Functional expression and activity of the recombinant antifungal defensin PvD(1)r from Phaseolus vulgaris L. (common bean) seeds

BACKGROUND: Defensins are basic, cysteine-rich antimicrobial peptides that are important components of plant defense against pathogens. Previously, we isolated a defensin, PvD(1), from Phaseolus vulgaris L. (common bean) seeds. RESULTS: The aim of this study was to overexpress PvD(1) in a prokaryotic system, verify the biologic function of recombinant PvD(1) (PvD(1)r) by comparing the antimicrobial activity of PvD(1)r to that of the natural defensin, PvD(1), and use a mutant Candida albicans strain that lacks the gene for sphingolipid biosynthesis to unravel the target site of the PvD(1)r in C. albicans cells. The cDNA encoding PvD(1), 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 (DE(3)) pLysS) leading to recombinant protein expression. After expression had been induced, PvD(1)r 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 PvD(1)r, including cleavage with the enterokinase, was successful. Additionally, modeling revealed that PvD(1)r had a structure that was similar to the defensin isolated from plants. Purified PvD(1) and PvD(1)r 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. PvD(1)r 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 PvD(1)r retains full biological activity after recombinant production, enterokinase cleavage and purification. Additionally, our results from the antimicrobial assay, the microscopic analysis and the PvD(1)r-FITC labeling assays corroborate each other and lead us to suggest that the target of PvD(1) in C. albicans cells is the sphingolipid glucosylceramide.