Direct PCR of Cryptococcus neoformans MATalpha and MATa pheromones to determine mating type, ploidy, and variety: a tool for epidemiological and molecular pathogenesis studies

Abstract

Cryptococcus neoformans MATalpha and MATa pheromones were amplified by direct PCR. Nucleotide sequence analyses revealed unique restriction enzyme sites. Sixty strains were used to devise a restriction fragment length polymorphism typing scheme that yielded three variety-specific patterns. Additionally, pheromone-specific PCR allowed easier identification of diploid C. neoformans strains than flow cytometry.

FIG. 1

C. neoformans pheromone fragments analyzed by PCR-RFLP to determine α and a mating types (A) and three varieties (B). (A) A φX174 DNA size marker was digested with HaeIII (lane i). Strain ATCC 28957 α mating type 101-bp amplicon (lane ii) was digested with EarI to reveal 50- and 51-bp bands migrating together (lane iii) or was digested with AluI to get 53- and 48-bp bands (lane iv); the 117-bp a mating type amplicon of strain ATCC 28958 (lane v) was digested with EarI to yield 71- and 46-bp fragments (lane vi) or was digested with HgaI to reveal 73- and 44-bp bands (lane vii). (B) A φX174 DNA size marker was digested with HaeIII (lane 1). All other lanes have the 101-bp α mating type amplicon. The gel shows the following: a strain NYSD 1649 amplicon (lane 2), an NYSD 1649 amplicon digested with Tsp45I to reveal 67- and 34-bp bands (lane 3) and digested with HpaII, yielding a visible 78-bp band and an invisible 23-bp band (lane 4); a strain ATCC 28957 amplicon (lane 5), an AluI digest with 53- and 48-bp bands (lane 6), an AciI digest yielding a 68-bp band and a faint 33-bp band (lane 7), and a strain ATCC 32609 amplicon (lane 8), no restriction with Tsp45I (lane 9), an HpaII digest with a 78-bp fragment and a 23-bp invisible fragment (lane 10), no restriction with AluI (lane 11), and an AciI digest with 65- and 36-bp bands (lane 12).

FIG. 2

Ploidy of three C. neoformans strains analyzed by flow cytometry (A) and PCR (B) for MATα/MATa. (A) Haploid strain NYSD 1649 with nuclear DNA fluorescence intensities distributed from channels 50 to 240, the first peak being near channel 85 and the second peak near channel 164 (graph i); diploid strain UM15 with fluorescence intensities distributed from channels 100 to 440 with two peaks near channels 160 and 388 (graph ii). Graph iii shows that the second peak of NYSD 1649 coincided with the first peak of UM15, thereby indicating a doubling of cellular DNA in the latter strain. (B) Control strains NYSD 1649 (MATα) and ATCC 28958 (MATa) are positive for one of the two pheromone PCR amplicons that correlate with haploid DNA; the diploid strain UM15 was positive for both MATα and MATa amplicons, which confirmed its diploidy as seen with flow cytometry.