Jean-Christophe Piard

Evidence for two bacteriocins produced by Carnobacterium piscicola and Carnobacterium divergens isolated from fish and active against Listeria monocytogenes

Lactic acid bacteria (LAB) isolated from fish products (fresh fish, smoked and marinated fish, fish intestinal tract) were screened for bacteriocin production and immunity in conditions eliminating the effects of organic acids and hydrogen peroxide. Twenty-two isolates which were found to produce bacteriocin-like compounds were identified as Carnobacteria, Lactococci and Enterococci on the basis of morphological examination, gas production from glucose, growth temperatures, configuration of lactic acid, carbohydrates fermentation and deamination of arginine. Two Carnobacteria named V1 and V41 were selected for further studies and identified by DNA-DNA hybridization as Carnobacterium piscicola and Carnobacterium divergens , respectively. Their respective bacteriocins named piscicocin V1 and divercin V41 were heat-resistant and sensitive to various proteolytic enzymes. These bacteriocins were active against Listeria monocytogenes and exhibited a different spectrum of activity against LAB. Both bacteriocins had a bactericidal and non-bacteriolytic mode of action. Maximum production of piscicocin V1 and divercin V41 in Man Rogosa Sharpe (MRS) medium broth occurred at the beginning of the stationary phase and was higher at 20°C than at 30°C. When the cultures were maintained at pH 6.5, bacteriocin production was significantly increased.

Design of a Protein-Targeting System for Lactic Acid Bacteria

We designed an expression and export system that enabled the targeting of a reporter protein (the staphylococcal nuclease Nuc) to specific locations in Lactococcus lactis cells, i.e., cytoplasm, cell wall, or medium. Optimization of protein secretion and of protein cell wall anchoring was performed with L. lactis cells by modifying the signals located at the N and C termini, respectively, of the reporter protein. Efficient translocation of precursor (approximately 95%) is obtained using the signal peptide from the lactococcal Usp45 protein and provided that the mature protein is fused to overall anionic amino acids at its N terminus; those residues prevented interactions of Nuc with the cell envelope. Nuc could be covalently anchored to the peptidoglycan by using the cell wall anchor motif of the Streptococcus pyogenes M6 protein. However, the anchoring step proved to not be totally efficient in L. lactis, as considerable amounts of protein remained membrane associated. Our results may suggest that the defect is due to limiting sortase in the cell. The optimized expression and export vectors also allowed secretion and cell wall anchoring of Nuc in food-fermenting and commensal strains of Lactobacillus. In all strains tested, both secreted and cell wall-anchored Nuc was enzymatically active, suggesting proper enzyme folding in the different locations. These results provide the first report of a targeting system in lactic acid bacteria in which the final location of a protein is controlled and biological activity is maintained.

Metabolic engineering of lactic acid bacteria for the production of nutraceuticals

Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal sugar metabolism to nutritional end-products other than lactic acid such as L-alanine, several low-calorie sugars and oligosaccharides or to enhancement of sugar metabolism for complete removal of (undesirable) sugars from food materials. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to the production of the B-vitamins folate and riboflavin. An overview of these metabolic engineering activities can be found on the website of the Nutra Cells 5th Framework EU-project (www.nutracells.com). Finally, the impact of the developments in the area of genomics and corresponding high-throughput technologies on nutraceutical production will be discussed.

Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis.

ABSTRACT Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.

Biofilm-associated persistence of food-borne pathogens

Microbial life abounds on surfaces in both natural and industrial environments, one of which is the food industry. A solid substrate, water and some nutrients are sufficient to allow the construction of a microbial fortress, a so-called biofilm. Survival strategies developed by these surface-associated ecosystems are beginning to be deciphered in the context of rudimentary laboratory biofilms. Gelatinous organic matrices consisting of complex mixtures of self-produced biopolymers ensure the cohesion of these biological structures and contribute to their resistance and persistence. Moreover, far from being just simple three-dimensional assemblies of identical cells, biofilms are composed of heterogeneous sub-populations with distinctive behaviours that contribute to their global ecological success. In the clinical field, biofilm-associated infections (BAI) are known to trigger chronic infections that require dedicated therapies. A similar belief emerging in the food industry, where biofilm tolerance to environmental stresses, including cleaning and disinfection/sanitation, can result in the persistence of bacterial pathogens and the recurrent cross-contamination of food products. The present review focuses on the principal mechanisms involved in the formation of biofilms of food-borne pathogens, where biofilm behaviour is driven by its three-dimensional heterogeneity and by species interactions within these biostructures, and we look at some emergent control strategies.

Characterization of the melA Locus for α-Galactosidase in Lactobacillus plantarum

ABSTRACT Alpha-galactosides are abundant sugars in legumes such as soy. Because of the lack of α-galactosidase (α-Gal) in the digestive tract, humans are unable to digest these sugars, which consequently induce flatulence. To develop the consumption of the otherwise highly nutritional soy products, the use of exogenous α-Gal is promising. In this framework, we characterized the melA gene for α-Gal in Lactobacillus plantarum. The melA gene encodes a cytoplasmic 84-kDa protein whose enzymatically active form occurs as oligomers. The melA gene was cloned and expressed in Escherichia coli, yielding an active α-Gal. We show that melA is transcribed from its own promoter, yielding a monocistronic mRNA, and that it is regulated at the transcriptional level, i.e., it is induced by melibiose but is not totally repressed by glucose. Posttranscriptional regulation by the carbon source could also occur. Upstream of melA, a putative galactoside transporter, designated RafP, was identified that shows high homology to LacS, the unique transporter for both α- and β-galactosides in Streptococcus thermophilus. rafP is also expressed as a monocistronic mRNA. Downstream of melA, the lacL and lacM genes were identified that encode a heterodimeric β-galactosidase. A putative galM gene identified in the same cluster suggests the presence of a galactose operon. These results indicate that the genes involved in galactoside catabolism are clustered in L. plantarum ATCC 8014. This first genetic characterization of melA and of its putative associated transporter, rafP, in a lactobacillus opens doors to various applications both in the manufacture of soy-derived products and in probiotic and nutraceutical issues.

Only one of four oligopeptide transport systems mediates nitrogen nutrition in Staphylococcus aureus

Oligopeptides internalized by oligopeptide permease (Opp) transporters play key roles in bacterial nutrition, signaling, and virulence. To date, two opp operons, opp-1 and opp-2, have been identified in Staphylococcus aureus. Systematic in silico analysis of 11 different S. aureus genomes revealed the existence of two new opp operons, opp-3 and opp-4, plus an opp-5A gene encoding a putative peptide-binding protein. With the exception of opp-4, the opp operons were present in all S. aureus strains. Within a single strain, the different opp operons displayed little sequence similarity and distinct genetic organization. Transcriptional studies showed that opp-1, opp-2, opp-3, and opp-4 operons were polycistronic and that opp-5A is monocistronic. We designed a minimal chemically defined medium for S. aureus RN6390 and showed that all opp genes were expressed but at different levels. Where tested, OppA protein production paralleled transcriptional profiles. opp-3, which encodes proteins most similar to known peptide transport proteins, displayed the highest expression level and was the only transporter to be regulated by specific amino acids, tyrosine and phenylalanine. Defined deletion mutants in one or several peptide permeases were constructed and tested for their capacity to grow in peptide-containing medium. Among the four putative Opp systems, Opp-3 was the only system able to provide oligopeptides for growth, ranging in length from 3 to 8 amino acids. Dipeptides were imported exclusively by DtpT, a proton-driven di- and tripeptide permease. These data provide a first complete inventory of the peptide transport systems opp and dtpT of S. aureus. Among them, the newly identified Opp-3 appears to be the main Opp system supplying the cell with peptides as nutritional sources.

Ability of Lactococcus lactis To Export Viral Capsid Antigens: a Crucial Step for Development of Live Vaccines

ABSTRACT Thefood grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract. As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV). IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality. The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L. lactis. Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane. This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane. Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens. The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci.

Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides

Aims:  To test seven selected putative signal peptides from Lactobacillus plantarum WCFS1 in terms of their ability to drive secretion of two model proteins in Lact. plantarum, and to compare the functionality of these signal peptides with that of well‐known heterologous signal peptides (Usp45, M6).

The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis : location of the thioether bridges

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether‐bridging lanthionine and 3‐methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A‐FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.