Metabolitos de Aspergillus fumigatus endofítico e seu efeito in vitro contra o agente causal da tuberculose

Tuberculosis (TB) remains one of the most deadly communicable infectious diseases, causing 1.4 million deaths in 2015 worldwide due to many conditions, including the inadequate treatment and the emergence of multidrug-resistant strains of the causal agent, Mycobacterium tuberculosis. Therefore, drugs developed from natural sources, as microorganisms and plant extracts, are a frequent target for the research and discovery of antimicrobial compounds. The current study started the characterization of compounds produced by an Aspergillus fumigatus isolated from copaíba (Copaifera multijuga) that efficiently inhibits M. tuberculosis by releasing the compounds into the fermentation broth under specific culture conditions. A preliminary assay was carried out with a correlate species, M. smegmatis, aiming to detect an antimicrobial effect related to A. fumigatus fermentation broth. The direct use of this substrate in antibiosis assays againstM. tuberculosis H37Rv strain (ATCC 27294) allowed the detection of antimicrobial activity with a minimal inhibitory concentration of 256 μg mL-1, demonstrating that purification processes developed by the Biotage Flash Chromatography System are robust and reliable techniques for purification of compounds from natural sources. Also, this chromatographic system can be used in combination with specific biochemical tests, improving the search for reliable results. We conclude that this fraction can express a broad action range, inhibiting both Mycobacterium species used as target organisms.A tuberculose continua a ser uma das doenças infecciosas transmissíveis mais mortais, causando 1,4 milhão de mortes em 2015 em todo o mundo devido a vários fatores, incluindo o tratamento inadequado e o surgimento de cepas multirresistentes do agente causal, Mycobacterium tuberculosis. Portanto, as drogas desenvolvidas a partir de fontes naturais, como micro-organismos e extratos de plantas, são um alvo freqüente para a pesquisa e descoberta de compostos antimicrobianos. O presente estudo foi um ponto de partida para caracterizar compostos produzidos por um Aspergillus fumigatus isolado de copaíba (Copaifera multijuga) que inibe eficientemente M. tuberculosis, liberando os compostos no caldo de fermentação em condições de cultura específicas. Realizou-se um ensaio preliminar com uma espécie correlata, M. smegmatis, com o objetivo de detectar um efeito antimicrobiano relacionado ao caldo de fermentação de A. fumigatus. O uso direto deste substrato em ensaios de antibiose contra a estirpe H37Rv de M. tuberculosis (ATCC 27294) permitiu a detecção de atividade antimicrobiana com uma concentração inibitória mínima de 256 μg mL-1, demonstrando que os processos de purificação desenvolvidos pelo Biotage Flash Chromatography System são técnicas robustas e confiáveis para purificar compostos de fontes naturais. Além disso, este sistema cromatográfico pode ser usado em combinação com testes bioquímicos específicos, melhorando a busca de resultados confiáveis. Concluímos que esta fração pode expressar uma ampla gama de ação, inibindo ambas as espécies de Mycobacterium utilizadas como organismos-alvo

Structural basis for the differential binding affinities of the HsfBD1 and HsfBD2 domains in the Haemophilus influenzae Hsf adhesin

Haemophilus influenzae is a human-specific gram-negative coccobacillus that causes a variety of human infections ranging from localized respiratory infections to invasive diseases. Hsf is the major nonpilus adhesin in encapsulated strains of H. influenzae and belongs to the trimeric autotransporter family of proteins. The Hsf protein contains two highly homologous binding domains, designated HsfBD1 and HsfBD2. In this study we characterized the differential binding properties of HsfBD1 and HsfBD2. In assays using HeLa cells, we found that bacteria expressing either full-length Hsf or HsfBD1 by itself adhered at high levels, while bacteria expressing HsfBD2 by itself adhered at low levels. Immunofluorescence microscopy and a cellular enzyme-linked immunosorbent assay using purified proteins revealed that the binding affinity was significantly higher for HsfBD1 than for HsfBD2. Purified HsfBD1 was able to completely block adherence by bacteria expressing either HsfBD1 or HsfBD2, while purified HsfBD2 was able to block adherence by bacteria expressing HsfBD2 but had minimal activity against bacteria expressing HsfBD1. Conversion of the residue at position 1935 in the HsfBD1 binding pocket from Asp to Glu resulted in HsfBD2-like binding properties, and conversion of the residue at position 569 in the HsfBD2 binding pocket from Glu to Asp resulted in HsfBD1-like binding properties, as assessed by adherence assays with recombinant bacteria and by immunofluorescence microscopy with purified proteins. This work demonstrates the critical role of a single amino acid in the core of the binding pocket in determining the relative affinities of the HsfBD1 and HsfBD2 binding domains