Fontes preferenciais e mecanismos moleculares envolvidos na homeostase de ferro em Paracoccidioides spp.

Tese (Doutorado)—Universidade de Brasília, Faculdade de Medicina, Pós-Graduação em Patologia Molecular, 2014.Paracoccidioides spp. is a thermodimorphic fungus, which causes paracoccidioidomycosis (PCM), an endemic disease in Latin America. Currently, the Paracoccidioides genus is considered a phylogenetic group containing two distinct species: P. brasiliensis and P. lutzii. However, all isolates identified to date are able to cause PCM. Iron is an essential micronutrient for all eukaryotes, since it participates as a cofactor in several metabolic pathways. However, the amount of available iron inside the host is limited to the pathogen. The host iron sources that are used by Paracoccidioides spp. and the molecular mechanisms used by the fungus to acquire iron has not been investigated. In this study, a robust growth of Paracoccidioides spp. in the presence of hemin or hemoglobin was observed. This fact was supported by the ability of the fungus to internalize protoporphyrin rings and to lyse erythrocytes. Transcriptional and proteomic analysis indicated the induction of possible hemoglobin receptors in the presence of hemoglobin and the possibility of internalization of the entire molecule by the fungus. One of these potential receptors, Rbt5, was characterized in this work. Rbt5 is a GPI-anchored protein, present at the Paracoccidioides sp. cell surface. The protein is able to bind hemin, hemoglobin and protoporphyrin and presents antigenic properties. A Paracoccidioides sp.rbt5knockdown strain was obtained in this work using antisense RNA and Agrobacterium tumefaciens-mediated transformation. This strain showed lower survival inside macrophages and in mouse spleen, indicating that Rbt5 may be important for the establishment of Paracoccidioides spp. infection. Furthermore, the reductive iron uptake pathway was investigated in this fungus. In silico analysis pointed that Paracoccidioides spp. could use ferric reductases, ferroxidases and zinc and iron transporters to uptake free iron or iron bound to certain molecules, such as transferrin. The genes encoding those molecules possibly involved in iron uptake were activated in the absence of the metal, which points to the importance of this pathway in conditions of iron deprivation. Genes involved in intracellular iron homeostasis were also investigated in this work and presented regulation by the metal. A ferric reductase activity was demonstrated for Paracoccidioides sp.; however, 59Fe uptake assays showed that the reductive pathway is functional only in conditions of iron deprivation and that the Paracoccidioides spp. ferric reductases are not strongly affected by platinum. Surface molecules involved in iron uptake by the fungus may be important targets for vaccines and/or antifungal drugs. Thus, this work can contribute to the development of alternative therapies for PCM treatment.Paracoccidioides spp. é um fungo termodimórfico, causador da paracoccidioidomicose (PCM), uma doença endêmica da América Latina. Atualmente o gênero Paracoccidioides é considerado um grupo filogenético, contendo duas espécies distintas: P. brasiliensis e P. lutzii. Porém, todos os isolados identificados até o momento são capazes de causar a PCM. O ferro é um micronutriente essencial para todos os eucariotos, pois participa como cofator de diversas vias metabólicas. Porém, dentro do hospedeiro, a quantidade de ferro disponível para captação pelo patógeno é limitada. As fontes de ferro do hospedeiro preferenciais de Paracoccidioides spp. e os mecanismos moleculares utilizados pelo fungo para captar ferro ainda não foram investigados. Neste trabalho, foi constatado um robusto crescimento de Paracoccidioides spp. na presença de hemina ou hemoglobina, corroborado pela capacidade do fungo de internalizar uma protoporfirina e de lisar hemácias. Análises transcricionais e proteômicas apontaram para a indução de possíveis receptores de hemoglobina na presença desta fonte de ferro e para a possível internalização de toda a molécula de hemoglobina pelo fungo. Um desses possíveis receptores, Rbt5, foi caracterizado neste trabalho. Rbt5 é uma proteína GPI-ancorada presente na superfície de Paracoccidioides sp., capaz de se ligar à hemina, protoporfirina e hemoglobina e apresenta propriedades antigênicas. Uma linhagem silenciada para o gene rbt5 foi obtida através de RNA antisentido e de transformação mediada por Agrobacterium tumefaciens. Esta linhagem apresentou menor sobrevivência dentro de macrófagos e em baço de camundongo, indicando que Rbt5 pode ser importante para o estabelecimento da infecção por Paracoccidioides spp. Além disso, a via redutiva de captação de ferro foi investigada neste fungo. Análises in silico apontaram que Paracoccidioides spp. possa utilizar redutases férricas, ferroxidases e transportadores de ferro e zinco para captar ferro livre ou ligado a certas moléculas, como a transferrina. Os genes codificantes para essas moléculas possivelmente envolvidas com a captação de ferro foram ativados na ausência desse metal, o que aponta para a importância dessa via em condições de privação de ferro. Genes envolvidos com a homeostase intracelular de ferro também foram investigados neste trabalho e apresentaram-se regulados pelo metal. A atividade de redutase férrica foi demonstrada para Paracoccidioides sp., porém ensaios de captação com 59Fe apontaram que a via redutiva só é funcional em condições de privação do metal e que as redutases férricas de Paracoccidioides spp. não são fortemente afetadas por platina. Moléculas de superfície envolvidas com a captação de ferro pelo fungo podem ser importantes alvos de vacinas e/ou de drogas antifúngicas. Dessa forma, este trabalho pode contribuir para o desenvolvimento de terapias alternativas para o tratamento da PCM

Hydroxamate production as a high affinity iron acquisition mechanism in Paracoccidioides Spp.

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity

Hemoglobin Uptake by Paracoccidioides spp. Is Receptor-Mediated

Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciensmediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms

Urban Occupation Increases Water Toxicity of an Important River in Central Brazil

Meia Ponte River supplies water for two million people in Goiás State, Brazil. Despite its importance, the Meia Ponte River faces serious environmental problems such as the disposal of domestic and industrial effluents, what could impact the aquatic biota and the health of people who consumes its water. In this sense, here we aimed to evaluate the environmental quality and toxicity of surface water along the course of this river. Physicochemical analyses of water at Goiânia urban perimeter were higher than the limits of Brazil environmental regulations for fresh water. In relation to the diversity of species, phytoplankton classes associated to polluted environments were detected closer to urban perimeter. Allium cepa bioassay suggested that this river may contain substances with mitogenic activity. This result is in accordance with genotoxic analysis, because it was observed a significant increase in chromosomal aberrations. This data reveal the genotoxic potential of Meia Ponte River water. This genotoxicity represents a risk for aquatic biota and humans, once the genotoxic agents in water samples might cause the loss of DNA integrity, inducing damages and DNA breaks. In this context, the water utilization from Meia Ponte River without any treatment should be avoided and public policies need to be formulated and implemented to depollute this important river for Goiás State.O rio Meia Ponte fornece água para dois milhões de pessoas no estado de Goiás, Brasil. Apesar de sua importância, o rio Meia Ponte enfrenta sérios problemas ambientais, como o lançamento de efluentes domésticos e industriais, o que pode impactar a biota aquática e a saúde das pessoas que consomem sua água. Nesse sentido, neste trabalho tivemos como objetivo avaliar a qualidade e a toxicidade das águas superficiais ao longo deste rio. As análises físico-químicas da água no perímetro urbano de Goiânia apresentaram alguns parâmetros fora dos limites das regulamentações ambientais do Brasil. Em relação à diversidade de espécies, foram detectadas classes de fitoplâncton associadas a ambientes poluídos. O ensaio com Allium cepa sugere que este rio pode conter substâncias com atividade mitogênica. Este resultado está de acordo com a análise genotóxica, pois se observou um aumento significativo nas aberrações cromossômicas. Estes dados revelam o potencial genotóxico da água do rio Meia Ponte. Esta genotoxicidade representa um risco para a biota aquática e os seres humanos, uma vez que os agentes genotóxicos em amostras de água podem causar a perda de integridade do DNA, induzindo danos e rupturas de DNA. Nesse contexto, a utilização da água do Rio Meia Ponte, sem qualquer tratamento, deve ser evitada, e políticas públicas precisam ser formuladas e implementadas para despoluir este importante rio para o estado de Goiás

The chick embryo chorioallantoic membrane assay as a model for the study of angiogenesis

Angiogenesis is a fundamental physiological process with strong implications in tissue homeostasis. Animal models helping to identify how angiogenesis is regulated are fundamental to answer many biological questions. Chick embryo chorioallantoic membrane (CAM) assay is one of the most employed methods to study angiogenesis. In this study we applied a scientometric approach to evaluate the employment of CAM assay in published articles. Temporal trends indicated that CAM assay was the preferred method to investigate angiogenesis over time. The publications had a significant number of citations and the impact factor of journals publishing articles is relevant for the scientific community. A total of 52 different research areas have articles published using this particular technique. Oncology is the research field in which CAM assay was mostly used. Accordingly, tumor-derived cell lines were the most frequent sample tested on CAM. We also identified that 73,6% of articles published used only CAM assay to answer questions concerning angiogenesis. We concluded that although the CAM assay is a classical approach, that does not need so much infrastructure and financial support to be performed, it is a well-accepted technique by the scientific community. In addition, this methodology has gain attention in scientific community because no pain is experienced by the chick and they are minor ethical concerns to employ this method. Moreover, this data can help researchers who are unfamiliar with the CAM assay to identify if this particular method is suitable for their research

In vivo and in vitro analysis of Paracoccidioides brasiliensis Beta-1,3-glucanosyltransferase 1 intermolecular interactions

Made available in DSpace on 2014-07-29T15:16:35Z (GMT). No. of bitstreams: 1 Dissertacao FINAL_EF_corrigida_BC-UFG.pdf: 1485117 bytes, checksum: ecdc9729cc08da2832a6566c39052f65 (MD5) Previous issue date: 2010-01-28The cell wall of pathogenic microbes acts as an initial barrier that is in contact with hostile environments. Besides functioning as a mechanical barrier, it harbours an immunogenic macromolecules arsenal. One of the ways that proteins can be associated to the cell wall, it is through GPI anchor. The hydrophobic C-terminal end of the β-1,3-glucanosyltransferase enzyme of the human pathogenic fungus Paracoccidioides brasiliensis is characteristic of GPI anchored proteins. The β-1,3-glucan assembling and rearrangement are essential since this molecule acts as a scaffold to support cell wall proteins and polysaccharides. In the thermodimorphic fungus P. brasiliensis, β-1,3-glucan is found predominantly in mycelium form and α-1,3-glucan is predominant in the yeast form. In this work, it was screened possible protein-protein interactions performed by β-1,3-glucanosyltransferase 1 of P. brasiliensis (PbGel1p). To obtain these results, a P. brasiliensis cDNA library was screened with PbGel1p using the Saccharomyces cerevisiae two hybrid system. In addition, pull-down assay was used as an in vitro complementary technique to isolate proteins that interact direct or indirectly with PbGel1p. It was screened 38 gene products using two hybrid system and it was identified 3 proteins using the pull-down assay associated with mass spectrometry. The PbGel1p role in the cell wall maintenance and remodeling was indicated through the analysis of screened interactions, like alpha-glucosides permease, acid phosphatase, GDSL lipase, septin, actin, tubulin, HSP90 and pyruvate kinase. Furthermore, nuclear localization of PbGel1p and its role in the locus-specific transcriptional silencing were suggested based on such interactions: Qde2 argonaute, transcription elongation factor spt6, others transcription factors and ATP-citrate synthase. Therefore, this study indicated, for the first time, that PbGel1p has multiple location and it participates either in roles classically described for glucanosyltransferases, as the cell wall remodeling, or in recently described functions for this family of proteins, as the locus-specific transcriptional silencing.A parede celular de microrganismos patogênicos atua como uma barreira inicial no contato entre o parasito e o hospedeiro. Além de funcionar como uma barreira mecânica, ela abriga um arsenal de macromoléculas imunogênicas. Uma forma pela qual as proteínas estão associadas à parede celular é por meio de âncoras-GPI. A extremidade carboxila hidrofóbica da enzima β-1,3-glicanosiltransferase do fungo patogênico humano Paracoccidioides brasiliensis é característica de proteínas GPI-ancoradas. A montagem e o rearranjo de β-1,3- glicana são de fundamental importância porque esta molécula serve como um esqueleto sobre o qual outros polissacarídeos e proteínas da parede celular estão associados. No fungo termodimórfico P. brasiliensis, β-1,3-glicana é encontrada prioritariamente em micélio, sendo α-1,3-glicana predominante em levedura. Foram rastreadas neste trabalho possíveis interações proteína-proteína realizadas pela β-1,3-glicanosiltransferase 1 de P. brasiliensis (PbGel1p). Para isso utilizou-se a técnica de duplo-híbrido em Saccharomyces cerevisiae, rastreando-se uma biblioteca de cDNA do fungo P. brasiliensis com a enzima estudada. Adicionalmente, foi utilizado, como técnica complementar, o ensaio de pull-down, que isolou in vitro proteínas que interagem direta ou indiretamente com a PbGel1p. Foi possível rastrear 38 produtos gênicos através do sistema de duplo híbrido e isolar três proteínas pelo ensaio de pull-down, identificadas por espectrometria de massas. O papel da PbGel1p na manutenção e no remodelamento da parede celular do fungo foi indicado através da análise das interações rastreadas, como permease de α-glicosídeos, fosfatase ácida, lipase da família GDSL, septina, actina, tubulina, HSP90 e piruvato quinase. Além disso, foram sugeridos a localização da PbGel1p no núcleo das células do fungo e seu papel no silenciamento gênico mediado por alterações estruturais, por meio do rastreamento das seguintes proteínas ligantes a PbGel1p: argonauta Qde2, fator de alongamento transcricional spt6, outros fatores transcricionais e ATP-citrato sintase. Portanto, este estudo indicou, pela primeira vez, que PbGel1p tem localização múltipla e participa tanto de funções classicamente descritas para glicanosiltransferases, como o remodelamento da parede celular, quanto de funções recentemente descritas para essa família de proteínas, como o silenciamento transcricional sítio-específico

ESTUDO CIENCIOMÉTRICO DA ESPÉCIE VEGETAL DO CERRADO Physocalymma scaberrimum

O Cerrado destaca-se entre os biomas brasileiros, pois apresenta uma rica biodiversidade, o que estimula diversos autores a proporem trabalhos sobre diferentes espécies vegetais e animais. Isso colabora para o desenvolvimento sustentável e conservação desse bioma. Physocalymma scaberrimum Pohl (Lythraceae), conhecida popularmente como "pau-de-rosas", é uma espécie arbórea encontrada no Cerrado Brasileiro, reconhecida por seu potencial madeireiro e de reflorestamento. Recentemente, a utilização desta planta pela medicina popular foi investigada. O presente artigo propôs o levantamento dos trabalhos já publicados sobre P. scaberrimum, destacando a área de concentração e a finalidade destes estudos. A pesquisa bibliográfica foi feita nas bases de dados SciELO, Science Direct, Scopus e ISI Web of Knowledge, utilizando os seguintes termos: Physocalymma scaberrimum, Physocalymma, scaberrimum e Lythraceae. Esta pesquisa revelou a existência de poucos trabalhos a respeito desta espécie vegetal, com a maioria dos trabalhos concentrados nas áreas de Filogenia e Palinologia

PROSPECÇÃO FITOQUÍMICA E ANÁLISE CITOGENÉTICA DE Physocalymma scaberrimum (POHL) LYTHRACEAE

Introdução e objetivos: Physocalymma scaberrimum é uma espécie arbórea do Cerrado pouco estudada, mas com utilização popular. Este trabalho objetivou determinar as principais classes de metabólitos secundários de extratos de folhas e cascas do caule dessa espécie, bem como avaliar possíveis efeitos cito/genotóxicos ocasionados pelo uso destes. Metodologia: Inicialmente foram realizados ensaios de controle de qualidade. Testes qualitativos para a determinação de alcalóides, heterosídeos flavonóides, compostos fenólicos e saponinas também foram realizados. A análise citogenética consistiu na exposição de bulbos de Allium cepa à três concentrações de chá de folhas e de cascas do caule: 0,75; 1,00 e 1,25 g/mL. Após determinados tempos, raízes foram coletadas para análises micro e macroscópicas. Resultados e discussões: Nos ensaios fitoquímicos dos extratos foram detectados compostos fenólicos, saponinas e heterosídeos flavonóides. A pesquisa foi negativa para a presença de alcalóides. O índice de intumescência foi 3 para folha e 2,5 para casca, já o teor de compostos voláteis foi, em média, 10,06% + 2,93% e 10,25% + 1,84%, respectivamente. Os organismos submetidos ao tratamento com o chá apresentaram padrões indicadores de toxicidade da substância, como raízes curvas, de coloração marrom e com formação de tumores. As análises microscópicas estão em andamento. Conclusões: O presente estudo aumenta o conhecimento a respeito de uma espécie do Cerrado, fortalecendo políticas de uso sustentável do mesmo

Bioactive Compounds Found in Brazilian Cerrado Fruits

Functional foods include any natural product that presents health-promoting effects, thereby reducing the risk of chronic diseases. Cerrado fruits are considered a source of bioactive substances, mainly phenolic compounds, making them important functional foods. Despite this, the losses of natural vegetation in the Cerrado are progressive. Hence, the knowledge propagation about the importance of the species found in Cerrado could contribute to the preservation of this biome. This review provides information about Cerrado fruits and highlights the structures and pharmacologic potential of functional compounds found in these fruits. Compounds detected in Caryocar brasiliense Camb. (pequi), Dipteryx alata Vog. (baru), Eugenia dysenterica DC. (cagaita), Eugenia uniflora L. (pitanga), Genipa americana L. (jenipapo), Hancornia speciosa Gomes (mangaba), Mauritia flexuosa L.f. (buriti), Myrciaria cauliflora (DC) Berg (jabuticaba), Psidium guajava L. (goiaba), Psidium spp. (araçá), Solanum lycocarpum St. Hill (lobeira), Spondias mombin L. (cajá), Annona crassiflora Mart. (araticum), among others are reported here

Paracoccidioides spp .ferrous and ferric iron assimilation pathways

Submitted by Jaqueline Silva ([email protected]) on 2018-05-14T19:24:11Z No. of bitstreams: 2 Artigo - Elisa Flávia Luiz Cardoso Bailão - 2015.pdf: 2352117 bytes, checksum: ad80fe6f836e11b501d4c14b1c2ac915 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Approved for entry into archive by Luciana Ferreira ([email protected]) on 2018-05-15T13:18:32Z (GMT) No. of bitstreams: 2 Artigo - Elisa Flávia Luiz Cardoso Bailão - 2015.pdf: 2352117 bytes, checksum: ad80fe6f836e11b501d4c14b1c2ac915 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2018-05-15T13:18:32Z (GMT). No. of bitstreams: 2 Artigo - Elisa Flávia Luiz Cardoso Bailão - 2015.pdf: 2352117 bytes, checksum: ad80fe6f836e11b501d4c14b1c2ac915 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-08Iron is an essential micronutrient for almost all organisms, including fungi. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA). Traditionally, the RIA pathway consists of ferric reductases (Fres), ferroxidase (Fet3) and a high-affinity iron permease (Ftr1). Paracoccidioides spp. genomes do not present an Ftr1 homolog. However, this fungus expresses zinc regulated transporter homologs (Zrts), members of the ZIP family of membrane transporters that are able in some organisms to transport zinc and iron. A 2,3,5-triphenyltetrazolium chloride (TTC)-overlay assay indicates that both Pb01 and Pb18 express a ferric reductase activity; however, 59Fe uptake assays indicate that only in Pb18 is this activity coupled to a reductase-dependent iron uptake pathway. In addition, Zrts are up-regulated in iron deprivation, as indicated by RNAseq and qRT-PCR using Pb01 transcripts. RNAseq strategy also demonstrated that transcripts related to siderophore uptake and biosynthesis are up-regulated in iron-deprived condition. The data suggest that the fungus could use both a non-classical RIA, comprising ferric reductases and Fe/Zn permeases (Zrts), and siderophore uptake pathways under iron-limited conditions. The study of iron metabolism reveals novel surface molecules that could function as accessible targets for drugs to block iron uptake and, consequently, inhibit pathogen's proliferation