Comparison of gene expression in 23 Cryptococcus gattii isolates. Cryptococcus bacillisporus

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. Overall design: Dual-colour hybridization (each sample was hybridized against pooled RNA), one array per isolate, 23 biological samples, no technical replicates.

本研究针对加蒂隐球菌（C. gattii，曾用名C. bacillosporus）开展芯片阵列分析（array analysis），旨在筛选与温哥华岛暴发株（Vancouver Island Outbreak，VIO）高毒力相关的基因位点。共纳入23株加蒂隐球菌分离株，涵盖VIO毒株与对照菌株，将其于哺乳动物巨噬细胞（macrophages）中培养24小时。随后提取总RNA，并以全部23份样本的混合RNA（pooled RNA）作为参照，定量各菌株的基因表达水平。采用线性回归（linear regression）分析，筛选与“细胞内增殖速率（intracellular proliferation rate）”——即毒力的替代衡量指标——呈正相关或负相关的基因位点。本分析所得数据收录于Ma等人2009年发表于《美国国家科学院院刊》（Proceedings of the National Academy of Sciences，PNAS）的论文：106(31): 12980-12985。以下为该论文摘要：
1999年，加拿大温哥华岛地区暴发由高毒力加蒂隐球菌谱系引发的致命真菌病，随后该病原体蔓延至加拿大本土及太平洋西北地区，但此次暴发的分子机制至今仍未明确。本研究显示，相较于其他加蒂隐球菌菌株，VIO分离株在哺乳动物免疫系统巨噬细胞内的增殖能力显著提升。进一步实验证实，这种增强的胞内寄生能力与小鼠隐球菌感染模型中的毒力直接相关，表明该表型可能是此次暴发的致病根源。最后，针对24株加蒂隐球菌的芯片微阵列（microarray）分析结果显示，VIO分离株的高毒力特征表现为大量基因的上调表达，其中许多基因由线粒体基因组编码，或与线粒体功能活动相关。该表达谱与VIO菌株在吞噬作用后呈现的异常线粒体形态相契合。综上，本研究证实巨噬细胞胞内寄生是引发人类疾病暴发的关键驱动因素，这一发现对诸多其他人类病原体的相关研究具有重要启示意义。
整体实验设计：采用双色杂交（dual-colour hybridization）实验方案，每份样本与混合RNA进行杂交，每株分离株对应一张芯片，共包含23份生物学重复样本（biological samples），无技术重复（technical replicates）。