Candida albicans TLOs and fitness: phenotypic analysis of a TLO null strain of C. albicans generated via CRISPR-Cas9 mutagenesis

Candida albicansis a fungus that is both a commensal and opportunistic pathogenic in humans. Genomic analysis has highlighted the expansion of the telomere associated ORF (TLO) gene family as unique in C. albicans. In C. albicans there are 15 different members of the TLO family present, compared to only two in its closest relative, C. dubliniensis. Here we show that deleting all TLOs from C. albicans reduces fitness and effects multiple phenotypes.

A guide RNA (gRNA) with sequence with homology to the TLOgenes was introduced to C. albicans AHY940 (aLEU2/Δleu2), along with the other components of the CRISPR-Cas9 (Nguyen et al. 2017).

The Δtlo strain was found to be generally pseudohyphal in morphology, compared to the wild type. It also showed defective growth in nutrient rich YEPD and YEP-Galactose. Growth on YEPD agar produced colonies similar in appearance to WT, however hyphal induction via growth on Spider agar was greatly reduced in the mutant strain compared to WT. Resistance to oxidative stress was examined and the Δtlostrain was more susceptible to stress induced by both H₂O₂ and tBOOH. In tests to determine the resistance of the Δtlostrain to cell wall perturbating compounds it was seen that this strain is much less resistant to Congo Red and Calcofluor white than WT. Biofilm formation on plastic surfaces also was reduced in the Δtlo strain compared to that of the WT.

Deletion of the TLO genes in C. albicans greatly impacts phenotypes associated with virulence, and generally results in a less fit strain of C. albicans.