Antimicrobial peptides from wild bee venom and antibiotics: Synergistic effect against Pseudomonas aeruginosa

Antimicrobial peptides (AMPs) from the venom of Hymenoptera insect are known for their broad spectrum of antimicrobial activity including Gram-negative and Gram-positive bacteria as well as fungi [1,2]. Although the exact mechanism of their action is still not exactly known, it is accepted that these positively charged peptides target the anionic bacterial cell envelope and then disrupt its structure by various modes. In the present work we describe a novel peptide designated HYL-I, which we isolated by RP-HPLC from the extract of venom reservoirs of wild bee Hylaeus signatus (Hymenoptera: Colletidae). The amino acid sequence of this peptide was determined by Edman degradation and ESI-QTOF mass spectrometry as H-Gly-Ile-Met-Ser-Ser-Leu-Met-LysLys-Leu-Ala-Ala-His-Ile-Ala-Lys-NH2 (1696.98 Da). HYL-I and its analogs were prepared by solid phase peptide synthesis using the protocol of Fmoc chemistry. CD spectra of HYL-I measured in the presence of helix promoting additives indicate the tendency to adopt an amphipathic -helical secondary structure in the anisotropic environment such as bacterial cell membrane. HYL-I exhibited strong antimicrobial activity against Gram-positive bacteria Micrococcus luteus and Bacillus subtilis, but very weak activity against Staphylococcus aureus. The activity against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa and against Candida albicans was moderate while the peptide was not hemolytic. Some of the analogs of HYL-I showed remarkable increase in antimicrobial activity against all bacteria tested, which was however accompanied by the increase of hemolytic activity considered as a measure of toxicity. The further design of the analogs resulted in the elimination of this impediment. In addition, we studied possible synergism between this series of AMPs and three different antibiotics which act on intracellular targets of bacteria (tetracycline, rifampicin and clarithromycin). As a studied microorganism we used pathogenic Gram-negative bacterium Pseudomonas aeruginosa.