Project description:Thermally dimorphic human fungal pathogens undergo a reversible program of cellular differentiation in response to their environment that is essential for infectivity and pathogenicity. In the soil, these organisms grow as highly polarized, multicellular hyphal filaments that produce infectious particles. When inhaled by a mammalian host, these cells switch to a unicellular yeast form that causes disease even in healthy hosts. Temperature is considered to be the primary environmental cue that promotes reversible cellular differentiation; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. In a search for other signals that regulate morphogenesis, we considered the monosaccharide N-acetylglucosamine (GlcNAc), which is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc was a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift, indicating that fungal cells sense GlcNAc to promote filamentation. The synchronous morphologic transition stimulated by low temperature and GlcNAc allowed us to examine the temporal regulation of the transcriptome during morphogenesis to reveal candidate genes involved in establishing the filamentous growth program. Through this analysis, we identified two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes.

Project description:Thermally dimorphic human fungal pathogens undergo a reversible program of cellular differentiation in response to their environment that is essential for infectivity and pathogenicity. In the soil, these organisms grow as highly polarized, multicellular hyphal filaments that produce infectious particles. When inhaled by a mammalian host, these cells switch to a unicellular yeast form that causes disease even in healthy hosts. Temperature is considered to be the primary environmental cue that promotes reversible cellular differentiation; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. In a search for other signals that regulate morphogenesis, we considered the monosaccharide N-acetylglucosamine (GlcNAc), which is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc was a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift, indicating that fungal cells sense GlcNAc to promote filamentation. The synchronous morphologic transition stimulated by low temperature and GlcNAc allowed us to examine the temporal regulation of the transcriptome during morphogenesis to reveal candidate genes involved in establishing the filamentous growth program. Through this analysis, we identified two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes. For each time-course sample, cDNA was coupled to Cy5 and a reference cDNA pool was made by combining RNA from t = 0 and all late time course samples, which was coupled to Cy3. For end point microarray experiments (i.e., established yeast samples compared to established filamentous samples), G217B yeast cDNA was coupled to Cy5 and filament cDNA was coupled to Cy3.

Project description:The exoskeleton gives to insects its shape, waterproofing and a range of other roles, besides serving as means of locomotion, the motor function being accomplished by the attachment of somatic muscles. Exoskeleton is renewed every time the insect molts under the control of ecdysteroid hormones. The last molt, which in the holometabolous transforms pupae in adults, involves biosynthesis of the definitive exoskeleton by the subjacent epidemis, followed by progressive differentiation. We used whole genome-based oligonucleotide microarray hybridization and RNA extracted from the honey bee thoracic dorsum, at three time-points of the pupal-to-adult molt, as a strategy to screen genes involved in exoskeleton formation. Gene ontology analysis separated the differentially expressed genes (DEGs) in distinct Biological Processes and Molecular Functions dependently on the time-point approached, thus revealing the functional categories required for adult exoskeleton formation. Approximately 546 of a list of 1253 unique DEGs were up-regulated in the thoracic dorsum during adult cuticle formation, including 23 among the 24 identified cuticle protein (CP) genes, and 29 muscle-related genes. Conserved sequence motifs allowed to include the CP genes in the CPR, Tweedle, Apidermin, CPF, CPLCP1 and Analogous-to-Peritrophins families. Two genes, which do not pertain to any known CP families, were abundantly expressed in the epidermis during adult cuticle formation as shown by in situ hybridization, thus strongly suggesting that they are genuine CP genes. The search for cis-regulatory motifs in the 5M-bM-^@M-^Yuntranslated region of the DEGs revealed potential binding sites for known transcript factors. Several of them were shared by CP genes and thoracic muscle genes, thus allowing to infer that they are co-regulated during differentiation of the thoracic exoskeleton. Together, these data add new information on molecular aspects of exoskeleton formation in the context of the ecdysteroid-coordinated pupal-to-adult molt. The microarray experiments compared differential gene expression in the thoracic integuments of newly-ecdysed pupae (Pw phase), pupae-in-apolysis (Pp phase) and pharate-adults (Pbl phase) collected at the same time from a single honey bee colony. Two separate pools of 10 thoracic integuments each were prepared for each developmental phase (Pw, Pp and Pbl). Dye swaps were done for each comparison and two slides were used to evaluate the differential expression.

Project description:The exoskeleton gives to insects its shape, waterproofing and a range of other roles, besides serving as means of locomotion, the motor function being accomplished by the attachment of somatic muscles. Exoskeleton is renewed every time the insect molts under the control of ecdysteroid hormones. The last molt, which in the holometabolous transforms pupae in adults, involves biosynthesis of the definitive exoskeleton by the subjacent epidemis, followed by progressive differentiation. We used whole genome-based oligonucleotide microarray hybridization and RNA extracted from the honey bee thoracic dorsum, at three time-points of the pupal-to-adult molt, as a strategy to screen genes involved in exoskeleton formation. Gene ontology analysis separated the differentially expressed genes (DEGs) in distinct Biological Processes and Molecular Functions dependently on the time-point approached, thus revealing the functional categories required for adult exoskeleton formation. Approximately 546 of a list of 1253 unique DEGs were up-regulated in the thoracic dorsum during adult cuticle formation, including 23 among the 24 identified cuticle protein (CP) genes, and 29 muscle-related genes. Conserved sequence motifs allowed to include the CP genes in the CPR, Tweedle, Apidermin, CPF, CPLCP1 and Analogous-to-Peritrophins families. Two genes, which do not pertain to any known CP families, were abundantly expressed in the epidermis during adult cuticle formation as shown by in situ hybridization, thus strongly suggesting that they are genuine CP genes. The search for cis-regulatory motifs in the 5’untranslated region of the DEGs revealed potential binding sites for known transcript factors. Several of them were shared by CP genes and thoracic muscle genes, thus allowing to infer that they are co-regulated during differentiation of the thoracic exoskeleton. Together, these data add new information on molecular aspects of exoskeleton formation in the context of the ecdysteroid-coordinated pupal-to-adult molt.

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N-acetylglucosamine (GlcNAc) sensor Ngs1 is essential for GlcNAc-involved biological control potential of fungal insecticide Beauveria bassiana

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