, Volume 173, Issue 5–6, pp 347–357 | Cite as

Heterozygosis and Pathogenicity of Cryptococcus neoformans AD-Hybrid Isolates

  • M. Cogliati
  • F. Barchiesi
  • E. Spreghini
  • Anna Maria Tortorano


Nineteen Cryptococcus neoformans AD-hybrid isolates were investigated to assess whether hybrid genomic background could affect virulence in a mouse model. The level of heterozygosity of each strain was analyzed using primers specific for allele A and D of 15 polymorphic genes. Virulence was tested in a mouse model of systemic infection by measuring time of survival. In addition, the putative virulence attributes, melanin, phospholipase, and capsule production, as well as growth at 39°C and UV sensitivity were investigated. Eight strains showed to be heterozygous in up to 70% of loci, other eight strains were heterozygous in less than 60% of loci, while the remaining three strains were homozygous at all tested loci. Mice infected with hybrids with a high percentage of heterozygosis showed significantly (P < 0.01) shorter survival than mice infected with the other hybrids. Mortality was not correlated with the mating-type locus pattern, as well as it was not correlated with the level of expression of the different virulence attributes investigated. The present study confirms that hybridization in C. neoformans could represent an important evolutionary driving force in increasing the fitness of this yeast in the environment and in the host.


Cryptococcusneoformans AD hybrids Heterozygosis Virulence factors Mating type 


  1. 1.
    Heitman J, Kozel R, Kwon-Chung KJ, Perfect JR, Casadevall A. Cryptococcus. From human pathogen to model yeast. Washington, DC: ASM Press; 2011.Google Scholar
  2. 2.
    Capoor MR, Mandal P, Deb M, Aggarwal P, Banerjee U. Current scenario of cryptococcosis and antifungal susceptibility pattern in India: a cause for reappraisal. Mycoses. 2008;51(3):258–65.PubMedCrossRefGoogle Scholar
  3. 3.
    Hakim JG, Gangaidzo IT, Heyderman RS, Mielke J, Mushangi E, Taziwa A, et al. Impact of HIV infection on meningitis in Harare, Zimbabwe: a prospective study of 406 predominantly adult patients. AIDS. 2000;14:1401–7.PubMedCrossRefGoogle Scholar
  4. 4.
    Helbok R, Pongpakdee S, Yenjun S, Dent W, Beer R, Lackner P, et al. Chronic meningitis in Thailand—Clinical characteristics, laboratory data and outcome in patients with specific reference to tuberculosis and cryptococcosis. Neuroepidemiol. 2006;26:37–44.CrossRefGoogle Scholar
  5. 5.
    Ikeda R, Shinoda T, Fukazawa Y, Kaufman L. Antigenic characterization of Cryptococcus neoformans serotypes and its application to serotyping of clinical isolates. J Clin Microbiol. 1982;16:22–9.PubMedGoogle Scholar
  6. 6.
    Viviani MA, Wen H, Roverselli A, Caldarelli-Stefano R, Cogliati M, Ferrante P, et al. Identification by polymerase chain reaction fingerprinting of Cryptococcus neoformans serotype AD. J Med Vet Mycol. 1997;35:355–60.PubMedCrossRefGoogle Scholar
  7. 7.
    Boekhout T, Theelen B, Diaz M, Fell JW, Hop WCJ, Abeln ECA, et al. Hybrid genotypes in the pathogenic yeast Cryptococcus neoformans. Microbiology. 2001;147:891–907.PubMedGoogle Scholar
  8. 8.
    Meyer W, Castaneda A, Jackson S, Huynh M, Castaneda E. IberoAmerican Cryptococcal study group. Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerg Infect Dis. 2003;9:189–95.PubMedGoogle Scholar
  9. 9.
    Franzot SP, Salkin IF, Casadevall A. Cryptococcus neoformans var. grubii: separate varietal status for Cryptococcus neoformans serotype A isolates. J Clin Microbiol. 1999;37:838–40.PubMedGoogle Scholar
  10. 10.
    Meyer W, Aanensen DM, Boekhout T, Cogliati M, Diaz MR, Esposto MC, et al. Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii. Med Mycol. 2009;47:561–70.PubMedCrossRefGoogle Scholar
  11. 11.
    Cogliati M, Esposto MC, Clarke DL, Wickes BL, Viviani MA. Origin of Cryptococcus neoformans var. neoformans diploid strains. J Clin Microbiol. 2001;39:3889–94.PubMedCrossRefGoogle Scholar
  12. 12.
    Lengeler KB, Cox GM, Heitman J. Serotype AD strains of Cryptococcus neoformans are diploid or aneuploid and are heterozygous at the mating-type locus. Infect Immun. 2001;69:115–22.PubMedCrossRefGoogle Scholar
  13. 13.
    Viviani MA, Antinori S, Cogliati M, Esposto MC, Pinsi G, Casari S, et al. European Confederation of Medical Mycology (ECMM) prospective survey of cryptococcosis: report from Italy. Med Mycol. 2002;40:507–17.Google Scholar
  14. 14.
    Viviani MA, Cogliati M, Esposto MC, Lemmer K, Tintelnot K, Valiente MFC, et al. Molecular analysis of 311 Cryptococcus neoformans isolates from a 30-month ECMM survey of cryptococcosis in Europe. FEMS Yeast Res. 2006;6:614–9.PubMedCrossRefGoogle Scholar
  15. 15.
    Kohno S, Varma A, Kwon-Chung KJ, Hara K. Epidemiology studies of clinical isolates of Cryptococcus neoformans of Japan by restriction fragment length polymorphism. Kansenshogaku Zasshi. 1994;68:1512–7.PubMedGoogle Scholar
  16. 16.
    Yan Z, Li XG, Xu JP. Geographic distribution of mating type alleles of Cryptococcus neoformans in four areas of the United States. J Clin Microbiol. 2002;40:965–72.PubMedCrossRefGoogle Scholar
  17. 17.
    Lin XR, Litvintseva AP, Nielsen K, Patel S, Floyd A, Mitchell TG, et al. αADα hybrids of Cryptococcus neoformans: evidence of same-sex mating in nature and hybrid fitness. PLoS Genet. 2007;3:e186.CrossRefGoogle Scholar
  18. 18.
    Chaturvedi V, Fan JJ, Stein B, Behr MJ, Samsonoff WA, Wickes BL, et al. Molecular genetic analyses of mating pheromones reveal intervariety mating or hybridization in Cryptococcus neoformans. Infect Immun. 2002;70:5225–35.PubMedCrossRefGoogle Scholar
  19. 19.
    Barchiesi F, Cogliati M, Esposto MC, Spreghini E, Schimizzi M, Wickes BL, et al. Comparative analysis of pathogenicity of Cryptococcus neoformans serotypes A, D and AD in murine cryptococcosis. J Infect. 2005;51:10–6.PubMedCrossRefGoogle Scholar
  20. 20.
    Lin XR, Nielsen K, Patel S, Heitman J. Impact of mating type, serotype, and ploidy on the virulence of Cryptococcus neoformans. Infect Immun. 2008;76:2923–38.PubMedCrossRefGoogle Scholar
  21. 21.
    Shahid M, Han S, Yoell H, Xu JP. Fitness distribution and transgressive segregation across 40 environments in a hybrid progeny population of the human-pathogenic yeast Cryptococcus neoformans. Genome. 2008;51:272–81.PubMedCrossRefGoogle Scholar
  22. 22.
    Mallet J. Hybridization as an invasion of the genome. Trends Ecol Evol. 2005;20:229–37.PubMedCrossRefGoogle Scholar
  23. 23.
    Cogliati M, Esposto MC, Tortorano AM, Viviani MA. Cryptococcus neoformans population includes hybrid strains homozygous at mating-type locus. FEMS Yeast Res. 2006;6:608–13.PubMedCrossRefGoogle Scholar
  24. 24.
    Kwon-Chung KJ, Polacheck I, Popkin TJ. Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice. J Bacteriol. 1982;150:1414–21.PubMedGoogle Scholar
  25. 25.
    Price MF, Wilkinson ID, Gentry LO. Plate method for detection of phospholipase activity in Candida albicans. 1982:7–14.Google Scholar
  26. 26.
    Zaragoza O, Casadevall A. Experimental modulation of capsule size in Cryptococcus neoformans. Biol Proced Online. 2004;6:10–5.PubMedCrossRefGoogle Scholar
  27. 27.
    Sun S, Xu JP. Genetic analyses of a hybrid cross between serotypes A and D strains of the human pathogenic fungus Cryptococcus neoformans. Genetics. 2007;177:1475–86.PubMedCrossRefGoogle Scholar
  28. 28.
    Sun S, Xu J. Chromosomal rearrangements between serotype A and D in Cryptococcus neoformans. PlosOne. 2009;4:1–17.Google Scholar
  29. 29.
    Li W, Ni M, Averette AF, Desnos-Ollivier M, Dromer F, Heitman J. Genetic diversity and genomic plasticity of Cryptococccus neoformans AD hybrids. In: 8th international conference on Cryptococcus and Cryptococcosis, 1–5 May 2011, Charleston, SC, USA. Poster 15.Google Scholar
  30. 30.
    Wang P, Nichols CB, Lengeler MB, Cardenas ME, Cox GM, Perfect JR, et al. Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans. Eukaryot Cell. 2002;1:257–72.PubMedCrossRefGoogle Scholar
  31. 31.
    Nielsen K, Marra RE, Hagen F, Boekhout T, Mitchell TG, Cox GM, et al. Interaction between genetic background and the mating-type locus in Cryptococcus neoformans virulence potential. Genetics. 2005;171:975–83.PubMedCrossRefGoogle Scholar
  32. 32.
    Hicks JK, D’Souza CA, Cox GM, Heitman J. Cyclic AMP-dependent protein kinase catalytic subunits have divergent roles in virulence factor production in two varieties of the fungal pathogen Cryptococcus neoformans. Eukaryot Cell. 2004;3:14–26.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • M. Cogliati
    • 1
  • F. Barchiesi
    • 2
  • E. Spreghini
    • 2
  • Anna Maria Tortorano
    • 1
  1. 1.Lab. Micologia Medica, Dipartimento di Sanità Pubblica, Microbiologia, VirologiaUniversità degli Studi di MilanoMilanItaly
  2. 2.Dipartimento di Scienze Biomediche, Clinica Malattie InfettiveUniversità Politecnica delle MarcheAnconaItaly

Personalised recommendations