Microplate alamarblue assay for paracoccidioides susceptibility testing

CLSI method M27-A3 is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this study, we developed a microdilution method and added the alamarBlue reagent to test the responses of Paracoccidioides brasiliensis and Paracoccidioides lutzii against amphotericin B and itraconazole antifungals. The test proved to be sensitive, practical, and inexpensive and can be used to monitor the activity of low-growth microorganisms and their response to various drugs. © 2013, American Society for Microbiology.

Intermolecular interactions of the malate synthase of Paracoccidioides spp

Background: The fungus Paracoccidioides spp is the agent of paracoccidioidomycosis (PCM), a pulmonary mycosis acquired by the inhalation of fungal propagules. Paracoccidioides malate synthase (PbMLS) is important in the infectious process of Paracoccidioides spp because the transcript is up-regulated during the transition from mycelium to yeast and in yeast cells during phagocytosis by murine macrophages. In addition, PbMLS acts as an adhesin in Paracoccidioides spp. The evidence for the multifunctionality of PbMLS indicates that it could interact with other proteins from the fungus and host. The objective of this study was to identify and analyze proteins that possibly bind to PbMLS (PbMLS-interacting proteins) because protein interactions are intrinsic to cell processes, and it might be possible to infer the function of a protein through the identification of its ligands. Results: The search for interactions was performed using an in vivo assay with a two-hybrid library constructed in S. cerevisiae; the transcripts were sequenced and identified. In addition, an in vitro assay using pull-down GST methodology with different protein extracts (yeast, mycelium, yeast-secreted proteins and macrophage) was performed, and the resulting interactions were identified by mass spectrometry (MS). Some of the protein interactions were confirmed by Far-Western blotting using specific antibodies, and the interaction of PbMLS with macrophages was validated by indirect immunofluorescence and confocal microscopy. In silico analysis using molecular modeling, dynamics and docking identified the amino acids that were involved in the interactions between PbMLS and PbMLS-interacting proteins. Finally, the interactions were visualized graphically using Osprey software. Conclusion: These observations indicate that PbMLS interacts with proteins that are in different functional categories, such as cellular transport, protein biosynthesis, modification and degradation of proteins and signal transduction. These data suggest that PbMLS could play different roles in the fungal cell. © 2013 de Oliveira et al.; licensee BioMed Central Ltd.

Early endosome antigen 1 (EEA1) decreases in macrophages infected with Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM) is a chronic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis, endemic in Latin America. P. brasiliensis has been observed in epithelial cells in vivo and in vitro, as well as within the macrophages. The identification of the mechanism by which it survives within the host cell is fertile ground for the discovery of its pathogenesis since this organism has the ability to induce its own endocytosis in epithelial cells and most likely in macrophages. The study of the expression of endocytic proteins pathway and co-localization of microorganisms enable detection of the mechanism by which microorganisms survive within the host cell. The aim of this study was to evaluate the expression of the endocytic protein EEA1 (early endosome antigen 1) in macrophages infected with P. brasiliensis. For detection of EEA1, three different techniques were employed: immunofluorescence, real-time polymerase chain reaction (PCR) and immunoblotting. In the present study, decreased expression of EEA1 as well as the rearrangement of the actin was observed when the fungus was internalized, confirming that the input mechanism of the fungus in macrophages occurs through phagocytosis. © 2013 ISHAM.

Expressão gênica das adesinas e fatores de virulência da fase miceliar e leveduriforme do complexo paracoccidioides e de sua interação com células epiteliais e fagócitos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Detecção de Paracoccidioides sp. em amostras ambientais aerossóis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito da interleucina-15 sobre a atividade fungicida, metabolismo oxidativo e produção de citocinas por monócitos humanos, infectados in vitro com Paracoccidioides brasiliensis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito da suplementação nutricional com cogumelos A. blazei sobre a infecção experimental murina aguda por Paracoccidioides brasiliensis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito da IL-15 sobre a atividade fungicida de neutrófilos humanos contra Paracoccidioides brasiliensis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Participação do gene Alc11a1 na infecção por Paracoccidioides brasiliensis em linhagens de camundongos selecionados segundo a alta ou baixa reatividade inflamatória aguda

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito de um inibidor de leucotrienos no processo inflamatório decorrente da infecção por Paracoccidioides brasiliensis em camundongos geneticamnte selecionados

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção de prostaglandinas e leucotrienos por Paracoccidioides brasiliensis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito de citocinas sobre a atividade fungicida e fungistática de neutrófilos humanos contra o Paracoccidioides brasiliensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação por métodos fenotípicos e proteômicos de galatos de alquila com atividade anti-complexo Paracoccidioides

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Espécies crípticas em Paracoccidioides brasiliensis: diferenciação antigênica e de resposta a fatores estressantes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização da transição micélio-levedura em Paracoccidioides brasiliensis e sua relação com a expressão do gene do choque térmico 70(HSP70)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)