Project description:Mucormycosis, caused by Mucoralean fungi, is among the most lethal fungal diseases, and a deeper understanding of its pathogenesis is urgently needed. In this study, we examined the early stages of fungal interaction with host macrophages using Mucor lusitanicus and Rhizopus microsporus as model organisms. Transcriptomic profiling of virulent (WT) and an RNAi-deficient strain (r3b2Δ) of M. lusitanicus strains during phagocytosis uncovered thousands of differentially expressed genes (DEGs), highlighting early metabolic activation as a key survival strategy inside the phagosome. Enriched pathways included amino acid transport, nucleotide metabolism, and translation, reflecting an adaptive fungal response to nutrient deprivation and host immune stress. Integrative analyses of mRNA and sRNA profiles also revealed a critical role of the RNAi pathways in modulating gene expression during infection. Building on these observations, we identified four chromatin- and transcription-related candidate virulence genes—brca1, box, hist1, and hda10—which were strongly upregulated during phagocytosis and regulated by RNAi. Functional validation through gene deletion in M. lusitanicus and disruption in R. microsporus revealed that while loss of these genes in M. lusitanicus did not significantly affect virulence, R. microsporus mutants for brca1, hist1, and hda10 showed attenuated virulence in a murine model. Our findings suggest that although M. lusitanicus remains a valuable tool for genetic manipulation, species-specific differences must be considered when studying virulence. The study also underscores the importance of using multiple Mucorales models to uncover conserved and divergent strategies employed by pathogenic fungi. These insights contribute to a broader understanding of fungal adaptation, immune evasion, and the identification of novel targets for antifungal intervention.
Project description:Mucorales are basal fungi that opportunistically cause a fatal infection known as mucormycosis (black fungus disease), which poses a significant threat to human health due to its high mortality rate and its recent association with SARS-CoV-2 infections. On the other hand, histone methylation is a regulatory mechanism with pleiotropic effects, including the virulence of several pathogenic organisms. However, the role of epigenetic changes at the histone level never has been studied in Mucorales. Here, we dissected the functional role of Set1, a histone methyltransferase that catalyzes the methylation of H3K4, which is associated with the activation of gene transcription and virulence. A comparative analysis of the Mucor lusitanicus genome (previously known as Mucor circinelloides f. lusitanicus) identified only one homolog of Set1 from Candida albicans and Saccharomyces cerevisiae that contains the typical SET domain. Knockout strains in the gene set1 lacked H3K4 monomethylation, dimethylation, and trimethylation enzymatic activities. These strains also showed a significant reduction in vegetative growth and sporulation. Additionally, set1 null strains were more sensitive to SDS, EMS, and UV light, indicating severe impairment in the repair process of the cell wall and DNA lesions and a correlation between Set1 and these processes. During pathogen-host interactions, strains lacking the set1 gene exhibited shortened polar growth within the phagosome and attenuated virulence both in vitro and in vivo. Our findings suggest that the histone methyltransferase Set1 coordinates several cell processes related to the pathogenesis of M. lusitanicus and may be an important target for future therapeutic strategies against mucormycosis.
Project description:The silkworm, Bombyx mori, is a complete metamorphosis insect and an economically important for silk production, the model to study insect physiology and biochemistry. Bombyx mori nucleopolyhedrovirus (BmNPV) is a principal pathogen of the silkworm and its host range is restricted to silkworm larvae, requiring interaction with silkworm larvae to accomplish virus replication. Prothoracic glands (PGs) are a model for synthetic ecdysone with regulating insect growth and development. In this study, day-4 fifth instar silkworm larvae were infected by BmNPV, the wandering silkworms appeared in the infected groups were 12 hours earlier than that in the control groups, and the ecdysone titer in infected larvae was significantly higher than that of the control larvae. Then, we used RNA sequencing (RNA-seq) to analyze silkworm PGs 48 h after BmNPV infection. The classifications of the 15 differential expression genes (DEGs) were mainly involved in the metabolic processes and pathways. The RT-qPCR results of the DEGs in the PGs of BmNPV-infected at 24, 48, and 72 h were generally consistent with the transcriptome data. The transcripts of BmTrypsin-1 and BmACSS3 were significantly increased from 24 to 72 h after BmNPV infection that they may be involved in the maturation process in the latter half of silkworm fifth instar larvae. These findings will help to address the interactions between BmNPV infection and host developmental response.
Project description:Silkworms show a reproductive behavior induced by sex pheromone. To elucidate the neral mechanism of sex pheromone induced sexual behavior in the silkworm, we attempted to use the neural activity-induced gene as a neural activity marker. Since no neural activity-induced gene was identified in the silkworm, we conducted screening of neural activity-induced gene using the male silkworm brain. By the screening, we identified Bhr38 as a novel neural activity-induced gene, and succeded to comprehensively map the active neruons in the silkworm brain in response to the sex pheromone exposure. Further, we found that Dhr38, the Drosophila homologue of Bhr38, also expressed in a neural activity dependent manner. These results strongly suggest that Hr38 is a highly conserved neural activity-induced gene.
Project description:Background: MicroRNA (miRNA) and other small regulatory RNAs contribute to the modulation of a large number of cellular processes. We sequenced three total RNA libraries prepared from the whole body, and the anterior and posterior silk glands of Bombyx mori, with a view to expanding the repertoire of silkworm miRNAs and exploring transcriptional differences in miRNAs between segments of the silk gland. Results: With the aid of large-scale Solexa sequencing technology, we validated 244 unique miRNA genes, including 191 novel and 53 previously reported genes, corresponding to 309 loci in the silkworm genome. Interestingly, 24 unique miRNAs were widely conserved from invertebrates to vertebrates; 12 unique ones were limited to invertebrates and 33 were confined to insects; whereas the majority of the newly identified miRNAs were silkworm-specific. We identified 21 clusters and 42 paralogs of miRNAs in the silkworm genome. However, sequence tags showed that paralogs or clusters are not prerequisites for coordinated transcription and accumulation. The majority of silkworm-specific miRNAs are located in transposable elements, and display significant differences in abundance between the anterior and posterior silk glands. Conclusions: Conservative analysis revealed that miRNAs serve as phylogenetic markers and function in evolutionary signaling. The newly identified miRNAs greatly enriched the repertoire of insect miRNAs, and provide insights into miRNA evolution, biogenesis, and expression in insects. The differential expression of miRNAs in the anterior and posterior silk glands supports their involvement as new layers in the regulation of the silkworm silk gland.
Project description:Background: MicroRNA (miRNA) and other small regulatory RNAs contribute to the modulation of a large number of cellular processes. We sequenced three total RNA libraries prepared from the whole body, and the anterior and posterior silk glands of Bombyx mori, with a view to expanding the repertoire of silkworm miRNAs and exploring transcriptional differences in miRNAs between segments of the silk gland. Results: With the aid of large-scale Solexa sequencing technology, we validated 244 unique miRNA genes, including 191 novel and 53 previously reported genes, corresponding to 309 loci in the silkworm genome. Interestingly, 24 unique miRNAs were widely conserved from invertebrates to vertebrates; 12 unique ones were limited to invertebrates and 33 were confined to insects; whereas the majority of the newly identified miRNAs were silkworm-specific. We identified 21 clusters and 42 paralogs of miRNAs in the silkworm genome. However, sequence tags showed that paralogs or clusters are not prerequisites for coordinated transcription and accumulation. The majority of silkworm-specific miRNAs are located in transposable elements, and display significant differences in abundance between the anterior and posterior silk glands. Conclusions: Conservative analysis revealed that miRNAs serve as phylogenetic markers and function in evolutionary signaling. The newly identified miRNAs greatly enriched the repertoire of insect miRNAs, and provide insights into miRNA evolution, biogenesis, and expression in insects. The differential expression of miRNAs in the anterior and posterior silk glands supports their involvement as new layers in the regulation of the silkworm silk gland. Sequencing three total RNA pools of the whole silkworm body from 5th-instar day-3 larvae, and anterior and posterior silkworm silk glands, using the latest sequencing Solexa technology