Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.IMPORTANCE Isolates of Cryptococcus gattii are currently causing an outbreak of cryptococcosis in western North America, and most of the cases occurred in the absence of coinfection with HIV. This pattern is therefore in stark contrast to the current global burden of one million annual cases of cryptococcosis, caused by the related species Cryptococcus neoformans, in the HIV/AIDS population. The genome sequences of two outbreak-associated major genotypes of C. gattii reported here provide insights into genome variation within and between cryptococcal species. These sequences also provide a resource to further evaluate the epidemiology of cryptococcal disease and to evaluate the role of pathogen genes in the differential interactions of C. gattii and C. neoformans with immunocompromised and immunocompetent hosts.

A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5′-monophosphate (cAMP)–dependent protein kinase A (PKA) is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential targets for anti-cryptococcal therapy.

The mechanisms by which pathogens sense and transport iron are important during infection, because of the low availability of free iron in the mammalian host. Iron is a key nutritional cue for the pathogen Cryptococcus neoformans, because it influences expression of the polysaccharide capsule that is the major virulence factor of the fungus. In this study, C. neoformans mutants were constructed with a defect in the iron-regulated gene SIT1 that encodes a putative siderophore iron transporter. Analysis of mutants in serotype A and D strains demonstrated that SIT1 is required for the use of siderophore-bound iron, and for growth in a low-iron environment. The sit1 mutants also showed changes in melanin formation and cell wall density, and it was found that mutants defective in protein kinase A, which is known to influence melanization and capsule formation, showed elevated SIT1 transcripts in both the serotype A and the serotype D backgrounds. Finally, the mutants were tested for virulence in a murine model of cryptococcosis, and it was found that SIT1 was not required for virulence. Overall, these studies establish links between iron acquisition, melanin formation and cAMP signalling in C. neoformans. INTRODUCTIONCryptococcus neoformans is the leading cause of fungal meningitis in immunocompromised individuals (Casadevall & Perfect, 1998). Five serotypes (A, B, C, D and AD) are recognized, based on the antigenicity of the polysaccharide capsule, and three varieties have been described: neoformans (D), grubii (A) and gattii (B and C). Several virulence factors have been identified for the fungus, including the polysaccharide capsule, production of melanin by the enzyme laccase, the ability to grow at 37 u C, and survival within macrophages (Casadevall & Perfect, 1998). The polysaccharide capsule is antiphagocytic and suppresses the immune response, while the expression of laccase and melanin formation are necessary for survival within alveolar macrophages, resistance to oxidative stress, and extrapulmonary dissemination to the brain (Bose et al., 2003;Casadevall & Perfect, 1998;Gomez & Nosanchuk, 2003;Janbon, 2004;Liu et al., 1999;Noverr et al., 2004;Perfect, 2005;Williamson, 1997). Capsule and melanin production are regulated by several factors. For example, capsule size is influenced by iron and CO 2 levels, serum, and the location of the fungus in host tissue (Bose et al., 2003;Janbon, 2004;Vartivarian et al., 1993;Zaragoza et al., 2003). Melanin synthesis is regulated by iron and copper, and by low glucose levels (Alspaugh et al., 1997;Jacobson & Compton, 1996;Polacheck et al., 1982; Salas et al., 1996;Zhu et al., 2001;Zhu & Williamson, 2004). The cAMP pathway is known to regulate both capsule and melanin, and the PKC1/MAP kinase pathway has also been implicated in melanin production, because loss of the C1 domain of PKC1 leads to reduced laccase activity (Alspaugh et al., 1997;D'Souza et al., 2001;Heung et al., 2005;Hicks et al., 2004).We are interested in the mechanisms of iron regulation and uptake in C. n...