Transcriptome analysis of Paracoccidioides brasiliensis cells undergoing the Mycelium-to-Yeast transition
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ABSTRACT: 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:Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), the most common systemic mycosis in Latin America. The infection is initiated by inhalation of environmental dispersed conidia produced by the saprophytic phase of the fungus. In the lungs, P. brasiliensis assumes the parasitic yeast form and must cope with the adverse conditions imposed by cells of the host immune system, which includes a harsh environment highly concentrated in reactive oxygen species (ROS). In this work, we used the ROS-generating agent paraquat to experimentally simulate oxidative stress conditions in order to evaluate the stress-induced modulation in gene expression of cultured P. brasiliensis yeast cells using a microarray hybridization approach.
Project description:Phenothiazines are antipsychotic drugs used in the treatment of schizophrenia, which have been shown to present antimicrobial activity (in vitro and in vivo) against a great variety of pathogenic microorganisms, including bacteria, parasites (protozoa and helmints) and fungi. In this study, we used competitive microarray hybridization to evaluate the effects of increasing concentrations of the piperidinic phenothiazine derivative thioridazine (TR) on the transcriptome of Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM) - the most common systemic mycosis in Latin America. These analyses showed that the presence of TR affected expression of more than 1,800 genes from this fungus, including genes related to cellular processes such as cell wall metabolism and drug resistance.
Project description:Examination and comparison of the transcriptional profile of bone marrow derived dendritic cells (BMDCs) in response to infection by the fungus Paracoccidioides brasiliensis in resistant/susceptible mice
Project description:Paracoccidioides spp. is the etiological agent of Paracoccidioidomycosis (PCM), a systemicinfection with wide distribution in Latin America. Macrophages are very important cells during the response to infection by Paracoccidoides brasiliensis and understanding the interaction between the fungus and immune cells is very relevant for understanding the disease. In this study, we performed a proteomic analysis to assess the consequences of P. brasiliensis yeast infection on the THP-1 macrophage proteome and to verify whether there are differences between the proteome of cells infected with the live fungus. We identified 443 upregulated and 2247 downregulated proteins in macrophages infected with live P. brasiliensis yeasts, compared to uninfected macrophages. Proteins differentially expressed in cells infected are related to metabolism and energy production, protein synthesis and processing, transcription, cell cycle, DNA processing, cell signaling, oxidative stress, immune response, among other processes . Proteomic analysis revealed that P. brasiliensis, causes metabolic alterations in infected cells, drastically affecting energy production pathways. In addition, macrophages showed many upregulated, mostly downregulated, immune system proteins. Thus, the present work contributes to elucidate the changes that occur in immune cells in response to infection by P. brasiliensis and may help to better understand PCM.
Project description:Phenothiazines are antipsychotic drugs used in the treatment of schizophrenia, which have been shown to present antimicrobial activity (in vitro and in vivo) against a great variety of pathogenic microorganisms, including bacteria, parasites (protozoa and helmints) and fungi. In this study, we used competitive microarray hybridization to evaluate the effects of increasing concentrations of the piperidinic phenothiazine derivative thioridazine (TR) on the transcriptome of Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM) - the most common systemic mycosis in Latin America. These analyses showed that the presence of TR affected expression of more than 1,800 genes from this fungus, including genes related to cellular processes such as cell wall metabolism and drug resistance. Microarray hybridizations were carried out with amplified RNA (aRNA), which was produced starting with total RNA extracted from cells treated with thioridazine (15, 20 or 25 M-BM-5M) for 168 hours. The aRNA synthesized from cells unexposed to TR was taken as reference. Each treatment was analyzed with two independent hybridizations (a pair of hybridizations, with dye-swaps within each pair). Since each biochip carried two replicas of the arrayed genes, a total of four intensity readings were generated for each element in the microarray.The data has been filtered, normalized and adjusted as described in the "data processing" box for each individual sample. Dye swap consistency checking has been performed between equivalent experimental pairs with the aid of the software TIGR MIDAS v2.19 and inconsistent spots have been flagged and excluded from further analyses. The values obtained from each dye swap pair have been averaged and consolidated into a single data file, generating a total of 8 hybridization files (four from each treatment concentration). These files have been loaded into the software TIGR Multi-Experiment Viewer, v.3.1 Experiments were then normalized and genes that displayed statistically significant modulation were identified by one-way ANOVA, considering p < 0.01 as a cutoff value. A list of statisticaly significant, modulated genes was created, showing the average log ratio for each one of these genes, within each time point.
Project description:The fungi of Paracoccidioides spp. genus are the causative agents of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. This fungus is considered a facultative intracellular pathogen that is able to survive and replicate inside macrophages. The survival of the fungus during infection depends on its adaptability to various conditions, such as nitrosative/oxidative stress produced by the host immune cells, particularly alveolar macrophages. Currently, there is little knowledge about the P. brasiliensis signaling pathways involved in the fungus evasion mechanism of the host defense response. However, it is known that some of these pathways are triggered by reactive oxygen species and reactive nitrogen species (ROS/RNS) produced by host cells. Considering that the effects of NO (nitric oxide) on pathogens are concentration dependent, such effects could alter the redox state of cysteine residues by influencing (activating or inhibiting) a variety of protein functions, notably S-nitrosylation, a highly important NO-dependent posttranslational modification that regulates cellular functions and signaling pathways. It has been demonstrated by our group that P. brasiliensis yeast cells proliferate when exposed to low NO concentrations. Thus, this work investigated the modulation profile of S-nitrosylated proteins of P. brasiliensis, as well as identifying S-nitrosylation sites after treatment with RNS. Through mass spectrometry analysis (LC-MS/MS) and label-free quantification, it was possible to identify 474 proteins in the S-nitrosylated proteome study. With this approach, we observed that proteins treated with NO at low concentrations presented a proliferative response pattern, with several proteins involved in cellular cycle regulation and growth being activated. These proteins appear to play important roles in fungal virulence. On the other hand, proteins stimulated by high NO concentrations exhibited a survival response pattern, which may help to elucidate RNS antifungal properties and identify potential molecular targets for the development of new drugs in the future
Project description:Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America. Few virulence factors have been identified for this pathogen. In this study, we quantitatively evaluated the protein composition of P. brasiliensis in the yeast phase using minimal and rich media to obtain a better understanding of its virulence and to gain new insights into pathogen adaptation strategies. This analysis was performed on two isolates of the Pb18 strain showing distinct infection profiles in B10.A mice. Using liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis, we identified and quantified 317 proteins in minimal medium, 29 of which were overexpressed in virulent Pb18. In rich medium, 29 out of 214 identified proteins were over-expressed in the virulent fungus. Alcohol dehydrogenase, mitochondrial peroxiredoxin PRX1 and protein vacuolar ATP synthase catalytic subunit A were up-regulated in virulent Pb18 in both media, suggesting potential roles of these proteins in virulence regulation in P. brasiliensis. Proteins up-regulated in both isolates were grouped according to their functional categories. Virulent Pb18 undergoes metabolic reorganization and increased expression of proteins involved in fermentative respiration. This approach allowed us to identify potential virulence regulators and to understand how Paracoccidioides modulates the host-pathogen interaction to its advantage. Data are available via ProteomeXchange Consortium with identifier PXD000804.
Project description:Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), the most common systemic mycosis in Latin America. The infection is initiated by inhalation of environmental dispersed conidia produced by the saprophytic phase of the fungus. In the lungs, P. brasiliensis assumes the parasitic yeast form and must cope with the adverse conditions imposed by cells of the host immune system, which includes a harsh environment highly concentrated in reactive oxygen species (ROS). In this work, we used the ROS-generating agent paraquat to experimentally simulate oxidative stress conditions in order to evaluate the stress-induced modulation in gene expression of cultured P. brasiliensis yeast cells using a microarray hybridization approach. Microarray hybridizations were carried out with RNA obtained from cells treated with paraquat (0.5 or 5.0 mM) for 5 hours. The RNA obtained from cells unexposed to paraquat was taken as reference. Each treatment was analyzed with four independent hybridizations (two pairs of dye-swapped hybridizations), using RNA from two biological replicas. The data has been filtered, normalized and adjusted as described in the "data processing" box for each individual sample. Dye swap consistency checking has been performed between equivalent experimental pairs with the aid of the software TIGR MIDAS v2.19 and inconsistent spots have been flagged and excluded from further analyses. The values obtained from each dye swap pair have been averaged and consolidated into a single data file, generating a total of 8 hybridization files (four from each treatment concentration). These files have been loaded into the software TIGR Multi-Experiment Viewer, v.3.01. Experiments were then normalized and genes that displayed statistically significant modulation were identified by one-way ANOVA, considering p < 0.01 as a cutoff value. A list of statisticaly significant, modulated genes was created, showing the average log ratio for each one of these genes, within each time point