Project description:An array analysis of C. gattii (C. bacillosporus), intended to identify loci associated with the hypervirulence of the Vancouver Island Outbreak (VIO). 23 C. gattii isolates, representing both VIO strains and control strains, were grown for 24 hours in mammalian macrophages. RNA was isolated and gene expression for each strain quantified relative to pooled RNA from all 23 samples. Linear regression was used to identify loci showing positive or negative correlation with "intracellular proliferation rate", a proxy measure for virulence. Data from this analysis is included in Ma et al, 2009, PNAS 106(31) 12980-12985. The abstract is included below. In 1999, the population of Vancouver Island, Canada, began to experience an outbreak of a fatal fungal disease caused by a highly virulent lineage of Cryptococcus gattii. This organism has recently spread to the Canadian mainland and Pacific Northwest, but the molecular cause of the outbreak remains unknown. Here we show that the Vancouver Island outbreak (VIO) isolates have dramatically increased their ability to replicate within macrophages of the mammalian immune system in comparison with other C. gattii strains. We further demonstrate that such enhanced intracellular parasitism is directly linked to virulence in a murine model of cryptococcosis, suggesting that this phenotype may be the cause of the outbreak. Finally, microarray studies on 24 C. gattii strains reveals that the hypervirulence of the VIO isolates is characterized by the up-regulation of a large group of genes, many of which are encoded by mitochondrial genome or associated with mitochondrial activities. This expression profile correlates with an unusual mitochondrial morphology exhibited by the VIO strains after phagocytosis. Our data thus demonstrate that the intracellular parasitism of macrophages is a key driver of a human disease outbreak, a finding that has significant implications for a wide range of other human pathogens.
Project description:An array analysis of C. gattii (C. bacillosporus), intended to identify loci associated with the hypervirulence of the Vancouver Island Outbreak (VIO). 23 C. gattii isolates, representing both VIO strains and control strains, were grown for 24 hours in mammalian macrophages. RNA was isolated and gene expression for each strain quantified relative to pooled RNA from all 23 samples. Linear regression was used to identify loci showing positive or negative correlation with "intracellular proliferation rate", a proxy measure for virulence. Data from this analysis is included in Ma et al, 2009, PNAS 106(31) 12980-12985. The abstract is included below. In 1999, the population of Vancouver Island, Canada, began to experience an outbreak of a fatal fungal disease caused by a highly virulent lineage of Cryptococcus gattii. This organism has recently spread to the Canadian mainland and Pacific Northwest, but the molecular cause of the outbreak remains unknown. Here we show that the Vancouver Island outbreak (VIO) isolates have dramatically increased their ability to replicate within macrophages of the mammalian immune system in comparison with other C. gattii strains. We further demonstrate that such enhanced intracellular parasitism is directly linked to virulence in a murine model of cryptococcosis, suggesting that this phenotype may be the cause of the outbreak. Finally, microarray studies on 24 C. gattii strains reveals that the hypervirulence of the VIO isolates is characterized by the up-regulation of a large group of genes, many of which are encoded by mitochondrial genome or associated with mitochondrial activities. This expression profile correlates with an unusual mitochondrial morphology exhibited by the VIO strains after phagocytosis. Our data thus demonstrate that the intracellular parasitism of macrophages is a key driver of a human disease outbreak, a finding that has significant implications for a wide range of other human pathogens. Dual-colour hybridization (each sample was hybridized against pooled RNA), one array per isolate, 23 biological samples, no technical replicates.
Project description:Our previous investigation indicated that high-virulence C. gattii (C. gattii R265) tend to reside in the alveoli, whereas low-virulence C. gattii (C. gattii 5815) tend to be washed out from the alveoli and move into the central side of the respiratory system of the mice. Furthermore, C. gattii R265 and 5815 infected mice showed much lesser macrophage response than C. neoformans H99 infected mice. To elucidate the mechanism of this phenomenon from the viewpoint of genetic analysis, we performed this microarray assay.
Project description:Our previous investigation indicated that high-virulence C. gattii (C. gattii R265) tend to reside in the alveoli, whereas low-virulence C. gattii (C. gattii 5815) tend to be washed out from the alveoli and move into the central side of the respiratory system of the mice. Furthermore, C. gattii R265 and 5815 infected mice showed much lesser macrophage response than C. neoformans H99 infected mice. To elucidate the mechanism of this phenomenon from the viewpoint of genetic analysis, we performed this microarray assay. C. gattii R265, C. gattii 5815, and C. neoformans H99 were prepared for the study. The mice were anesthetized and the spore suspension was intratracheally injected into each mouse. Mice were sacrificed 15 days after the infection and then examined. Lungs of the mice infected with C. gattii R265 (n=4), C. gattii 5815 (n=4), and C. neoformans H99 (n=3) and of a normal control (n=1) were obtained. RNA targets preparation was performed according to the manufacturer's protocol using GeneChip(R) 3' IVT Express Kit (Affymetrix). One hundred nanograms of total RNA were converted into double-stranded cDNA template for transcription. In vitro transcription synthesized amplified RNA (aRNA) and incorporated a biotin-conjugated nucleotide. After purification and fragmentation of aRNA, 12.5 ug of them was hybridized to GeneChip(R) Mouse Genome 430 2.0 Array (Affymetrix).The Probe Array was scanned using a GeneChip(R) Scanner 3000 7G.