Presence of alpha and a mating types in environmental and clinical collections of Cryptococcus neoformans var. gattii strains from Australia

Abstract

Cryptococcus neoformans var. gattii lives in association with certain species of eucalyptus trees and is a causative agent of cryptococcosis. It exists as two mating types, MATalpha and MATa, which is determined by a single-locus, two-allele system. In the closely related C. neoformans var. neoformans, the alpha mating type has been found to outnumber its a counterpart by at least 30:1, but there have been very limited data on the proportions of each mating type in C. neoformans var. gattii. In the present study, specific PCR primers were designed to amplify two separate alpha-mating-type genes from C. neoformans var. gattii strains. These were used to survey for the presence of the two mating types in clinical and environmental collections of C. neoformans var. gattii strains from Australia. Sixty-eight of 69 clinical isolates produced both alpha mating type-specific bands and were assumed to be of the alpha mating type. The majority of environmental isolates were also of the alpha mating type, but the a mating type was located in two separate areas. In one area, the a mating type outnumbered the alpha mating type by 27:2, but in the second area, the ratio of the two mating types was close to the 50:50 ratio expected for sexual recombination.

FIG. 1

Distribution of environmental and clinical isolates used in the study: 1, Balranald; 2, Hay; 3, Adelaide; 4, Gold Coast; 5, Renmark; 6, Sydney; 7, Coffs Harbour; 8, Pilliga; 9, Breza/Tamworth; 10, Port Macquarie; 11, West Wyalong; 12, Perth; 13, Dubbo; 14, Newcastle; 15, Alice Springs; 16, Darwin; 17, Townsville; 18, Melbourne; 19, Arnhem Land; 20, Brisbane; 21, Toowomba.

FIG. 2

Representative gel of DNA from clinical and environmental C. neoformans var. gattii isolates coamplified with the MFα and 660 primers. Lanes: 1 and 24, pGEM size marker (Promega); 2, GC5; 3, GC12; 4, GC17; 5, GC22; 6, GC27; 7, Ad5; 8, Ad12; 9, Ad17; 10, Ad22; 11, Ad26; 12, 1408; 13, 571 146; 14, 571 067; 15, 1409; 16, 571 178; 17, H28; 18, H23; 19, H13; 20, H8; 21, CBS 5757 (α); 22, CBS 6998 (a); 23, negative control for PCR amplification. The upper band of 216 bpl of 109 bp was amplified by the 660 primers; the lower band was amplified by the MFα primers. Primer dimer can be seen below many of the amplified fragments. Lanes 12, 15, and 18 show slight variation in size of 660 fragment.

FIG. 3

Representative gel of DNA from Balranald isolates coamplified by the MFα and 660 primers (a) and the STE12α primers (b). Lanes 1: pGEM size marker; 2, Bal 23; 3, Bal 24; 4, Bal 25; 5, Bal 26; 6, Bal 27; 7, Bal 28; 8, Bal 29; 9, Bal 30; 10, 401 Bal 2; 11, 402 Bal 6; 12, 402/1; 13, 403 Bal 8; 14, 405 Bal 2c; 15, 406 Bal 2d; 16, 407 Bal 2f; 17, 408 B1; 18, 409 B2; 19, 410 B5; 20, 404 H22b; 21, CBS 5757 (α); 22, CBS 6998 (a). Primer dimers can be seen in many of the lanes.

FIG. 4

Sequence alignments of the 109-bp MFα fragment with the MFα gene (GenBank accession no. S56460), which covers nucleotide positions 10 to 118 (a), and the 149-bp STE12α fragment with the corresponding segment of the STE12α GenBank sequence (accession no. AF012924), which covers nucleotide positions 1194 to 1343 (b). Both GenBank sequences were from C. neoformans var. neoformans isolates. The primer sequences are shown in bold, and asterisks indicate positions which are identical in all isolates.