Judith Berman

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/α2 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.

Candida albicans : A molecular revolution built on lessons from budding yeast

Candida albicans is an opportunistic fungal pathogen that is found in the normal gastrointestinal flora of most healthy humans. However, in immunocompromised patients, blood-stream infections often cause death, despite the use of anti-fungal therapies. The recent completion of the C. albicans genome sequence, the availability of whole-genome microarrays and the development of tools for rapid molecular-genetic manipulations of the C. albicans genome are generating an explosion of information about the intriguing biology of this pathogen and about its mechanisms of virulence. They also reveal the extent of similarities and differences between C. albicans and its benign relative, Saccharomyces cerevisiae.

A Human-Curated Annotation of the Candida albicans Genome

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.

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.

Genotypic Evolution of Azole Resistance Mechanisms in Sequential Candida albicans Isolates

ABSTRACT TAC1 (for transcriptional activator of CDR genes) is critical for the upregulation of the ABC transporters CDR1 and CDR2, which mediate azole resistance in Candida albicans. While a wild-type TAC1 allele drives high expression of CDR1/2 in response to inducers, we showed previously that TAC1 can be hyperactive by a gain-of-function (GOF) point mutation responsible for constitutive high expression of CDR1/2. High azole resistance levels are achieved when C. albicans carries hyperactive alleles only as a consequence of loss of heterozygosity (LOH) at the TAC1 locus on chromosome 5 (Chr 5), which is linked to the mating-type-like (MTL) locus. Both are located on the Chr 5 left arm along with ERG11 (target of azoles). In this work, five groups of related isolates containing azole-susceptible and -resistant strains were analyzed for the TAC1 and ERG11 alleles and for Chr 5 alterations. While recovered ERG11 alleles contained known mutations, 17 new TAC1 alleles were isolated, including 7 hyperactive alleles with five separate new GOF mutations. Single-nucleotide-polymorphism analysis of Chr 5 revealed that azole-resistant strains acquired TAC1 hyperactive alleles and, in most cases, ERG11 mutant alleles by LOH events not systematically including the MTL locus. TAC1 LOH resulted from mitotic recombination of the left arm of Chr 5, gene conversion within the TAC1 locus, or the loss and reduplication of the entire Chr 5. In one case, two independent TAC1 hyperactive alleles were acquired. Comparative genome hybridization and karyotype analysis revealed the presence of isochromosome 5L [i(5L)] in two azole-resistant strains. i(5L) leads to increased copy numbers of azole resistance genes present on the left arm of Chr 5, among them TAC1 and ERG11. Our work shows that azole resistance was due not only to the presence of specific mutations in azole resistance genes (at least ERG11 and TAC1) but also to their increase in copy number by LOH and to the addition of extra Chr 5 copies. With the combination of these different modifications, sophisticated genotypes were obtained. The development of azole resistance in C. albicans is therefore a powerful instrument for generating genetic diversity.

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.

Transcriptional profiling in Candida albicans reveals new adaptive responses to extracellular pH and functions for Rim101p

The human pathogen Candida albicans grows and colonizes sites that can vary markedly in pH. The pH response in C. albicans is governed in part  by the Rim101p pathway. In Saccharomyces cerevisiae, Rim101p promotes alkaline responses by repressing expression of NRG1, itself a transcriptional repressor. Our studies reveal that in C. albicans, Rim101p‐mediated alkaline adaptation is not through repression of CaNRG1. Furthermore, our studies suggest that Rim101p and Nrg1p act in parallel pathways to regulate hyphal morphogenesis, an important contributor to virulence. To determine the wild‐type C. albicans transcriptional response to acidic and alkaline pH, we utilized microarrays and identified 514 pH‐responsive genes. Of these, several genes involved in iron acquisition were upregulated at pH 8, suggesting that alkaline pH induces iron starvation. Microarray analysis of rim101–/– cells indicated that Rim101p does not govern transcriptional responses at acidic pH, but does regulate a subset of transcriptional responses at alkaline pH, including the iron acquisition genes. We found that rim101–/– cells are sensitive to iron starvation, which suggests that one important aspect of the Rim101p‐dependent alkaline pH response is to adapt to iron starvation conditions.

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 (FluR). Comparative genome hybridization arrays revealed that the presence of two extra copies of Chr5L, on the isochromosome, conferred increased FluR and that partial truncation of Chr5L reduced FluR. 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 FluR 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.

Acquisition of Aneuploidy Provides Increased Fitness during the Evolution of Antifungal Drug Resistance

The evolution of drug resistance is an important process that affects clinical outcomes. Resistance to fluconazole, the most widely used antifungal, is often associated with acquired aneuploidy. Here we provide a longitudinal study of the prevalence and dynamics of gross chromosomal rearrangements, including aneuploidy, in the presence and absence of fluconazole during a well-controlled in vitro evolution experiment using Candida albicans, the most prevalent human fungal pathogen. While no aneuploidy was detected in any of the no-drug control populations, in all fluconazole-treated populations analyzed an isochromosome 5L [i(5L)] appeared soon after drug exposure. This isochromosome was associated with increased fitness in the presence of drug and, over time, became fixed in independent populations. In two separate cases, larger supernumerary chromosomes composed of i(5L) attached to an intact chromosome or chromosome fragment formed during exposure to the drug. Other aneuploidies, particularly trisomies of the smaller chromosomes (Chr3–7), appeared throughout the evolution experiment, and the accumulation of multiple aneuploid chromosomes per cell coincided with the highest resistance to fluconazole. Unlike the case in many other organisms, some isolates carrying i(5L) exhibited improved fitness in the presence, as well as in the absence, of fluconazole. The early appearance of aneuploidy is consistent with a model in which C. albicans becomes more permissive of chromosome rearrangements and segregation defects in the presence of fluconazole.

Candida albicans hyphae have a Spitzenkörper that is distinct from the polarisome found in yeast and pseudohyphae.

Fungi grow with a variety of morphologies: oval yeast cells, chains of elongated cells called pseudohyphae and long, narrow, tube-like filaments called hyphae. In filamentous fungi, hyphal growth is strongly polarised to the tip and is mediated by the Spitzenkörper, which acts as a supply centre to concentrate the delivery of secretory vesicles to the tip. In the budding yeast Saccharomyces cerevisiae, polarised growth is mediated by the polarisome, a surface cap of proteins that nucleates the formation of actin cables delivering secretory vesicles to the growing tip. The human fungal pathogen, Candida albicans, can grow in all three morphological forms. Here we show the presence of a Spitzenkörper at the tip of C. albicans hyphae as a ball-like localisation of secretory vesicles, together with the formin Bni1 and Mlc1, an ortholog of an S. cerevisiae myosin regulatory light chain. In contrast, in C. albicans yeast cells, pseudohyphae and hyphae Spa2 and Bud6, orthologs of S. cerevisiae polarisome components, as well as the master morphology regulator Cdc42, localise predominantly, but not exclusively, to a surface cap resembling the polarisome of S. cerevisiae yeast cells. A small amount of Cdc42 also localises to the Spitzenkörper. Furthermore, we show differences in the genetic and cytoskeletal requirements, and cell cycle dynamics of polarity determinants in yeast, pseudohyphae and hyphae. These results, together with the cytological differences between the cell types, suggest that the Spitzenkörper and polarisome are distinct structures, that the polarisome and Spitzenkörper coexist in hyphae, and that polarised growth in hyphae is driven by a fundamentally different mechanism to that in yeast and pseudohyphae.