Map of dimorphic switching-related signaling pathways in Sporothrix schenckii based on its transcriptome
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ABSTRACT: Sporotrichosis, an implantation mycosis caused by the dimorphic fungus Sporothrix schenckii, gained attention over the two last decades due to its broad geographic range and prevalence in tropical and subtropical areas. The dimorphic switching from hyphal form to the yeast form is essential for the pathogenesis of S. schenckii. During the mycelium-to-yeast transition, there are many changes in asexual development, yeast-phase cell formation, cell wall integrity, and melanin synthesis. However, the mechanisms of the signaling pathways associated with these process remain unclear. In this study, transcriptome sequencing of both the yeast stage and mycelium of S. schenckii was carried out. The results showed that 12217 genes, including genes involved in signal transduction and chitin synthesis, were expressed differentially between the two stages. According to these results, a map of the signaling pathways, related to the dimorphic switch, was drawn. Overall, the transcriptome data and analysis presented here lay the foundation for further research into the molecular mechanisms controlling the dimorphic switch of S. schenckii and support the development of anti-S. schenckii strategies targeting genes related to signaling pathways.
Project description:Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), a prevalent systemic mycosis in South America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and transform into the yeast parasitic form. Thus, the mycelium-to-yeast transition is of particular interest because conversion to yeast is essential for infection. We have used a P. brasiliensis biochip, carrying sequences of 4,692 genes from this fungus to monitor gene expression at several time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). Keywords: Time Course
Project description:Histoplasma capsulatum is a thermally dimorphic fungus with worldwide distribution, and high incidence in the Americas. It is the etiologic agent of histoplasmosis, an important life-threatening systemic mycosis. Dimorphism is an important feature for fungal survival in different environments and it has been related to the virulence of H. capsulatum, and essential to the establishment of infection. Proteomic profiles have brought important contributions to the knowledge of metabolism and pathogenicity in several biological models. However, studies of the H. capsulatum proteome have been underexplored. In the present study, we report the first proteomic comparison between the mycelium and the yeast cells of H. capsulatum. Liquid chromatography coupled to mass spectrometry was used to evaluate the proteomic profile of the two phases of H. capsulatum. In summary, 214 proteins were only detected/or preferentially abundant in mycelium, while the same occurred to 335 proteins in yeast cells. In mycelium, enzymes related to the glycolytic pathway and to the alcoholic fermentation showed greater abundance, suggesting a higher use of anaerobic pathways for energy production. In yeast cells, proteins related to the tricarboxylic acid cycle and response to temperature stress showed high abundance. Proteins related to oxidative stress response or involved with cell wall metabolism were identified with differential abundance in both conditions. Validation of proteomic data was performed by enzymatic activity determination, western blot assays, or immunofluorescence microscopy. These experiments corroborated, directly or indirectly, the abundance of isocitrate lyase, 2-methylcitrate synthase, catalase B, and mannosyl-oligosaccharide-1,2-alpha-mannosidase in the mycelium and heat shock protein (HSP) 30, HSP60, glucosamine-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, and N-acetylglucosamine-phosphate mutase in yeast-cells. The proteomic profile associated functional classification analyzes of proteins provided a better understanding of the metabolic reorganization and cell wall remodeling on the yeast form of H. capsulatum.
Project description:Although many studies have focused on dimorphic switching, noncoding RNAs in Talaromyces marneffei have been neglected until now. rRNA depletion RNA-seq with or without RNase R treatment were performed in mycelium and yeast conditions of Talaromyces marneffei by to unveil the functions of circRNAs in dimorphic switching.
Project description:Tremella fuciformis is a dimorphic fungus that can undertake the reversible transition between yeast-like spores and hypha forms. In this project, we attempted to explore the differential proteins profile of dikaryotic yeast-like spores (FBMDS) and dikaryotic mycelium (DM) by applying the HRMS1-DIA full proteomics and PRM target proteomics synthetically. The results showed that a total of 5687 proteins were quantified, and 2220 of them (39.01%) showed more than a two-fold change in expression. The functional analysis of differential expression proteins (DEPs) confirmed that the DEPs were mainly located on membrane and nucleus, and FBMDS tended to express proteins involved in DNA replication and transcription, DNA damage repair, biosynthesis, and metabolism. At the same time, DM exhibited increased expression of signal transduction such as MAPK signaling pathway, Ras signaling pathway.
Project description:Paracoccidioides brasiliensis, a thermally dimorphic fungus, is the causative agent of paracoccidioidomycosis, a systemic mycosis that is widespread in Latin America. P. brasiliensis infection occurs when conidia or mycelial fragments are inhaled by the host, which causes these cells to transition to the yeast form. The development of disease requires conidia inside host alveoli to differentiate into yeast cells in a temperature-dependent manner. This fungus is a facultative intracellular pathogen able to survive and replicate inside non-activated macrophages. Therefore, the survival of P. brasiliensis inside the host depends on the ability to adapt to oxidative stress induced by immune cells, especially alveolar macrophages. For several years, reactive oxygen species (ROS) were only associated with pathological processes. Currently, a plethora of roles for ROS in cell signaling have emerged. We have previously reported that low ROS concentrations cause cell proliferation in the human pathogenic fungus P. brasiliensis. In the present report, we investigated the influence of phosphorylation events in that process. Using a mass spectrometry-based approach, we mapped 440 phosphorylation sites in 230 P. brasiliensis proteins and showed that phosphorylation at different sites determines fungal responses to oxidative stress. Furthermore, we described, for the first time, the presence of a two-component signal transduction system in P. brasiliensis. These findings will help us to understand the phosphorylation events involved in the oxidative stress response.
Project description:Dimorphic fungi are temperature-sensitive organisms that couple their cell shape with their environment. One of these fungi, Histoplasma capsulatum, exists as both a soil-dwelling hypha and a host-associated yeast. Here we examine the role of the previously uncharacterized gene MSB2 in filamentous growth. We performed a genetic screen to identify insertion mutants that are unable to transition from the yeast form to the hyphal form. One yeast-locked mutant has an insertion upstream of the MSB2 gene. This mutant strain (SG1) fails to express MSB2, whose ortholog in the model yeast Saccharomyces cerevisiae is a known signaling component in the high osmolarity glycerol (HOG) pathway and filamentous growth (FG) pathway. Here, we profiled gene expression by RNA-seq to characterize transcriptional differences between wild type and msb2 mutant strains during the yeast to hyphal transition.
Project description:Penicillium marneffei (Talaromyces marneffei) is an opportunistic human pathogen that can grow in a multicellular hyphal form at 25°C or a unicellular fission yeast form at 37°C, and can switch between these two forms in response to temperature. The yeast form is found in infected individuals and represents the pathogenic phase. In response to specific environmental cues, the hyphal form can undergo asexual development (conidiation) the produce a differentiated multicellular structure called a conidiophore which produces dormant spores (conidia). Inhaled conidia initiate infection. To identify genes that are important during the early stages in the transition from hyphal to yeast and yeast to hyphal cells transcriptional profiling was performed with a custom microarray consisting of ~42% of the predicted P. marneffei genes and RNA from P. marneffei hyphal cells switched to growth at 37°C for 6 hours (hyphal-yeast switch) and yeast cells switched to growth at 25°C for 6 hours (yeast-hyphal switch).
Project description:Paracoccidioides brasiliensis is a thermally dimorphic fungus, which causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has been rarely addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25% of the organism’s genome, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared gDNA and known control genes were printed onto glass slides to generate a microarray of over 12,000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from mycelia, especially at earlier time-points, and that mycelial gene expression changed less than it did on yeasts over time. Genes, upregulated in yeasts, were found to be involved in methionine/cysteine metabolism, respiratory processes, metabolic processes (sugars, amino acids, proteins and lipids), transporters (small peptides, sugar, ions and toxin), regulatory proteins and transcription factors. Mycelia genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transporters showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analyzed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes coding for ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand knowledge about the different morphologic forms of P. brasiliensis during growth in culture.
Project description:Penicillium marneffei (Talaromyces marneffei) is an opportunistic human pathogen that can grow in a multicellular hyphal form at 25M-BM-0C or a unicellular fission yeast form at 37M-BM-0C, and can switch between these two forms in response to temperature. The yeast form is found in infected individuals and represents the pathogenic phase. In response to specific environmental cues, the hyphal form can undergo asexual development (conidiation) the produce a differentiated multicellular structure called a conidiophore which produces dormant spores (conidia). Inhaled conidia initiate infection. To identify genes that are important during the early stages in the transition from hyphal to yeast and yeast to hyphal cells transcriptional profiling was performed with a custom microarray consisting of ~42% of the predicted P. marneffei genes and RNA from P. marneffei hyphal cells switched to growth at 37M-BM-0C for 6 hours (hyphal-yeast switch) and yeast cells switched to growth at 25M-BM-0C for 6 hours (yeast-hyphal switch). A custom microarray consisting of short, random genomic fragments from P. marneffei (~42% of the predicted genes) was generated using PCR products of the inserts from two independent DNA libraries constructed from genomic DNA of the P. marneffei type strain FRR2161. PCR products from previously cloned P. marneffei genes with known expression profiles were included as controls on the microarray. Total RNA from hyphal cells before and after switching to growth at 37M-BM-0C for 6 hours (hyphal-yeast switch) and yeast cells before and after switching to growth at 25M-BM-0C for 6 hours (yeast-hyphal switch) were used in pairwise combinations on the microarrays. Three biological replicate cultures were prepared for each experiment.
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.