Purification of Antilisterial Bacteriocins Jean Marc Berjeaud and Yves Cenatiempo

1. Introduction

In recent years, numerous contamination outbreaks, involving various pathogens (i.e., Listeria and Salmonella), have increased concern over food preservation. Research efforts have focused on the discovery of new molecules targeting such foodborne pathogens and therefore able to inhibit and or kill them. Lactic acid bacteria (LAB) extensively used in fermented foods for thousands of years not only improve their flavor and texture but also inhibit pathogenic and spoilage microorganisms. LAB inhibitory activity is primarily owing to pH decrease and competition for substrates. Antagonistic activity of LAB also depends on secreted antimicrobial compounds with a poor selectivity, such as metabolic compounds (i.e., hydrogen peroxide, acetoin, and others) or more specific ones like bacteriocins. The latter are proteina-ceous compounds, ribosomally synthesized and subsequently secreted by Gram-positive as well as Gram-negative bacteria. Their antimicrobial activity is generally restricted to strains phylogenetically related to the producers.

A classification of bacteriocins produced by LAB was first proposed by Klaenhammer in 1993 (1) and was modified by Nes et al. in 1996 (2); class I and class II bacteriocins are the most abundant and thoroughly studied (3,4). Bacteriocins from both classes exhibit antilisterial activity. Class I bacteriocins, namely, lantibiotics, have been widely studied, and among them, nisin is used in many countries as a preservative in food products (5). These bacteriocins are characterized by the presence, in their primary structure, of post-translationally modified amino acid residues (i.e., lanthionine and methylanthionine) that are formed (3). Class II bacteriocins, containing three subclasses, consist of small peptides that do not bear any modified amino acid residue. The most studied subclass corresponds to class IIa, also termed anti-Listeria bacteriocins (4). These peptides share strong structural homologies in their N-terminal domain (Table 1), with the presence of one disulfide bond and a net positive charge. Their C-terminal domain is more variable but appears quite hydrophobic.

From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ

Moreover, some of these bacteriocins, namely, sakacin G (6), pediocin PA-1 (7), enterocin A (8), coagulin (9), and divercin V41 (10), are characterized by the presence of a second disulfide bond in the C-terminal region (Table 1).

According to studies on pediocin PA-1 (11) and mesentericin Y105 (12), these peptides act by pore formation in the cytoplasmic membrane of target strains and consecutive dissipation of proton motive force. Recently, the presence of a mannose-spe-cific phosphotransferase, as a putative receptor, in the sensitivity of Listeria to leucocin A (13) or Enterococcus (14) and Listeria (15) to mesentericin Y105 was demonstrated. Analysis of the genetic determinants of several class Ila bacteriocins revealed that genes involved in the production and transport of the bacteriocin and immunity are organized in one or two operon-like structures. Most of them are located on a plasmid and possess at least two genes that encode proteins homologous to ABC transporters and accessory proteins, probably involved in the transport of class IIa bacteriocins (7,16,17). One noticeable exception is divercin V41, whose operon is located on a bacterial chromosome and that seems to be devoid of any accessory protein gene (10).

The number of such peptidic compounds may be limited since many of the newly detected strains express previously described bacteriocins (4). Consequently, detection and characterization of still unknown antagonistic peptides is becoming difficult. One of the more time-consuming steps in such studies consists of the purification of antagonistic compounds. In a previous work (18), we described an efficient purification method of mesentericin Y105, our class Ila bacteriocin model. We applied this rapid and efficient method, with some improvements, first to other known class IIa bacteriocins from their producer strain cultures and second to any culture supernatant showing antilisterial activity. Since all class Ila bacteriocins displayed a positive charge and a hydrophobic C-terminal part (Table 1), we thus used cation exchange and reverse phase chromatography to purify these peptides. The method developed consists of a straightforward four-step process. After removal of bacterial cells and heating of the culture supernatant, a cation exchange chromatography, eluted with increasing NaCl concentrations, was performed, followed by solid-phase extraction on a C18 reverse phase and finally reverse-phase high-performance liquid chromatography (HPLC) on a C8 column.

2. Materials

1. Culture medium (MRS broth, DIFCO), sterilized for 12 min at 110°C and stored at 4°C for up to 1 mo (see Note 1).

2. Elution solutions for cation exchange chromatography: acidified H2O, acidified 0.1 M NaCl, and acidified 0.5 M NaCl (see Note 2). The cation exchange phase chosen consisted of carboxy-methyl cellulose (see Note 3). After usage, the column was filled with 2% sodium azide and stored at room temperature.

3. For solid phase extraction, prior to sample deposit, each cartridge (C18 Sep-pak plus, Waters) was washed with 10 mL of ethanol (99%), 5 mL of acetonitrile (HPLC grade), and H2O (milliQ). Sample washing and elution were realized with 5 mL of 20 mM ammonium acetate (see Note 4) (final concentration) containing increasing quantities of acetonitrile (HPLC grade). Acetonitrile-containing buffers were stored at room temperature for up to 2 mo.

Table 1

Sequence Alignment of Class IIa Bacteriocins

Residue number*

Bacteriocina 1 10 20 30 40 Refs

Sakacin G Mesentericin Y105 Leucocin A Leucocin C Sakacin P Mundticin Sakacin A Piscicocin V1a Bacteriocin BM1 Bacteriocin B2 Bavaricin MN Enterocin P Bacteriocin 31 Bifidocin B Pediocin PA-1 Coagulin Enterocin A Divercin V41 Consensusc

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