Djamel Drider

The Continuing Story of Class IIa Bacteriocins

SUMMARY Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.

The Continuing Story of Endoribonuclease III

Endoribonuclease III (RNase III) plays an important role in the processing of rRNA and mRNAs. It is timely to summarize the most relevant insights obtained during the last years stemming from RNase III. With this aim, the present mini-review provides a wealth of new information focusing on the distribution and architecture of RNase III, substrate recognition, cleavage mechanisms and regulation of gene expression.

Prokaryotic Antimicrobial Peptides

Prokaryotic antimicrobial peptides , Prokaryotic antimicrobial peptides , کتابخانه مرکزی دانشگاه علوم پزشکی تهران

Long-Term Survival of Campylobacter jejuni at Low Temperatures Is Dependent on Polynucleotide Phosphorylase Activity

ABSTRACT Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Infection generally occurs after ingestion of contaminated poultry products, usually conserved at low temperatures. The mechanisms promoting survival of C. jejuni in the cold remain poorly understood despite several investigations. The present study provides insight into the survival mechanism by establishing the involvement of polynucleotide phosphorylase (PNPase), a 3′-5′ exoribonuclease with multiple biological functions in cold survival. The role of PNPase was demonstrated genetically using strains with altered pnp genes (which encode PNPase) created in C. jejuni F38011 and C. jejuni 81-76 backgrounds. Survival assays carried out at low temperatures (4 and 10°C) revealed a difference of 3 log CFU/ml between the wild-type and the pnp deletion (Δpnp) strains. This did not result from a general requirement for PNPase because survival rates of the strains were similar at higher growth temperatures (37 or 42°C). trans-Complementation with plasmid pNH04 carrying the pnp gene under the control of its natural promoter restored the cold survival phenotype to the pnp deletion strains (at 4 and 10°C) but not to the same level as the wild type. In this study we demonstrate the role of PNPase in low-temperature survival of C. jejuni and therefore attribute a novel biological function to PNPase directly related to human health.

Antibacterial activity of class I and IIa bacteriocins combined with polymyxin E against resistant variants of Listeria monocytogenes and Escherichia coli.

Development of resistance could render antimicrobial peptides ineffective as bio-preservatives in food. Variants resistant to nisin A or pediocin PA-1 were developed from Listeria monocytogenes and a variant resistant to polymyxin E was developed from Escherichia coli RR1. Inhibition of these organisms by these agents alone or in combination was assessed using critical dilution microassay and optical density measurement. The combination of pediocin or nisin with polymyxin E was synergistic against all five strains. The polymyxin/nisin combination at 9.3/32, 4.7/62.5 and 0.6/15.6 μg/ml inhibited growth of nisin-resistant L. monocytogenes, pediocin-resistant L. monocytogenes and polymyxin-resistant E. coli, respectively, by factors of 94%, 97% and 74% compared to controls. The pediocin/nisin combination was effective against L. monocytogenes and its variants, but not E. coli or its variant. Polymyxin (0.21 μg/ml) and polymyxin/nisin (0.3/7.8 μg/ml) reduced E. coli growth measured in the log phase by, respectively, 31.25% and 93.54%. L. monocytogenes growth in the logarithmic and stationary phases was reduced, respectively, by 90.46% and 77.52% by polymyxin/pediocin at 4.7/25 μg/ml. Our results suggest that the effective concentration of bacteriocin for control of resistant L. monocytogenes and E. coli variants could be lowered considerably by combination with polymyxin E. This suggests searching for polymyxin-like compounds to increase the efficiency of bacteriocins and slow the emergence of resistant mutants. This could be an important step towards the expanded use of bacteriocins in the medical arena.

In Vivo Activities of Recombinant Divercin V41 and Its Structural Variants against Listeria monocytogenes

ABSTRACT Recombinant divercin RV41 (DvnRV41) and its structural variants were used in this study to assess their antilisterial activities in vivo in mice challenged intravenously with Listeria monocytogenes EGDe. Treatment with DvnRV41 before and after infection permitted a conclusion as to the capacities of this peptide to retain activity and reduce growth of L. monocytogenes EGDe. Moreover, the use of structural variants for the first time in vivo and the reductions of their activities confirmed the importance of certain amino acids in antilisterial activity.

Insights into Structure-Activity Relationships in the C-Terminal Region of Divercin V41, a Class IIa Bacteriocin with High-Level Antilisterial Activity

ABSTRACT Divercin V41 (DvnV41) is a class IIa bacteriocin with potent antilisterial activity isolated from Carnobacterium divergens V41. Previously, we expressed from a synthetic gene, in Escherichia coli Origami, a recombinant DvnV41 designated DvnRV41, which possesses four additional amino acids (AMDP) in the N-terminal region that result from enzymatic cleavage and retains the initial DvnV41 activity. To unravel the relationship between the structure of DvnRV41 and its particularly elevated activity, we produced by site-directed mutagenesis eight variants in which a single amino acid replacement was specifically introduced into the sequence. The point mutations were designed to change either conserved residues in class IIa bacteriocins or residues specific to DvnV41 located mainly in the C-terminal region. The fusion proteins were purified from the cytosoluble fractions by immobilized affinity chromatography. DvnRV41 and its variants were released from the fusion proteins by enzymatic cleavage, using enterokinase. The purity of DvnRV41 and of the variants was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance liquid chromatography, and mass spectrometry. The antibacterial activity of DvnRV41 and its variants was assessed using different indicator strains, including Listeria monocytogenes EGDe and Enterococcus faecalis JH2-2. The activity of all of the variants appeared to be less than the activity of DvnRV41. The decrease in activity did not appear to be related to a global conformational change, as determined by circular dichroism. Overall, the variants of DvnRV41 produced in the present study provide interesting insights into structure-activity relationships of class IIa bacteriocins.

Class IIa Bacteriocins: Current Knowledge and Perspectives

Lactic acid bacteria (LAB) are known to produce antibacterial peptides and small proteins called bacteriocins, which enable them to compete against other bacteria in the environment. Bacteriocins fall structurally and chemically into three different classes, I, II, and III. Bacteriocins are ribosomally synthesized peptides with antagonism against closely related bacteria. This late observation has evolved because bacteriocins active against Gram-negative bacteria have recently been reported. Members of class IIa bacteriocins, referred to as pediocin-like bacteriocins, are among the most studied bacteriocins. This chapter is aimed at providing an updated review on the biology of class IIa bacteriocins.

RNA processing is involved in the post-transcriptional control of the citQRP operon from Lactococcus lactis biovar diacetylactis

Abstract The importance of Lactococcus lactis biovar diacetylactis (L. diacetylactis) in the dairy industry is due to its ability to produce aroma compounds, such as acetoin and diacetyl, from citrate. The first step in citrate utilization is its uptake by the cells. In L. diacetylactis, the citrate transport system is encoded by the citQRP operon. We have previously proposed that expression of citQRP operon is regulated at the post-transcriptional level. In this paper, we show that the cit mRNA is processed at a complex secondary structure in L. diacetylactis and Escherichia coli. This secondary structure includes the 5′-terminal two-thirds of citQ and the overlap between citQ and citR. Primer-extension analysis revealed that the major cleavage sites are located upstream of citR and within citQ. In an attempt to identify the enzyme(s) responsible for this cleavage, we have analyzed this processing in E.␣coli mutants deficient in endoribonucleases. A comparative analysis of cit mRNA degradation was performed in RNase E and RNase III mutants and in wild-type strains using Northern blot hybridization. This analysis revealed that the cit transcript is degraded into several breakdown products, which are significantly stabilized in the mutant lacking RNase III. Our results indicate that the complex secondary structure has a critical role in the control of the expression of cit mRNA. A model for processing is discussed.

Generation and utilization of polyclonal antibodies to a synthetic C-terminal amino acid fragment of divercin V41, a class IIa bacteriocin.

ABSTRACT Polyclonal antibodies have been generated by immunization of rabbits with a chemically synthesized C-terminal 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.