Identification of an active RNAi pathway in Candida albicans.

RNA interference (RNAi) is a fundamental regulatory pathway with a wide range of functions, including regulation of gene expression and maintenance of genome stability. Although RNAi is widespread in the fungal kingdom, well-known species, such as the model yeast Saccharomyces cerevisiae, have lost the RNAi pathway. Until now evidence has been lacking for a fully functional RNAi pathway in Candida albicans, a human fungal pathogen considered critically important by the World Health Organization. Here, we demonstrated that the widely used C. albicans reference strain (SC5314) contains an inactivating missense mutation in the gene encoding for the central RNAi component Argonaute. In contrast, most other C. albicans isolates contain a canonical Argonaute protein predicted to be functional and RNAi-active. Indeed, using high-throughput small and long RNA sequencing combined with seamless CRISPR/Cas9-based gene editing, we demonstrate that an active C. albicans RNAi machinery represses expression of subtelomeric gene families. Thus, an intact and functional RNAi pathway exists in C. albicans, highlighting the importance of using multiple reference strains when studying this dangerous pathogen.

Stress combined with loss of the Candida albicans SUMO protease Ulp2 triggers selection of aneuploidy via a two-step process.

A delicate balance between genome stability and instability ensures genome integrity while generating genetic diversity, a critical step for evolution. Indeed, while excessive genome instability is harmful, moderated genome instability can drive adaptation to novel environments by maximising genetic variation. Candida albicans, a human fungal pathogen that colonises different parts of the human body, adapts rapidly and frequently to different hostile host microenvironments. In this organism, the ability to generate large-scale genomic variation is a key adaptative mechanism triggering dangerous infections even in the presence of antifungal drugs. Understanding how fitter novel karyotypes are selected is key to determining how C. albicans and other microbial pathogens establish infections. Here, we identified the SUMO protease Ulp2 as a regulator of C. albicans genome integrity through genetic screening. Deletion of ULP2 leads to increased genome instability, enhanced genome variation and reduced fitness in the absence of additional stress. The combined stress caused by the lack of ULP2 and antifungal drug treatment leads to the selection of adaptive segmental aneuploidies that partially rescue the fitness defects of ulp2Δ/Δ cells. Short and long-read genomic sequencing demonstrates that these novel genotypes are selected via a two-step process leading to the formation of novel chromosomal fragments with breakpoints at microhomology regions and DNA repeats.

On and Off: Epigenetic Regulation of C. albicans Morphological Switches.

The human fungal pathogen Candida albicans is a dimorphic opportunistic pathogen that colonises most of the human population without creating any harm. However, this fungus can also cause life-threatening infections in immunocompromised individuals. The ability to successfully colonise different host niches is critical for establishing infections and pathogenesis. C. albicans can live and divide in various morphological forms critical for its survival in the host. Indeed, C. albicans can grow as both yeast and hyphae and can form biofilms containing hyphae. The transcriptional regulatory network governing the switching between these different forms is complex but well understood. In contrast, non-DNA based epigenetic modulation is emerging as a crucial but still poorly studied regulatory mechanism of morphological transition. This review explores our current understanding of chromatin-mediated epigenetic regulation of the yeast to hyphae switch and biofilm formation. We highlight how modification of chromatin structure and non-coding RNAs contribute to these morphological transitions.

Candida pathogens induce protective mitochondria-associated type I interferon signalling and a damage-driven response in vaginal epithelial cells.

Vaginal candidiasis is an extremely common disease predominantly caused by four phylogenetically diverse species: Candida albicans; Candida glabrata; Candida parapsilosis; and Candida tropicalis. Using a time course infection model of vaginal epithelial cells and dual RNA sequencing, we show that these species exhibit distinct pathogenicity patterns, which are defined by highly species-specific transcriptional profiles during infection of vaginal epithelial cells. In contrast, host cells exhibit a homogeneous response to all species at the early stages of infection, which is characterized by sublethal mitochondrial signalling inducing a protective type I interferon response. At the later stages, the transcriptional response of the host diverges in a species-dependent manner. This divergence is primarily driven by the extent of epithelial damage elicited by species-specific mechanisms, such as secretion of the toxin candidalysin by C. albicans. Our results uncover a dynamic, biphasic response of vaginal epithelial cells to Candida species, which is characterized by protective mitochondria-associated type I interferon signalling and a species-specific damage-driven response.

Susceptibility to Medium-Chain Fatty Acids Is Associated with Trisomy of Chromosome 7 in Candida albicans.

Fatty acids have known antifungal effects and are used in over-the-counter topical treatments. Screening of a collection of gene knockouts in Candida albicans revealed that one strain, carrying a deletion of the transcription factor DAL81, is very susceptible to the medium-chain fatty acid undecanoic acid. However, reintroducing DAL81 does not restore resistance, and editing DAL81 in a different background does not introduce sensitivity. Whole-genome sequencing revealed that the C. albicansdal81Δ/Δ strain has an extra copy of chromosomes 5 and 7. Reversion to resistance to undecanoic acid was induced by growing the sensitive strain in yeast extract-peptone-dextrose with 60 µg/ml undecanoic acid for up to 9 days. Nine isolates that regained some resistance to undecanoic acid lost one copy of chromosome 7. The copy number of chromosome 5 does not appear to affect resistance to fatty acids. Moreover, the sensitivity may be related to having two copies of haplotype B of chromosome 7. In addition, we find that C. albicans strain SN152, used to delete DAL81 and many other genes, has undergone a major loss of heterozygosity event on chromosome 2 and a smaller one on chromosome 3.IMPORTANCE Aneuploidy (changes in chromosome number) and loss of heterozygosity (LOH) occur frequently in the human-pathogenic yeast Candida albicans and are associated with adaptation to stress and to antifungal drugs. Aneuploidy and LOH can also be induced during laboratory manipulations, such as during genetic transformation. We find that C. albicans strain SN152, commonly used to generate gene deletions, has undergone a major LOH event on chromosome 2. One deletion strain generated in this background has acquired extra copies of chromosomes 5 and 7. We find that trisomy (three copies) of chromosome 7 is associated with sensitivity to fatty acids.

Correlating Genotype and Phenotype in the Asexual Yeast Candida orthopsilosis Implicates ZCF29 in Sensitivity to Caffeine.

Candida orthopsilosis is diploid asexual yeast that causes human disease. Most C. orthopsilosis isolates arose from at least four separate hybridizations between related, but not identical, parents. Here, we used population genomics data to correlate genotypic and phenotypic variation in 28 C. orthopsilosis isolates. We used cosine similarity scores to identify 65 variants with potential high-impact (deleterious effects) that correlated with specific phenotypes. Of these, 19 were Single Nucleotide Polymorphisms (SNPs) that changed stop or start codons, or splice sites. One variant resulted in a premature stop codon in both alleles of the gene ZCF29 in C. orthopsilosis isolate 185, which correlated with sensitivity to nystatin and caffeine. We used CRISPR-Cas9 editing to introduce this polymorphism into two resistant C. orthopsilosis isolates. Introducing the stop codon resulted in sensitivity to caffeine and to ketoconazole, but not to nystatin. Our analysis shows that it is possible to associate genomic variants with phenotype in asexual Candida species, but that only a small amount of genomic variation can be easily explored.

Identification of an Exceptionally Long Intron in the HAC1 Gene of Candida parapsilosis.

The unfolded protein response (UPR) in the endoplasmic reticulum (ER) is well conserved in eukaryotes from metazoa to yeast. The transcription factor HAC1 is a major regulator of the UPR in many eukaryotes. Deleting HAC1 in the yeast Candida parapsilosis rendered cells more sensitive to DTT, a known inducer of the UPR. The deletion strain was also sensitive to Congo red, calcofluor white, and the antifungal drug ketoconazole, indicating that HAC1 has a role in cell wall maintenance. Transcriptomic analysis revealed that treatment of the wild type with DTT resulted in the increased expression of 368 genes. Comparison with mutant cells treated with DTT reveals that expression of 137 of these genes requires HAC1 Enriched GO term analysis includes response to ER stress, cell wall biogenesis and glycosylation. Orthologs of many of these are associated with UPR in Saccharomyces cerevisiae and Candida albicans Unconventional splicing of an intron from HAC1 mRNA is required to produce a functional transcription factor. The spliced intron varies in length from 19 bases in C. albicans to 379 bases in Candida glabrata, but has not been previously identified in Candida parapsilosis and related species. We used RNA-seq data and in silico analysis to identify the HAC1 intron in 12 species in the CTG-Ser1 clade. We show that the intron has undergone major contractions and expansions in this clade, reaching up to 848 bases. Exposure to DTT induced splicing of the long intron in C. parapsilosisHAC1, inducing the UPR.IMPORTANCE The unfolded protein response (UPR) responds to the build-up of misfolded proteins in the endoplasmic reticulum. The UPR has wide-ranging functions from fungal pathogenesis to applications in biotechnology. The UPR is regulated through the splicing of an unconventional intron in the HAC1 gene. This intron has been described in many fungal species and is of variable length. Until now it was believed that some members of the CTG-Ser1 clade such as C. parapsilosis did not contain an intron in HAC1, suggesting that the UPR was regulated in a different manner. Here we demonstrate that HAC1 plays an important role in regulating the UPR in C. parapsilosis We also identified an unusually long intron (626 bp) in C. parapsilosisHAC1 Further analysis showed that HAC1 orthologs in several species in the CTG-Ser1 clade contain long introns.