Abstract
BackgroundLantibiotics are small lanthionine-containing bacteriocins produced by lactic acid bacteria. Salivaricin 9 is a newly discovered lantibiotic produced by Streptococcus salivarius. In this study we present the mechanism of action of salivaricin 9 and some of its properties. Also we developed new methods to produce and purify the lantibiotic from strain NU10.Methodology / Principal FindingsSalivaricin 9 was found to be auto-regulated when an induction assay was applied and this finding was used to develop a successful salivaricin 9 production system in liquid medium. A combination of XAD-16 and cation exchange chromatography was used to purify the secondary metabolite which was shown to have a molecular weight of approximately 3000 Da by SDS-PAGE. MALDI-TOF MS analysis indicated the presence of salivaricin 9, a 2560 Da lantibiotic. Salivaricin 9 is a bactericidal molecule targeting the cytoplasmic membrane of sensitive cells. The membrane permeabilization assay showed that salivaricin 9 penetrated the cytoplasmic membrane and induced pore formation which resulted in cell death. The morphological changes of test bacterial strains incubated with salivaricin 9 were visualized using Scanning Electron Microscopy which confirmed a pore forming mechanism of inhibition. Salivaricin 9 retained biological stability when exposed to high temperature (90-100°C) and stayed bioactive at pH ranging 2 to 10. When treated with proteinase K or peptidase, salivaricin 9 lost all antimicrobial activity, while it remained active when treated with lyticase, catalase and certain detergents.ConclusionThe mechanism of antimicrobial action of a newly discovered lantibiotic salivaricin 9 was elucidated in this study. Salivaricin 9 penetrated the cytoplasmic membrane of its targeted cells and induced pore formation. This project has given new insights on lantibiotic peptides produced by S. salivarius isolated from the oral cavities of Malaysian subjects.
Highlights
Different genera of lactic acid bacteria (LAB) can produce different kinds of antimicrobial peptides and bacteriocins such as plantaricin produced by Lactobacillus plantarum [1], enterococcin produced by Enterococcus faecium [2], leucocin produced by Leuconostoc carnosum [3], pediocin produced by Pediococcus acidilactici [4] and others
Distribution of salA and sivA structural genes in strains NU10 and YU10. Both strains NU10 and YU10 were shown to harbour the structural genes sivA and salA encoding the production of salivaricin 9 and A respectively. sivA from strain NU10 was sequenced and translated to protein using in silico analysis [28] (Figure 2)
Salivaricin 9 produced by strain S. salivarius NU10 isolated from a Malaysian subject was produced in liquid medium using an induction method
Summary
Different genera of lactic acid bacteria (LAB) can produce different kinds of antimicrobial peptides and bacteriocins such as plantaricin produced by Lactobacillus plantarum [1], enterococcin produced by Enterococcus faecium [2], leucocin produced by Leuconostoc carnosum [3], pediocin produced by Pediococcus acidilactici [4] and others. In recent years methods to enhance and optimize bacteriocin production have been developed [20,21,22] due to the potential importance of bacteriocin-producing strains in replacement therapy [23]. Lantibiotics are small lanthionine-containing bacteriocins produced by lactic acid bacteria. We developed new methods to produce and purify the lantibiotic from strain NU10. The membrane permeabilization assay showed that salivaricin 9 penetrated the cytoplasmic membrane and induced pore formation which resulted in cell death. Conclusion: The mechanism of antimicrobial action of a newly discovered lantibiotic salivaricin 9 was elucidated in this study. Salivaricin 9 penetrated the cytoplasmic membrane of its targeted cells and induced pore formation. This project has given new insights on lantibiotic peptides produced by S. salivarius isolated from the oral cavities of Malaysian subjects
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