| Abstrakt: |
Background: Defensins are basic, cysteine-rich antimicrobial peptides that are important components of plant defense against pathogens. Previously, we isolated a defensin, Pv D1, from Phaseolus vulgaris L. (common bean) seeds. Results: The aim of this study was to overexpress Pv D1 in a prokaryotic system, verify the biologic function of recombinant Pv D1 (Pv D1r) by comparing the antimicrobial activity of Pv D1r to that of the natural defensin, Pv D1, and use a mutant Candida albicans strain that lacks the gene for sphingolipid biosynthesis to unravel the target site of the Pv D1r in C. albicans cells. The cDNA encoding Pv D1, which was previously obtained, was cloned into the pET-32 EK/LIC vector, and the resulting construct was used to transform bacterial cells (Rosetta Gami 2 (DE3) pLysS) leading to recombinant protein expression. After expression had been induced, Pv D1r was purified, cleaved with enterokinase and repurified by chromatographic steps. N-terminal amino acid sequencing showed that the overall process of the recombinant production of Pv D1r, including cleavage with the enterokinase, was successful. Additionally, modeling revealed that Pv D1r had a structure that was similar to the defensin isolated from plants. Purified Pv D1 and Pv D1r possessed inhibitory activity against the growth of the wild-type pathogenic yeast strain C. albicans. Both defensins, however, did not present inhibitory activity against the mutant strain of C. albicans. Antifungal assays with the wild-type C. albicans strains showed morphological changes upon observation by light microscopy following growth assays. Pv D1r was coupled to FITC, and the subsequent treatment of wild type C. albicans with DAPI revealed that the labeled peptide was intracellularly localized. In the mutant strain, no intracellular labeling was detected. Conclusion: Our results indicate that Pv D1r retains full biological activity after recombinant production, enterokinase cleavage and purification. Additionally, our results from the antimicrobial assay, the microscopic analysis and the Pv D1r-FITC labeling assays corroborate each other and lead us to suggest that the target of Pv D1 in C. albicans cells is the sphingolipid glucosylceramide. [ABSTRACT FROM AUTHOR] |
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