title: Functional expression and activity of the recombinant antifungal defensin PvD1r from Phaseolus vulgaris L. (common bean) seeds.
abstract: BACKGROUND
  Defensins are basic, cysteine-rich antimicrobial peptides that are important components of plant defense against pathogens. Previously, we isolated a defensin, PvD1, from Phaseolus vulgaris L. (common bean) seeds.
  
  
  RESULTS
  The aim of this study was to overexpress PvD1 in a prokaryotic system, verify the biologic function of recombinant PvD1 (PvD1r) by comparing the antimicrobial activity of PvD1r to that of the natural defensin, PvD1, and use a mutant Candida albicans strain that lacks the gene for sphingolipid biosynthesis to unravel the target site of the PvD1r in C. albicans cells. The cDNA encoding PvD1, 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, PvD1r 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 PvD1r, including cleavage with the enterokinase, was successful. Additionally, modeling revealed that PvD1r had a structure that was similar to the defensin isolated from plants. Purified PvD1 and PvD1r 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. PvD1r 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.
  
  
  CONCLUSIONS
  Our results indicate that PvD1r retains full biological activity after recombinant production, enterokinase cleavage and purification. Additionally, our results from the antimicrobial assay, the microscopic analysis and the PvD1r-FITC labeling assays corroborate each other and lead us to suggest that the target of PvD1 in C. albicans cells is the sphingolipid glucosylceramide.
authors.forename: Erica de O
authors.initials: Ede O
authors.lastname: Mello
authors.forename: Izabela S
authors.initials: IS
authors.lastname: dos Santos
authors.forename: André de O
authors.initials: Ade O
authors.lastname: Carvalho
authors.forename: Luísa S
authors.initials: LS
authors.lastname: de Souza
authors.forename: Gonçalo A
authors.initials: GA
authors.lastname: de Souza-Filho
authors.forename: Viviane V
authors.initials: VV
authors.lastname: do Nascimento
authors.forename: Olga L T
authors.initials: OL
authors.lastname: Machado
authors.forename: Umberto
authors.initials: U
authors.lastname: Zottich
authors.affiliation: Laboratório de Fisiologia e Bioquímica de Microrganismos do Centro de Biociências e Biotecnologia da Universidade Estadual do Norte Fluminense Darcy Ribeiro, Avenida Alberto Lamego 2000, Campos dos Goytacazes, RJ, Brazil. valmg@uenf.br.
authors.forename: Valdirene M
authors.initials: VM
authors.lastname: Gomes
mesh_terms: D000935:Antifungal Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D002176:Candida albicans / Q000187:drug effects / Q000254:growth & development; D003001:Cloning, Molecular; D023082:Defensins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D027805:Phaseolus / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D012639:Seeds / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D023082:Defensins; D011994:Recombinant Proteins