Antimicrobial activity of linear lipopeptides derived from BP100 towards plant pathogens.

Àngel Oliveras,Marta Planas,Lidia Feliu,Emilio Montesinos,Laura Montesinos,Esther Badosa,Aina Baró,Vitaly Citovsky

doi:10.1371/journal.pone.0201571

Abstract

A collection of 36 lipopeptides were designed from the cecropin A-melittin hybrid peptide BP100 (H-Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-NH2) previously described with activity against phytopathogenic bacteria. These lipopeptides were synthesized on solid-phase and screened for their antimicrobial activity, toxicity and proteolytic stability. They incorporated a butanoyl, a hexanoyl or a lauroyl group at the N-terminus or at the side chain of a lysine residue placed at each position of the sequence. Their antimicrobial activity and hemolysis depended on the fatty acid length and its position. In particular, lipopeptides containing a butanoyl or a hexanoyl chain exhibited the best biological activity profile. In addition, we observed that the incorporation of the acyl group did not induce the overexpression of defense-related genes in tomato. Best lipopeptides were BP370, BP378, BP381, BP387 and BP389, which were highly active against all the pathogens tested (minimum inhibitory concentration of 0.8 to 12.5 μM), low hemolytic, low phytotoxic and significantly stable to protease degradation. This family of lipopeptides might be promising functional peptides useful for plant protection.

Highlights

A collection of 36 lipopeptides were designed from the cecropin A-melittin hybrid peptide BP100 (H-Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-NH2) previously described with activity against phytopathogenic bacteria
Best lipopeptides were BP370, BP378, BP381, BP387 and BP389, which were highly active against all the pathogens tested, low hemolytic, low phytotoxic and significantly stable to protease degradation
We described synthetic cyclic lipodecapeptides derived from the lead antimicrobial peptide BPC194 that were active against plant pathogenic bacteria and fungi, exhibiting differential hemolysis and phytotoxicity [10]

Summary

Materials and methods

Manual peptide synthesis was performed in polypropylene syringes (2 or 5 mL) fitted with a polyethylene porous disk. For lipopeptides BP367, BP379 and BP391 the N-terminal deprotected resin was acylated by treatment with the corresponding fatty acid (10 equiv.), DIC (10 equiv.) and Oxyma (10 equiv.) in NMP for 1 h under stirring. After this time, the resin was washed with NMP (6 × 1 min) and CH2Cl2 (6 × 1 min), and air dried. For antifungal activity 20 μL of each stock solution were mixed in a microtiter plate well with 80 μL of the corresponding suspension of the fungal pathogen and 100 μL of double concentrated Potato Dextrose Broth (PDB) to a total volume of 200 μL containing 0.003% w/v of chloramphenicol to prevent bacterial contamination. Digestion was estimated as the percentage of degraded lipopeptide calculated from the decrease of the HPLC peak area of the native peptide

Results

Discussion

Conclusions