Conservation of intracellular pathogenic strategy among distantly related Cryptococcal species. Conservation of intracellular pathogenic strategy among distantly related Cryptococcal species

We compared the interaction between Cryptococcus gattii species complex strain R265 and Cryptococcus neoformans species complex strain H99, molecular type VNI, with murine macrophages to ascertain similarities and differences in their intracellular pathogenic strategy. Parameters analyzed include non-lytic exocytosis using time-lapse microscopy, phagolysosomal pH by dual fluorescent microscopy, intracellular capsular growth, phagolysosomal membrane permeabilization by flow cytometry, and the macrophage transcriptional response by gene expression microarray analysis. Using microarrays, we compared the transcriptional response of Bone Marrow-derived macrophages (BMDM) infected with C. gattii species complex strain R265 or C. neoformans species complex strain H99, relative to uninfected BMDM collected from the same time intervals, 2hr and 24 hr post-infection. For analysis following normalization and summarization steps, a 2-way Analysis of Variance (ANOVA) with linear contrasts for treatment (infection/strain) and time (2 h or 24 h) vs. Control (uninfected) was performed with outputs of P value, Fold Change, and Mean Ratio. A linear contrast of uninfected controls (24 h uninfected vs 2 h uninfected) was also performed to generate a ‘time only’ gene list. Cutoff criteria for filtering gene lists was significant P values (p<0.01) with fold changes greater that 2 or less than –2. Ten genes from the filtered gene lists were selected for validation by qPCR, with four housekeeping genes for normalization and comparison. Overall design: Control (uninfected) and C. gattii- or C. neoformans-infected BMDM were harvested at 2hr and 24hr time points and processed in triplicate for each condition.

本研究比较了格特隐球菌（Cryptococcus gattii）物种复合体菌株R265与新型隐球菌（Cryptococcus neoformans）物种复合体菌株H99（分子型VNI）分别与小鼠巨噬细胞的相互作用，旨在明确二者在细胞内致病策略上的异同。本研究分析的检测指标包括：采用延时显微镜（time-lapse microscopy）观测的非裂解胞吐（non-lytic exocytosis）现象、通过双荧光显微镜（dual fluorescent microscopy）检测的吞噬溶酶体pH值、细胞内荚膜生长情况、经流式细胞术（flow cytometry）分析的吞噬溶酶体膜通透性，以及通过基因表达微阵列（gene expression microarray）分析得到的巨噬细胞转录应答特征。利用基因表达微阵列技术，我们对比了感染格特隐球菌物种复合体菌株R265或新型隐球菌物种复合体菌株H99的骨髓来源巨噬细胞（Bone Marrow-derived macrophages, BMDM）的转录应答，并以相同感染时间点（感染后2小时与24小时）收集的未感染BMDM作为对照。在完成数据标准化与汇总处理后，我们采用双向方差分析（two-way Analysis of Variance, ANOVA），针对处理因素（感染组别/菌株类型）与时间因素（2 h或24 h）vs. 对照组（未感染）开展线性对比分析，输出结果包含P值、倍数变化（Fold Change）与平均比值（Mean Ratio）。此外，我们还针对未感染对照组（24 h未感染组 vs. 2 h未感染组）进行线性对比，以生成“仅受时间调控”的基因列表。基因列表的筛选阈值为：P值具有统计学显著性（p<0.01）且倍数变化大于2或小于-2。我们从筛选得到的基因列表中选取10个基因，通过定量聚合酶链反应（qPCR）进行验证，并以4个持家基因（housekeeping genes）作为标准化内参用于比较分析。总体实验设计如下：分别在感染后2小时与24小时收集对照组（未感染）以及格特隐球菌或新型隐球菌感染的BMDM，每种实验条件设置3次生物学重复。