Aneuploid Chromosomes Are Highly Unstable during DNA Transformation of Candida albicans

Abstract

Candida albicans strains tolerate aneuploidy, historically detected as karyotype alterations by pulsed-field gel electrophoresis and more recently revealed by array comparative genome hybridization, which provides a comprehensive and detailed description of gene copy number. Here, we first retrospectively analyzed 411 expression array experiments to predict the frequency of aneuploidy in different strains. As expected, significant levels of aneuploidy were seen in strains exposed to stress conditions, including UV light and/or sorbose treatment, as well as in strains that are resistant to antifungal drugs. More surprisingly, strains that underwent transformation with DNA displayed the highest frequency of chromosome copy number changes, with strains that were initially aneuploid exhibiting ϳ3-fold more copy number changes than strains that were initially diploid. We then prospectively analyzed the effect of lithium acetate (LiOAc) transformation protocols on the stability of trisomic chromosomes. Consistent with the retrospective analysis, the proportion of karyotype changes was highly elevated in strains carrying aneuploid chromosomes. We then tested the hypothesis that stresses conferred by heat and/or LiOAc exposure promote chromosome number changes during DNA transformation procedures. Indeed, a short pulse of very high temperature caused frequent gains and losses of multiple chromosomes or chromosome segments. Furthermore, milder heat exposure over longer periods caused increased levels of loss of heterozygosity. Nonetheless, aneuploid chromosomes were also unstable when strains were transformed by electroporation, which does not include a heat shock step. Thus, aneuploid strains are particularly prone to undergo changes in chromosome number during the stresses of DNA transformation protocols.

Phenotypic Consequences of a Spontaneous Loss of Heterozygosity in a Common Laboratory Strain of Candida albicans

“…The high degree of heterozygosity of the C. albicans genome, as exemplified by sequencing efforts of SC5314 and WO-1 (Butler et al 2009), can be irreversibly modified by LOH events. LOH occurs spontaneously with 10 26 events per cell per generation and is elevated by one to two orders of magnitude when cells are exposed to external stressors or undergo transformation protocols utilizing heat shock (Wellington and Rustchenko 2005;Bouchonville et al 2009;Forche et al 2011). During the construction of CAI4, cells were subject to two independent heat shocks with counterselections each (Fonzi and Irwin 1993) and subsequent derivatives constructed for additional auxotrophic markers were again exposed to such stresses (Alonso-Monge et al 2003;Noble and Johnson 2005).…”

“…Some 5-FOA R segregants had acquired the LR-LOH on Chr2L (indicated by SNP56 and SNP60 homozygosity) ( Figure S6B) but conserved the CAF4-2 MMS rsistance ( Figure S6C). This is likely a reflection of the intrinsic genomic instability of C. albicans, which has shown to be enhanced by stresses including 5-FOA (Wellington and Rustchenko 2005;Rustchenko 2007;Bouchonville et al 2009;Gerstein and Berman 2015) and from random LOH or mutational events . We infer that Chr2L homozygosis is neutral in isolation but can significantly enhance MMS sensitivity in conjunction with Chr3R LOH and is the first report of sign epistasis in C. albicans.…”

“…Locus SNF5 was PCR amplified from both CAF2-1 and CAI4 genomic DNAs using a set of primers (Table S2). Most primers used for SNP amplification are described in Forche et al 2009. Heterozygosity in the locus POL1 was detected by PCR amplification of a 360-bp internal fragment using primers 2021-F and 2380-R (sequences listed in Table S2) followed by digestion with endonuclease HincII (allele a cut, allele b no cut).…”

“…In C. albicans, the frequency of spontaneous LOH is 10 24 -10 26 (Forche et al 2009b;Lephart and Magee 2006) and increases significantly under stress conditions in vitro (Forche et al 2011), in vivo (Forche et al 2009b, or during molecular manipulations such as construction of modified laboratory strains (Selmecki et al 2006;Arbour et al 2009;Bouchonville et al 2009;Abbey et al 2011). Importantly, short-or long-range LOH events yield new genotypes and, potentially, new phenotypes that are heritable.…”

“…In animal cells, LR-LOH causes genetic instability, and along with aneuploidy, is associated with human disease and observed in .90% of solid tumors. C. albicans exploits these natural occurrences to generate new phenotypes, including but not limited to auxotrophy (Gómez-Raja et al 2008), mating proficiency (Magee and Magee 2000), and antifungal drug resistance (Selmecki et al 2006(Selmecki et al , 2008Niimi et al 2010;Sasse et al 2012).In C. albicans, the frequency of spontaneous LOH is 10 24 -10 26 (Forche et al 2009b;Lephart and Magee 2006) and increases significantly under stress conditions in vitro (Forche et al 2011), in vivo (Forche et al 2009b, or during molecular manipulations such as construction of modified laboratory strains (Selmecki et al 2006;Arbour et al 2009;Bouchonville et al 2009;Abbey et al 2011). Importantly, short-or long-range LOH events yield new genotypes and, potentially, new phenotypes that are heritable.…”