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

• Published in: BMC biochemistry Vol. 15; no. 1; p. 7
• Main Authors: de O Mello, Erica, dos Santos, Izabela S, de O Carvalho, Andre, de Souza, Luisa S, de Souza-Filho, Goncalo A, do Nascimento, Viviane V, Machado, Olga LT, Zottich, Umberto, Gomes, Valdirene M
• Format: Journal Article
• Language: English
• Published: 01.01.2014
• Subjects: Candida albicans; Phaseolus vulgaris
• ISSN: 1471-2091; 1471-2091
• DOI: 10.1186/1471-2091-15-7

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