The emerging human fungal pathogen Candida auris has been recognized as a multidrug resistant species and is associated with high mortality. This fungus was first described in Japan in 2009 and has been reported in at least 18 countries on five continents. In this study, we report the first isolate of C. auris from the bronchoalveolar lavage fluid (BALF) of a hospitalized woman in China. Interestingly, this isolate is susceptible to all tested antifungals including amphotericin B, fluconazole, and caspofungin. Copper sulfate (CuSO4) also has a potent inhibitory effect on the growth of this fungus. Under different culture conditions, C. auris exhibits multiple morphological phenotypes including round-to-ovoid, elongated, and pseudohyphal-like forms. High concentrations of sodium chloride induce the formation of a pseudohyphal-like form. We further demonstrate that C. auris is much less virulent than Candida albicans in both mouse systemic and invertebrate Galleria mellonella models.

Morphological plasticity has historically been an indicator of increased virulence among fungal pathogens, allowing rapid adaptation to changing environments. Candida auris has been identified as an emerging multidrug-resistant human pathogen of global importance. Since the discovery of this species, it has been thought that C. auris is incapable of filamentous growth. Here, we report the discovery of filamentation and three distinct cell types in C. auris: typical yeast, filamentation-competent (FC) yeast, and filamentous cells. These cell types form a novel phenotypic switching system that contains a heritable (typical yeast-filament) and a nonheritable (FC-filament) switch. Intriguingly, the heritable switch between the typical yeast and the FC/filamentous phenotype is triggered by passage through a mammalian body, whereas the switch between the FC and filamentous phenotype is nonheritable and temperature-dependent. Low temperatures favor the filamentous phenotype, whereas high temperatures promote the FC yeast phenotype. Systemic in vivo and in vitro investigations were used to characterize phenotype-specific variations in global gene expression, secreted aspartyl proteinase (SAP) activity, and changes in virulence, indicating potential for niche-specific adaptations. Taken together, our study not only sheds light on the pathogenesis and biology of C. auris but also provides a novel example of morphological and epigenetic switching in fungi.

Candida auris is a newly emerging fungal pathogen of humans and has attracted considerable attention from both the clinical and basic research communities. Clinical isolates of C. auris are often resistant to one or more antifungal agents. To explore how antifungal resistance develops, we performed experimental evolution assays in the current study using a fluconazole-susceptible isolate of C. auris (BJCA001). After a series of passages through medium containing increased concentrations of fluconazole, fungal cells acquired resistance. By sequencing and comparing the genomes of the parental fluconazole-susceptible strain and 26 experimentally evolved strains of C. auris, we found that a portion of fluconazole-resistant strains carried one extra copy of Chromosome V. In the absence of fluconazole, C. auris cells rapidly became susceptible and lost the extra copy of Chromosome V. Genomic and RNA-Seq analyses indicate that this chromosome carries a number of drug resistance-related genes, which were transcriptionally up-regulated in the resistant, aneuploid strains. Moreover, missense mutations were identified in genes TAC1B, RRP6, and SFT2 in all experimentally evolved strains. Our findings suggest that the gain of an extra copy of chromosome V is associated with the rapid acquisition of fluconazole-resistance and may represent an important evolutionary mechanism of antifungal resistance in C. auris.

The conserved cAMP-dependent protein kinase (PKA) is composed of the regulatory and catalytic subunits and acts as the central component of the cAMP signaling pathway. In the human fungal pathogen Candida albicans, the PKA regulatory subunit Bcy1 plays a critical role in the regulation of cell differentiation and death. It has long been considered that Bcy1 is essential for cell viability in C. albicans. In the current study, surprisingly, we found that Bcy1 is not required for cell growth, and we successfully generated a bcy1/bcy1 null mutant in C. albicans. Deletion of BCY1 leads to multiple cellular morphologies and promotes the development of filaments. Filamentous and smooth colonies are two typical morphological types of the bcy1/bcy1 mutant, which can undergo spontaneous switching between the two types. Cells of filamentous colonies grow better on a number of different culture media and have a higher survival rate than cells of smooth colonies. In addition, deletion of BCY1 significantly increased the frequency of white-to-opaque switching on N-acetylglucosamine (GlcNAc)-containing medium. The bcy1/bcy1 null mutant generated herein provides the field a new resource to study the biological functions of the cAMP signaling pathway in C. albicans.

While sexual reproduction is pervasive in eukaryotic cells, the strategies employed by fungal species to achieve and complete sexual cycles is highly diverse and complex. Many fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, are homothallic (able to mate with their own mitotic descendants) because of homothallic switching (HO) endonuclease-mediated mating-type switching. Under laboratory conditions, the human fungal pathogen Candida albicans can undergo both heterothallic and homothallic (opposite- and same-sex) mating. However, both mating modes require the presence of cells with two opposite mating types (MTLa/a and α/α) in close proximity. Given the predominant clonal feature of this yeast in the human host, both opposite- and same-sex mating would be rare in nature. In this study, we report that glucose starvation and oxidative stress, common environmental stresses encountered by the pathogen, induce the development of mating projections and efficiently permit same-sex mating in C. albicans with an “a” mating type (MTLa/a). This induction bypasses the requirement for the presence of cells with an opposite mating type and allows efficient sexual mating between cells derived from a single progenitor. Glucose starvation causes an increase in intracellular oxidative species, overwhelming the Heat Shock transcription Factor 1 (Hsf1)- and Heat shock protein (Hsp)90-mediated stress-response pathway. We further demonstrate that Candida TransActivating protein 4 (Cta4) and Cell Wall Transcription factor 1 (Cwt1), downstream effectors of the Hsf1–Hsp90 pathway, regulate same-sex mating in C. albicans through the transcriptional control of the master regulator of a-type mating, MTLa2, and the pheromone precursor-encoding gene Mating α factor precursor (MFα). Our results suggest that mating could occur much more frequently in nature than was originally appreciated and that same-sex mating could be an important mode of sexual reproduction in C. albicans.

A striking feature of pathogenic Candida species is morphological plasticity that facilitates environmental adaptation and host infection. Candida auris is an emerging multidrug-resistant fungal pathogen first described in Japan in 2009. In this study, we demonstrate that clinical isolates of C. auris have multiple colony and cellular morphologies including the yeast, filamentous, aggregated, and elongated forms. This phenotypic diversity has been observed in eight clinical isolates of C. auris representing four major genetic clades, suggesting that it could be a general characteristic. We further demonstrate that different cell types of C. auris exhibit distinct antifungal resistance and virulence properties in a Galleria mellonella infection model. Our findings imply that morphological diversity is an important biological feature of C. auris and could be a contributor to its emergence and rapid prevalence worldwide. Lay Summary Candida auris is an emerging multidrug-resistant fungal pathogen. Morphological analyses indicate that filamentation is a general feature of clinical isolates of C. auris. This ability is associated with antifungal resistance and virulence.

Phenotypic switching is a strategy by which microbial organisms adapt to environmental changes. The human fungal pathogens, Candida albicans and Candida tropicalis, are closely related species and capable of undergoing morphological transitions. C. albicans primarily exists in human or warm-blooded animals as a commensal, whereas C. tropicalis not only exists as a commensal but also is widely distributed in the environment. In this study, we describe the environmental and genetic regulatory mechanisms of white-opaque switching in C. tropicalis, which is associated with virulence and sexual mating. A comparative study with C. albicans demonstrated that C. tropicalis responds to environmental stimuli, such as elevated CO levels and pH changes, in opposite manners. An acidic pH and elevated CO levels promote the opaque phenotype in C. albicans but have an opposite effect in C. tropicalis, whereas alkaline pH conditions facilitate white-to-opaque switching and sexual mating in C. tropicalis. The conserved Rim101-mediated pH sensing and Ras1-cAMP/PKA signaling pathways are involved in this regulation. By screening an overexpression library of transcription factors, we identified 26 white-opaque regulators, including WOR1, AHR1, EFG1, CUP9, BCR1 and SFL2. Transcriptional analysis indicated that the pH sensing and Ras1-cAMP/PKA signaling pathways and transcriptional regulators coordinately regulate white-to-opaque switching.

Sexual reproduction is a universal mechanism for generating genetic diversity in eukaryotes. Fungi exhibit diverse strategies for sexual reproduction both in nature and in the laboratory. In this study, we report the discovery of same-sex (homothallic) mating in the human fungal pathogen Candida tropicalis. We show that same-sex mating occurs between two cells carrying the same mating type (MTLa/a or α/α) and requires the presence of pheromone from the opposite mating type as well as the receptor for this pheromone. In ménage à trois mating mixes (i.e., “a x a + α helper” or “α x α + a helper” mixes), pheromone secreted by helper strains promotes diploid C. tropicalis cells to undergo same-sex mating and form tetraploid products. Surprisingly, however, the tetraploid mating products can then efficiently mate with cells of the opposite mating type to generate hexaploid products. The unstable hexaploid progeny generated from this coupled process of same- and opposite-sex mating undergo rapid chromosome loss and generate extensive genetic variation. Phenotypic analysis demonstrated that the mating progeny-derived strains exhibit diverse morphologies and phenotypes, including differences in secreted aspartic proteinase (Sap) activity and susceptibility to the antifungal drugs. Thus, the coupling of same- and opposite-sex mating represents a novel mode to generate polyploidy and genetic diversity, which may facilitate the evolution of new traits in C. tropicalis and adaptation to changing environments.