Food spoilage is a complex process in which foods become unsafe or undesirable for human consumption. Microbial spoilage causes food deterioration that may lead to slime and/or off-odors and off-flavors. In addition, human pathogens can be transmitted by food vehicles and the consumption of contaminated foods may lead to foodborne illnesses. The Centers for Disease Control and Prevention (CDC) estimated that foodborne pathogens caused 9.4 million illnesses, 55,961 hospitalizations and 1,351 deaths annually in the United States. Many food preservation and processing technologies have been developed to minimize the microbiological hazards in foods. Conventional chemical preservatives such as nitrites, and natural antimicrobial agents, such as nisin and essential oils, are used to preserve foods by preventing or retarding the growth of foodborne pathogens and spoilage microorganisms. The increasing use of nisin by food processors has promoted many investigators to search for other natural antimicrobial peptides with improved properties.
The widespread of antibiotic-resistant pathogens has become a global public health concern. Most of current antibiotics in the market are derived from the chemical scaffolds discovered between mid-1930s and early 1960s. After the “golden era” of discovery, only four new classes of antibiotics were introduced to the market in the past 40 years. The emergence of drug resistant pathogenic strains has increased mortality rate due to infections with these particular pathogens. For example, the methicillin-resistant Staphylococcus aureus (MRSA) caused ~19,000 deaths each year in the United States. Therefore, new and potent antibiotics are urgently needed against emerging antibiotic-resistant pathogens. Peptide antibiotics, including polymyxins and daptomycin, have received great attention in recent years for treating infections caused by drug-resistant pathogens.
The overall objective of this study was to search for safe and effective natural antimicrobial agents from microbial sources. To better use the newly found antimicrobials, we aimed to elucidate the chemical structure of the new compounds, examine the mechanisms of action and identify the biosynthetic pathway of the new antimicrobial agents. Additionally, we investigated the efficacy of of new discovered compounds using different model food systems.
In this current study, we described three antimicrobial peptides, including a nonribosomal peptide, paenibacterin, and two ribosomal peptides, paenibacillin and enterocin RM6. Paenibacterin is a lipopeptide antibiotic that is active against both Gram-negative and Gram-positive bacteria, including antibiotic-resistant pathogens. Paenibacterin is a promising antibiotic scaffold for developing new antibiotics targeting drug-resistant pathogens. On the other hand, paenibacillin and enterocin RM6 are anti-Gram positive agents that belong to the bacteriocin family, which are ribosomally synthesized peptides. Paenibacillin is lanthionine-containing lantibiotic (class I bacteriocin) whereas enterocin RM6 is a cyclic unmodified class II bacteriocin. Paenibacillin and enterocin RM6 are naturally occurring antimicrobial peptides with great potential for food preservation.
Paenibacterin is produced by a soil isolate, Paenibacillus thiaminolyticus OSY-SE. The compound was extracted from cells with acetonitrile and purified to homogeneity by high performance liquid chromatography (HPLC). The chemical structure of paenibacterin was elucidated using mass spectrometry (MS) and nuclear magnetic resonance (NMR). Paenibacterin is a cyclic lipopeptide consisting of 13 amino acids and an N-terminal C15 fatty acyl (FA) chain. The deduced sequence is: FA-Orn-Val-Thr-Orn-Ser-Val-Lys-Ser-Ile-Pro-Val-Lys-Ile. The carboxyl terminal Ile is connected with Thr by an ester linkage forming a macrolactone ring. The mechanisms of action of paenibacterin involve direct cell membrane damage and indirect oxidative cellular damage. Paenibacterin is a cationic lipopeptide with four positive charges. The electrostatic interaction between paenibacterin and LPS can displace the divalent cations on the LPS network and promote the uptake of paenibacterin. The cytoplasmic membrane is the direct target of paenibacterin. Paenibacterin depolarized cell membrane, triggered K+ release and increased the uptake of hydrophobic nucleic acid stain, propidium iodide. In addition, paenibacterin led to production of hydroxyl radicals in bacterial cells. The radical scavenger, thiourea, and the iron chelator, 2, 2’-dipyridyl, reduced the killing effect of paenibacterin against Escherichia coli and Staphylococcus aureus.
The presence of non-proteinogeneic amino acids (Orn, ornithine) in the peptide sequence suggested that paenibacterin is synthesized by nonribosomal synthetases. In order to determine the genes for paenibacterin biosynthesis, we sequenced the whole genome of the producer strain using the next-generation sequencing technology. The gene cluster was identified within 52-kb region, encoding three non-ribosomal peptide synthetases (PbtA, PbtB and PbtC) and two ABC-transporters (PbtD and PbtE). As deduced from the sequence data, each PbtA and PbtB enzyme consists of five modules, whereas PbtC is composed of three modules. Each of the 13 modules assembles one amino acid into the paenibacterin peptide. Sequence analysis revealed that Orn1, Orn4, Lys7 and Ser8 in paenibacterin may have D-configuration. The absolute configuration of two ornithine residues was confirmed by chirality analysis using Marfey's reagents. In addition, the substrate specificities of selected adenylation domains were confirmed by overexpression in Escherichia coli and in vitro protein function analyses.
The lantibiotic, paenibacillin, was discovered by our group and its chemical structure has been elucidated in 2007-2008. In the current study, we investigated the mechanism of action and biosynthesis of this lantibiotic. Paenibacillin depolarized cell membrane and triggered potassium ions efflux, leading to cell death. The gene cluster of paenibacillin in Paenibacillus polymyxa OSY-DF was identified using a PCR-based method combined with whole genome sequencing. The paenibacillin gene cluster (11.7 kb) consisted of 11 open reading frames (ORFs) encoding proteins for production, modification, regulation, immunity and transportation of the lantibiotic. Disruption of the lantibiotic dehydratase (PaenB) by targeted mutagenesis completely eliminated the production of paenibacillin.
Additionally, we tested the efficacy of paenibacillin against Listeria in meat products. The crude extract of paenibacillin inhibited or delayed the growth of Listeria monocytogenes in sausage and irradiated ground beef products.
Enterocin RM6 is produced by Enterococcus faecalis isolated from raw milk. In this study, we purified enterocin RM6 by HPLC and identified its chemical structure by mass spectrometry. This compound is a 70-residue cyclic peptide with a head-to-tail linkage between the N-terminal methionine and C-termianl tryptophan. The peptide sequence was further confirmed by sequencing the structural gene of the peptide. Enterocin RM6 (final concentration, 80 AU/ml) caused a rapid 4-log reduction of L. monocytogenes in cottage cheese within 30 min, and no viable cells were detected after 26 hrs of treatment. Therefore, enterocin RM6 is potentially useful in inhibiting L. monocytogenes in foods.