FEMS Microbiology Letters 192 (2000) 79^83 www.fems-microbiology.org E¡ect of Enterocin CRL35 on Listeria monocytogenes cell membrane Carlos J. Minahk a;*, Mar|¨a E. Far|¨as b, Fernando Sesma b, Roberto D. Morero a a Departamento de Bioqu|¨mica de la Nutricio¨n, Instituto Superior de Investigaciones Biolo¨gicas, Instituto de Qu|¨mica Biolo¨gica - `Dr. B. Bloj', Facultad de Bioqu|¨mica, Qu|¨mica y Farmacia (CONICET/UNT), Chacabuco 461, 4000 San Miguel de Tucuma¨n, Argentina b Centro de Referencias para lactobacilos (CERELA, CONICET), Chacabuco 145, 4000 San Miguel de Tucuma¨n, Argentina Downloaded from https://academic.oup.com/femsle/article/192/1/79/634204 by guest on 30 September 2021 Received 31 July 2000; received in revised form 2 September 2000; accepted 2 September 2000 Abstract The antimicrobial peptide Enterocin CRL35, a class II bacteriocin, produces at high concentrations (8 Wgml31) localized holes in the wall and cellular membrane of Listeria monocytogenes, reflected in the efflux of macromolecules such as proteins and other ultraviolet-absorbing materials. At lower concentrations (0.5 Wgml31), neither ultra structural changes nor macromolecules efflux were observed, however potassium and phosphate ions were released, dissipating the proton motive force. As a result the bacteria were killed. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Enterocin; Antimicrobial peptide; Bacteriocin 1. Introduction tained and showed that it can be categorized in the group II [4]. Bacteriocins are antimicrobial peptides lethal to closely To use bacteriocins in the most e¡ective way, it is im- related bacteriocin-producing species. According to Moll portant to determine their mode of action against food- et al. [1], bacteriocins can be classi¢ed in three groups: (I) spoilage and food-borne bacteria. The purpose of this lantibiotics, small peptides ( 6 5 kDa) that undergo post- study was to obtain a general view of the mechanism of translational modi¢cations, (II) small heat-stable peptides action of Enterocin CRL 35. ( 6 10 kDa) and (III) large heat labile bacteriocins ( s 30 The investigations were performed on whole L. mono- kDa). The group II is subdivided in four subgroups: (IIa) cytogenes cells, examining the e¡ect on the microscopic Listeria active peptides, which have an N-terminal consen- structure and the e¥ux of di¡erent molecules. sus sequence, YGNGVXC. (IIb) Bacteriocins that require two di¡erent peptides for activity. (IIc) Sec dependent bac- teriocins and (IId) class II bacteriocins that do not belong 2. Materials and methods to the other groups. Class IIa bacteriocins, which seem to have a common mechanism of action, would act by dis- 2.1. Bacterial strains and media sipating the membrane proton motive gradient [2]. Enterococcus faecium CRL35, a strain isolated from re- E. faecium CRL35 belongs to the CERELA Stock col- gional Argentinean cheese (Ta¢ cheese) produces a bacter- lection and the sensitive strain L. monocytogenes LS01 was iocin called Enterocin CRL35, which was recently de- provided by Ca¨tedra de Bacteriolog|¨a, Facultad de Bioqu|¨- scribed [3]. Enterocin CRL35 possess activity against the mica, Qu|¨mica y Farmacia (UNT). Both strains were food borne pathogen Listeria monocytogenes, and because grown at 30³C in Laptg broth, without Tween 80 [5]. of its e¤ciency this peptide has potential as antimicrobial agent in food. The partial N-terminal sequence was ob- 2.2. Puri¢cation of Enterocin CRL35 E. faecium CRL35 was grown in 1 l of media for 18 h. * Corresponding author. Tel.: +54 (81) 248 921; The cells were removed by centrifugation and the peptide Fax: +54 (81) 248 025; E-mail: [email protected] present in the supernatant was precipitated by adding (NH ) SO to a ¢nal concentration of 60% (w/v). The Abbreviations: cfu, colony forming units; DiSC3(5), 3,3P-dipropylthia- 4 2 4 dicarbocyanine iodide pellet, obtained by centrifugation at 12 000Ug, was dis- 0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0378-1097(00)00412-2 FEMSLE 9633 12-10-00 80 C.J. Minahk et al. / FEMS Microbiology Letters 192 (2000) 79^83 solved in 100 ml of distilled water and passed through a 2.6. Electron microscopy C18 cartridge (Impaq RG1080 C18) which was washed with several volumes of distilled water and then eluted with Mid-exponential phase cultures of L. monocytogenes increasing concentrations of acetonitrile. The fractions were inoculated with di¡erent amounts of Enterocin showing antibiotic activity were pooled, concentrated CRL35 to 30 Wgml31. Non-inoculated samples were and applied to a CM cellulose cation exchanger column used as a control. After 30 min of incubation at 30³C, equilibrated with 10 mM acetate bu¡er pH 5.2. The col- cells were pelleted, and ¢xed for 3 h with 4% glutaralde- umn was washed with the same bu¡er until the OD at 280 hyde in 0.1 M phosphate bu¡er, pH 7.4, 1 mM CaCl2. The nm decreased to zero. The bacteriocin was eluted with 300 ¢xed material washed with the same bu¡er was included in mM of NaCl in the same bu¡er. The active fractions were 2% agar and subjected to an overnight post ¢xation with concentrated and loaded on a HPLC system utilizing a 1% osmium tetroxide in the same phosphate bu¡er, and 300U4.6 mm Bonda Pack C18 reversed-phase column then to a 40-min ¢xation with 2% uranyl acetate. Dehy- (Waters). The column was equilibrated with 0.1% tri- dration was carried out in a graded ethanol series, ex- Downloaded from https://academic.oup.com/femsle/article/192/1/79/634204 by guest on 30 September 2021 £uoroacetic acid (TFA) in water and eluted with a linear changed through acetone and embedded in Spurr resin gradient of 0^100% acetonitrile containing 0.1% TFA, at a (Pelco Inc.). Blocks were sectioned on a Sorvall Porter £ow rate of 1 ml min31. This procedure yields approxi- Blum MT1 ultramicrotome. Silver gray sections were mately 0.2 mg of Enterocin CRL35 per liter of culture, stained with uranyl-acetate and lead citrate and examined which appeared homogeneous on analytical RP-HPLC with a Zeiss EM 109 transmission electron microscope. and SDS-PAGE [6]. 2.7. Other determinations 2.3. E¡ects of Enterocin CRL35 on sensitive cells UV absorbing materials were determined in a Gilford Sensitive cells were grown 4 h, harvested, washed twice spectrophotometer measuring OD at 260 and 280 nm. Po- and suspended to approximately 107 cells per ml in 10 mM tassium was measured by £ame photometry [8]. The inor- HEPES^Na bu¡er pH 7.2. The bacteriocin was added at ganic phosphorus was determined by the method of Ames di¡erent concentrations and samples were taken at appro- [9] and proteins with the Bio-Rad protein assay. priate times to determine the colony forming units (cfu). 2.4. E¥ux of di¡erent cellular materials 3. Results L. monocytogenes LS01 harvested by centrifugation 3.1. E¡ects of bacteriocin CRL35 on the viability of were washed three times and suspended in 5 mM sensitive cells HEPES^Na bu¡er pH 7.2 at a concentration of 108 cells ml31. After incubation with a determinate concentration Experiments in which L. monocytogenes were incubated of Enterocin CRL35, the samples were subjected to ¢ltra- tion through a Millipore ¢lter (Millipore 0.22 Wm pore size). The extracellular K, phosphate and UV absorbing materials in each ¢ltrated were measured. Two controls were carried out; one of them consisted of cells incubated in the absence of Enterocin CRL35 and the other a com- plete cellular lysis by sonication after incubation. 2.5. Measurement of the transmembrane electrical potential The transmembrane electrical potential, vi, was deter- mined as described by Bennik et al. [7]. Brie£y: cells were grown until mid-exponential phase, harvested by centrifu- gation, washed twice and resuspended in 50 mM HEPES^ K bu¡er pH 7.4 (0.05 DO, 1.5U108 cells ml31) containing 10 mM glucose and 0.5 WM of 3,3P-dipropylthiadicarbo- cyanine iodide (DiSC3(5)), a potential sensitive £uorescent probe (Molecular Probes). Fluorescence was measured with a SLM 4048c spectro£uorometer at 30³C, excitation Fig. 1. E¡ect of Enterocin CRL35 on the viability of L. monocytogenes. Viability of a 4-h culture of L. monocytogenes after exposure to 0.16 Wg and emission wavelength were 622 and 674 nm respec- ml31 (squares) or 0.3 Wgml31 (circles). Open symbols represent cells tively. A completely dissipated vi was achieved with suspended in 10 mM HEPES bu¡er pH 7.2. Closed symbols represent 1 WM of valinomycin. vpH was dissipated with nigericin. cells suspended in the same bu¡er supplemented with 10 mM glucose. FEMSLE 9633 12-10-00 C.J. Minahk et al. / FEMS Microbiology Letters 192 (2000) 79^83 81 cells suspended in an energizing medium containing glu- cose were more sensitive than cells in a non-energized one. In this case, a few percent of cells remain alive even after a long time of exposure. 3.2. E¡ects of Enterocin CRL35 on the release of cellular material The ultraviolet absorbance of the supernatant of sensi- tive cell suspensions is important as lysis indicator, and should re£ect the e¥ux of macromolecules as proteins and other substances [10]. L. monocytogenes treated with pure Enterocin CRL35 resulted in the leakage of ultravio- Downloaded from https://academic.oup.com/femsle/article/192/1/79/634204 by guest on 30 September 2021 let-absorbing materials, compared with controls (Fig.