Strain background of Candida albicans interacts with SIR2 to alter phenotypic switching

Abstract

The genetic background between strains of a single species and within a single strain lineage can significantly impact the expression of biological traits. This genetic variation may also reshape epigenetic mechanisms of cell identity and environmental responses that are controlled by interconnected transcriptional networks and chromatin-modifying enzymes. Histone deacetylases, including sirtuins, are critical regulators of chromatin state and have been directly implicated in governing the phenotypic transition between the 'sterile' white state and the mating-competent opaque state in Candida albicans, a common fungal commensal and pathogen of humans. Here, we found that a previously ambiguous role for the sirtuin SIR2 in C. albicans phenotypic switching is likely linked to the genetic background of mutant strains produced in the RM lineage of SC5314. SIR2 mutants in a specific lineage of BWP17 displayed increased frequencies of switching to the opaque state compared to the wild-type. Loss of SIR2 in other SC5314-derived backgrounds, including newly constructed BWP17 sir2Δ/Δ mutants, failed to recapitulate the increased white-opaque switching frequencies observed in the original BWP17 sir2Δ/Δ mutant background. Whole-genome sequencing revealed the presence of multiple imbalanced chromosomes and large loss of heterozygosity tracts that likely interact with SIR2 to increase phenotypic switching in this BWP17 sir2Δ/Δ mutant lineage. These genomic changes are not found in other SC5314-derived sir2Δ/Δ mutants that do not display increased opaque cell formation. Thus, complex karyotypes can emerge during strain construction that modify mutant phenotypes and highlight the importance of validating strain background when interpreting phenotypes.

Keywords: Candida; aneuploidy; epigenetics; white opaque switching.

Fig. 1.. Strain-specific loss of SIR2 increases white-to-opaque switching. (a) Construction diagram of SIR2 mutants in the BWP17 (yellow for the original set, orange for the CRISPR-derived set), SN152 (magenta) and CRISPR-derived SC5314 (cyan) backgrounds. MTL, mating type-like locus. (b) Opaque sectors and the corresponding cells are displayed from wild-type and sir2Δ/Δ mutants grown on solid SCD medium at room temperature for 7 days. ‘a’ and ‘α‘ indicate the MTL genotype. Grey arrows indicate opaque sectors. Scale bars, 10 µm. (c) One hundred cells were plated from three to six pure white colonies onto solid SCD medium and grown at room temperature for 7 days and the frequency of opaque sectors or colonies was quantified. Each dot represents an independent switching assay for the indicated MTL and SIR2 genotype and follows the same colouring scheme as the construction diagram. n≥3 biological replicates. *, P<0.05 (Kruskal–Wallis test with Dunn’s post-hoc against wild-type). **, P<0.01 (Kruskal–Wallis test with Dunn’s post-hoc against wild-type). WT, wild-type.

Fig. 2.. No evidence exists for altered SIR2 function or slow growth in the original BWP17 sir2Δ/Δ mutants. (a) Transcript abundance of four subtelomeric TLO genes and the non-subtelomeric gene TEF1 in the original BWP17 and SC5314 SIR2 wild-type and sir2Δ/Δ mutants was measured by qRT-PCR. Overnight cultures grown in liquid YPD medium at 30 °C were diluted into fresh liquid YPD medium and grown for 3–4 h at 30 °C prior to harvesting RNA. Expression was normalized to housekeeping gene ACT1 with a minimum of four biological replicates. *, P<0.05 (Mann–Whitney U test); **, P<0.01 (Mann–Whitney U test). (b) The doubling time of each strain was calculated in SCD liquid medium during logarithmic phase growth at room temperature. The doubling time and white–opaque switching frequency of each strain is plotted with whiskers indicating the standard deviation. n≥4 biological replicates. Each shape denotes the MTL configuration. The best fit line for all data points is given in black. For both panels, yellow denotes BWP17-derived strains, magenta denotes SN152 strains and cyan denotes SC5314-derived strains.

Fig. 3.. The original BWP17 sir2Δ/Δ mutants exhibit complex karyotypes. Whole-genome sequencing of each indicated strain was performed to an average depth of 150× and visualized using Y_MAP against Assembly 21 [70]. The height of the solid black bars indicates copy number in 10 kb bins (flat black line=2 N). Grey, cyan and magenta colours represent heterozygous, homozygous homologue A and homozygous homologue B regions, respectively. Blue indicates A/A/B allelic balance and purple indicates A/B/B allelic balance. Red indicates homozygous regions not matching either homologue.