Purification, De Novo Characterization and Antibacterial Properties of a Novel, Narrow-Spectrum Bacteriostatic Tripeptide from Geotrichum candidum OMON-1

Abstract

Antibiotic resistance and need for novel antibiotics have seen focus on antimicrobial peptides (AMPs) increase in recent years. The study investigated purification and de novo characterization of an AMP compound produced by bioactive Geotrichum candidum OMON-1. Antimicrobial extract of G. candidum OMON-1 was purified via reverse-phase high-performance liquid chromatography (RP-HPLC). Purified compound (GP-2B) was characterized on liquid chromatography–mass spectrometry (LC–MS) and MALDI-TOF tandem mass spectrometry (MS/MS), and spectra were interpreted using de novo peptide sequencing protocols. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of GP-2B were determined in vitro against Gram-positive clinical isolates and time-kill kinetics against Staphylococcus aureus investigated, while antimicrobial activity was also observed on scanning electron microscope (SEM). GP-2B was successfully purified on RP-HPLC with trifluoroacetic acid-buffered acetonitrile. LC–MS confirmed GP-2B as pure and positively charged with molecular mass 409.23 amu, while characterization of MS/MS spectrum via de novo peptide sequencing described it a tripeptide with carboxymethylcystyl–asparagyl–aspartate amino acid sequence. GP-2B had MIC 8 and 32 µg/mL against antibiotic-sensitive and -resistant S. aureus, respectively, and 64 µg/mL for Enterococci strains. MBC was fourfold MIC, while GP-2B reduced S. aureus by 21.8% and 30% at 5× and 10× MIC, respectively, in time-kill kinetics. SEM images also confirmed bacteriostasis of S. aureus cells. This study successfully purified and characterized via de novo sequencing, a novel bacteriostatic AMP from G. candidum OMON-1.