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...

The basidiomycete fungus Cryptococcus neoformans is an important opportunistic pathogen of humans that poses a significant threat to immunocompromised individuals. Isolates of C. neoformans are classified into serotypes (A, B, C, D, and AD) based on antigenic differences in the polysaccharide capsule that surrounds the fungal cells. Genomic and EST sequencing projects are underway for the serotype D strain JEC21 and the serotype A strain H99. As part of a genomics program for C. neoformans, we have constructed fingerprinted bacterial artificial chromosome (BAC) clone physical maps for strains H99 and JEC21 to support the genomic sequencing efforts and to provide an initial comparison of the two genomes. The BAC clones represented an estimated 10-fold redundant coverage of the genomes of each serotype and allowed the assembly of 20 contigs each for H99 and JEC21. We found that the genomes of the two strains are sufficiently distinct to prevent coassembly of the two maps when combined fingerprint data are used to construct contigs. Hybridization experiments placed 82 markers on the JEC21 map and 102 markers on the H99 map, enabling contigs to be linked with specific chromosomes identified by electrophoretic karyotyping. These markers revealed both extensive similarity in gene order (conservation of synteny) between JEC21 and H99 as well as examples of chromosomal rearrangements including inversions and translocations. Sequencing reads were generated from the ends of the BAC clones to allow correlation of genomic shotgun sequence data with physical map contigs. The BAC maps therefore represent a valuable resource for the generation, assembly, and finishing of the genomic sequence of both JEC21 and H99. The physical maps also serve as a link between map-based and sequence-based data, providing a powerful resource for continued genomic studies.

In 1991, a matched case-control study was performed in Norwegian freshwater hatcheries on risk factors for infection with Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis. The study was based on repl~es to a questionnaire mailed to smolt producers, and included 30 infected and 66 non-infected hatcheries, matched by county. The odds ratios for infection with A, salmonicjda subsp. salmonicida In hatcheries associated with certaln management and envlronmental factors were analyzed using a conditional logistic regression analysis. The study revealed that the main risk factors for infection with A. salmonicida subsp. salmonicida in freshwater hatcheries were: (1) migration of anadromous fish into the freshwater supply of the hatchery, (2) sharing of personnel with other fish farms, and (3) a high concentration of fish farms infected with A. salmonicida subsp salmoniada near the hatchery. Results indicate that the high prevalence of furunculosis in Norwegian seawater farms has great lmpact on the risk of infection with A. salmonicida subsp. salmonicida in hatcheries, and also that the bacterium may be transmitted between fish farms by humans.

Wood sapstain, a cosmetic defect that results in significant economical loss to forest-products industries, is caused by mycelial melanization of the wood-colonizing ophiostomatoid fungi. To improve our understanding of how melanin biosynthesis is regulated in the cosmopolitan sapstaining fungus, Ophiostoma piceae, we used insertional mutagenesis. Insertional mutants were generated by restriction enzyme-mediated integration (REMI) and Agrobacterium-mediated integration (AMI). We screened 1,053 REMI and 1,083 AMI transformants and found 30 mutants with impaired growth or pigmentation. We characterized four AMI transformants in more detail, in which the T-DNA integrated at a single locus. The albino mutant TOPA45 had incorporated the T-DNA in a polyketide synthase gene (PKS1). The mutants TOPA1 and TOPA1076 displayed reduced pigmentation. In TOPA1, the T-DNA was inserted into a gene that encodes a putative protein kinase activator whereas, for TOPA1076, it was inserted into a gene that encodes a protein with unknown function. Finally, the vegetative hyphae of mutant TOPA814 were not melanized, whereas the synnemata displayed the same level of pigmentation as the wild type. In the TOPA814 mutant, segregation analysis revealed that the mutant phenotype was not linked to the T-DNA insertion locus but to a translocation from the PIG1 locus to the left border of the T-DNA. The protein predicted for the PIG1 locus had a middle homology region that was specific to fungal transcription factors.

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