Origin of Cryptococcus neoformans var. neoformans Diploid Strains

doi:10.1128/JCM.39.11.3889-3894.2001

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Journal of Clinical Microbiology, November 2001, p. 3889-3894, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.3889-3894.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Origin of Cryptococcus neoformans var. neoformans Diploid Strains

Massimo Cogliati,¹ Maria C. Esposto,¹ David L. Clarke,² Brian L. Wickes,² and Maria A. Viviani¹^,^*

Laboratorio di Micologia Medica, Istituto di Igiene e Medicina Preventiva, Università degli Studi, IRCCS Ospedale Maggiore, 20122 Milan, Italy,¹ and Department of Microbiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900²

Received 8 May 2001/Returned for modification 18 June 2001/Accepted 12 August 2001

The basidiomycetous yeast Cryptococcus neoformans is an important human fungal pathogen. Two varieties, C. neoformans var.neoformans and C. neoformans var. gattii, have been identified.Both are heterothallic with two mating types, MATa and MAT $alpha$ . Somerare isolates are self-fertile and are considered occasional diploidor aneuploid strains. In the present study, 133 isolates, mostlyfrom Italian patients, were investigated to detect the presenceof diploid strains in the Igiene Università Milano culture collection.All of the diploid isolates were further investigated by differentmethods to elucidate their origins. Forty-nine diploid strainswere identified by flow cytometry. PCR fingerprinting using the(GACA)₄ primer showed that the diploid state was associated withtwo specific genotypes identified as VN3 and VN4. Determinationof mating type on V8 juice medium confirmed that the majorityof the strains were sterile. PCR and dot blotting using the twopheromone genes (MFa and MF $alpha$ ) as probes identified 36 of the 49diploid isolates as MATa/ $alpha$ . The results of pheromone gene sequencingshowed that two allelic MF $alpha$ genes exist and are distinct for serotypesA and D. In contrast, the MFa gene sequence was conserved in bothserotype alleles. Amplification of serotype-specific STE20 allelesdemonstrated that the diploid strains contained one mating locusinherited from a serotype A parent and one inherited from a serotypeD parent. The present results suggest that diploid isolates maybe common among the C. neoformans population and that in Italyand other European countries serotype A and D populations arenot genetically isolated but are able to recombine by sexualreproduction.

^* Corresponding author. Mailing address: Laboratorio di Micologia Medica, Istituto di Igiene e Medicina Preventiva, Universitàdegli Studi di Milano, Via Sforza 35, 20122 Milan, Italy. Phone:39 02 55188373. Fax: 39 02 55191561. E-mail: marianna.viviani{at}unimi.it.

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