Demonstration of Loss of Heterozygosity by Single-Nucleotide Polymorphism Microarray Analysis and Alterations in Strain Morphology in Candida albicans Strains during Infection

Abstract

Candida albicans is a diploid yeast with a predominantly clonal mode of reproduction, and no complete sexual cycle is known. As a commensal organism, it inhabits a variety of niches in humans. It becomes an opportunistic pathogen in immunocompromised patients and can cause both superficial and disseminated infections. It has been demonstrated that genome rearrangement and genetic variation in isolates of C. albicans are quite common. One possible mechanism for generating genome-level variation among individuals of this primarily clonal fungus is mutation and mitotic recombination leading to loss of heterozygosity (LOH). Taking Candida albicans is a commensal diploid yeast and inhabits a variety of niches in human populations. An opportunistic pathogen, it can cause both superficial and disseminated infections. The recently published complete diploid genome sequence of SC5314, a clinical isolate, showed a high degree of heterozygosity, including more than 55,700 single-nucleotide polymorphisms (SNPs) in the 32-Mb diploid genome (21). Clinical isolates of C. albicans show wide variations in karyotype, suggesting that genome rearrangement is relatively common in the host (30). Although C. albicans can be made to mate in the laboratory, there is no evidence for a complete sexual cycle either in vivo or in vitro (4,20,29) and the importance of recombination and rearrangement in generating genetic variation is not clear.One possible mechanism for generating genome-level variation among individuals of this primarily clonal fungus is mutation and mitotic recombination leading to loss of heterozygosity (LOH). For example, Tavanti et al. (44) found evidence for the recombination generating a heterogenous array of haplotypes in clinical isolates of C. albicans. However, the frequency or rate of recombination, and thus its importance, cannot be evaluated by analysis of static haplotypes alone. To that end, we used strains heterozygous at the GAL1 locus to demonstrate that mitotic recombination occurs at a measurable level during the course of experimental infections of mice (15, 16).Counter-selectable markers such as GAL1 are powerful but of limited utility for studying genome-wide recombination, chromosomal dynamics, or genome rearrangement. For these purposes, we turned to the use of SNP markers. SNPs are the most frequently observed differences at the DNA sequence level across individuals and chromosomes of diploid organisms and thus can be used to study processes involved in the evolution of virulence in C. albicans. To examine LOH across the entire genome, we recently developed an SNP map for strain SC5314 containing 150 marker sequences comprising 561 SNPs and 9 indels (15). The SNPs are spaced about 100 kb apart across the eight chromosomes.In this paper, we report the development and use of SNP markers, in a microarray format, on the basis of hybridization of allele-specific oligonucleotides to PCR-generated probes from test and control (SC5314) genomes. Our approach is similar to that employed for the high-through...

Evolution inCandida albicansPopulations During a Single Passage Through a Mouse Host

“…The AF values are normalized to give an expected value of 0.5 for heterozygous loci. The distribution of AF values for homozygous and heterozygous loci and the cutoff values to score homozygous states were as described previously (Forche et al 2005). For each individual SNP locus, AF values between 0.40 and 0.60, inclusive, were scored as heterozygous, AF values ,0.40 were scored as allele 1 homozygous, and AF values .0.60 were scored as allele 2 homozygous.…”

“…The method accounts for differences of in vitro and in vivo growth rates and permits direct comparison of LOH rates at GAL1. SNP microarray analysis: We expanded a previously described SNP microarray (Forche et al 2005), adding 98 SNP loci to cover a total of 123 SNP loci (Table 1). The SNP loci are positioned $100 kb apart across the eight C. albicans chromosomes except for regions of low polymorphism in the fully sequenced genome of strain SC5314 (Chr3 right arm, Chr7 left arm, and telomere distal regions of ChrR (van het Hoog et al 2007).…”

“…The SNP genotypes were determined using the allelic fraction (AF), which describes the relative intensity of the control probe (SC5314: labeled with Cy5) compared to the experimental probe (test strain: labeled with Cy3) in competitive hybridizations to the microarray according to the protocol detailed in Forche et al (2005). The AF values are normalized to give an expected value of 0.5 for heterozygous loci.…”

“…The SNP loci are positioned $100 kb apart across the eight C. albicans chromosomes except for regions of low polymorphism in the fully sequenced genome of strain SC5314 (Chr3 right arm, Chr7 left arm, and telomere distal regions of ChrR (van het Hoog et al 2007). Design of allele-specific oligonucleotides, probe generation, slide preparation and hybridization, and data analysis was performed as described earlier (Forche et al 2005). Four multiplex PCR reactions with 28-34 primer pairs/ reaction (Table S1) were carried out for each C. albicans isolate, using 60 ng of genomic DNA and a Qiagen Multiplex PCR kit following the manufacturer's instructions (Qiagen, Valencia, CA).…”

“…Finally, we determined the rates and types of phenotypic variation in colony growth that arose during in vivo and in vitro propagation. To conduct the analyses, we exploited the counterselectable marker GAL1, measured recombination as LOH using genomewide SNPs, and evaluated changes in chromosome copy number using competitive genome hybridization (CGH) (Forche et al 2005;Selmecki et al 2005). We found fivefold lower population growth rates and distinctly different genome dynamics arising in response to growth in vivo compared to growth in vitro.…”