Evolution of Fluconazole-Resistant Candida albicans Strains by Drug-Induced Mating Competence and Parasexual Recombination

Abstract

The clonal population structure of Candida albicans suggests that (para)sexual recombination does not play an important role in the lifestyle of this opportunistic fungal pathogen, an assumption that is strengthened by the fact that most C. albicans strains are heterozygous at the mating type locus (MTL) and therefore mating-incompetent. On the other hand, mating might occur within clonal populations and allow the combination of advantageous traits that were acquired by individual cells to adapt to adverse conditions. We have investigated if parasexual recombination may be involved in the evolution of highly drug-resistant strains exhibiting multiple resistance mechanisms against fluconazole, an antifungal drug that is commonly used to treat infections by C. albicans. Growth of strains that were heterozygous for MTL and different fluconazole resistance mutations in the presence of the drug resulted in the emergence of derivatives that had become homozygous for the mutated allele and the mating type locus and exhibited increased drug resistance. When MTLa/a and MTLα/α cells of these strains were mixed in all possible combinations, we could isolate mating products containing the genetic material from both parents. The initial mating products did not exhibit higher drug resistance than their parental strains, but further propagation under selective pressure resulted in the loss of the wild-type alleles and increased fluconazole resistance. Therefore, fluconazole treatment not only selects for resistance mutations but also promotes genomic alterations that confer mating competence, which allows cells in an originally clonal population to exchange individually acquired resistance mechanisms and generate highly drug-resistant progeny. IMPORTANCE Sexual reproduction is an important mechanism in the evolution of species, since it allows the combination of advantageous traits of individual members in a population. The pathogenic yeast Candida albicans is a diploid organism that normally propagates in a clonal fashion, because heterozygosity at the mating type locus (MTL) inhibits mating between cells. Here we show that C. albicans cells that have acquired drug resistance mutations during treatment with the commonly used antifungal agent fluconazole rapidly develop further increased resistance by genome rearrangements that result in simultaneous loss of heterozygosity for the mutated allele and the mating type locus. This enables the drug-resistant cells of a population to switch to the mating-competent opaque morphology and mate with each other to combine different individually acquired resistance mechanisms. The tetraploid mating products reassort their merged genomes and, under selective pressure by the drug, generate highly resistant progeny that have retained the advantageous mutated alleles. Parasexual propagation, promoted by stress-induced genome rearrangements that result in the acquisition of mating competence in cells with adaptive mutations, may therefore be an important mechanism in the evolution of C. albicans populations.

Candida albicans exhibits distinct cytoprotective responses to anti-fungal drugs that facilitate the evolution of drug resistance

“…This data, and additional whole genome sequencing (single cell or bulk) of survivor populations, would allow us to distinguish between epigenetic versus genetic survival mechanisms. The next phase of our work will be to combine the transcriptomic data with whole genome DNA sequencing to identify plausible molecular mechanisms for rapid evolution perhaps based on genomic neoplasticity and instability [18][19][20]22,24,25,69 .…”

“…Importantly, recent unbiased genomic profiling of 43 clinical isolates identified loss of heterozygosity events and single nucleotide polymorphisms in over 240 genes involved in adherence, filamentation, virulence and other processes, suggesting that genetic acquired resistance may be achieved in many ways including via genomic instability 17 . There is now a substantial literature suggesting that C. albicans , like S. cerevisiae , generate large scale genomic variation as a means of adaptation [18][19][20][21][22][23] , and there is some evidence that this could be facilitated by the parasexuality of the fungus 18,20,[24][25][26][27] .…”

Sexual Processes in Microbial Eukaryotes

“…Instead a reduction from four copies of the genome (tetraploidy) to two copies (diploidy) occurs by random chromosome loss during the mitotic cell division subsequent to meiotic recombination [37]. Although C. albicans populations are largely clonal, parasexual recombination can facilitate the evolution of resistance to the antifungal agent fluconazole upon exposure to the agent over successive generations [38].…”

“…This masking benefit of out-crossing is generally recognized as underlying such concepts as heterosis, hybrid vigor or the avoidance of "inbreeding depression" [81]. Also, sexual processes can produce genetic variation that may be beneficial, as in the case of C. albicans populations where parasexual recombination can apparently facilitate, over successive generations, the evolution of resistance to the antifungal agent fluconazole [38].…”

Ploidy determines the consequences of antifungal-induced mutagenesis in Candida albicans, a human fungal pathogen

“…However in vitro, tetraploids rapidly, yet transiently, arise in response to high doses of fluconazole by decoupling cell growth and DNA replication, leading to trimeric structures and defects in budding (Harrison et al 2014). Tetraploids can also arise in vitro via mating and potentially combine drug-resistant alleles that arose independently in diploid cells to generate mating products with even greater increases in their fluconazole MIC, (Popp et al 2019).…”