RESUMO
Histoplasma capsulatum is a major endemic mycosis. Our laboratories have demonstrated that H. capsulatum produces extracellular vesicles (EV) that are loaded with diverse compounds that influence virulence. We have further shown that H. capsulatum dynamically regulates the loading and release of fungal EV in response to stimuli and growth conditions. This chapter details the current knowledge of EV biology in H. capsulatum and the impact of this information on our understanding of this important process that is closely linked to pathogenesis.
Assuntos
Vesículas Extracelulares , Micoses , Histoplasma , Humanos , VirulênciaRESUMO
Oxidative/nitrosative stress may be important in the pathology of Chagas' disease. Experimental animals infected by Trypanosoma cruzi showed an early rise in myocardial and peripheral protein-3-nitrotyrosine (3NT) and protein-carbonyl formation that persisted during the chronic stage of disease. In comparison, experimental chronic ethanol-induced cardiomyopathy was slow to develop and presented with a moderate increase in oxidative stress and minimal to no nitrosative stress after long-term alcohol feeding of animals. The oxidative stress in both chagasic animals and animals with ethanol-induced cardiomyopathy correlated with the persistence of reactive oxygen species-producing inflammatory intermediates. Protein-3NT formation in T. cruzi-infected animals was associated with enhanced nitric oxide expression (inferred by nitrite/nitrate levels) and myeloperoxidase activity, suggesting that both peroxynitrite- and myeloperoxidase-mediated pathways contribute to increased protein nitration in Chagas' disease. We used one- and two-dimensional gel electrophoresis and Western blot analysis to identify disease-specific plasma proteins that were 3NT-modified in T. cruzi-infected animals. Nitrated protein spots (56 in total) were sequenced by matrix-assisted laser desorption ionization/time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry and identified by a homology search of public databases. Clustering of 3NT-modified proteins according to their functional characteristics revealed that the nitration of immunoglobulins, apolipoprotein isoforms, and other proteins might perturb their functions and be important in the pathology of Chagas' disease. We also showed that nitrated peptides derived from titin and alpha-actin were released into the plasma of patients with Chagas' disease. Such modified proteins may be useful biomarkers of Chagas' disease.
Assuntos
Proteínas Sanguíneas/metabolismo , Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/patologia , Carbonilação Proteica , Tirosina/análogos & derivados , Animais , Biomarcadores/sangue , Western Blotting , Cardiomiopatia Chagásica/imunologia , Ensaio Cometa , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Camundongos , Miocárdio/química , Miocárdio/metabolismo , Miocárdio/patologia , Nitrosação , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trypanosoma cruzi/imunologia , Tirosina/sangueRESUMO
Histone tails provide sites for a variety of post-translational modifications implicated in the control of gene expression and chromatin assembly. As both histones and control of gene expression in trypanosomes are highly divergent compared to most eukaryotes, post-translational modifications of Trypanosoma cruzi histones were investigated. After in vivo incubation of live parasites with radiolabeled precursors, histone H4 mainly incorporates [(3)H]-acetyl, and to a lesser extent [(3)H]-methyl residues. In contrast, histone H3 preferentially incorporates [(3)H]-methyl residues. The modifications of histone H4 were further characterized by mass spectrometry. MALDI-TOF-TOF-MS analysis revealed that peptides from histone H4 amino-terminus, obtained by either endoproteinase Glu-C or endoproteinase Arg-C digestion, contain isoforms with 14 and 42Da additions, suggesting the presence of simultaneous acetylations and/or methylations. Tandem mass spectrometry analysis demonstrated that the N-terminal alanine is methylated, and lysine residues at positions 4, 10, 14 and 57 are acetylated; lysine at position 18 is mono-methylated, while arginine at position 53 is dimethylated. Immunoblotting analyses using specific antibodies raised against synthetic and acetylated peptides of T. cruzi histone H4 indicate that lysine 4 is acetylated in the majority of histone H4, while other acetylations at the N-terminus portion of histone H4 are less abundant.