A new Kunitz trypsin inhibitor from Erythrina poeppigiana exhibits antimicrobial and antibiofilm properties against bacteria

Erythrina poeppigiana belongs to Fabaceae family (subfamily Papillionoideae) and is commonly found in tropical and subtropical regions in Brazil. Herein, we described the purification and characterization of a new Kunitz-type inhibitor, obtained from E. poeppigiana seeds (EpTI). EpTI is composed by three isoforms of identical aminoterminal sequences with a molecular weight ranging from 17 to 20 kDa. The physicochemical features showed by EpTI are common to Kunitz inhibitors, including the dissociation constant (13.1 nM), stability against thermal (37–100 ◦C) and pH (2–10) ranging, and the presence of disulfide bonds stabilizing its reactive site. Furthermore, we investigated the antimicrobial, anti-adhesion, and anti-biofilm properties of EpTI against Grampositive and negative bacteria. The inhibitor showed antimicrobial activity with a minimum inhibitory concentration (MIC, 5–10 μM) and minimum bactericidal concentration (MBC) of 10 μM for Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus haemolyticus. The combination of EpTI with ciprofloxacin showed a marked synergistic effect, reducing the antibiotic concentration by 150%. The increase in crystal violet uptake for S. aureus and K. pneumoniae strains was approximately 30% and 50%, respectively, suggesting that the bacteria plasma membrane is targeted by EpTI. Treatment with EpTI at 1x and 10 x MIC significantly reduced the biofilm formation and prompted the disruption of a mature biofilm. At MIC/2, EpTI decreased the bacterial adhesion to polystyrene surface within 2 h. Finally, EpTI showed low toxicity in animal model Galleria mellonella. Given its antimicrobial and anti-biofilm properties, the EpTI sequence might be used to design novel drug prototypes

Purification and characterization of a Kunitz inhibitor from Poincianella pyramidalis with insecticide activity against the Mediterranean flour moth

CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFINEP - FINANCIADORA DE ESTUDOS E PROJETOSFUNDECT - FUNDAÇÃO DE APOIO AO DESENVOLVIMENTO DO ENSINO, CIÊNCIA E TECNOLOGIA DO ESTADO DO MATO GROSSO DO SULThis paper describes the characterization of a trypsin inhibitor from Poincianella pyramidalis seeds (PpyTI). The partial sequencing of PpyTI revealed homology to Kunitz inhibitors, clustered as a member of Family 103 in MEROPS database. PpyTI has a single polypeptide chain of 19,042 Da and presents stability at high temperatures (up to 70 degrees C) and a wide range of pH. In vitro assays showed that disulfide bridges have an important stabilization role of reactive site in PpyTI, a characteristic shared among several Kunitz inhibitors. Bioassays carried out with the Mediterranean flour moth (Anagasta kuehniella) revealed a significant decrease in both larval weight and survival of PpyTI-fed larvae, besides a larval stage extension. Through biochemical analysis, we demonstrated that the PpyTI insecticide effects were triggered by digestion process commitment, through the inhibition of trypsin and chymotrypsin activities, the major digestive enzymes in this species. The insecticide effects and biochemical characterization of PpyTI encourage further studies using this inhibitor for insect pest control. (C) 2014 Elsevier Inc. All rights reserved.This paper describes the characterization of a trypsin inhibitor from Poincianella pyramidalis seeds (PpyTI). The partial sequencing of PpyTI revealed homology to Kunitz inhibitors, clustered as a member of Family 103 in MEROPS database. PpyTI has a single polypeptide chain of 19,042 Da and presents stability at high temperatures (up to 70 degrees C) and a wide range of pH. In vitro assays showed that disulfide bridges have an important stabilization role of reactive site in PpyTI, a characteristic shared among several Kunitz inhibitors. Bioassays carried out with the Mediterranean flour moth (Anagasta kuehniella) revealed a significant decrease in both larval weight and survival of PpyTI-fed larvae, besides a larval stage extension. Through biochemical analysis, we demonstrated that the PpyTI insecticide effects were triggered by digestion process commitment, through the inhibition of trypsin and chymotrypsin activities, the major digestive enzymes in this species. The insecticide effects and biochemical characterization of PpyTI encourage further studies using this inhibitor for insect pest control11819CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFINEP - FINANCIADORA DE ESTUDOS E PROJETOSFUNDECT - FUNDAÇÃO DE APOIO AO DESENVOLVIMENTO DO ENSINO, CIÊNCIA E TECNOLOGIA DO ESTADO DO MATO GROSSO DO SULCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFINEP - FINANCIADORA DE ESTUDOS E PROJETOSFUNDECT - FUNDAÇÃO DE APOIO AO DESENVOLVIMENTO DO ENSINO, CIÊNCIA E TECNOLOGIA DO ESTADO DO MATO GROSSO DO SULsem informaçãosem informaçãosem informaçã