Experimental evolution ofCandida albicansunder hypoxia and heat shock reveals nuclear genome variants and mitochondrial methylome alterations

Abstract

Infection by Candida albicans requires its adaption to physical constraints in the human body, such as low oxygen tension (hypoxia), increased temperature (37°C) and different carbon sources. Previous studies demonstrated that the genetic variability of C. albicans isolates is an important adaptive mechanism, although little is known about the dynamics of this genetic diversity, and the influence of these environmental conditions on its mitochondrial genome (mtDNA). To test the synergistic effect of these stress conditions on C. albicans genome, reference strain SC5314 was subjected to an in vitro evolution scheme under hypoxia and 37°C, with two different carbon sources (glycerol and dextrose) for up to 48 weeks (approximately 4,000 generations). Experimental evolution results showed no sequence or copy number changes in the mtDNA, although sequence variants were detected on its nuclear genome by Multilocus sequence typing (MLST) and whole genome sequencing (WGS). After 12 weeks of experimental evolution, sample GTH12, grown under hypoxia at 37°C in glycerol, showed inferior growth and respiratory rates as compared to other conditions tested. Although WGS of GTH12 revealed no variants in its mtDNA, WGS with sodium bisulfite showed a significant reduction in mtDNA methylation in GTH12 in both non-coding and coding regions.Our results provide the first whole mitochondrial genome methylation map of C. albicans and show that environmental conditions promote the selective growth of specific variants and affect the methylation patterns of the mtDNA in a strain-specific manner.

Experimental evolution and genome data analysis ofCandida albicansreveals cryptic bacteria in single yeast colonies

“…An in vitro evolution model was developed by our research group (Bartelli et al, 2017) and consists in serial culturing of C. albicans yeasts (strain SC5314) for prolonged periods under hypoxia, YPG medium (non-fermentative) at 37°C, (GTH strains). Results from this experiment showed a gradual increase in bacterial cells in aging cultures ( Figure 4).…”

“…Among the five different C. albicans strains tested, L757 was the only that did not show sufficient 16S rRNA gene amplification after 8 days ( Figure 3). However, after 12 weeks of continuous growth in YPG medium at 37°C under hypoxic condition, named GTH (Bartelli et al, 2017), it was possible to amplify 16S rRNA for sequencing and species identification. Sequencing of the amplified 16S rRNA gene from total DNA extracted from cultures and BLASTn searches in the 16S ribosomal RNA sequences database, revealed different bacterial species (Table 1).…”

“…Once a week, 0.1% of cell cultures were transferred to fresh 10mL medium for created an evolution in vitro. To generate a hypoxic environment, cells were maintained in hermetic closed jars with a hypoxia generator (Microaerobac, Probac do Brasil) with an atmosphere of 5-15% O2, while cells under normoxia were kept under 150 rpm agitation with O2 levels close to 21% (Bartelli et al, 2017). The other strains 9117, ATCC 24433 and ATCC 90029 of C. albicans, grown on YPD broth at 28°C, 150 rpm for 48 hours, only to be performed to antifungal susceptibility testing.…”