Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur

<p>Abstract</p> <p>Background</p> <p>Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from <it>M. bovis </it>BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection.</p> <p>Results</p> <p>The 2DE proteomic map of <it>M. bovis </it>BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern.</p> <p>Conclusions</p> <p>Here we report the detailed 2DE profile of CFPs from <it>M. bovis </it>BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.</p

A Phosphoproteomic Approach towards the Understanding of the Role of TGF-β in Trypanosoma cruzi Biology

Transforming growth factor beta (TGF-β) plays a pivotal role in Chagas disease, not only in the development of chagasic cardiomyopathy, but also in many stages of the T. cruzi life cycle and survival in the host cell environment. The intracellular signaling pathways utilized by T. cruzi to regulate these mechanisms remain unknown. To identify parasite proteins involved in the TGF-β response, we utilized a combined approach of two-dimensional gel electrophoresis (2DE) analysis and mass spectrometry (MS) protein identification. Signaling via TGF-β is dependent on events of phosphorylation, which is one of the most relevant and ubiquitous post-translational modifications for the regulation of gene expression, and especially in trypanosomatids, since they lack several transcriptional control mechanisms. Here we show a kinetic view of T. cruzi epimastigotes (Y strain) incubated with TGF-β for 1, 5, 30 and 60 minutes, which promoted a remodeling of the parasite phosphorylation network and protein expression pattern. The altered molecules are involved in a variety of cellular processes, such as proteolysis, metabolism, heat shock response, cytoskeleton arrangement, oxidative stress regulation, translation and signal transduction. A total of 75 protein spots were up- or down-regulated more than twofold after TGF-β treatment, and from these, 42 were identified by mass spectrometry, including cruzipain–the major T. cruzi papain-like cysteine proteinase that plays an important role in invasion and participates in the escape mechanisms used by the parasite to evade the host immune system. In our study, we observed that TGF-β addition favored epimastigote proliferation, corroborating 2DE data in which proteins previously described to be involved in this process were positively stimulated by TGF-β

Mycobacterium tuberculosis acquires iron by cell-surface sequestration and internalization of human holo-transferrin

Mycobacterium tuberculosis (M.tb), which requires iron for survival, acquires this element by synthesizing iron-binding molecules known as siderophores and by recruiting a host iron-transport protein, transferrin, to the phagosome. The siderophores extract iron from transferrin and transport it into the bacterium. Here we describe an additional mechanism for iron acquisition, consisting of an M.tb protein that drives transport of human holo-transferrin into M.tb cells. The pathogenic strain M.tb H37Rv expresses several proteins that can bind human holo-transferrin. One of these proteins is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rv1436), which is present on the surface of M.tb and its relative Mycobacterium smegmatis. Overexpression of GAPDH results in increased transferrin binding to M.tb cells and iron uptake. Human transferrin is internalized across the mycobacterial cell wall in a GAPDH-dependent manner within infected macrophages

Análise de antígenos de tuberculinas PPD bovinas de diferentes potências por Western blot e ELISA

Orientadora: Lucy OnoCo-orientadora: Luciene Airy NagashimaTrabalho de Conclusão de Curso (Bacharelado) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Biomedicin