Background is the most important thermal dimorphic fungus causing systemic mycosis
Background is the most important thermal dimorphic fungus causing systemic mycosis in China and Southeast Asia. in mycelial and seven in yeast phase. Two genes and of was more closely related to the homologues in other thermal dimorphic pathogenic fungi than to and spp. suggesting the co-evolution of among the thermal dimorphic fungi. Moreover demonstrated higher mRNA expression levels in mycelial BG45 than yeast phase by 7 folds (and and deletion mutants we showed that the biogenesis of both milRNAs were dependent on but not or were upregulated in knockdown strain is the BG45 most important thermal dimorphic pathogenic fungus in Southeast Asia. Despite findings on diverse genes and mechanisms involved in dimorphic switching the key to signally pathways governing the switch is still unknown. Since miRNAs are important regulatory molecules in eukaryotes we attempt to define if miRNAs are expressed in different growth phases of and of was more closely related to the homologues in other thermal dimorphic pathogenic fungi than to and spp.. demonstrated higher mRNA levels in mycelial than LEFTYB yeast phase. Northern blot analysis confirmed expression of two milRNAs and but not or were upregulated in knockdown strain is the most important thermal dimorphic fungus causing respiratory skin and systemic mycosis in Southeast Asia -. Recently it has been renamed as based on phylogenetic analyses . The fungus was first discovered in Chinese bamboo rats infections in Southeast Asia where the fungus is endemic. Penicilliosis is the third most common indicator disease of AIDS In northern Thailand . In Hong Kong about 10% of HIV patients are infected with infections have also been reported from countries outside Southeast Asia  . In addition infections are increasingly reported in other immunocompromised patients such as transplant recipients and others on immunosuppressant therapy -. Despite its medical importance the mode of transmission and dimorphic and pathogenic mechanisms of remain largely unknown. In particular exhibits distinct cellular morphologies in different temperatures in mycelial phase at 25°C and yeast phase at 37was discovered in in 1993 . However it was only until 2000 that the second miRNA and the human pathogenic yeast genome project in an attempt to expedite the study of biology epidemiology and virulence factors of this dimorphic fungus -. Based on the available genome sequence data potential genes encoding proteins important for miRNA biogenesis can be identified in and involved in the regulation of thermal dimorphism. We attempted to identify milRNAs in in both mycelial and yeast phase using high-throughput Illumina DNA sequencing. Sequence analysis revealed 24 potential milRNA BG45 candidates which were more abundantly expressed in mycelial than yeast phase of and and but not or strain PM1 was obtained from an already-existing collection from the clinical microbiology laboratory in Queen Mary Hospital and the strain was anonymized. strains BG45 and growth conditions strain PM1 was isolated from a patient with culture-documented penicilliosis in Hong Kong. Knock-out mutant strains including double mutant (were generated as described below. All strains were grown on Sabouraud dextrose agar (SDA) (Oxoid Cambridge UK) at 25°C for 7 days for the collection of conidia BG45 as described previously . Conidia were collected by scraping and resuspension in 0.1% Tween-20 with PBS followed by three washes in sterile PBS before subculturing into liquid cultures in BHI medium (Difco NJ USA) in a shaker at 37°C for yeasts BG45 or at 25°C for mycelia for 48 hours. Cells were enumerated using a hemocytometer. Small RNA purification library preparation and sequencing Small RNA libraries were constructed for mycelial and yeast phases of PM1 genome sequence . Small RNA sequences between 17-30 nt were selected to identify perfect matches to the genome using Bowtie (0.12.8) . Identification of milRNAs and milRNA loci To identify milRNA candidates other non-coding RNAs including rRNAs and tRNAs were first excluded. Potential milRNA candidates were predicted with miRDeep  based on draft PM1 genome. Analysis was performed with the following adjustments: (1) Filtering ubiquitous alignments keeping only reads that were perfectly mapped to no more than 5 different regions in the genome; (2) Potential precursor sequences were excised from the genome with the.