Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China.
College of Life and Health Sciences, Northeastern University, Liaoning, China.
Front Cell Infect Microbiol. 2020 Jun 23;10:256. doi: 10.3389/fcimb.2020.00256. eCollection 2020.
Membrane vesicles are considered virulence cargoes as they carry capsular and melanin components whose secretory transport is critical for the virulence of the human fungal pathogen species. However, other components of the vesicles and their function in the growth and virulence of the fungus remain unclear. We have previously found that the cryptococcal intersectin protein Cin1 governs a unique Cin1-Wsp1-Cdc42 endocytic pathway required for intracellular transport and virulence. Using RNA sequencing, we compared the profiles of extracellular RNA (exRNA), including microRNA (miRNA), small interference RNA (siRNA), long noncoding RNA (lncRNA), and messenger RNA (mRNA) between the wild-type (WT), and derived Δ mutant strains of . Seven hundred twelve miRNAs and 88 siRNAs were identified from WT, whereas 799 miRNAs and 66 siRNAs were found in Δ. Also, 572 lncRNAs and 7,721 mRNAs were identified from WT and 584 lncRNAs and 7,703 mRNAs from Δ. Differential expression analysis revealed that the disruption of results in many important cellular changes, including those in exRNA expression, transport, and function. First, for miRNA target genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that cellular processes, components, and macromolecular functions are the most affected pathways. A higher number of genes were involved in the intracellular transport of endocytosis. Second, the results of GO term and KEGG analysis of differentially expressed lncRNA target genes and mRNA genes were consistent with those of miRNA targets. In particular, protein export is the topmost affected pathway among lncRNA target genes and one of the affected pathways among mRNA genes. The result of quantitative real-time reverse transcription PCR (qRT-PCR) from 12 mRNAs tested is largely agreeable with that of RNA-Seq. Taken together, our studies provide a comprehensive reference that secretes abundant RNAs and that Cin1 plays a critical role in regulating their secretion. Given the growing clinical importance of exRNAs, our studies illuminate the significance of exploring this cutting-edge technology in studies of cryptococcal pathogenesis for the discovery of novel therapeutic strategies.
膜泡被认为是毒力货物，因为它们携带荚膜和黑色素成分，其分泌运输对于人类真菌病原体的毒力至关重要。然而，囊泡的其他成分及其在真菌生长和毒力中的作用尚不清楚。我们之前发现，隐球菌的 intersectin 蛋白 Cin1 控制着一种独特的 Cin1-Wsp1-Cdc42 内吞途径，该途径对于细胞内运输和毒力是必需的。使用 RNA 测序，我们比较了野生型 (WT) 和衍生的 Δ突变株之间的细胞外 RNA (exRNA) 谱，包括 microRNA (miRNA)、small interference RNA (siRNA)、long noncoding RNA (lncRNA) 和 messenger RNA (mRNA)。从 WT 中鉴定出 712 个 miRNA 和 88 个 siRNA，而从 Δ中鉴定出 799 个 miRNA 和 66 个 siRNA。此外，从 WT 中鉴定出 572 个 lncRNA 和 7721 个 mRNA，而从 Δ中鉴定出 584 个 lncRNA 和 7703 个 mRNA。差异表达分析表明， 的破坏导致许多重要的细胞变化，包括 exRNA 表达、运输和功能的变化。首先，对于 miRNA 靶基因，GO 和 KEGG 通路富集分析显示，细胞过程、成分和大分子功能是受影响最大的途径。更多的基因参与了内吞作用的细胞内运输。其次，差异表达 lncRNA 靶基因和 mRNA 基因的 GO 术语和 KEGG 分析结果与 miRNA 靶基因的结果一致。特别是，蛋白质输出是 lncRNA 靶基因中受影响最大的途径之一，也是 mRNA 基因中受影响的途径之一。从 12 个测试的 mRNA 进行的定量实时逆转录 PCR (qRT-PCR) 的结果与 RNA-Seq 的结果基本一致。总之，我们的研究提供了一个全面的参考，表明 分泌丰富的 RNA，而 Cin1 在调节它们的分泌中起着关键作用。鉴于外泌 RNA 的临床重要性日益增加，我们的研究阐明了在探索隐球菌发病机制的这一前沿技术中探索这一技术的意义，以发现新的治疗策略。
