RESUMO
Parkinson's disease (PD) is a chronic and progressive neurodegenerative disease with the major symptoms comprising loss of movement coordination (motor dysfunction) and non-motor dysfunction, including gastrointestinal symptoms. Alterations in the gut microbiota composition have been reported in PD patients vs. controls. However, it is still unclear how these compositional changes contribute to disease etiology and progression. Furthermore, most of the available studies have focused on European, Asian, and North American cohorts, but the microbiomes of PD patients in Latin America have not been characterized. To address this problem, we obtained fecal samples from Colombian participants (n = 25 controls, n = 25 PD idiopathic cases) to characterize the taxonomical community changes during disease via 16S rRNA gene sequencing. An analysis of differential composition, diversity, and personalized computational modeling was carried out, given the fecal bacterial composition and diet of each participant. We found three metabolites that differed in dietary habits between PD patients and controls: carbohydrates, trans fatty acids, and potassium. We identified six genera that changed significantly in their relative abundance between PD patients and controls, belonging to the families Lachnospiraceae, Lactobacillaceae, Verrucomicrobioaceae, Peptostreptococcaceae, and Streptococcaceae. Furthermore, personalized metabolic modeling of the gut microbiome revealed changes in the predicted production of seven metabolites (Indole, tryptophan, fructose, phenylacetic acid, myristic acid, 3-Methyl-2-oxovaleric acid, and N-Acetylneuraminic acid). These metabolites are associated with the metabolism of aromatic amino acids and their consumption in the diet. Therefore, this research suggests that each individual's diet and intestinal composition could affect host metabolism. Furthermore, these findings open the door to the study of microbiome-host interactions and allow us to contribute to personalized medicine.
RESUMO
BACKGROUND: The Plasmodium vivax Duffy binding protein (PvDBP) has been the most studied ligand binding human reticulocytes to date. This molecule has a cysteine-rich domain in region II (RII) which has been used as control for evaluating the target cell binding activity of several parasite molecules. However, obtaining rPvDBP-RII in a soluble form using the Escherichia coli expression system usually requires laborious and time-consuming steps for recovering the molecule's structure and function, considering it is extracted from inclusion bodies. The present study describes an easy and fast method for expressing and obtaining several PvDBP fragments which should prove ideal for use in protein-cell interaction assays. RESULTS: Two PvDBP encoding regions (rii and riii/v) were cloned in pEXP5-CT vector and expressed in E. coli and extracted from the soluble fraction (rPvDBP-RIIS and rPvDBP-RIII/VS) using a simple freezing/thawing protocol. After the purification, dichroism analysis enabled verifying high rPvDBP-RIIS and rPvDBP-RIII/VS secondary structure α-helix content, which was lowered when molecules were extracted from inclusion bodies (rPvDBP-RIIIB and rPvDBP-RIII/VIB) using a denaturing step. Interestingly, rPvDBP-RIIS, but not rPvDBP-RIIIB, bound to human reticulocytes, while rPvDBP-RIII/VS and rPvDBP-RIII/VIB bound to such cells in a similar way to negative control (cells incubated without recombinant proteins). CONCLUSIONS: This research has shown for the first time how rPvDBP-RII can be expressed and obtained in soluble form using the E. coli system and avoiding the denaturation and refolding steps commonly used. The results highlight the usefulness of the rPvDBP-RIII/VS fragment as a non-binding control for protein-cell target interaction assays. The soluble extraction protocol described is a good alternative to obtain fully functional P. vivax proteins in a fast and easy way, which will surely prove useful to laboratories working in studying this parasite's biology.
