Biology subjects

Kusuya, Y.

Publications and source records attributed to Kusuya, Y..

4 recordsLinked to original sources

bioRxiv2021

Intimate genetic relationships and fungicide resistance in multiple strains of human pathogenic fungus Aspergillus fumigatus isolated from a plant bulb

Fungal infections are increasingly dangerous because of environmentally-dispersed resistance to antifungal drugs. Azoles are commonly used antifungal drugs, but they are also used as fungicides in agriculture, which may enable enrichment of azole-resistant strains of the human pathogen Aspergillus fumigatus in the environment. Understanding of environmental dissemination and enrichment of genetic variation associated with azole resistance in A. fumigatus is required to suppress resistant strains. Here, we focused on eight strains of azole-resistant A. fumigatus isolated from a single tulip bulb for sale in Japan. This set includes strains with TR34/L98H/T289A/I364V/G448S and TR46/Y121F/T289A/S363P/I364V/G448S mutations in the cyp51A gene, which showed higher tolerance to several azoles than strains harboring TR46/Y121F/T289A mutation. The strains were typed by microsatellite typing, single nucleotide polymorphism profiles, and mitochondrial and nuclear genome analyses. The strains grouped differently using each typing method, suggesting historical genetic recombination among the strains. Our data also revealed that some strains isolated from the tulip bulb showed tolerance to other classes of fungicide, such as QoI and carbendazim, followed by related amino acid alterations in the target proteins. Considering spatial-temporal factors, plant bulbs are an excellent environmental niche for fungal strains to encounter partners, and to obtain and spread resistance-associated mutations.

microbiology↗

bioRxiv2020

Ribosome profiling in Streptococcus pneumoniae reveals the role of methylation of 23S rRNA nucleotide G748 on ribosome stalling.

2.(1) BackgroundMany nucleotides in 23S rRNA are methylated post-transcriptionally by methyltransferases and cluster around the peptidyltransferase center (PTC) and the nascent peptidyl exit tunnel (NPET) located in 50S subunit of 70S ribosome. Biochemical interactions between a nascent peptide and the tunnel may stall ribosome movement and affect expression levels of the protein. However, no studies have shown a role for NPET on ribosome stalling using an NPET mutant. (2) ResultsA ribosome profiling assay in Streptococcus pneumoniae demonstrates for the first time that an NPET mutant exhibits completely different ribosome occupancy compared to wild-type. We demonstrate, using RNA footprinting, that changes in ribosome occupancy correlate with changes in ribosome stalling. Further, statistical analysis shows that short peptide sequences that cause ribosome stalling are species-specific and evolutionarily selected. NPET structure is required to realize these specie-specific ribosome stalling. (3) ConclusionsResults support the role of NPET on ribosome stalling. NPET structure is required to realize the species-specific and evolutionary conserved ribosome stalling. These findings clarify the role of NPET structure on the translation process.

genomics↗

bioRxiv2020

Identification of novel mutations contributing to azole tolerance of Aspergillus fumigatus through in vitro exposure to tebuconazole

Azole resistance of Aspergillus fumigatus is a global problem. The major resistant mechanism is a cyp51A alteration such as mutation(s) in the gene and the acquisition of a tandem repeat in the promoter. Although other azole tolerances and resistant mechanisms such as hmg1 mutation are known, few reports describe studies elucidating non-cyp51A resistance mechanisms. This study explored genes contributing to azole tolerance in A. fumigatus by in vitro mutant selection with tebuconazole, an azole fungicide. After three-round selection, we obtained four isolates with low susceptibility to tebuconazole. These isolates also showed low susceptibility to itraconazole and voriconazole. Comparison of the genome sequences of the obtained isolates and the parental strain revealed a non-synonymous mutation in MfsD (Afu1g11820, R337L mutation) in all isolates. Furthermore, non-synonymous mutations in AgcA (Afu7g05220, E535Stop mutation), UbcD (Afu3g06030, T98K mutation), AbcJ (Afu3g12220, G297E mutation), and RttA (Afu7g04740, A83T mutation), a protein responsible for tebuconazole tolerance, were found in at least one isolate. Clarification by constructing the MfsD R337L mutant suggests that the mutation contributes to azole tolerance. Disruption of the agcA gene and reconstruction of the A83T point mutation in RttA led to decreased susceptibility to azoles. The reversion of T98K mutation to wild type in UbcD led to the level of azole susceptibility comparable to the parental strain. These results suggest that these mutations contribute to lowered susceptibility to medical azoles and to agricultural azole fungicides.

microbiology↗

bioRxiv2020

Comparative genome and transcriptome analyses revealing interspecies variations in the expression of fungal biosynthetic gene clusters

Filamentous fungi produce various bioactive compounds that are biosynthesized by a set of proteins encoded in biosynthetic gene clusters (BGCs). For an unknown reason, large parts of the BGCs are transcriptionally silent under laboratory conditions, which has hampered the discovery of novel fungal compounds. The transcriptional regulation of fungal secondary metabolism is not fully understood from an evolutionary viewpoint. To address this issue, we conducted comparative genomic and transcriptomic analyses using five closely related species of the Aspergillus section Fumigati: Aspergillus fumigatus, Aspergillus lentulus, Aspergillus udagawae, Aspergillus pseudoviridinutans, and Neosartorya fischeri. From their genomes, 298 secondary metabolite (SM) core genes were identified, with 27.4% to 41.5% being unique to a species. Compared with the species-specific genes, a set of section-conserved SM core genes was expressed at a higher rate and greater magnitude, suggesting that their expression tendency is correlated with the BGC distribution pattern. However, the section-conserved BGCs showed diverse expression patterns across the Fumigati species. Thus, not all common BGCs across species appear to be regulated in an identical manner. A consensus motif was sought in the promoter region of each gene in the 15 section-conserved BGCs among the Fumigati species. A conserved motif was detected in only two BGCs including the gli cluster. The comparative transcriptomic and in silico analyses provided insights into how the fungal SM gene cluster diversified at a transcriptional level, in addition to genomic rearrangements and cluster gains and losses. This information increases our understanding of the evolutionary processes associated with fungal secondary metabolism. Author summaryFilamentous fungi provide a wide variety of bioactive compounds that contribute to public health. The ability of filamentous fungi to produce bioactive compounds has been underestimated, and fungal resources can be developed into new drugs. However, most biosynthetic genes encoding bioactive compounds are not expressed under laboratory conditions, which hampers the use of fungi in drug discovery. The mechanisms underlying silent metabolite production are poorly understood. Here, we attempted to show the diversity in fungal transcriptional regulation from an evolutionary viewpoint. To meet this goal, the secondary metabolisms, at genomic and transcriptomic levels, of the most phylogenetically closely related species in Aspergillus section Fumigati were compared. The conserved biosynthetic gene clusters across five Aspergillus species were identified. The expression levels of the well-conserved gene clusters tended to be more active than the species-specific, which were not well-conserved, gene clusters. Despite highly conserved genetic properties across the species, the expression patterns of the well-conserved gene clusters were diverse. These findings suggest an evolutionary diversification at the transcriptional level, in addition to genomic rearrangements and gains and losses, of the biosynthetic gene clusters. This study provides a foundation for understanding fungal secondary metabolism and the potential to produce diverse fungal-based chemicals.

genomics↗