Influence of Physico-Chemical Factors on the Oligomerization and ...

CURRENT MICROBIOLOGY Vol. 42 (2001), pp. 89 –95 DOI: 10.1007/s002840010184

Current Microbiology An International Journal © Springer-Verlag New York Inc. 2001

Influence of Physico-Chemical Factors on the Oligomerization and Biological Activity of Bacteriocin AS-48 Hikmate Abriouel,1 Eva Valdivia,1 Antonio Gálvez,2 Mercedes Maqueda1 1

Dpto. Microbiología, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, E-18071-Granada, Spain Area de Microbiología, Facultad de Ciencias Experimentales, Universidad de Jaén, E-23071 Jaén, Spain

2

Received: 3 July 2000 / Accepted: 11 August 2000

Abstract. Bacteriocin AS-48 forms a mixture of monomers and oligomers in aqueous solutions. Such oligomers can be clearly differentiated by SDS-PAGE after formaldehyde crosslinking, and we have verified that these associates are stable to acid treatment after fixation. In addition, they show antimicrobial activity and are recognized by anti-AS-48 antibodies. AS-48 oligomers can be dissociated by the detergents SDS and Triton X-100. The degree of oligomerization of AS-48 depends on the pH of the solution and the protein concentration. At pH below 5, AS-48 is in the monomeric state at protein concentrations below 0.55.mg ml21, but it also forms dimers above this protein concentration. This bacteriocin forms oligomers at pH values above 5, in agreement with the observation that it is also more hydrophobic at neutral pH. AS-48 is stable to mild heat treatments irrespectively of pH. At 120°C it is more heat resistant under acidic conditions, but it inactivates at neutral pH. Activity of AS-48 against E. faecalis is highest at neutral pH, but it is highest at pH 4 for E. coli. The influence of pH on bacteriocin activity could be owing to changes in the conformation/oligomerization of the bacteriocin peptide as well as to changes in the surface charge of the target bacteria.

The bacteriocin AS-48 produced by Enterococcus faecalis subsp. liquefaciens S-48 is unique in its cyclic structure and its broad antimicrobial spectrum [1, 4, 6, 7, 15, 21]. AS-48 is a strongly basic molecule (pI close to 10.5), and it contains a high proportion (49%) of hydrophobic amino acids (Ala, Pro, Val, Met, Ile, Leu, and Phe) and uncharged hydrophilic residues (Ser, Gly, Thr, and Tyr). Therefore, the combination of a net positive charge with a large proportion of hydrophobic residues suggests an amphipathic structure for AS-48 [4, 5]. This bacteriocin exerts bactericidal and bacteriolytic effects against most of the Gram-positive bacteria and some Gram-negative bacteria [4, 6]. Its bactericidal effect on Escherichia coli and Salmonella is well documented [1, 7, 8], although the bacteriocin concentrations required are much higher, probably because of the protective effect of the bacterial outer membrane. Lactic acid bacteria produce strongly cationic peptides with potent antimicrobial activity like nisin [9, 10], Correspondence to: M. Maqueda; email: [email protected]

lacticin 481 [18], carnocin UI49 [22], lactocin S [17], pep-5 [20], or bacteriocin C3603 [11], among others. All of them are similar in molecular sizes (3– 6 kDa), isoelectric points (ca. 10), and biological activities [12]. Together, these inhibitory substances may represent the conservation in the course of evolution of a general mechanism of antibiosis by means of basic peptides. Bacteriocin AS-48 shows striking similarities to these cationic antimicrobial peptides, but the lack of lanthionine and its capacity to inhibit several Gram-negative bacteria represent two major differences. Because of its stability and solubility over a wide pH range and its broad antimicrobial spectrum, AS-48 is a good candidate to be used as a natural food preservative. In this respect, its activity against several food-borne bacteria such as E. coli, Salmonella, and Listeria monocytogenes is noteworthy [16]. In this context, it is important to gain knowledge into the effect of several factors (especially those that may be encountered in food systems) on the physico-chemical behavior, stability, and biological activity of this bacteriocin, which was the aim of this work.

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Fig. 1. Electrophoretic separation of formaldehyde-crosslinked AS-48 in aqueous solution. A) Coomassie blue-stained SDS-PAGE gel showing the different multimers. AS-48 has a molecular mass of 7.1 kDa (lane 2). Bands corresponding to various mono- and multimeric forms of AS-48 after formaldehyde crosslinking are shown in lane 3. Lane 1: standard markers (in kDa). B) Antimicrobial activity against E. faecalis S-47 of formaldehyde-crosslinked AS-48. C) Immunological detection of AS-48 in Western blotting by using specific anti-AS-48 antibodies. Lane 1: formaldehyde-crosslinked AS-48. Lane 2: untreated AS-48.

Preliminary data on AS-48 purification indicated that this bacteriocin showed an abnormal behavior on size-exclusion chromatography, since multiple forms seemed to be present (unpublished results), and the bacteriocin eluted as a peak of low-molecular mass only under acidic pH [4]. In this work we present data about formation of oligomers, its biological activity, and the effect of different physico-chemical factors on these features. Materials and Methods Strains and growth conditions. Enterococcus faecalis A-48-32 was used to produce AS-48 E. faecalis S-47 and Escherichia coli U-9 from our collection were used as indicator strains. The bacteria were propagated in brain heart infusion broth (BHI, Oxoid). AS-48 preparation and activity assay. Bacteriocin preparations were obtained as described elsewhere [5]. Protein concentration of stock solution (2.2 mg.ml21) was determined by the method of Bradford [3]. The inhibitory activity of AS-48 was determined by the agar-well diffusion method [4]. For the assay of antimicrobial activity at different pH values, exponential-phase cells of the bacteria were used as described by Abriouel et al. [1]. The logarithmic reduction factor (LRF) was calculated as log10 CFU/ml in controls minus log10 CFU/ml in treated samples. Effects of heat treatments. Bacteriocin solutions (50 mg.ml21) in different buffers were held at different temperatures (60°C for 30 min; 70°C for 10 min; 80°C for 5 min; 120°C for 15 min; and 140°C for 4 s). After heat treatments, samples were cooled rapidly and tested for remaining antimicrobial activity. Positive controls were held at room temperature for similar periods of time at the desired pH values. Buffer solutions without bacteriocin were used as negative controls.

Buffers. Sodium citrate (pH 3.0 and 4.0), sodium acetate (pH 5 and 5.6), and sodium phosphate (pH 6 – 8) buffers were used at 0.1 M concentration. Electrophoretic techniques. AS-48 samples were separated by SDSPAGE on 10% slab gels as described by Laemmli [13] and transferred to a nitrocellulose (NC) membrane according to Towbin et al. [23]. Immunoblotting was carried out with specific anti-AS-48 antibodies [14]. The antimicrobial activity of proteins separated by SDS-PAGE was carried out according to the method reported by Bhunia et al. [2]. Chemical cross-linking. Bacteriocin solutions in different buffers were cross-linked by incubation with 1% formaldehyde (from paraformaldehyde powder, Taap, Altermaston, England) for 1 h at room temperature before they were separated by SDS-PAGE. Alternatively, samples crosslinked by formaldehyde were incubated at room temperature for 2 h with 10% trichloroacetic acid. The precipitated proteins were collected by centrifugation (12,000 g for 10 min) in a microfuge, redissolved in sample buffer, and separated by SDS-PAGE. Peptide hydrophobocity. The hydrophobicity of AS-48 was evaluated by phase-partition between the aqueous phase and n-octanol (Sigma) according to Puyal et al. [19].

Results and Discussion The bacteriocin AS-48 shows several interesting features that make it an attractive candidate for food application, and also to study protein-protein interactions, especially when these are accompanied by changes in the biological activity of the molecule. In this study, we present data confirming earlier observations about oligomerization of

H. Abriouel et al.: Oligomerization and Biological Activity of Bacteriocin AS-48

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Fig. 2. Effect of detergents on AS-48 association. A) Aqueous solutions of AS-48 (0.54 mgzml21) were added of Triton X-100 (1%) or SDS (1%) before or after formaldehyde fixation. Lane 1: Control (untreated AS-48); 2: formaldehyde-fixed AS-48; 3: AS-48 treated with Triton X-100 alone; 4: AS-48 treated with Triton X-100 before fixation with formaldehyde; 5: AS-48 fixed before treatment with Triton X-100; 6: AS-48 treated with SDS before fixation; 7:AS-48 fixed before addition of SDS. B) Effect of SDS on AS-48 association. Aqueous solutions of AS-48 (0.51 mgzml21) were added of SDS at a final concentration of 0% (lane 2), 0.1% (lane 3), 0.2% (lane 4), 0.4% (lane 5), 0.8% (lane 6) or 1% (lane 7) before formaldehyde fixation. Lane 1: AS-48 without formaldehyde fixation (control). M: Standard markers (in kDa).

bacteriocin AS-48, and suggesting how this phenomenon affects AS-48 stability and biological activity. Oligomerization of AS-48 in aqueous solutions. The degree of oligomerization of AS-48 in aqueous solution (1.3 mg.ml21) was studied by SDS-PAGE after crosslinking with formaldehyde. The results obtained revealed that AS-48 forms a mixture of monomers and different multimers (associates of 13, 8, 5, 4, 3, and 2 molecules of AS-48), with M1 ranging from 91 to 7 kDa (Fig. 1A). Non-fixed AS-48 solutions yielded a single monomeric band when separated under the same conditions. Gels prepared as above were treated to remove SDS and then incubated with a soft agar overlay seeded with the indicator strain E. faecalis S-47. After incubation, the agar overlay showed zones of inhibited growth that corresponded to the different bacteriocin protein bands in the gel (Fig. 1B). The sizes of the inhibition zones correlated with the intensity of the protein bands. According to the amino acid composition of AS-48, the residues involved in methylene bridge formation after fixation with formaldehyde could be two residues of

arginine, one residue of asparagine, and another one of tyrosine, and these bridges remained stable after acid treatment. To verify this fact, crosslinked bacteriocin samples were treated with 10% trichloroacetic acid, and the acid-precipitated proteins were collected by centrifugation, dissolved in buffer, and separated by SDSPAGE. In these conditions, we still got the same types of oligomers as in crosslinked samples without acid treatment (data not shown). These results clearly indicate that associates crossslinking by formaldehyde were stable to acidic conditions. Therefore, the antimicrobial activity detected on gels should correspond to the oligomers and not to dissociated forms originated by acid treatment. Immunological studies show that AS-48 monomer and also oligomers and multimers were recognized by anti-AS-48 antibodies after being transferred to a NC membrane (Fig. 1C). This behavior is comprehensive because rabbits were immunized with concentrated bacteriocin solutions containing the different oligomeric forms [14], and reactive sera probably contain a mixture

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Fig. 3. Effect of pH on AS-48 association. Aliquots of AS-48 (0.54 mgzml21) buffered at pH values of 3 (lane 2), 4 (lane 3), 5 (lane 4), 6 (lane 5), 7 (lane 6), and 8 (lane 7) fixed with 1% formaldehyde and separated by SDS-PAGE. Lane 1: AS-48 without formaldehyde fixation. M: Standard markers (in kDa).

of antibodies that can recognize both the bacteriocin monomer and the oligomers.

neutral, and the degree of association increased markedly as pH rose from 6 to 8.

Effect of detergents on bacteriocin association. Bacteriocin oligomerization in aqueous solutions is a reversible process, and it probably arises from the attraction between hydrophobic groups. In order to investigate the effect of detergents on AS-48 association, aqueous bacteriocin solutions (0.54 mg.ml21) at neutral pH were treated with SDS or Triton X-100 (1% final concentration) and then fixed with formaldehyde before separation by SDS-PAGE. In samples treated with SDS, the bacteriocin dissociated completely into monomers (Fig. 2A). The minimal concentration of SDS required for complete dissociation of AS-48 oligomers (0.8%; Fig. 2B) was much higher than the critical micellar concentration of this detergent (ca. 0.27%), suggesting that the hydrophobic interactions between AS-48 molecules must be quite strong. As expected, neither of the detergents tested was able to dissociate AS-48 oligomers if they were previously fixed with formaldehyde.

Effect of the protein concentration on AS-48 association-dissociation at different pH values. Association of AS-48 was also influenced by bacteriocin concentration. In fact, formation of bacteriocin dimers could also be observed under acidic conditions (pH 3–5) at protein concentrations above 0.55 mg.ml21 (Fig. 4A). Dilution of protein samples below this concentration under acidic conditions resulted in dissociation to the monomeric form. These data are consistent with the fact that dilution can perturb subunit interactions in oligomeric proteins by changing the association-dissociation equilibrium. However, at pH 7 all of the samples showed oligomers regardless of bacteriocin concentration (Fig. 4B), although the proportion of oligomers was lower as the concentration of AS-48 decreased.

Effect of pH. Bacteriocin solutions at different pHs (3– 8) were crosslinked with formaldehyde and separated by SDS-PAGE to study the influence of pH on bacteriocin association. Samples cross-linked at acidic pH (3–5) showed a single protein band on the gels corresponding to momoners (Fig. 3). Since AS-48 is highly cationic (pI 10.5), the molecule should be highly positively charged under acidic conditions, and therefore the hydrophobic interactions should be weaker than they are at higher values of pH. There was a strong correlation between bacteriocin oligomerization and pH change from acid to

Peptide hydrophobicity. Hydrophobic interactions should play a key role in AS-48 oligomerization. Therefore, we studied the hydrophobicity of AS-48 as a function of pH by phase partition into n-octanol (Fig. 5). At pH 3 and 4, most of the bacteriocin (63%) remained in the aqueous phase. At higher pH, larger amounts of AS-48 migrated to the octanolic phase. Migration was highest at pH 7 and 8 (at which only 9% and 11% of the initial amount of protein remained in the aqueous phase, respectively). These results indicate that AS-48 is highly hydrophilic at pH 4 or below, while it becomes gradually hydrophobic from pH 5 to 8. Accordingly, bacteriocin oligomerization may provide a mechanism to increase its solubility in aqueous systems under conditions in which


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Fig. 4. Concentration-dependent associationdissociation equilibrium of AS-48 at different pHs. Different dilutions of buffered solutions of AS-48 (2.2 mg.ml21) fixed with formaldehyde before SDS-PAGE. Lanes 1–5: solutions containing different AS-48 concentrations: 2.2, 1.1, 0.73, 0.55, and 0.44 mg.ml21 respectively; (A) fixed at pH 3, (B) at pH 7. M: Standard markers (in kDa).

certain regions of the molecule become more hydrophobic. Effect of pH on the antimicrobial activity of AS-48. To test whether the changes in pH could have any influence on the antimicrobial activity of AS-48 (in addition to their influence on bacteriocin hydrophobicity and antimicrobial activity, as described above), bacterial cell suspensions at different pHs were incubated with AS-48 for 30 min, and then the number of survivors was determined. The results obtained indicated that the antimicrobial activity of AS-48 was influenced markedly by the pH of the solution. Bacteriocin activity against E. faecalis S-47 was higher in the range of pH from 5 to 7 (Table 1). The highest activity was obtained at pH 6, and the lowest at pH 4. Some decrease in antimicrobial activity was also detected at alkaline pH. The results obtained by using E. coli U-9 as indicator strain were much different. In this case, some antimicrobial activity

was detected at pH 6 (followed by that of pH 7 and 8) by using tenfold higher bacteriocin concentrations. Nevertheless, the activity of AS-48 against this bacterium increased markedly by lowering the pH from 5 to 4 (Table 1). Exposure to pH 4 alone did not have much effect on the viability of E. coli cells (LRF 5 0.41; data not shown). These results indicate that AS-48 is more active against Gram-positive bacteria when the bacteriocin forms oligomers, while it is more active on E. coli at acidic pH, when it is in the monomeric state. In addition to this, we should take into account that pH also modifies the surface charge of the cell wall/membrane of target cells, and that changes in sensitivity of Grampositive and Gram-negative bacteria over a pH range may also be owing to the great differences in their cell wall composition. This phenomenon may be of great importance in food preservation, since the antimicrobial activity of AS-48 against Gram-negative bacteria could

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Fig. 5. Phase partition of AS-48 into n-octanol in function of pH. Solutions of AS-48 in different buffers (pH 3– 8) mixed with n-octanol as described in Materials and Methods. Percentage of protein (bars) and the activity against E. faecalis (m) remaining in the aqueous phase. Table 1. Effect of pH on the antimicrobial activity of bacteriocin AS-48. Cell suspensions in different pH buffers were incubated for 30 min at 37°C with buffer alone or with bacteriocin AS-48 (final concentration of 5 mg.ml21 for E. faecalis S-47 and 70 mg.ml21 for E. coli U-9). The loss of viability is expressed as the logarithmic reduction factor (LRF) pH Indicator strain

4

5

6

7

8

E. faecalis S-47 E. coli U-9

0.54 4.16

1.73 0.09

2.03 0.42

1.58 0.27

1.28 0.17

be enhanced by lowering the food pH without compromising its antimicrobial activity on Gram-positives to a large extent. Effect of pH on heat stability of AS-48. The physicochemical characteristics of AS-48 as well as its broad antimicrobial spectrum make it a good candidate for use in food preservation. In this context, it should be of interest to investigate the effects of changes in pH (and therefore in the degree of oligomerization) on bacteriocin stability to heat, and especially to heat treatments applied in the food industry. In order to determine the stability of AS-48, bacteriocin solutions buffered at pH 3– 8 were treated by heat as described above (Fig. 6). We did not detect any loss of activity in this range of pH at temperatures of 80°C or lower. Heating at 120°C for 15 min caused a marked loss of bacteriocin activity at pH 6 as well as a complete loss of activity at pH 7 and 8. Since these pH values induce the highest degree of oligomerization, these results suggest that oligomers are much less stable than monomers. In this treatment, some loss

Fig. 6. Heat stability of AS-48 as a function of pH. Bacteriocin solutions at different pHs were heated for different times at various temperatures. The remaining antimicrobial activity is shown.

of activity was also observed at pH 4 (but not at pH 3 or 5). Short-time ultra-high temperature treatments caused an almost complete inactivation of the bacteriocin at pH 4 and pH 8. For other pH values, this treatment caused only partial inactivation, and the remaining activity of samples ranged from 60 to 80% of controls (Fig. 6). Altogether, these results predict that incorporation of bacteriocin into foods should be compatible with mild heat treatments irrespectively of the food pH. The broad antimicrobial spectrum of this bacteriocin and its stability at moderate temperatures over a broad range of pH make it a suitable candidate to be used as a biopreservative in pasteurized as well as in minimally processed foods and foodstuffs. Application of more severe heat treatment, however, should have to be studied in more detail, because the pH of the solution may have a marked effect on bacteriocin stability. ACKNOWLEDGMENTS This research was supported by a grant from the Comisión Interministerial de Ciencia y Tecnología (BIO95-0466) of the Spanish Ministry of Education and Science.

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