Antibodies to a synthetic 1–9-N-terminal amino acid fragment of mature pediocin PA-1: sensitivity and specificity for pediocin PA-1 and cross-reactivity against Class IIa bacteriocins

Immunoassays with specific antibodies offer higher sensitivity than do bioassays with indicator strains in the detection and quantification of several bacteriocins. Here we present the purification of lacticin RM and the production of specific polyclonal antibodies to a synthetic peptide resembling an internal fragment of the mature bacteriocin. The specificity and sensitivity of the generated polyclonal antibodies were evaluated in various immunoassays. The detection limits of lacticin RM were found to be 1.9, 0.16, and 0.18 g ml ؊1 for Western blot, immuno-dot blot, and noncompetitive indirect enzyme-linked immunosorbent assays, respectively. Immunoassay sensitivities were 12.5-fold higher than that of the agar diffusion test (ADT). The production of lacticin RM showed temperature dependency, with 3, 4.2, 12.7, 28.9, 37.8, and 12 g ml ؊1 at 37, 30, 20, 15, 10, and 4°C, respectively. Temperature-stability analysis demonstrated that lacticin RM is sensitive to mild temperature, but the loss of activity does not seem to result from protein degradation. Tween 80 increased the concentration of lacticin RM eightfold and probably affected the results of the ADT either by enhancing the activity of lacticin RM or by increasing the sensitivity of the indicator strain. The use of antibodies for the specific detection and quantification of lacticin RM can expand our knowledge of its production and stability, with important implications for further investigation and future application.Bacteriocins are antibacterial proteins produced by bacteria that kill or inhibit the growth of closely related bacteria. Many lactic acid bacteria (LAB) produce a high diversity of bacteriocins, with a fairly broad inhibitory spectrum (19). In recent years, many bacteriocins from LAB belonging to different groups have been described, characterized, and purified. Nisin, produced by several strains of Lactococcus lactis, is the most studied bacteriocin, and it is used as a commercial food preservative (6,11,16). The role of LAB and their bacteriocins as food biopreservatives is expected to grow in the future as a result of consumer awareness of the potential risks derived from food-borne pathogens as well as from the artificial chemical preservatives currently used to control them (14).Lacticin RM is a bacteriocin produced by Lactococcus lactis subsp. lactis EZ26 (DSM ID-95-131), isolated from goat's milk. It displays a wide range of inhibition, including Listeria monocytogenes and Staphylococcus aureus (36). The operon encoding lacticin RM is located on a 2.5-kb fragment in the plasmid pHU1, and it is rather simple in comparison to other bacteriocin systems, including only four genes. lacA is the bacteriocin structural gene, while lacF, lacG, and lacI participate in immunity against lacticin RM (36). Preliminary results indicate that lacticin RM is optimally produced at low growth temperatures, with maximal inhibition zones against an indicator strain at 10 and 15°C. Although most of the characterized bacteriocins are produced at the producer...

Section: Discussionmentioning

confidence: 99%

“…with ELISA but only 5 g ml Ϫ1 with immuno-slot blot assay (27). Immunodetection of enterocin A and pediocin PA-1 was able to detect the low concentration of 0.09 g ml Ϫ1 (26).…”

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confidence: 95%

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confidence: 99%

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Immunodetection of the Bacteriocin Lacticin RM: Analysis of the Influence of Temperature and Tween 80 on Its Expression and Activity

Keren

Yarmus

Halevy

et al. 2004

“…Such applications could be greatly facilitated with the development of efficient procedures for detection, quantification, and purification of bacteriocins (34). Up to now, bioassays that assess the inhibitory effect of bacteriocins on indicator microorganisms have been most commonly used for detection and quantification of bacteriocin activity.…”

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confidence: 99%

Heterologous Coproduction of Enterocin A and Pediocin PA-1 by Lactococcus lactis : Detection by Specific Peptide-Directed Antibodies

Martínez

Kok

Sanders

et al. 2000

Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N-and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (

“…Several reports describing generation of antibodies against class IIa bacteriocins were focused on pediocin (6,7,25,26,27,28), while only one report dealt with enterocin A (27). Antibodies generated by immunization using the whole class IIa bacteriocin molecule either alone or conjugated to carriers (5, 7) have been scarcer than antibodies generated by using a chemically synthesized fragment derived from the C-or N-terminal region of the bacteriocin (25,26,27,28). Our investigations are focused on divercin V41 as a model class IIa bacteriocin.…”

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confidence: 99%

Generation and Utilization of Polyclonal Antibodies to a Synthetic C-Terminal Amino Acid Fragment of Divercin V41, a Class IIa Bacteriocin

Richard

Drider

Fliss

et al. 2004

Polyclonal antibodies have been generated by immunization of rabbits with a chemically synthesized Cterminal part of divercin V41 (DvnCt) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and reactivity of the DvnCt-KLH-generated antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) using supernatant from cultures of 13 representative lactic acid bacterium strains, and specificity was confirmed by Western blot analysis. Anti-DvnCt-KLH antibodies were able to recognize not only divercin V41 but also enterocin P and piscicocin V1b, two other members of the class IIa bacteriocins. Production and activity of DvnV41 were evaluated by ELISA and activity tests during the growth of Carnobacterium divergens V41 in MRS medium containing or not containing Tween 80. Divercin V41, enterocin P, and piscicocin V1b were therefore purified by a single-step immunoaffinity chromatography method using a Sepharose matrix CNBr-activated directed binding of anti-DvnCt-KLH polyclonal antibodies.Many lactic acid bacteria (LAB) are known to secrete small, ribosomally synthesized antimicrobial peptides referred to as bacteriocins (20-22, 29, 32). Bacteriocins are of major interest to the food industry, as they can be used against pathogen and spoilage flora such as Brochothrix spp., Clostridium spp., Bacillus spp., and Staphylococcus spp. (11,12,20), and especially against food-borne Listeria monocytogenes, which is responsible for serious listeriosis outbreaks (15, 31). Four classes of bacteriocins have been defined on the basis of common characteristics, mainly structural ones (22). Various reports indicated that all class IIa bacteriocins have high-efficiency antiListeria activity. Among these classes, the class IIa bacteriocins contain the amino acid sequence YGNGV within their Nterminal regions (13,22,34) and are heat-stable small peptides (37 to 48 amino acids). Class IIa bacteriocins are promising candidates for industrial applications due to their high biological activity and their physicochemical properties (11,14).Increasing applications of class IIa bacteriocins as food preservatives could be facilitated by development and use of polyclonal antibodies generated against these antimicrobial peptides in sensitive and specific detection methods, such as immunoblotting and enzyme-linked immunosorbent assay (ELISA) (28).Antibodies offer potential alternative methods of bacteriocin purification based on immunoaffinity strategies (37). Several reports describing generation of antibodies against class IIa bacteriocins were focused on pediocin (6,7,25,26,27,28), while only one report dealt with enterocin A (27). Antibodies generated by immunization using the whole class IIa bacteriocin molecule either alone or conjugated to carriers (5, 7) have been scarcer than antibodies generated by using a chemically synthesized fragment derived from the C-or N-terminal region of the bacteriocin (25,26,27,28). Our investigations are focused on divercin V41 as a model class IIa bacteriocin. It has been...