A Novel Broad-spectrum Lipopeptide Antimicrobial Agent, Paenibacterin, against Drug-resistant Bacteria: Structural Elucidation, Biosynthesis, and mechanisms of action
Food, Agricultural, and Environmental Sciences (FAES): 3rd Place (The Ohio State University Edward F. Hayes Graduate Research Forum)The ongoing explosion of infections caused by antibiotic-resistant bacteria continues to challenge the global public health. However, the discovery and development of novel antibacterial drugs are on the decline in the past decades. Therefore, it is urgent to develop potent and safe antimicrobial agents. Here we report the chemical structure, biosynthesis and modes of action of a novel antimicrobial agent, paenibacterin, from a strain of Paenibacillus thiaminolyticus OSY-SE. The producer microorganism was isolated from a soil sample collected in Columbus, OH in 2011. The potent antimicrobial agent was extracted by acetonitrile from the producer cells and purified using HPLC. The core peptide structure of paenibacterin was elucidated by mass-spectrometry (MS) and nuclear magnetic resonance (NMR) while the lipid tail of compound was determined by GC-MS. In order to identify the biosynthetic pathway of the compound, the whole genome of the producer strain OSY-SE was sequenced using the high throughput Illumina sequencing technology. The paenibacterin gene cluster (pbt) was further identified by bioinformatic analyses and confirmed by in vitro protein functional analyses. Furthermore, the mechanisms of action of paenibacterin were studied by fluorescence microscopy, membrane integrity assays, and hydroxyl radical production assays. Paenibacterin is a broad-spectrum antimicrobial agent with potent activity against foodborne pathogens and clinical drug-resistant isolates. Paenibacterin generally yielded a minimum inhibitory concentration (MIC) at 2-8 μg/ml against Gram-negative strains, and 8-64 μg/ml against Gram-positive strains. Paenibacterin is a cyclic lipopeptide, consisting of 13 amino acids and a C15 fatty acid side chain. Among the amino acids, some are D-amino acids and non-proteinogenic amino acid (ornithine). Paenibacterin is biosynthesized by the producer strain through non-ribosomal peptide synthetases (NRPS). The biosynthetic gene cluster was identified within 52-kb region, encoding three NRPSs (PbtA, PbtB and PbtC) and two ABC-transporters (PbtD and PbtE). Paenibacterin damages bacterial cell membrane, resulting in cytoplasmic membrane deporlarization and potassium ions release. Furthermore, paenibacterin triggers radical production via Fenton reaction and subsequently leads to cell death. This study reported a unique and potent antimicrobial agent with activity against drug-resistant bacteria. The structure of the compound could serve a scaffold for designing even more potent compounds by chemical synthesis. In addition, the elucidation of the biosynthetic pathway expedites the effort to produce paenibacterin derivatives by genetic engineering. This new and potent compound is a very promising candidate for agricultural and clinical application.A five-year embargo was granted for this item
