Abstract
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.
Highlights
Cryptococcus spp. are encapsulated basidiomycetous fungi that infect both the healthy people and immunocompromised individuals, causing meningoencephalitis (Perfect, 1989)
Endocytosis is a process in which living cells uptake foreign materials through the invagination of the plasma membrane (PM) to form vesicles, whereas exocytosis is the release of vesicle contents to the cell exterior through vesicle fusion with PM (Oka and Krieger, 2005)
We showed that Cryptococcus secretes abundant RNAs, including microRNA, small interference RNAs, long noncoding RNA, and messenger RNA and that Cin1 plays a regulatory role including that in secretion
Summary
Cryptococcus spp. are encapsulated basidiomycetous fungi that infect both the healthy people and immunocompromised individuals, causing meningoencephalitis (Perfect, 1989). Virulence of Cryptococcus is multifaceted, with the production of the polysaccharide capsule, melanin pigment, and extracellular proteinases characterized as the common virulence factors (Kozel, 1995; Buchanan and Murphy, 1998; Lengeler et al, 2000). The elaboration of these virulence factors depends on intact intracellular transport, including exocytosis and endocytosis, which is a highly conserved and essential cellular process. The secretory transport of glucuronoxylomannan (GXM) and aspartic proteinases (SAPs) is considered to be important for capsule formation, a virulence factor of Cryptococcus spp. and the virulence of Candida albicans, respectively (Schaller et al, 2005)
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