Identification of microRNA in penicillium marneffei

Abstract

Penicillium marneffei is the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, the existence of miRNAs in fungi was less well studied and their potential roles in fungal dimorphism were largely unknown. Based on available genome sequence of P. marneffei, it is hypothesized that miRNA-like small RNAs (milRNAs) may be expressed in the dimorphic fungus and dicer- or argonuate-like proteins may be involved in dimorphism or virulence in P. marneffei. I attempted to identify milRNAs in P. marneffei in both mycelial and yeast phase using high-throughput sequencing technology. Small RNAs were more abundantly expressed in mycelial than yeast phase. Sequence analysis revealed 24 potential milRNA candidates, including 17 (2502 reads) candidates in mycelial and seven (232 reads) in yeast phase. Two genes, dcl-1 and dcl-2, encoding putative Dicer-like proteins and the gene, qde-2, encoding Argonaute-like protein, were identified in P. marneffei. Phylogenetic analysis showed that dcl-2 and qde-2 of P. marneffei were more closely related to the homologues in the thermal dimorphic pathogenic fungi, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Coccidioides immitis than to Penicillium chrysogenum and Aspergillus spp., suggesting the co-evolution of dcl-2 and qde-2 among other thermal dimorphic fungi. Moreover, dcl-2 and qde-2 demonstrated higher mRNA expression levels in mycelial than yeast phase by 7 folds and 2 folds respectively (P<0.001). Northern blot analysis confirmed the expression of two milRNAs, PM-milR-M1 and PM-milR-M2, only in mycelial phase. Using dcl-1KO, dcl-2KO, dclDKO and qde-2KO deletion mutants, it was shown that the biogenesis of both milRNAs were dependent on dcl-2 but not dcl-1 or qde-2. While deletion of qde-2, but not the two dcl genes, was found to decrease the virulence level of P. marneffei in mice model, the deaths of the qde-2KO conidia challenged mice were delayed for over 10 days. The qde-2KO conidia have lower recovery rate both in human THP1 and murine J774 macrophage cell lines and also reduced resistance to hydrogen peroxide than the wild type. This study provided the first evidence for differential expression of milRNAs in different growth phases of thermal dimorphic fungi and shed light on the evolution of fungal proteins involved in milRNA biogenesis and possible role of post-transcriptional control in governing thermal dimorphism. This is also the first study to reveal the relationship between argonuate-like QDE-2 protein and virulence in P. marneffei in mice model. This study provides a foundation for the milRNAs study in pathogenic thermal dimorphic fungi.