TECHNICAL NOTE Guanggan Hu Æ James W. Kronstad Gene disruption in Cryptococcus neoformans and Cryptococcus gattii by in vitro transposition Received: 7 November 2005 / Revised: 7 December 2005 / Accepted: 10 December 2005 / Published online: 6 January 2006 Ó Springer-Verlag 2006 Abstract Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous fungi that infect immuno- compromised and immunocompetent people. We devel- oped an insertional mutagenesis strategy for these species based on in vitro transposition and we tested the method by disrupting the URA5 gene in a strain of C. neoformans and the CAP10 gene in three strains of C. gattii. We targeted plasmid DNA containing the URA5 gene or plasmid DNA containing the CAP10 gene from genomic libraries from the shotgun sequencing project for the C. gatti strain WM276. In the latter case, the availability of the end sequences of the clones from the assembled genomic sequence allows rapid selection of target genes for disruption. Modified transposons containing the nourseothricin (NAT) or neomycin (Neo) resistance cassettes were randomly inserted into the target DNA by in vitro transposition. The disrupted genes were used for biolistic transformation and homologous integration was subsequently confirmed by PCR and Southern blot analysis. These results demonstrate that the emerging genomic resources, combined with in vitro transposition into plasmid DNAs from shotgun sequencing libraries or cloned PCR products, will facilitate high-throughput genetic analysis in Cryptococcus species. Keywords Gene disruption Æ Transposon Æ Basidiomycetes Æ Virulence factors Introduction The basidiomycete fungal pathogens Cryptococcus neoformans and Cryptococus gattii infect both immu- nocompetent and immunocompromised individuals to cause cryptococcosis. Cryptococcus strains infect the central nervous system and cause meningioencephalitis that can be fatal if not aggressively treated. Five dif- ferent serotypes (A, B, C, D and AD) have been clas- sified based on antigenic differences in the capsule of the fungus. Strains of the B and C serotypes have re- cently been recognized as a separate species called C. gattii, while serotypes A and D are considered dif- ferent varieties called C. neoformans var. grubii and C. neoformans var. neoformans, respectively (Kwon- Chung et al. 2002). Serotype A is the most clinically prevalent in cryptococcosis patients in North America, followed by serotype D. Serotype B isolates (C. gattii) were considered to be limited to tropical and subtrop- ical environments (Casadevall and Perfect 1998) but this idea has been called into question by the emer- gence of infections caused by C. gattii in immuno- competent individuals on Vancouver Island (Hoang et al. 2004). The fungus produces a polysaccharide capsule that is a well-characterized virulence factor for the pathogen. Other virulence factors include the ability to grow at 37°C, melanin synthesis, and production of urease and phospholipase (Casadevall and Perfect 1998; Cox et al. 2000, 2001; Kwon-Chung and Rhodes 1986). There is considerable interest in determining the genetic and bio- chemical basis of capsule formation in C. neoformans and C. gattii because the capsule is antiphagocytic and exerts a negative influence on the host immune response (Bu- chanan and Murphy 1998). By isolating and comple- menting acapsular mutants, Kwon-Chung and colleagues identified four genes (CAP10, CAP59, CAP60 and CAP64) involved in capsule biosynthesis (Chang et al. 1996; Chang and Kwon-Chung 1994, 1998, 1999). These CAP genes are essential for capsule formation and for virulence in the murine model. Further studies demon- strated that the CAP10 gene product was located in the cytoplasm while Cap60 was located at the nuclear mem- brane (Chang and Kwon-Chung 1998, 1999). CAP10 is expressed at high levels in late-stationary-phase cells, and Communicated by J. Heitman G. Hu Æ J. W. Kronstad (&) The Michael Smith Laboratories, The University of British Columbia, 2185 East Mall, Vancouver, BC, Canada, V6T 1Z4 E-mail: kronstad@interchange.ubc.ca URL: http://www.kronstadlab.biotech.ubc.ca/ Tel.: +1-604-8224732 Fax: +1-604-8222214 Curr Genet (2006) 49: 341–350 DOI 10.1007/s00294-005-0054-x