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Cytotoxic effects of pyocin S2 produced by Pseudomonas aeruginosa on the growth of three human cell lines

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Abstract:

Pyocin typing of 82 Pseudomonas aeruginosa strains, collected from different Iranian clinical sources, revealed that one isolate, P. aeruginosa 42A, produced pyocin S2, a protease-sensitive bacteriocin. Pyocin S2 production was induced by mitomycin C (2 µg/mL) in the pyocin S2 producer P. aeruginosa 42A. Pyocin S2 was purified using ion exchange chromatography with CM-Sepharose CL-6B and sodium phosphate buffer (pH 8) from an 80% ammonium sulfate precipitate of whole-cell lysates. Pyocin activity of the fractions was detected using the Govan spot testing method. The purity of the active fraction was confirmed by SDS–PAGE, where a single band with a molecular mass of 74 kDa was detected. Cytotoxic effects of purified pyocin S2 and partially purified pyocin from P. aeruginosa 42A on the human tumor cell lines HepG2 and Im9 and the normal human cell line HFFF (Human Foetal Foreskin Fibroblast) were studied by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results demonstrated that partially purified pyocin and pyocin S2 exhibited substantial inhibitory effects on the growth of the tumor cell lines HepG2 and Im9, while no inhibitory effects were observed on the normal cell line HFFF. Pure lipopolysaccharide was used as a control and was found to have no inhibitory effect on any of the cell lines tested.Key words: Pseudomonas aeruginosa, pyocin, cytotoxicity, MTT assay.

Le typage de la pyocine de 82 souches de Pseudomonas aeruginosa, recueillies de diverses sources cliniques iraniennes, a révélé qu'un isolat, P. aeruginosa 42A, produisait la pyocine S2, une bactériocine sensible aux protéases. La production de la pyocine S2 fut induite par la mitomycine C (2 µg/mL) chez la souche P. aeruginosa 42A, un producteur de pyocine S2. La pyocine S2 a été purifiée par chromatographie à échange d'ion avec du CM-sépharose CL-6B et du tampon phosphate de sodium (pH 8) à partir d'une précipitation au sulfate d'ammonium à 80 % d'un lysat cellulaire total. L'activité pyocine des fractions a été détectée en utilisant la méthode d'essai à la touche de Govan. La pureté de la fraction active a été confirmée par la détection d'une seule bande en SDS–PAGE d'un poids moléculaire de 74 kDA. Les effets cytotoxiques de la pyocine S2 purifiée et de la pyocine partiellement purifiée de P. aeruginosa 42A sur les lignées cellulaires humaines tumorales HepG2 et Im9 de même que sur la lignée cellulaire humaine normale HFFF (« Human Foetal Foreskin Fibroblast ») ont été étudiées à l'aide du test MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Les résultats démontrèrent que la pyocine partiellement purifiée et la pyocine S2 affectaient négativement la croissance des lignées tumorales HepG2 et Im9, alors qu'aucun effet n'a été noté sur la lignée cellulaire normale HFFF. Le LPS purifié a été utilisé comme témoin et n'a pas entraîné d'effet inhibiteur sur aucune des lignées cellulaires analysées.Mots clés : Pseudomonas aeruginosa, pyocine, cytotoxicité, test MTT.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: May 1, 2004

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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