Genomic and population analyses of the mating type loci in Coccidioides species reveal evidence for sexual reproduction and gene acquisition

Abstract

Coccidioides species, the fungi responsible for the valley fever disease, are known to reproduce asexually through the production of arthroconidia that are the infectious propagules. The possible role of sexual reproduction in the survival and dispersal of these pathogens is unexplored. To determine the potential for mating of Coccidioides, we analyzed genome sequences and identified mating type loci characteristic of heterothallic ascomycetes. Coccidioides strains contain either a MAT1-1 or a MAT1-2 idiomorph, which is 8.1 or 9 kb in length, respectively, the longest reported for any ascomycete species. These idiomorphs contain four or five genes, respectively, more than are present in the MAT loci of most ascomycetes. Along with their cDNA structures, we determined that all genes in the MAT loci are transcribed. Two genes frequently found in common sequences flanking MAT idiomorphs, APN2 and COX13, are within the MAT loci in Coccidioides, but the MAT1-1 and MAT1-2 copies have diverged dramatically from each other. Data indicate that the acquisition of these genes in the MAT loci occurred prior to the separation of Coccidioides from Uncinocarpus reesii. An analysis of 436 Coccidioides isolates from patients and the environment indicates that in both Coccidioides immitis and C. posadasii, there is a 1:1 distribution of MAT loci, as would be expected for sexually reproducing species. In addition, an analysis of isolates obtained from 11 soil samples demonstrated that at three sampling sites, strains of both mating types were present, indicating that compatible strains were in close proximity in the environment.

FIG. 1.

Mating type idiomorphs of Coccidioides spp. (A) Schematic representation of the two mating type idiomorphs of Coccidioides are shown in the boxed regions. The horizontal lines to the left and right of the idiomorphs indicate the common flanking sequences. The 8,071-bp MAT1-1 idiomorph contains four predicted genes (gray arrows with white dots), and the 9,046-bp MAT1-2 idiomorph contains five predicted genes (white arrows with black dots). There are COX13 and APN2 genes in both idiomorphs; the levels of amino acid identity between the products of these idiomorph genes in C. immitis are indicated in brackets. (B) Sequence identity of the idiomorph-flanking regions of C. immitis strains RS and H538.4. The flanking regions 10 kb on either side of the idiomorphs are represented by horizontal lines. Notice the reduced identity in the proximal region of the left flank.

FIG. 2.

Determination of mating types of Coccidioides spp. PCR amplification of C. immitis strains RMSCC 3703 and RMSCC 2394 and C. posadasii strains RMSCC 1040, RMSCC 3488, RMSCC 1037, and Silveira with primers common to both mating types in the region to the left of the loci (+) and with idiomorph-specific primers (arrowheads below the MAT1-1-1 and MAT1-2-1 genes) revealed that strains have either the MAT1-1 (1-1) or MAT1-2 (1-2) mating type.

FIG. 3.

Transcription analysis of Coccidioides spp. mating type genes. (A) Schematic representation of the MAT1-1-1, MAT1-1-5, MAT1-1-6, MAT1-1-7, and MAT1-2-4 ORFs from C. posadasii strains RMSCC 1040 (MAT1-1) and Silveira (MAT1-2) and C. immitis strain RMSCC 3703 (MAT1-1). Shaded boxes represent exons within the ORFs. Boxes below the MAT1-1-6 and MAT1-1-7 genes indicate alternatively spliced transcripts in RMSCC 3703 and RMSCC 1040, respectively. Double arrows indicate PCR fragments amplified to demonstrate transcription. (B) Genomic DNA (g) and first-strand cDNA (c) from C. posadasii strains RMSCC 1040 and Silveira were amplified with primers that span one or two introns.

FIG. 4.

Phylogenetic trees of the COX13 and APN2 genes. Neighbor-joining trees for the COX13 (A) and APN2 (B) coding sequences found in each Coccidioides idiomorph indicate that the incorporation of these two genes into the mating type loci likely occurred in an ancestor common to both Uncinocarpus and Coccidioides. The numbers at the branches are percentages indicating the frequency at which each branch was obtained in the bootstrap analysis. CI, C. immitis; CP, C. posadasii.

FIG. 5.

Distribution of C. posadasii mating type idiomorphs among human isolates. (A) C. posadasii isolates from human patients sorted by date of collection. If the date was not available, strains were grouped as “unknown.” A trend toward greater numbers of MAT1-1 idiomorphs among the 2002 to 2005 data set was found; however, this difference was not significant (P = 0.08). (B) C. posadasii isolates from human patients sorted by infection type. If infection data were not available, strains were grouped as “undefined infection.”