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Heterologous Expression of the Class IIa Bacteriocins, Plantaricin 423 and Mundticin ST4SA, in Escherichia coli Using Green Fluorescent Protein as a Fusion Partner.

ABSTRACT: The antilisterial class IIa bacteriocins, plantaricin 423 and mundticin ST4SA, have previously been purified from the cell-free supernatants of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, respectively. Here, we present the fusions of mature plantaricin 423 and mundticin ST4SA to His-tagged green fluorescent protein (GFP) for respective heterologous expression in Escherichia coli. Fusion of plantaricin 423 and mundticin ST4SA to His-tagged GFP produced the fusion proteins GFP-PlaX and GFP-MunX, respectively. Both fusion proteins were autofluorescent, circumvented inclusion body formation and lowered the toxicity of class IIa bacteriocins during heterologous expression. Not only did GFP-class IIa fusion stabilize heterologous expression and boost yields, the fluorescent intensity of GFP-PlaX and GFP-MunX could be monitored quantitatively and qualitatively throughout expression and purification. This robust fluorometric property allowed rapid optimization of conditions for expression and bacteriocin liberation from GFP via the incorporated WELQut protease cleavage sequence. Incubation temperature and IPTG concentration had a significant effect on bacteriocin yield, and was optimal at 18°C and 0.1-0.2 mM, respectively. GFP-MunX was approximately produced at a yield of 153.30 mg/L culture which resulted in 12.4 mg/L active mundticin ST4SA after liberation and HPLC purification. While GFP-PlaX was produced at a yield of 121.29 mg/L culture, evidence suggests heterologous expression resulted in conformation isomers of WELQut liberated plantaricin 423.

SUBMITTER: Vermeulen RR

PROVIDER: S-EPMC7381239 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Heterologous Expression of the Class IIa Bacteriocins, Plantaricin 423 and Mundticin ST4SA, in Escherichia coli Using Green Fluorescent Protein as a Fusion Partner.

Vermeulen Ross Rayne RR Van Staden Anton Du Preez ADP Dicks Leon L

Frontiers in microbiology 20200714

The antilisterial class IIa bacteriocins, plantaricin 423 and mundticin ST4SA, have previously been purified from the cell-free supernatants of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, respectively. Here, we present the fusions of mature plantaricin 423 and mundticin ST4SA to His-tagged green fluorescent protein (GFP) for respective heterologous expression in Escherichia coli. Fusion of plantaricin 423 and mundticin ST4SA to His-tagged GFP produced the fus ...[more]

PMID: 32765464

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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.

Dataset Information

Heterologous Expression of the Class IIa Bacteriocins, Plantaricin 423 and Mundticin ST4SA, in Escherichia coli Using Green Fluorescent Protein as a Fusion Partner.

Publications

Heterologous Expression of the Class IIa Bacteriocins, Plantaricin 423 and Mundticin ST4SA, in <i>Escherichia coli</i> Using Green Fluorescent Protein as a Fusion Partner.

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