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Bioluminescence imaging in Paracoccidioides spp.: a tool to monitor the infectious processes
(2022) Leite, Vanessa Rafaela Milhomem Cruz; Tomazett, Mariana Vieira; Curcio, Juliana Santana de; Oliveira, Nicolau Sbaraini; Bailão, Alexandre Melo; Gonçales, Relber Aguiar; Moraes, Dayane; Pereira, Maristela; Vainstein, Marilene Henning; Schrank, Augusto
The genus Paracoccidioides comprises the species complex causing paracoccidioidomycoses (PCM). These fungi are a serious public health problem due to the long-term drug therapy, follow-up treatment, and frequent sequelae generated by the infection, such as pulmonary fibrosis. In this sense, the objective of this work was to generate bioluminescent reporter strains of Paracoccidioides spp. harboring a thermostable, red-shifted luciferase gene under the control of different constitutive promoters. The strains were generated by the integration of a species-specific codon-optimized luciferase gene upon actin or enolase promoter's control. The insertion of the constructs in Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells were performed through Agrobacterium tumefaciens-mediated transformation. The results demonstrated the presence of several transformants harboring the luciferase gene. These transformants were further confirmed by the expression of luciferase and by the presence of the hygromycin resistance gene. Moreover, the luciferase activity could be detected in in vitro bioluminescence assays and in vivo models of infection. In general, this work presents the methodology for the construction of bioluminescent strains of Paracoccidioides spp., highlighting potential promoters and proposing an in vivo model, in which those strains could be used for the systemic study of PCM.
Immunoproteomic approach of extracellular antigens from Paracoccidioides species reveals exclusive B-cell epitopes
(2020) Moreira, André Luís Elias; Oliveira, Milton Adriano Pelli de; Silva, Lana OHara Souza; Inácio, Moisés Morais; Bailão, Alexandre Melo; Rocha, Juliana Alves Parente; Leite, Vanessa Rafaela Milhomem Cruz; Paccez, Juliano Domiraci; Soares, Célia Maria de Almeida; Weber, Simone Schneider; Borges, Clayton Luiz
Fungi of the Paracoccidioides genus are the etiological agents of paracoccidioidomycosis (PCM), a systemic mycosis restricted to the countries of Latin America. Currently, the Paracoccidioides complex is represented by Paracoccidioides lutzii, Paracoccidioides americana, Paracoccidioides brasiliensis, Paracoccidioides restrepiensis, and Paracoccidioides venezuelensis. Even with advances in techniques used for diagnosing fungal diseases, high rates of false-positive results for PCM are still presented. Additionally, there is no efficient antigen that can be used to follow up the efficiency of patient treatment. The immunoproteomic is considered a powerful tool for the identification of antigens. In addition, antigens are molecules recognized by the immune system, which make them excellent targets for diagnostic testing of diseases caused by microorganisms. In this vein, we investigated which antigens are secreted by species representing Paracoccidioides complex to increase the spectrum of molecules that could be used for future diagnostic tests, patient follow-up, or PCM therapy. To identify the profile of antigens secreted by Paracoccidioides spp., immunoproteomic approaches were used combining immunoprecipitation, followed by antigen identification by nanoUPLC-MSE -based proteomics. Consequently, it was possible to verify differences in the exoantigen profiles present among the studied species. Through a mass spectrometry approach, it was possible to identify 79 exoantigens in Paracoccidioides species. Using bioinformatics tools, two unique exoantigens in P. lutzii species were identified, as well as 44 epitopes exclusive to the Paracoccidioides complex and 12 unique antigenic sequences that can differentiate between Paracoccidioides species. Therefore, these results demonstrate that Paracoccidioides species have a range of B-cell epitopes exclusive to the complex as well as specific to each Paracoccidioides species. In addition, these analyses allowed us the identification of excellent biomarker candidates for epidemiology screening, diagnosis, patient follow-up, as well as new candidates for PCM therapy.
Comparative proteomic analysis of histoplasma capsulatum yeast and mycelium reveals differential metabolic shifts and cell wall remodeling processes in the different morphotypes
(2021) Almeida, Marcos Abreu; Baeza, Lilian Cristiane; Paes, Rodrigo de Almeida; Bailão, Alexandre Melo; Borges, Clayton Luiz; Guimarães, Allan Jefferson; Soares, Célia Maria Almeida; Oliveira, Rosely Maria Zancopé
Histoplasma capsulatum is a thermally dimorphic fungus distributed worldwide, but with the highest incidence in the Americas within specific geographic areas, such as the Mississippi River Valley and regions in Latin America. This fungus is the etiologic agent of histoplasmosis, an important life-threatening systemic mycosis. Dimorphism is an important feature for fungal survival in different environments and is related to the virulence of H. capsulatum, and essential to the establishment of infection. Proteomic profiles have made important contributions to the knowledge of metabolism and pathogenicity in several biological models. However, H. capsulatum proteome studies 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 growth, mycelium, and yeast. In summary, 214 and 225 proteins were only detected/or preferentially abundant in mycelium or yeast cells, respectively. In mycelium, enzymes related to the glycolytic pathway and to the alcoholic fermentation occurred in 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 were in high abundance. Proteins related to oxidative stress response or involved with cell wall metabolism were identified with differential abundance in both conditions. Proteomic data validation 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 analyses of proteins provided new and interesting information regarding the differences in metabolism between the two distinct growth forms of H. capsulatum.
A glyphosate-based herbicide induces histomorphological and protein expression changes in the liver of the female guppy Poecilia reticulata
(2017) Santos, Ana Paula Rezende dos; Rocha, Thiago Lopes; Borges, Clayton Luiz; Bailão, Alexandre Melo; Soares, Célia Maria de Almeida; Saboia-Morais, Simone Maria Teixeira de
Glyphosate-based herbicides (GBH) are among the most common herbicides found in aquatic systems, but limited data are available about their mode of action and hepatotoxicity in fish. This study investigated the hepatotoxicity induced by GBH in the guppy Poecilia reticulata using a histopathological assessment associated with a proteomic approach. Guppies were exposed to GBH for 24 h at 1.8 mg of glyphosate L−1, corresponding to 50% of the LC50, 96 h. The results indicate that the GBH at 1.8 mg of glyphosate L−1 induce the development of hepatic damage in P. reticulata, which is exposure-time dependent. The histopathological indexes demonstrate that GBH cause inflammatory, regressive, vascular and progressive disorders in the liver of guppies. Using 2D gel electrophoresis associated with mass spectrometry, 18 proteins that changed by GBH were identified and were related to the cellular structure, motility and transport, energy metabolism and apoptosis. The results show that the acute exposure to GBH causes hepatic histopathological damage related to protein expression profile changes in P. reticulata, indicating that a histopathological assessment associated with a proteomic analysis provides a valuable approach to assess the toxic effects of GBH in sentinel fish species.
Mechanisms of copper and zinc homeostasis in pathogenic black fungi
(2018) Bailão, Mirelle Garcia Silva; Silva, Kassyo Lobato Potenciano da; Anjos, Laura Raniere Borges dos; Lima, Patrícia de Sousa; Teixeira, Marcus de Melo; Soares, Célia Maria de Almeida; Bailão, Alexandre Melo
Black fungi comprise a diverse group of melanized microorganisms, many of which are able to infect humans. One of the recognized diseases that arise with black fungi infection is chromoblastomycosis, a neglected implantation mycosis. Considering their ecology, black fungi may face conditions with distinct metal availability. Zinc and copper are essential transition metals, which become toxic in excess. During the interaction with host, fungi may face either metal deprivation or poisoning. Here we report an in silico analysis of four black fungi genomes concerning zinc and copper homeostasis. Overall, these organisms share apparatus of metal uptake, storage and detoxification with other pathogenic and non-pathogenic fungi. Genes coding plasma membrane and organelle transporters, as well as metal binding proteins were identified. Althought putatives zinc and copper responsive transcription factors have been found in the analyzed genomes, remarkable structural differences were perceived when compared to the already characterized regulators. Black fungi may harbor unique features concerning the regulation of zinc and copper homeostasis, which is probably a result of the niches they can inhabit. The data provided here add knowlegde to a still unexplored aspect of black fungi biology that may be useful in the understanding of their pathogenicity.
Osmotic stress adaptation of Paracoccidioides lutzii, Pb01, monitored by proteomics
(2016) Rodrigues, Leandro Nascimento da Silva; Brito, Wesley de Almeida; Parente, Ana Flávia Alves; Weber, Simone Schneider; Bailão, Alexandre Melo; Casaletti, Luciana; Borges, Clayton Luiz; Soares, Célia Maria de Almeida
The ability to respond to stressful conditions is essential for most living organisms. In pathogenic organisms, this response is required for effective transition from a saprophytic lifestyle to the establishment of pathogenic interactions within a susceptible host. Hyperosmotic stress has been used as a model to study signal transduction and seems to cause many cellular adaptations, including the alteration of protein expression and cellular volume as well as size regulation. In this work, we evaluated the proteomic profile of Paracoccidioides lutzii Pb01 yeast cells during osmotic stress induced by potassium chloride. We performed a high accuracy proteomic technique (NanoUPLC-MSE) to identify differentially expressed proteins during osmotic shock. The data describe an osmoadaptative response of this fungus when subjected to this treatment. Proteins involved in the synthesis of cell wall components were modulated, which suggested cell wall remodeling. In addition, alterations in the energy metabolism were observed. Furthermore, proteins involved in amino acid metabolism and hydrogen peroxide detoxification were modulated during osmotic stress. Our study suggests that P. lutzii Pb01. presents a vast osmoadaptative response that is composed of different proteins that act together to minimize the effects caused by osmotic stress.
The second International Symposium on Fungal Stress: ISFUS
(2018) Alder-Rangel, Alene Estelle; Bailão, Alexandre Mello; Cunha, Anderson Ferreira da; Soares, Célia Maria de Almeida; Chengshu, Wang; Bonatto, Diego; Dadachova, Ekaterina; Hakalehto, Elias; Eleutherio, Elis Cristina Araujo; Fernandes, Éverton Kort Kamp; Gadd, Geoffrey Michael; Braus, Gerhard H.; Braga, Gilberto Úbida Leite; Goldman, Gustavo Henrique; Malavazi, Iran; Hallsworth, John E.; Takemoto, Jon Y.; Fuller, Kevin K.; Selbmann, Laura; Corrochano Peláez, Luis María; Rangel, Drauzio Eduardo Naretto
The topic of ‘fungal stress’ is central to many important disciplines, including medical mycology, chronobiology, plant and insect pathology, industrial microbiology, material sciences, and astrobiology. The International Symposium on Fungal Stress (ISFUS) brought together researchers, who study fungal stress in a variety of fields. The second ISFUS was held in May 8-11 2017 in Goiania, Goiás, Brazil and hosted by the Instituto de Patologia Tropical e Saúde Pública at the Universidade Federal de Goiás. It was supported by grants from CAPES and FAPEG. Twenty-seven speakers from 15 countries presented their research related to fungal stress biology. The Symposium was divided into seven topics: 1. Fungal biology in extreme environments; 2. Stress mechanisms and responses in fungi: molecular biology, biochemistry, biophysics, and cellular biology; 3. Fungal photobiology in the context of stress; 4. Role of stress in fungal pathogenesis; 5. Fungal stress and bioremediation; 6. Fungal stress in agriculture and forestry; and 7. Fungal stress in industrial applications. This article provides an overview of the science presented and discussed at ISFUS-2017.
Paracoccidioides brasiliensis presents metabolic reprogramming and secretes a serine proteinase during murine infection
(2017) Pigosso, Laurine Lacerda; Baeza, Lilian Cristiane; Tomazett, Mariana Vieira; Faleiro, Mariana Batista Rodrigues; Moura, Veridiana Maria Brianezi Dignani de; Bailão, Alexandre Melo; Borges, Clayton Luiz; Rocha, Juliana Alves Parente; Fernandes, Gabriel da Rocha; Gauthier, Gregory M.; Soares, Célia Maria de Almeida
Paracoccidoides brasiliensis and Paracoccidioides lutzii, the etiologic agents of paracoccidioidomycosis, cause disease in healthy and immunocompromised persons in Latin America. We developed a method for harvesting P. brasiliensis yeast cells from infected murine lung to facilitate in vivo transcriptional and proteomic profiling. P. brasiliensis harvested at 6 h post-infection were analyzed using RNAseq and LC-MSE. In vivo yeast cells had 594 differentially expressed transcripts and 350 differentially expressed proteins. Integration of transcriptional and proteomic data indicated that early in infection (6 h), P. brasiliensis yeast cells underwent a shift in metabolism from glycolysis to b-oxidation, upregulated detoxifying enzymes to defend against oxidative stress, and repressed cell wall biosynthesis. Bioinformatics and functional analyses also demonstrated that a serine proteinase was upregulated and secreted in vivo. To our knowledge this is the first study depicting transcriptional and proteomic data of P. brasiliensis yeast cells upon 6 h post-infection of mouse lung.
Proteome characterization of Paracoccidioides lutzii conidia by using nanoUPLC-MSE
(2020) Moreira, André Luís Elias; Leite, Vanessa Rafaela Milhomem Cruz; Silva, Lana OHara Souza; Parente, Ana Flávia Alves; Bailão, Alexandre Melo; Soares, Célia Maria de Almeida; Rocha, Juliana Alves Parente; Hernandez Ruiz, Orville; Borges, Clayton Luiz
Fungi of the genus Paracoccidioides are the etiological agents of Paracoccidioidomycosis (PCM), the most prevalent mycosis in Latin America. Paracoccidioidomycosis infection is acquired by inhalation of Paracoccidioides conidia, which have first contact with the lungs and can subsequently spread to other organs/tissues. Until now, there have been no proteomic studies focusing on this infectious particle of Paracoccidioides. In order to identify the Paracoccidioides lutzii conidia proteome, conidia were produced and purified. Proteins were characterized by use of the nanoUPLC-MSE approach. The strategy allowed us to identify a total of 242 proteins in P. lutzii conidia. In the conidia proteome, proteins were classified in functional categories such as protein synthesis, energy production, metabolism, cellular defense/virulence processes, as well as other processes that can be important for conidia survival. Through this analysis, a pool of ribosomal proteins was identified, which may be important for the initial processes of dimorphic transition. In addition, molecules related to energetic and metabolic processes were identified, suggesting a possible basal metabolism during this form of resistance of the fungus. In addition, adhesins and virulence factors were identified in the P. lutzii conidia proteome. Our results demonstrate the potential role that these molecules can play during early cell–host interaction processes, as well as the way in which these molecules are involved in environmental survival during this form of propagation.
An efficient Agrobacterium tumefaciens-mediated transformation method for Simplicillium subtropicum (Hypocreales: Cordycipitaceae)
(2021) Oliveira, Nicolau Sbaraini; Tomazett, Mariana Vieira; Penteriche, Augusto Bartz; Gonçales, Relber Aguiar; Camargo, Matheus da Silva; Bailão, Alexandre Melo; Borges, Clayton Luiz; Schrank, Augusto; Soares, Célia Maria de Almeida; Staats, Charley Christian
Filamentous fungi are the organisms of choice for most industrial biotechnology. Some species can produce a variety of secondary metabolites and enzymes of commercial interest, and the production of valuable molecules has been enhanced through different molecular tools. Methods for genetic manipulation and transformation have been essential for the optimization of these organisms. The genus Simplicillium has attracted increased attention given several potential biotechnological applications. The Simplicillium genus harbors several entomopathogenic species and some isolates have been explored for bioremediation of heavy metal contaminants. Furthermore, the myriad of secondary metabolites isolated from Simplicillium spp. render these organisms as ideal targets for deep exploration and further biotechnological mining possibilities. However, the lack of molecular tools hampered the exploration of this genus. Thus, an Agrobacterium tumefaciens-mediated transformation method was established for Simplicillium subtropicum, employing the far-red fluorescent protein TURBOFP635/Katushka, as a visual marker, and the selection marker SUR gene, that confers resistance to chlorimuron ethyl. Notably, one round of transformation using the established method yielded almost 400 chlorimuron resistant isolates. Furthermore, these transformants displayed mitotic stability for, at least, five generations. We anticipate that this method can be useful for deep molecular exploration and improvement of strains in the Simplicillium genus.
Zinc at the Host-Fungus interface: how to uptake the metal?
(2020) Soares, Lucas Weba; Bailão, Alexandre Melo; Soares, Célia Maria de Almeida; Bailão, Mirelle Garcia Silva
Zinc is an essential nutrient for all living organisms. However, firm regulation must be maintained since micronutrients also can be toxic in high concentrations. This notion is reinforced when we look at mechanisms deployed by our immune system, such as the use of chelators or membrane transporters that capture zinc, when threatened with pathogens, like fungi. Pathogenic fungi, on the other hand, also make use of a variety of transporters and specialized zinc captors to survive these changes. In this review, we sought to explain the mechanisms, grounded in experimental analysis and described to date, utilized by pathogenic fungi to maintain optimal zinc levels.
Identification of membrane proteome of Paracoccidioides Lutzii and its regulation by zinc
(2017) Curcio, Juliana Santana de; Silva, Marielle Garcia; Bailão, Mirelle Garcia Silva; Báo, Sônia Nair; Casaletti, Luciana; Bailão, Alexandre Melo; Soares, Célia Maria de Almeida
The methodology of protein identification allows the characterization of biological processes performed by those molecules. Therefore, we performed a membrane proteomic analysis of Paracoccidioides lutzii and further evaluated the responses of the fungus to zinc deprivation. The results obtained in the work allowed the characterization of membrane proteins present in organelles that are related to different cellular functions. Zinc deprivation changes processes related to cellular physiology and metabolism. These results help us to understand the process of pathogen–host interaction, since zinc deprivation is a condition present during infection.
Antifungal resistance, metabolic routes as drug targets, and new antifungal agents: an overview about endemic dimorphic fungi
(2017) Rocha, Juliana Alves Parente; Bailão, Alexandre Melo; Amaral, Andre Correa; Taborda, Carlos Pelleschi; Paccez, Juliano Domiraci; Borges, Clayton Luiz; Pereira, Maristela
Diseases caused by fungi can occur in healthy people, but immunocompromised patients are the major risk group for invasive fungal infections. Cases of fungal resistance and the difficulty of treatment make fungal infections a public health problem. This review explores mechanisms used by fungi to promote fungal resistance, such as the mutation or overexpression of drug targets, efflux and degradation systems, and pleiotropic drug responses. Alternative novel drug targets have been investigated; these include metabolic routes used by fungi during infection, such as trehalose and amino acid metabolism and mitochondrial proteins. An overview of new antifungal agents, including nanostructured antifungals, as well as of repositioning approaches is discussed. Studies focusing on the development of vaccines against antifungal diseases have increased in recent years, as these strategies can be applied in combination with antifungal therapy to prevent posttreatment sequelae. Studies focused on the development of a pan-fungal vaccine and antifungal drugs can improve the treatment of immunocompromised patients and reduce treatment costs.
Insights into Histoplasma capsulatum behavior on zinc deprivation
(2020) Assunção, Leandro do Prado; Moraes, Dayane; Soares, Lucas Weba; Bailão, Mirelle Garcia Silva; Siqueira, Janaina Gomes de; Baeza, Lilian Cristiane; Báo, Sônia Nair; Soares, Célia Maria de Almeida; Bailão, Alexandre Melo
Histoplasma capsulatum is a thermodimorphic fungus that causes histoplasmosis, a mycosis of global incidence. The disease is prevalent in temperate and tropical regions such as North America, South America, Europe, and Asia. It is known that during infection macrophages restrict Zn availability to H. capsulatum as a microbicidal mechanism. In this way the present work aimed to study the response of H. capsulatum to zinc deprivation. In silico analyses showed that H. capsulatum has eight genes related to zinc homeostasis ranging from transcription factors to CDF and ZIP family transporters. The transcriptional levels of ZAP1, ZRT1, and ZRT2 were induced under zinc-limiting conditions. The decrease in Zn availability increases fungicidal macrophage activity. Proteomics analysis during zinc deprivation at 24 and 48 h showed 265 proteins differentially expressed at 24 h and 68 at 48 h. Proteins related to energy production pathways, oxidative stress, and cell wall remodeling were regulated. The data also suggested that low metal availability increases the chitin and glycan content in fungal cell wall that results in smoother cell surface. Metal restriction also induces oxidative stress triggered, at least in part, by reduction in pyridoxin synthesis.
Comparative proteomics in the three major human pathogenic species of the genus Sporothrix
(2021) Bailão, Mirelle Garcia Silva; Lima, Patrícia de Sousa; Oliveira, Manoel Marques Evangelista; Oliveira, Luã Cardoso; Paes, Rodrigo Almeida; Borges, Clayton Luiz; Bailão, Alexandre Melo; Coelho, Alexandre Siqueira Guedes; Soares, Célia Maria de Almeida; Oliveira, Rosely Maria Zancopé
Sporotrichosis is a subcutaneous mycosis of humans and other mammals, caused by dimorphic species of the genus Sporothrix. In Brazil, human disease is broadly linked to transmission by infected cats and is mainly caused by Sporothrix brasiliensis, Sporothrix schenckii and Sporothrix globosa. In this study, we used a nanoscale liquid chromatography coupled with tandem mass spectrometry approach to provide the yeast proteomic profiles of S. brasiliensis, S. schenckii and S. globosa. From a total of 247 identified proteins, 137 were found as differentially expressed. Functional classification revealed that most are related to carbohydrate and amino acid metabolism as well as stress response. Our data indicate that S. brasiliensis metabolism is distinct of that of S. schenckii and S. globosa, mainly regarding amino acid metabolism and cell wall remodeling, which are induced in the former. Enzymes belonging to glycolytic pathway are, on the other hand, up-regulated in S. schenckii and S. globosa. These findings may explain the previously described more virulent character of S. brasiliensis. Besides complementing genomic comparisons already published, this first comparative proteomic study provided information that indicates new aspects of Sporothrix species metabolism as well as offers information that may be useful in the development of prospective functional studies.
Metabolic peculiarities of Paracoccidioides brasiliensis dimorphism as demonstrated by iTRAQ labeling proteomics
(2019) Araújo, Danielle Silva; Pereira, Maristela; Portis, Igor Godinho; Santos Júnior, Agenor de Castro Moreira dos; Fontes, Wagner; Sousa, Marcelo Valle de; Assunção, Leandro do Prado; Baeza, Lilian Cristiane; Bailão, Alexandre Melo; Ricart, Carlos Andre Ornelas
Paracoccidioidomycosis (PCM), a systemic mycosis with a high incidence in Latin America, is caused by thermodimorphic fungi of the Paracoccidioides genus. The contact with host occurs by the inhalation of conidia or mycelial propagules which once reaching the pulmonary alveoli differentiate into yeast cells. This transition process is vital in the pathogenesis of PCM allowing the fungus survival in the host. Thus, the present work performed a comparative proteome analysis of mycelia, mycelia-to-yeast transition, and yeast cells of Paracoccidioides brasiliensis. For that, tryptic peptides were labeled with iTRAQ and identified by LC–MS/MS and computational data analysis, which allowed the identification of 312 proteins differentially expressed in different morphological stages. Data showed that P. brasiliensis yeast cells preferentially employ aerobic beta-oxidation and the tricarboxylic acid cycle accompanied by oxidative phosphorylation for ATP production, in comparison to mycelia and the transition from mycelia-to-yeast cells. Furthermore, yeast cells show a metabolic reprogramming in amino acid metabolism and in the induction of virulence determinants and heat shock proteins allowing adaptation to environmental conditions during the increase of the temperature. In opposite of that, the alcoholic fermentation found to P. lutzii, at least under laboratory conditions, is strongly favored in mycelium compared to yeast cells. Thereby, the data strongly support substantial metabolic differences among members of the Paracoccidioides complex, when comparing the saprobiotic mycelia and the yeast parasitic phases.
Interaction with Pantoea agglomerans modulates growth and melanization of Sporothrix brasiliensis and Sporothrix schenckii
(2019) Paes, Rodrigo Almeida; Santos, Fabio Brito dos; Oliveira, Manoel Marques Evangelista; Bailão, Alexandre Melo; Borges, Clayton Luiz; Araújo, Glauber Ribeiro de Sousa; Frases Carvajal, Susana; Soares, Célia Maria de Almeida; Oliveira, Rosely Maria Zancopé
Sporothrix brasiliensis and Sporothrix schenckii stand as the most virulent agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. The origin of Sporothrix virulence seems to be associated with fungal interactions with organisms living in the same environment. To assess this hypothesis, the growth of these two species in association with Pantoea agglomerans, a bacterium with a habitat similar to Sporothrix spp., was evalu ated. Growth, melanization, and gene expression of the fungus were compared in the presence or absence of the bacterium in the same culture medium. Both S. brasiliensis and S. schenckii grew in contact with P. agglomerans yielding heavily melanized conidia after 5 days of incubation at 30 C in Sabouraud agar. This increased melanin production occurred around bacte rial colonies, suggesting that fungal melanization is triggered by a diffusible bacterial product, which is also supported by a similar pattern of melanin production during Sporothrix spp. growth in contact with heat-killed P. agglomerans. Growth of P. agglomerans was similar in the presence or absence of the fungus. However, the growth of S. brasiliensis and S. schenckii was initially inhibited, but further enhanced when these species were co-cultured with P. agglomerans. Moreover, fungi were able to use killed bacteria as both carbon and nitrogen sources for growth. Representational difference analysis identi- fied overexpressed genes related to membrane trans port when S. brasiliensis was co-cultured with the bacteria. The down-regulation of metabolism-related genes appears to be related to nutrient availability during bacterial exploitation. These findings can lead to a better knowledge on Sporothrix ecology and virulence.
Metabolic adaptation of Paracoccidioides brasiliensis in response to in vitro Copper deprivation
(2020) Pititto, Guilherme; Curcio, Juliana Santana de; Pereira, Maristela; Bailão, Alexandre Melo; Paccez, Juliano Domiraci; Tristão, Gabriel Brum; Morais, Camila Oliveira Barbosa de; Sousa, Agenor de Castro Moreira dos Santos Júnior Marcelo Valle de; Fontes, Wagner
Copper is an essential micronutrient for the performance of important biochemical processes such as respiration detoxification, and uptake of metals like iron. Studies have shown that copper deprivation is a strategy used by the host against pathogenic fungi such as Cryptoccocus neoformans and Candida albicans during growth and development of infections in the lungs and kidneys. Although there are some studies, little is known about the impact of copper deprivation in members of the Paracoccidioides genus. Therefore, using isobaric tag labeling (iTRAQ)-Based proteomic approach and LC-MS/MS, we analyzed the impact of in vitro copper deprivation in the metabolism of Paracoccidioides brasiliensis. One hundred and sixty-four (164) differentially abundant proteins were identified when yeast cells were deprived of copper, which affected cellular respiration and detoxification processes. Changes in cellular metabolism such as increased beta oxidation and cell wall remodeling were described.
Molecular characterization of siderophore biosynthesis in Paracoccidioides brasiliensis
(2020) Silva, Marielle Garcia; Curcio, Juliana Santana de; Bailão, Mirelle Garcia Silva; Lima, Raisa Melo; Tomazett, Mariana Vieira; Souza, Aparecido Ferreira de; Leite, Vanessa Rafaela Milhomem Cruz; Oliveira, Nicolau Sbaraini; Bailão, Alexandre Melo; Rodrigues, Fernando
Iron is an essential nutrient for all organisms. For pathogenic fungi, iron is essential for the success of infection. Thus, these organisms have developed high affinity iron uptake mechanisms to deal with metal deprivation imposed by the host. Siderophore production is one of the mechanisms that fungal pathogens employ for iron acquisition. Paracoccidioides spp. present orthologous genes encoding the enzymes necessary for the biosynthesis of hydroxamates, and plasma membrane proteins related to the transport of these molecules. All these genes are induced in iron deprivation. In addition, it has been observed that Paracoccidioides spp. are able to use siderophores to scavenge iron. Here we observed that addition of the xenosiderophore ferrioxamine B FOB) to P. brasiliensis culture medium results in repression (at RNA and protein levels) of the SidA, the first enzyme of the siderophore biosynthesis pathway. Furthermore, SidA activity was reduced in the presence of FOB, suggesting that P. brasiliensis blocks siderophores biosynthesis and can explore siderophores in the environment to scavenge iron. In order to support the importance of siderophores on Paracoccidioides sp. life and infection cycle, silenced mutants for the sidA gene were obtained by antisense RNA technology. The obtained AsSidA strains displayed decreased siderophore biosynthesis in iron deprivation conditions and reduced virulence to an invertebrate model.
Propionate metabolism in a human pathogenic fungus: proteomic and biochemical analyses
(2020) Santos, Luiz Paulo Araújo; Assunção, Leandro do Prado; Lima, Patrícia de Souza; Tristão, Gabriel Brum; Brock, Matthias; Borges, Clayton Luiz; Bailão, Mirelle Garcia Silva; Soares, Célia Maria de Almeida; Bailão, Alexandre Melo
Fungi of the complex Paracoccidioides spp. are thermodimorphic organisms that cause Paracoccidioidomycosis, one of the most prevalent mycoses in Latin America. These fungi present metabolic mechanisms that contribute to the fungal survival in host tissues. Paracoccidioides lutzii activates glycolysis and fermentation while inactivates aerobic metabolism in iron deprivation, a condition found during infection. In lungs Paracoccidioides brasiliensis face a glucose poor environment and relies on the beta-oxidation to support energy requirement. During mycelium to yeast transition P. lutzii cells up-regulate transcripts related to lipid metabolism and cell wall remodeling in order to cope with the host body temperature. Paracoccidioides spp. cells also induce transcripts/enzymes of the methylcitrate cycle (MCC), a pathway responsible for propionyl-CoA metabolism. Propionyl-CoA is a toxic compound formed during the degradation of odd-chain fatty acids, branched chain amino acids and cholesterol. Therefore, fungi require a functional MCC for full virulence and the ability to metabolize propionyl-CoA is related to the virulence traits in Paracoccidioides spp. On this way we sought to characterize the propionate metabolism in Paracoccidioides spp. The data collected showed that P. lutzii grows in propionate and activates the MCC by accumulating transcripts and proteins of methylcitrate synthase (MCS), methylcitrate dehydratase (MCD) and methylisocitrate lyase (MCL). Biochemical characterization of MCS showed that the enzyme is regulated by phosphorylation, an event not yet described. Proteomic analyses further indicate that P. lutzii yeast cells degrades lipids and amino acids to support the carbon requirement for propionate metabolism. The induction of a putative propionate kinase suggests that fungal cells use propionyl-phosphate as an intermediate in the production of toxic propionyl-CoA. Concluding, the metabolism of propionate in P. lutzii is under regulation at transcriptional and phosphorylation levels and that survival on this carbon source requires additional mechanisms other than activation of MCC.

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