MODE OF ACTION OF SALIVARICIN CRL 1328, A TWO PEPTIDE BACTERIOCIN PRODUCED BY LACTOBACILLUS SALIVARIUS CRL 1328 ISOLATED FROM HUMAN VAGINA

VERA PINGITORE E.; HEBERT E.M.; NADER-MACIAS M.E.; SESMA F.

Saint-Malo - Francia

Simposio; SECOND INTERNATIONAL SYMPOSIUM ON ANTIMICROBIAL PEPTIDES: FOOD, VETERINARY, MEDICAL AND NOVEL APPLICATIONS; 2009

French Research Institute INRA (Research Unit Food Safety and Microbiology, Nantes (UMR INRA 1014 SECALIM), en colaboración con French Food Safety Agency (AFSSA)

Salivaricin CRL 1328 is a heat-stable bacteriocin produced by Lactobacillus salivarius CRL 1328, a strain isolated from healthy human vagina, with potential applications for preventing urogenital infections [1]. The locus responsible for salivaricin CRL 1328 production was identified, sequenced and analyzed. The salivaricin CRL1328 biosynthetic cluster was found to be almost identical to the salivaricin ABP118 cluster, which codes for a two peptide bacteriocin [2]. The objective of this study was to determine the mechanism of action of salivaricin CRL 1328 against the uropathogenic strain E. faecalis MP97 as sensitive microorganism.The putative mature two peptides of salivaricin CRL 1328, Salα (krgpncvgnflgglfagaaagvplgpagivgganlgmvggaltc) and Salβ (kngyggsgnrwvhcgagivggaligaiggpwsavaggisggftscr), were chemically synthesized. Antimicrobial activity assays by using the synthetic Salα and Salβ peptides demonstrated that the two peptides exhibited a full activity synergistically at an equimolar ratio, whereas each peptide individually did not show antimicrobial activity. The minimal inhibitory concentration value for the combination of Salα and Salβ at a molar ratio of 1:1 was 1 µM.Transmission electron micrographies of E. faecalis MP97 exposed to the synthetic bacteriocin showed the presence of an electron-transparent layer between the cell membrane and the cell wall layer, which indicates the retraction of the cytoplasm. In addition, ultrastructural modifications of the cell wall were observed.Spectroscopic fluorescence assays were carried out by using the synthetic peptides in order to study the effect of salivaricin on the proton motive force. This bacteriocin showed to dissipate the trans-membrane electrical potential and the trans-membrane proton gradient, both components of proton motive force, probably originating pores in the membrane which results in the leakage of cellular materials.The results obtained support the scientific evidence for the application of this bacteriocin as a potential bioactive product to be included in a pharmaceutical product for the prevention of urogenital tract infections.References.[1] Ocaña, V.S., Pesce de Ruiz Holgado, A., Nader-Macías, M.E. (1999) Characterization of a bacteriocin-like substance produced by a vaginal Lactobacillus salivarius strain. Appl Environ Microbiol 65: 5631-5635.[2] Flynn, S., van Sinderen, D., Thornton, G.M., Holo, H., Nes, I.F., Collins, J.K. (2002) Characterization of the genetic locus responsible for the production of ABP-118, a novel bacteriocin produced by the probiotic bacterium Lactobacillus salivarius subsp. salivarius UCC118. Microbiology 148: 973-984.