such as fluphenazine, while hyperactive alleles result in constitutive high expression of CDR1 and CDR2. Here we recovered TAC1 alleles from two pairs of matched azole-susceptible (DSY294; FH1: heterozygous at mating-type locus) and azole-resistant isolates (DSY296; FH3: homozygous at mating-type locus). Two different TAC1 wild-type alleles were recovered from DSY294 (TAC1-3 and TAC1-4) while a single hyperactive allele (TAC1-5) was isolated from DSY296. A single amino acid (aa) difference between TAC1-4 and TAC1-5 (Asn977 to Asp or N977D) was observed in a region corresponding to the predicted activation domain of Tac1p. Two TAC1 alleles were recovered from FH1 (TAC1-6 and TAC1-7) and a single hyperactive allele (TAC1-7) was recovered from FH3. The N977D change was seen in TAC1-7 in addition to several other aa differences. The importance of N977D in conferring hyperactivity to TAC1 was confirmed by site-directed mutagenesis. Both hyperactive alleles TAC1-5 and TAC1-7 were codominant with wild-type alleles and conferred hyperactive phenotypes only when homozygous. The mechanisms by which hyperactive alleles become homozygous was addressed by comparative genome hybridization and single nucleotide polymorphism arrays and indicated that loss of TAC1 heterozygosity can occur by recombination between portions of chromosome 5 or by chromosome 5 duplication.

Haplotyping a Non-meiotic Diploid Fungal Pathogen Using Induced Aneuploidies and SNP/CGH Microarray Analysis.

The generation of haplotype information has recently become very attractive due to its utility for identifying mutations associated with human disease and for the development of personalized medicine. Haplotype information also is crucial for studying recombination mechanisms and genetic diversity, and for analyzing allele-specific gene expression. Classic haplotyping methods require the analysis of hundreds of meiotic progeny. To facilitate haplotyping in the non-meiotic human fungal pathogen Candida albicans, we exploited trisomic heterozygous chromosomes generated via the UAU1 selection strategy. Using this system, we obtained phasing information from allelic biases, detected by SNP/CGH microarray analysis. This strategy has the potential to be applicable to other diploid, asexual Candida species that are important causes of human disease.

Ploidy Variation in Fungi: Polyploidy, Aneuploidy, and Genome Evolution.

The ability of an organism to replicate and segregate its genome with high fidelity is vital to its survival and for the production of future generations. Errors in either of these steps (replication or segregation) can lead to a change in ploidy or chromosome number. While these drastic genome changes can be detrimental to the organism, resulting in decreased fitness, they can also provide increased fitness during periods of stress. A change in ploidy or chromosome number can fundamentally change how a cell senses and responds to its environment. Here, we discuss current ideas in fungal biology that illuminate how eukaryotic genome size variation can impact the organism at a cellular and evolutionary level. One of the most fascinating observations from the past 2 decades of research is that some fungi have evolved the ability to tolerate large genome size changes and generate vast genomic heterogeneity without undergoing canonical meiosis.