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 Cryptococcus 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 Δcin1 mutant strains of Cryptococcus deneoformans. Seven hundred twelve miRNAs and 88 siRNAs were identified from WT, whereas 799 miRNAs and 66 siRNAs were found in Δcin1. Also, 572 lncRNAs and 7,721 mRNAs were identified from WT and 584 lncRNAs and 7,703 mRNAs from Δcin1. Differential expression analysis revealed that the disruption of CIN1 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 Cryptococcus 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，其分泌运输对人类真菌病原体的毒力至关重要隐球菌物种。然而，囊泡的其他成分及其在真菌生长和毒力中的作用仍不清楚。我们之前发现隐球菌交叉蛋白 Cin1 控制着细胞内运输和毒力所需的独特 Cin1-Wsp1-Cdc42 内吞途径。使用 RNA 测序，我们比较了野生型 (WT) 之间的细胞外 RNA (exRNA) 谱，包括 microRNA (miRNA)、小干扰 RNA (siRNA)、长链非编码 RNA (lncRNA) 和信使 RNA (mRNA)，并导出 Δcin1突变株隐球菌. 从 WT 中鉴定出 712 个 miRNA 和 88 个 siRNA，而在 Δ 中发现 799 个 miRNA 和 66 个 siRNAcin1. 此外，从 WT 中鉴定出 572 个 lncRNA 和 7,721 个 mRNA，从 Δ 鉴定出 584 个 lncRNA 和 7,703 个 mRNA。cin1. 差异表达分析表明，CIN1导致许多重要的细胞变化，包括 exRNA 表达、运输和功能方面的变化。首先，对于 miRNA 靶基因，基因本体论 (GO) 和京都基因与基因组百科全书 (KEGG) 通路富集分析表明，细胞过程、成分和大分子功能是受影响最大的通路。更多的基因参与内吞作用的细胞内转运。其次，差异表达的lncRNA靶基因和mRNA基因的GO term和KEGG分析结果与miRNA靶点的结果一致。特别是，蛋白质输出是 lncRNA 靶基因中受影响最大的途径，也是 mRNA 基因中受影响的途径之一。来自 12 个 mRNA 的定量实时逆转录 PCR (qRT-PCR) 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，隐球菌分泌丰富的RNA，Cin1在调节其分泌中起关键作用。鉴于 exRNA 在临床上的重要性日益增加，我们的研究阐明了探索这一尖端技术在隐球菌发病机制研究中发现新治疗策略的重要性。