Mechanistic plasticity of sexual reproduction and meiosis in the Candida pathogenic species complex

Abstract

Background: Candida species are microbial pathogens originally thought to be asexual, but several are now recognized as sexual or parasexual. Candida albicans, the most common fungus infecting humans, is an obligate diploid with a parasexual cycle involving mating, recombination, and genome reduction but no recognized meiosis. Others (C. lusitaniae, C. guilliermondii) are haploid, and their mating produces spores, suggestive of complete meiotic sexual cycles. However, comparative genomic analysis reveals that these species lack key meiotic components, including the recombinase Dmc1 and cofactors (Mei5/Sae3), synaptonemal-complex proteins (Zip1-Zip4/Hop1), and the crossover interference pathway (Msh4/5).

Results: Here we elucidate the structure and functions of the mating-type (MAT) locus and establish that C. lusitaniae undergoes meiosis during its sexual cycle. The MAT-encoded a2 (high-mobility group) and alpha1 (alpha domain) factors specify a and alpha cell identity, whereas the a1 homeodomain protein drives meiosis and sporulation and functions without its canonical heterodimeric partner, alpha2. Despite the apparent loss of meiotic genes, C. lusitaniae undergoes meiosis during sexual reproduction involving diploid intermediates, frequent SPO11-dependent recombination, and whole-genome reduction generating haploid progeny. The majority of meiotic progeny are euploid, but approximately one-third are diploid/aneuploid.

Conclusions: The cell identity and meiotic pathways have been substantially rewired, and meiotic generation of both recombinant and aneuploid progeny may expand genetic diversity. These findings inform our understanding of sexual reproduction in pathogenic microbes and the evolutionary plasticity of the meiotic machinery, with implications for the sexual nature of C. albicans and the generation and consequences of aneuploidy in biology and medicine.

Figure 1. Mating type (MAT) locus of C. lusitaniae and C. guilliermondii are syntenic with C. albicans

a, Alignment of the MAT loci from the sexual species C. lusitaniae and C. guilliermondii with that of the parasexual species C. albicans. The colored genes are contained within the MAT locus (PAP1, OBP1, PIK1,a1, a2, and α1), while the white/striped genes (HIP1, MAS2, and RCY1) flank the locus. The intron and exon structure of the C. guilliermondii and C. lusitaniae a1 and a2 genes are shown. Crosses denote the loss of either the a1 or α2 transcription factor genes. The C. lusitaniae MATα allele is 7741bp and the MAT a allele is 7389 bp. The C. guilliermondii MATα allele is 9228 bp and the MAT a allele is 9534 bp. An independent C. guilliermondii MAT a allele was sequenced and found to be identical in gene content to the Broad sequence strain. b, Pustell DNA scoring matrices (created with MacVector^®) demonstrating the gene rearrangement and sequence divergence between the MAT locus alleles of a single species. Gene identity flanking the locus is 97 – 99%, while genes within the locus share 60 – 80% identity. The MATα allele is plotted on the y-axis and the MAT a allele is plotted on the x-axis for each species.

Figure 2. α1 and a2 are required for mating and control of cell identity

a, Representative images of C. lusitaniae matings at 24 hours showing a conjugated cell pair in the wild-type mating. In crosses with α1 or a2 mutants, cells continue to bud as though an opposite mating type partner were not present, and no conjugating cells or zygotes were observed. Scale bar, 10 μm. b, Plate matings with each parent alone flanking a mating mixture in the center. After 96 hours, the matings were replica plated to media to select for recombinant progeny. No recombinant progeny were formed in either mutant mating. c, Northern blot showing the expression of the C. lusitaniae STE3 homolog (a pheromone receptor) and STE2 homolog (α pheromone receptor). All strains were grown on dilute PDA for 24 hours prior to harvesting and RNA purification. Two independent a2Δ and α1Δ mutants are shown. The expression of both pheromone receptors is induced during mating, however minimal induction is seen in a2Δ × WTα or WTa × α1Δ matings. d, Model for the control of haploid specific genes and cell identity. In both C. albicans and C. lusitaniae a2 controls a cell identity, the default cell-state in S. cerevisiae. In all three species, α1 controls alpha cell identity, and in S. cerevisiae α2 functions to repress a cell specific genes. In both S. cerevisiae and C. albicans the a1/α2 heterodimer functions to repress haploid specific genes and additionally to promote entry into meiosis in S. cerevisiae. In C. lusitaniae, a1 is necessary for sporulation and meiosis, but not conjugation and lacks the canonical partner α2. Heterozygosity at the MAT locus is necessary for sporulation, indicating a MATα component, such as α1, also promotes sporulation and meiosis. Panel D adapted from Tsong et al.[30].

Figure 3. Ploidy and CGH analysis reveal euploid, aneuploid, and diploid progeny

a, Representative FACS plots of parental and progeny strains. The FACS plot of parental haploid α strain JLR610 is shown. Of the 27 progeny typing as a and α for at least one RFLP loci, 6 strains were haploid by FACS (representative strain, progeny p2 shown), and 21 were diploid by FACS analysis (representative strain, progeny p33 shown). b, CGH analysis plots of log₂ ratio of medians for each experimental strain versus parental strain JLR610 for which the array was designed. The 8 chromosomes are plotted in order and separated by thick black lines. For the mating-type specific primers, if only one strain hybridized to a particular probe the log₂ ratio was set to -1 (α1) or 1 (a1 or a2). Traditional CGH analysis plots a scanning window average of several genes, however due to the low-density nature of this array each probe is plotted individually. Aneuploid strains were apparent based on an increased log₂ ratio for all probes along an entire chromosome. Shown from top to bottom are parental strain CL16, haploid progeny p1, aneuploid progeny p19, diploid progeny p5, and diploid (2N+1) progeny p33. α denotes the α1 probe, and aa donates the a1 and a2 probes.

Figure 4. Complete RFLP typing of CL16 × JLR610 F1 progeny

All strains were typed at each RFLP locus as possessing the allele of the α parent (JLR610) (1) or the allele of the a parent (CL16) (0), or both (2). The parental strains are indicated by a bold box on the left. The first 67 progeny are haploid by FACS analysis, the next 6 enclosed within the box were haploid (H) by FACS and found to be aneuploid, and the last 21 strains were diploid (D) by FACS analysis. The cycloheximide (CHX) resistance of all strains was also tested and strain were scored as either sensitive (S) or resistant (R). Chromosome 1, marker 1106 is ~40 kb from the putative centromere, and chromosome 6, marker 303 is ~20 kb from the putative centromere. The paucity of (0) alleles seen on chromosome 5 is due to the linkage of these markers to the URA3 allele, which was selected for in the initial cross. Thus all progeny were selected to inherit at least one copy of URA3 from the α parent. The configuration of the diploid/ aneuploid strains at the URA3 locus was evaluated by growth of strains on both SD-ura and 5-FOA medium. All strains were capable of growth on SD-ura medium and thus contained at least one copy of the URA3 allele as expected. However, some diploid/ aneuploid strains readily papillated on 5-FOA media (converting to a ura- state) suggesting that these strains contained a copy of the ura3 gene from the a parent and could easily lose the wild-type URA3 allele becoming 5-FOA resistant. Thus, the diploid/ aneuploid strains that readily papillated on 5-FOA were scored as 2 at this locus.