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MDrop-seq: Massively parallel single-cell RNA-seq of Saccharomyces Cerevisiae and Candida Albicans

ABSTRACT: We present microbial Drop-seq or mDrop-seq, a high-throughput scRNA-seq technique that is used on two yeast species, Saccharomyces cerevisiae, a popular model organism and Candida albicans, a common opportunistic pathogen. We benchmarked mDrop-seq for sensitivity and specificity and used it to profile 35,109 S. cerevisiae cells to detect variation in mRNA levels between them. As a proof of concept, we quantified expression differences in heat-shocked S. cerevisiae using mDrop-seq. We detected differential activation of stress response genes within a seemingly homogenous population of S. cerevisiae under heat-shock. We also applied mDrop-seq to C. albicans cells, a polymorphic and clinically relevant yeast species with thicker cell wall compared to S. cerevisiae. Single cell transcriptomes in 39,705 C. albicans cells was characterized using mDrop-seq under different conditions, including exposure to fluconazole, a common anti-fungal drug.

ORGANISM(S): Candida albicans Saccharomyces cerevisiae

PROVIDER: GSE154515 | GEO | 2020/07/22

REPOSITORIES: GEO

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Project description:6-Nonadecynoic acid (6-NDA), a plant-derived acetylenic acid, exhibits strong inhibitory activity against the human fungal pathogens Candida albicans, Aspergillus fumigatus, and Trichophyton mentagrophytes. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model organism Saccharomyces cerevisiae to investigate the mechanism of action of this compound. 6-NDA elicited a transcriptome response indicative of fatty acid stress, altering the expression of genes known to be affected when yeast cells are grown in the presence of oleate. Mutants of S. cerevisiae lacking transcription factors that regulate fatty acid ?-oxidation showed increased sensitivity to 6-NDA. Fatty acid profile analysis indicated that 6-NDA inhibited the formation of fatty acids longer than 14 carbons in length. In addition, the growth inhibitory effect of 6-NDA was rescued in the presence of exogenously supplied oleate. To investigate the response of a pathogenic fungal species to 6-NDA, transcriptional profiling and biochemical analyses were also conducted in C. albicans. The transcriptional response and fatty acid profile of C. albicans were comparable to those obtained in S. cerevisiae, and the rescue of growth inhibition with exogenous oleate was also observed in C. albicans. In addition, 6-NDA enhanced the potency of the antifungal drug fluconazole in a fluconazole-resistant clinical isolate of C. albicans. Collectively, our results indicate that the antifungal activity of 6-NDA is mediated by a disruption in fatty acid homeostasis, and that this compound has potential utility in combination therapy in the treatment of drug-resistant fungal infections. Cultures of S. cerevisiae strain S288C were started at OD600 of 0.1, allowed to grow to OD600 of 0.2, then treated with either DMSO (0.25%) or 6-NDA at the IC50 concentration (2.2 ug/ml). When OD600 of 0.5 was reached, cells were harvested and frozen. Two biological replicate cultures were grown for each treatment, and RNA from each replicate was hybridized to 2 independent arrays, resulting in 4 hybridizations for each treatment. Cultures of C. albicans strain SC5314 were started at OD600 of 0.1, allowed to grow to OD600 of 0.2, then treated with either DMSO (0.25%) or 6-NDA at the IC50 concentration (4 ug/ml). When OD600 of 0.5 was reached, cells were harvested and frozen. Three biological replicate cultures were grown for each treatment, and RNA from each replicate was hybridized as dye-swap pairs, resulting in 6 hybridizations for each treatment.

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MDrop-seq: Massively parallel single-cell RNA-seq of Saccharomyces Cerevisiae and Candida Albicans

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