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Development of innovative pediocin PA-1 by DNA shuffling among class IIa bacteriocins.

ABSTRACT: Pediocin PA-1 is a member of the class IIa bacteriocins, which show antimicrobial effects against lactic acid bacteria. To develop an improved version of pediocin PA-1, reciprocal chimeras between pediocin PA-1 and enterocin A, another class IIa bacteriocin, were constructed. Chimera EP, which consisted of the C-terminal half of pediocin PA-1 fused to the N-terminal half of enterocin A, showed increased activity against a strain of Leuconostoc lactis isolated from a sour-spoiled dairy product. To develop an even more effective version of this chimera, a DNA-shuffling library was constructed, wherein four specific regions within the N-terminal half of pediocin PA-1 were shuffled with the corresponding sequences from 10 other class IIa bacteriocins. Activity screening indicated that 63 out of 280 shuffled mutants had antimicrobial activity. A colony overlay activity assay showed that one of the mutants (designated B1) produced a >7.8-mm growth inhibition circle on L. lactis, whereas the parent pediocin PA-1 did not produce any circle. Furthermore, the active shuffled mutants showed increased activity against various species of Lactobacillus, Pediococcus, and Carnobacterium. Sequence analysis revealed that the active mutants had novel N-terminal sequences; in active mutant B1, for example, the parental pediocin PA-1 sequence (KYYGNGVTCGKHSC) was changed to TKYYGNGVSCTKSGC. These new and improved DNA-shuffled bacteriocins could prove useful as food additives for inhibiting sour spoilage of dairy products.

SUBMITTER: Tominaga T

PROVIDER: S-EPMC1950993 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Development of innovative pediocin PA-1 by DNA shuffling among class IIa bacteriocins.

Tominaga Tatsuya T Hatakeyama Yoshinori Y

Applied and environmental microbiology 20070629 16

Pediocin PA-1 is a member of the class IIa bacteriocins, which show antimicrobial effects against lactic acid bacteria. To develop an improved version of pediocin PA-1, reciprocal chimeras between pediocin PA-1 and enterocin A, another class IIa bacteriocin, were constructed. Chimera EP, which consisted of the C-terminal half of pediocin PA-1 fused to the N-terminal half of enterocin A, showed increased activity against a strain of Leuconostoc lactis isolated from a sour-spoiled dairy product. T ...[more]

PMID: 17601819

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Project description:Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.

Project description:Class IIa bacteriocin antimicrobial peptides (AMPs) are a compelling alternative to current antimicrobials because of potential specific activity toward antibiotic-resistant bacteria, including vancomycin-resistant enterococci. Engineering of these molecules would be enhanced by a better understanding of AMP sequence-activity relationships to improve efficacy in vivo and limit effects of off-target activity. Toward this goal, we experimentally evaluated 210 natural and variant class IIa bacteriocins for antimicrobial activity against six strains of enterococci. Inhibitory activity was ridge regressed to AMP sequence to predict performance, achieving an area under the curve of 0.70 and demonstrating the potential of statistical models for identifying and designing AMPs. Active AMPs were individually produced and evaluated against eight enterococcus strains and four Listeria strains to elucidate trends in susceptibility. It was determined that the mannose phosphotransferase system (manPTS) sequence is informative of susceptibility to class IIa bacteriocins, yet other factors, such as membrane composition, also contribute strongly to susceptibility. A broadly potent bacteriocin variant (lactocin DT1) from a Lactobacillus ruminis genome was identified as the only variant with inhibitory activity toward all tested strains, while a novel enterocin variant (DT2) from an Enterococcus faecium genome demonstrated specificity toward Listeria strains. Eight AMPs were evaluated for proteolytic stability to trypsin, chymotrypsin, and pepsin, and three C-terminal disulfide-containing variants, including divercin V41, were identified as compelling for future in vivo studies, given their high potency and proteolytic stability.IMPORTANCE Class IIa bacteriocin antimicrobial peptides (AMPs), an alternative to traditional small-molecule antibiotics, are capable of selective activity toward various Gram-positive bacteria, limiting negative side effects associated with broad-spectrum activity. This selective activity is achieved through targeting of the mannose phosphotransferase system (manPTS) of a subset of Gram-positive bacteria, although factors affecting this mechanism are not entirely understood. Peptides identified from genomic data, as well as variants of previously characterized AMPs, can offer insight into how peptide sequence affects activity and selectivity. The experimental methods presented here identify promising potent and selective bacteriocins for further evaluation, highlight the potential of simple computational modeling for prediction of AMP performance, and demonstrate that factors beyond manPTS sequence affect bacterial susceptibility to class IIa bacteriocins.

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Development of innovative pediocin PA-1 by DNA shuffling among class IIa bacteriocins.

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Development of innovative pediocin PA-1 by DNA shuffling among class IIa bacteriocins.

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