Host-pathogen transcriptomics of Mucor circinelloides NCRIP deficient spores phagocytosed by mouse macrophages. Host-pathogen transcriptomics of Mucor circinelloides NCRIP deficient spores phagocytosed by mouse macrophages

Here in this work, we uncovered the role of NCRIP in regulating virulent processes and transposable movements through key components of the pathway, RdRP1 and R3B2. Mutants in these genes are unable to launch a proper virulent response to macrophage phagocytosis, resulting in a decreased virulence potential. The transcriptomic profile of rdrp1Δ and r3b2Δ mutants revealed a pre-exposure adaptation to the stressful phagosomal environment even when the strains are not confronted by macrophages. Overall design: 8 samples are provided and analyzed, comprising 2 replicates (blank and R) for each coculture interacting samples and each single culture, which are as follows: Mucor r3b2 mutant strain cocultured with mouse macrophages (cell line J774A.1) for 5h, Mucor rdrp1 mutant strain co-cultured with mouse macrophages (cell line J774A.1) for 5h, Mucor r3b2 mutant strain (R7B) single-cultured in cell media for 5h, and Mucor rdrp1 mutant strain singlecultured in cell media for 5h. Libraries were prepared with Illumina TruSeq strand-specific mRNA kits and sequenced with an Illumina HiSeq 2500 system to generate 50-bp, first-strand (or reverse) and single-end reads.

本研究揭示了NCRIP通过通路关键组分RdRP1与R3B2，调控病原菌致病进程与转座子移动的分子机制。上述基因的突变体无法对巨噬细胞吞噬作用产生正常的致病应答，进而导致其致病潜力下降。rdrp1Δ与r3b2Δ突变体的转录组分析显示，即便未暴露于巨噬细胞环境中，这些菌株已预先适应了吞噬体的应激环境。
整体实验设计：本研究共设置8个样本进行分析，每组共培养互作样本与单培养样本均设置2次生物学重复（空白组与R组），具体样本包括：与小鼠巨噬细胞（细胞系J774A.1）共培养5小时的毛霉r3b2突变菌株、与小鼠巨噬细胞（细胞系J774A.1）共培养5小时的毛霉rdrp1突变菌株、在细胞培养基中单培养5小时的毛霉r3b2突变菌株（R7B）、以及在细胞培养基中单培养5小时的毛霉rdrp1突变菌株。
文库构建采用Illumina TruSeq链特异性mRNA试剂盒（Illumina TruSeq strand-specific mRNA kits），随后使用Illumina HiSeq 2500测序系统进行测序，生成50bp单端正向（或反向）测序读段。