iTRAQ-based proteomic analysis of Paracoccidioides brasiliensis in response to hypoxia.

Aerobic organisms require oxygen for energy. In the course of the infection, adaptation to hypoxia is crucial for survival of human pathogenic fungi. Members of the Paracoccidioides complex face decreased oxygen tensions during the life cycle stages. In Paracoccidioides brasiliensis proteomic responses to hypoxia have not been investigated and the regulation of the adaptive process is still unknown, and this approach allowed the identification of 216 differentially expressed proteins in hypoxia using iTRAQ-labelling. Data suggest that P. brasiliensis reprograms its metabolism when submitted to hypoxia. The fungus reduces its basal metabolism and general transport proteins. Energy and general metabolism were more representative and up regulated. Glucose is apparently directed towards glycolysis or the production of cell wall polymers. Plasma membrane/cell wall are modulated by increasing ergosterol and glucan, respectively. In addition, molecules such as ethanol and acetate are produced by this fungus indicating that alternative carbon sources probably are activated to obtain energy. Also, detoxification mechanisms are activated. The results were compared with label free proteomics data from Paracoccidioides lutzii. Biochemical pathways involved with acetyl-CoA, pyruvate and ergosterol synthesis were up-regulated in both fungi. On the other hand, proteins from TCA, transcription, protein fate/degradation, cellular transport, signal transduction and cell defense/virulence processes presented different profiles between species. Particularly, proteins related to methylcitrate cycle and those involved with acetate and ethanol synthesis were increased in P. brasiliensis proteome, whereas GABA shunt were accumulated only in P. lutzii. The results emphasize metabolic adaptation processes for distinct Paracoccidioides species.

Therapeutic potential of using probiotics in the treatment and prevention of breast câncer / Potencial terapêutico do uso de probióticos no tratamento e prevenção de câncer de mama

Objective: This study aimed to evaluate the evidence available in the literature on probiotics on breast cancer. Methods: It is an integrative review of the literature, through the PICO strategy, with the guiding question "Does probiotics consumption act on oxidative stress and reduces the risk of breast cancer"? The selection of articles was made on MEDLINE, SciELO and PubMed, including those published between 2016 and january 2021, using the keywords: "breast cancer" AND "probiotics" AND "microbiota" their combinations in Portuguese and English, with previously established inclusion criteria. Results and Discussion: The sample consisted of ten articles, and the answers found were: scientific research in vivo and in vitro, with some variations of probiotics strains, where all demonstrate actions and/or effects on cancer cells or acted on metabolic pathways mitigating the development of tumors. Conclusions: The manifested potential for a possible insertion in the treatment and prevention of breast cancer, being considered an innovation for the scientific community that still fights against various types of problems related to human health, especially some types of cancers, of negative prognoses and with difficult therapeutic interventions.

Objetivo: Este estudo avaliou a literatura disponível sobre probióticos no câncer da mama. Métodos: É uma revisão integrativa da literatura, através da estratégia pico, com a pergunta orientadora "O consumo de probióticos atua sobre o stress oxidativo e reduz o risco de câncer da mama"? A seleção de artigos foi feita no MEDLINE, SciELO e PubMed, incluindo os publicados entre 2016 e janeiro de 2021, utilizando as palavras-chave: "câncer da mama" E "probióticos" E "microbiota" as suas combinações em português e inglês, com critérios de inclusão previamente estabelecidos. Resultados e Discussão: A amostra consistiu em dez artigos, e as respostas encontradas foram: investigação científica in vivo e in vitro, com algumas variações de estirpes de probióticos, onde todos demonstram ações e/ou efeitos sobre as células cancerosas ou atuaram sobre vias metabólicas mitigando o desenvolvimento de tumores. Conclusões: O potencial manifestado para uma possível inserção de probióticos no tratamento e prevenção do câncer da mama, sendo considerado uma inovação para a comunidade científica que ainda luta contra vários tipos de problemas relacionados com a saúde humana, especialmente alguns tipos de câncer de prognósticos negativos e com intervenções terapêuticas difíceis.

Metabolic Peculiarities of Paracoccidioides brasiliensis Dimorphism as Demonstrated by iTRAQ Labeling Proteomics.

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.

Proteomic Analysis of Paracoccidioides brasiliensis During Infection of Alveolar Macrophages Primed or Not by Interferon-Gamma.

Although members of the Paracoccidioides complex are not obligate intracellular pathogens, they present the ability to survive and multiply inside epithelial cells and phagocytes of mammals, which may favor the spread of the fungus in host tissues. Macrophages resident in the lung are the first line of defense against paracoccidioidomycosis (PCM), presenting mechanisms to control the pathogen dissemination through the granuloma formation or eliminating the fungus through phagocytosis. Phagocytosis triggers an oxidative burst, in which there is an increase in the production of toxic elements, derived from oxygen and nitrogen. The interior of the phagolysosome is a harsh environment to the internalized pathogens, since in addition to the oxygen and nitrogen reactive species, microorganisms face nutrient shortages and proteases activity. Through the NanoUPLC-MS E technology, we analyzed the proteomic response of Paracoccidioides brasiliensis during the infection of alveolar macrophages primed or not by interferon gamma (IFN-γ). At 6 hs post-infection, only (IFN-γ)-primed macrophages were able to kill the fungus. We observed the regulation of amino acids degradation, tricarboxylic acid cycle, respiratory chain, ATP synthesis, glyoxylate cycle, as well as an increase in the expression of defense proteins related to oxidative stress, heat shock, and virulence factors under both conditions analyzed. However, some pathways described as essential for the survival of pathogens inside macrophages were observed only or with higher intensity in yeast cells recovered from non-primed macrophages, as phosphate pentoses pathway, methylcitrate cycle, synthesis of cell wall components, and mitochondrial activity. The data indicate that the intracellular environment of non-primed macrophages could be more permissive to the survival and multiplication of P. brasiliensis. The identification of key molecules for the establishment of infection can help the understanding of the nature of the parasite-host relationship and pathogenesis of PCM.

Employing proteomic analysis to compare Paracoccidioides lutzii yeast and mycelium cell wall proteins.

Paracoccidioidomycosis is an important systemic mycosis caused by thermodimorphic fungi of the Paracoccidioides genus. During the infective process, the cell wall acts at the interface between the fungus and the host. In this way, the cell wall has a key role in growth, environment sensing and interaction, as well as morphogenesis of the fungus. Since the cell wall is absent in mammals, it may present molecules that are described as target sites for new antifungal drugs. Despite its importance, up to now few studies have been conducted employing proteomics in for the identification of cell wall proteins in Paracoccidioides spp. Here, a detailed proteomic approach, including cell wall-fractionation coupled to NanoUPLC-MSE, was used to study and compare the cell wall fractions from Paracoccidioides lutzii mycelia and yeast cells. The analyzed samples consisted of cell wall proteins extracted by hot SDS followed by extraction by mild alkali. In summary, 512 proteins constituting different cell wall fractions were identified, including 7 predicted GPI-dependent cell wall proteins that are potentially involved in cell wall metabolism. Adhesins previously described in Paracoccidioides spp. such as enolase, glyceraldehyde-3-phosphate dehydrogenase were identified. Comparing the proteins in mycelium and yeast cells, we detected some that are common to both fungal phases, such as Ecm33, and some specific proteins, as glucanase Crf1. All of those proteins were described in the metabolism of cell wall. Our study provides an important elucidation of cell wall composition of fractions in Paracoccidioides, opening a way to understand the fungus cell wall architecture.