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PubMed2004

Normal flora: living vehicles for non-invasive protein drug delivery.

Feasibility to use probiotic bacteria as a living protein delivery system through oral route was assessed in vitro. Lactococcus lactis transformed with a plasmid to express and secret beta-lactamase was used to deliver beta-lactamase through Caco-2 monolayer, an intestine epithelium. Transport of beta-lactamase through Caco-2 monolayer was carried out in the transwells. The viability and integrity of the cell monolayers co-cultured with L. lactis was examined by trypan blue exclusion method and by measuring the transport of mannitol and propranolol as well as the transepithelial electrical resistance (TEER). Results show that it is feasible to use cell culture technique to evaluate the drug delivery by normal flora. The transport rate of beta-lactamase when delivered by L. lactis was 2.0 +/- 0.1 x 10(-2)h(-1) (n = 9) and through free solution form was 1.0 +/- 0.1 x 10(-2)h-1. When co-cultured with L. lactis, Caco-2 cell viability decreased to 98, 96, and 94% at 6, 8, and 10h, respectively. Transport of mannitol through Caco-2 cell monolayer was significantly increased and the transport of propranolol through Caco-2 cell monolayer was significantly decreased in the presence of L. lactis. Increase in the amount of protein delivered is probably due to the concentrate of the protein by L. lactis on the monolayer (absorption surface) and the opening of the tight junction of Caco-2 monolayer by L. lactis.

Administration, Oral↗
PubMed2005

In vitro evaluation of coating polymers for enteric coating and human ileal targeting.

Recombinant interleukin-10 producing Lactococcus lactis is an alternative therapy for Crohn's disease. For in vivo interleukin-10 production, thymidine, the essential feed component of these recombinant bacteria should be coadministered. Different coating polymers were evaluated in vitro for enteric properties and targeting suitability to the ileum, the major site of inflammation in Crohn's disease. To guarantee ileal delivery, the polymer must dissolve from pH 6.8 and allow complete release within 40 min. Aqoat AS-HF coated pellets (15%) showed poor enteric properties and thymidine was released below pH 6.8. Eudragit FS30D coated pellets (15%) showed good enteric properties, but no thymidine was released within 40 min at pH 6.8. Eudragit S coated pellets (15%) showed good enteric properties after curing at elevated temperature while no thymidine was released within 40 min at pH 6.8. In another approach to pass the proximal small intestine intact, pellets were coated with 30% Eudragit L30D-55. At pH 6.0, they showed a lag-phase of 20 min. No influence of layer thickness was seen above pH 6.5. Alternatively, pellets were coated with a mixture of Eudragit FS30D/L30D-55 but they showed poor enteric properties and thymidine was released below pH 6.8. In conclusion, none of the tested polymers/mixtures ensured enteric properties and ileal targeting.

Humans↗
PubMed2005

Cloning and expression of the malolactic gene of Pediococcus damnosus NCFB1832 in Saccharomyces cerevisiae.

Wine production is characterized by a primary alcoholic fermentation, conducted by Saccharomyces cerevisiae, followed by a secondary malolactic fermentation (MLF). Although most lactic acid bacteria (LAB) have the ability to metabolize L-malate, only a few species survive the high ethanol and SO2 levels in wine. Wines produced in colder viticultural regions have a lower pH than wines produced in warmer regions. The decarboxylation of L-malate in these wines leads to an increase in pH, more organoleptic complexity and microbiological stability. MLF is, however, difficult to control and problems often occur during filtering of such wines. Pediococcus spp. are known to occur in high pH wines and have strong malolactic activity. However, some pediococci synthesize exocellular polysaccharides, which may lead to abnormal viscosity in wine. In this study, the malolactic gene from Pediococcus damnosus NCFB1832 (mleD) was cloned into S. cerevisiae and co-expressed with the malate permease gene (mae1) of Schizosaccharomyces pombe. Expression of the mleD gene was compared to the expression of two other malolactic genes, mleS from Lactococcus lactis MG1363 and mleA from Oenococcus oeni Lal1. The genetically modified strain of S. cerevisiae decreased the level of L-malate in grape must to less than 0.3 gl(-1) within 3 days. This is the first expression of a malolactic gene from Pediococcus in S. cerevisiae.

Bacterial Proteins↗
PubMed2004

Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes.

Mobile group II introns are site-specific retroelements that use a novel mobility mechanism in which the excised intron RNA inserts directly into a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Because the DNA target site is recognized primarily by base-pairing of the intron RNA with only a small number of positions recognized by the protein, it has been possible to develop group II introns into a new type of gene targeting vector ("targetron"), which can be reprogrammed to insert into desired DNA targets simply by modifying the intron RNA. Here, we used databases of retargeted Lactococcus lactis Ll.LtrB group II introns and a compilation of nucleotide frequencies at active target sites to develop an algorithm that predicts optimal Ll.LtrB intron-insertion sites and designs primers for modifying the intron to insert into those sites. In a test of the algorithm, we designed one or two targetrons to disrupt each of 28 Escherichia coli genes encoding DExH/D-box and DNA helicase-related proteins and tested for the desired disruptants by PCR screening of 100 colonies. In 21 cases, we obtained disruptions at frequencies of 1-80% without selection, and in six other cases, where disruptants were not identified in the initial PCR screen, we readily obtained specific disruptions by using the same targetrons with a retrotransposition-activated selectable marker. Only one DExH/D-box protein gene, secA, which was known to be essential, did not give viable disruptants. The apparent dispensability of DExH/D-box proteins in E.coli contrasts with the situation in yeast, where the majority of such proteins are essential. The methods developed here should permit the rapid and efficient disruption of any bacterial gene, the computational analysis provides new insight into group II intron target site recognition, and the set of E.coli DExH/D-box protein and DNA helicase disruptants should be useful for analyzing the function of these proteins.

Algorithms↗
PubMed2005

Genome analysis of lactic acid bacteria in food fermentations and biotechnological applications.

Lactic acid bacteria are an important group of microorganisms, several of which are used in fermented food processes. Lactococcus lactis is a non-pathogenic, non-invasive and non-colonising gram-positive lactic acid bacterium, the genome sequence of which has been established. A great deal is known about the genetics, vectors, gene expression systems and protein secretion apparatus of this bacterium. Recently, recombinant strains of L. lactis have been developed that might provide in vivo delivery of cytokines and specific antigens across mucosal surfaces to the immune system of animals.

Biotechnology↗
PubMed2005

Phage abortive infection in lactococci: variations on a theme.

Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance.

Bacterial Proteins↗
PubMed2004

Photometric assay for measuring the intracellular concentration of branched-chain amino acids in bacteria.

The changes in intracellular pool of branched-chain amino acids (BCAA) regulate different physiological processes in bacteria. Up to date, the only available photometric test for measuring BCAA concentration was adapted for blood and plasma samples in diagnostic purposes. We have modified this method for use on bacterial cells, and tested its applicability on several model organisms: Lactococcus lactis, Bacillus subtilis and Escherichia coli.

Amino Acids, Branched-Chain↗
PubMed2004

Resin straw as an alternative system to securely store frozen microorganisms.

Freezing of prokaryotic and eukaryotic microorganisms is the main interest in the study of cold stress responses of living organisms. In parallel, applications which arise from this approach are of two types: (i) optimization of the frozen starters used in food processing; and (ii) improvement of the ex situ preservation of microorganisms in collections. Currently, cryopreservation of microorganisms in collections is carried out in cryotubes, and bibliographical references related to freezing microorganisms packaged in straws are scarce. In this context, a preliminary study was completed to evaluate the technological potential of ionomeric resin straws compared to polycarbonate cryo-tubes. Survival under freezing stress was tested on three microorganisms selected for their biotechnological interest: two lactic acid bacteria, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus and a deuteromycete fungus, Geotrichum candidum. The stress was carried out by repeated freezing-thawing cycles to artificially accelerate the lethal effect of freezing on the microorganisms. Two main results were obtained: (i) the survival rate values (per freezing-thawing cycle) seems to depend on the thermal type of the studied microorganism, and (ii) there was no, under our experimental conditions, significant difference between straws and tubes. However, conservation in the resin straws lead to a slight increase in the survival of L. cremoris and G. candidum compared to microtubes. In those conditions, straws seems an alternative system to securely store frozen microorganisms with three main characteristics: (i) a high resistance to thermal stress, (ii) a safe closing by hermetic weld, and (iii) a system for inviolable identification.

Cold Temperature↗
PubMed2005

Application of reverse transcriptase PCR-based T-RFLP to perform semi-quantitative analysis of metabolically active bacteria in dairy fermentations.

A method consisting of reverse transcriptase (RT)-PCR amplification of 16S rRNA from the total microbial community, coupled with T-RFLP, was optimized for semi-quantitative characterization of the metabolically active population in defined strain cultures of Lactococcus lactis ssp. lactis and Leuconostoc citreum, two mesophilic lactic acid bacteria (LAB) species routinely used in cheese manufacture. The set of PCR primers selected efficiently amplified the 16S rRNA from both bacterial species. The digestion of the PCR products with DdeI yielded different terminal restriction fragments (T-RFs) for each species. Nevertheless, additional T-RFs due to formation of chimeric molecules and pseudo-T-RFs derived from partly single-stranded 16S rRNA amplicons were observed in both species, although in minor amounts. Twenty PCR cycles were determined as the optimum to minimize the presence of artifactual fragments and to avoid underestimation of populations due to the saturation effect on DNA quantification caused by a PCR product excess. T-RFLP analysis showed a good repeatability when applied to mixed dairy cultures. Dynamics of two defined mixed starters consisting of a L. lactis ssp. lactis strain and a L. citreum strain were studied by this method and results compared to those obtained by a culture-dependent technique. The data indicated the suitability of T-RFLP to perform semi-quantitative analyses of microbial populations. Some slight differences could be explained by the presence of metabolically active cells that could not be detected by colony counting. RT-PCR-based T-RFLP can be an alternative to classical methods in order to study dynamics of metabolically active populations in relatively simple microbial ecosystems, such as defined dairy starter cultures.

Animals↗
PubMed2005

pIH01, a small cryptic plasmid from Leuconostoc citreum IH3.

A small cryptic plasmid pIH01 from Leuconostoc citreum IH3 was characterized. This 1.8-kb sized plasmid contains single open reading frame that encodes a RepC class protein (342 amino acids) and a conserved pT181-type double strand origin, suggesting a rolling circle replication mode. This putative replicase protein shows the highest similarity to a replicase from pFR18 plasmid of Leuconostoc mesenteroides FR52 (64% identity), one of the pT181-type rolling circle plasmid family and contains a strictly conserved RepC-type active site sequence of pT181 family. A shuttle vector that was developed on the basis of this cryptic plasmid by insertion of both erythromycin resistance gene (ermC) from pE194 and Escherichia coli ColE1 origin was able to transform Leuconostoc strains, Lactobacillus plantarum, and Lactococcus lactis. Therefore, pIH01 derivative plasmids might be useful for the manipulation of Leuconostoc strains.

Amino Acid Sequence↗
PubMed2004

Lactovum miscens gen. nov., sp. nov., an aerotolerant, psychrotolerant, mixed-fermentative anaerobe from acidic forest soil.

An aerotolerant, psychrotolerant anaerobe, anNAG3, was isolated from an acidic forest floor solution (in situ pH of 4.5). Cells of anNAG3 stained Gram-positive did not form spores, and were not motile. Cells were ovoid, approximately 1 microm long and 0.7 microm wide, mostly in pairs, and contained a multi-layered cell wall and intracytoplasmic membranes. Growth was observed at pH 3.5-7.5 and 0-35 degrees C. Glucose, galactose, fructose, mannitol, glucosamine, N-acetylglucosamine, cellobiose, and maltose supported growth. Lactate, ethanol, formate, and acetate were end products. H(2) and CH(4) were not detected, and only very minor amounts of CO(2) were produced. The relative amount of a particular product was dependent on the substrate utilized, and product profiles indicated that (i) sugars were initially metabolized to pyruvate via glycolysis, and (ii) lactate dehydrogenase and pyruvate-formate lyase were responsible for the subsequent metabolism of pyruvate. O(2) was not significantly utilized and was not toxic to growth. anNAG3 did not contain detectable membranous or cytoplasmic cytochromes. Nitrate, sulfate, and Fe(III) were not dissimilated. Thus, anNAG3 was characterized as an aerotolerant, non-acetogenic chemoorganotroph with a mixed-fermentative metabolism. The G + C content of the DNA was 37.6 mol%. The similarity of the 16S rRNA gene sequence of anNAG3 to that of its closest phylogenetic relatives (which were in the genera Lactococcus and Streptococcus) approximated 88-89%, indicating that anNAG3 constitutes the type species of a new genus. Based on the collective properties of anNAG3, it is proposed that anNAG3 be termed Lactovum miscens.

Actinobacteria↗
PubMed2005

Testing of a whole genome PCR scanning approach to identify genomic variability in four different species of lactic acid bacteria.

Genomes can be markedly heterogeneous in conspecific bacterial strains. Genome sequences can be used to analyze genome plasticity via a PCR(2) (plasticity of chromosome revealed by PCR) approach. Small-sized chromosomes can indeed be fully amplified by long-range PCR with a set of primers designed using a reference strain and then applied to several other strains. Analysis of the resulting patterns can reveal genome plasticity. GenoFrag, a software package for the design of primers optimized for PCR(2) [N. Ben Zakour, M. Gautier, R. Andonov, D. Lavenier, M.F. Cochet, P. Veber, A. Sorokin, Y. Le Loir, GenoFrag: Software to design primers optimized for whole genome scanning by long-range PCR amplification, Nucleic Acids Res. 32 (2004) 17-24] was developed for the analysis of bacterial genome plasticity by whole genome amplification in approximately 10-kb-long fragments. By applying GenoFrag, we provide herewith evidence that genome plasticity can be analyzed in lactic acid bacteria using a PCR(2) approach. The genome sequences of Lactococcus lactis IL1403, Lactobacillus plantarum WCFS1, Lactobacillus bulgaricus ATCC11842 and Bifidobacterium longum NCC2705 were used to design four sets of primers. Each set was evaluated in silico to check that it ensured optimum coverage of the bacterial chromosome. To validate the primers generated by GenoFrag, a subset of primers was successfully used in LR-PCR experiments on genomic DNA from four L. bulgaricus strains.

Bacterial Typing Techniques↗
PubMed2005

Polyphasic characterization of the lactic acid bacteria in kefir.

The lactic acid bacteria of kefir were isolated and characterized using phenotypical, biochemical, and genotypical methods. Polyphasic analyses of results permitted the identification of the microflora to the strain level. The genus Lactobacillus was represented by the species Lb. kefir and Lb. kefiranofaciens. Both subspecies of Lactococcus lactis (lactis and cremoris) were isolated. Leuconostoc mesenteroides subsp. cremoris was also found. The kefir studied contained few species of lactic acid bacteria but showed a high number of different strains. We found that the polyphasic analysis approach increases the confidence in strain determination. It helped confirm strain groupings and it showed that it could have an impact on the phylogeny of the strains.

Cultured Milk Products↗
PubMed2005

Comparative and functional analysis of the rRNA-operons and their tRNA gene complement in different lactic acid bacteria.

The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5'-16S-23S-5S-3' structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three -10 and -35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.

Amino Acid Sequence↗
PubMed2006

Antimicrobial activity of lysostaphin and a Listeria monocytogenes bacteriophage endolysin produced and secreted by lactic acid bacteria.

The expression and secretion signals of the Sep protein from Lactobacillus fermentum BR11 were used to direct export of two peptidoglycan hydrolases by Lb. fermentum BR11, Lactobacillus rhamnosus GG, Lactobacillus plantarum ATCC 14917 and Lactococcus lactis MG1363. The production levels, hydrolytic and bacteriocidal activities of the Listeria monocytogenes bacteriophage N-acetylmuramoyl-l-alanine amidase endolysin Ply511 and the glycylglycine endopeptidase lysostaphin were examined. Buffering of the growth media to a neutral pH allowed detection of Ply511 and lysostaphin peptidoglycan hydrolytic activity from all lactic acid bacteria. It was found that purified Ply511 has a pH activity range similar to that of lysostaphin with both enzymes functioning optimally under alkaline conditions. Supernatants from lactobacilli expressing lysostaphin reduced viability of methicillin resistant Staphylococcus aureus (MRSA) by approximately 8 log(10) CFU/ml compared to controls. However, supernatants containing Ply511 were unable to control L. monocytogenes growth. In coculture experiments, both Lb. plantarum and Lb. fermentum synthesizing lysostaphin were able to effectively reduce MRSA cell numbers by >7.4 and 1.7 log(10)CFU/ml, respectively, while lactic acid bacteria secreting Ply511 were unable to significantly inhibit the growth of L. monocytogenes. Our results demonstrate that lysostaphin and Ply511 can be expressed in an active form from different lactic acid bacteria and lysostaphin showed superior killing activity. Lactobacilli producing lysostaphin may have potential for in situ biopreservation in foodstuffs or for prevention of S. aureus infections.

Anti-Bacterial Agents↗
PubMed2004

Engineering metabolic highways in Lactococci and other lactic acid bacteria.

Lactic acid bacteria (LAB) are widely used in industrial food fermentations and are receiving increased attention for use as cell factories for the production of food and pharmaceutical products. Glycolytic conversion of sugars into lactic acid is the main metabolic highway in these Gram-positive bacteria and Lactococcus lactis has become the model organism because of its small genome, genetic accessibility and simple metabolism. Here we discuss the metabolic engineering of L. lactis and the value of metabolic models compared with other LAB, with a particular focus on the food-grade production of metabolites involved in flavour, texture and health.

Energy Metabolism↗
PubMed2006

Novel methods of quantitative real-time PCR data analysis in a murine Helicobacter pylori vaccine model.

Monitoring of Helicobacter pylori in the stomach is important to assess the efficacy of new vaccines against the pathogen. To realise the full potential of quantitative real-time PCR (q-PCR), this technology has to offer accurate and easy models of post-PCR data analysis. In this work, we used a variety of absolute and relative approaches of q-PCR data analysis to monitor the H. pylori infection in the stomach of immunized mice. Relative quantification was performed with Ct-based methods, with the DART program, and with two methods based on the mathematical analysis of raw fluorescence kinetics, the LinReg program and the Sigmoidal Curve Fitting Method. The different calculation methods were validated in mice immunized with cell lysates of Lactococcus lactis expressing the H. pylori urease subunit B in combination with cholera toxin. The H. pylori load was found to be reduced in immunized mice by a factor of 50-144, depending on the calculation method employed. We found that relative quantification using DART, LinReg and Sigmoidal Curve Fitting methods generated similar results (infection ratios of 54-58) with absolute quantification results (54-65). Results were very different to those using relative quantification Ct-based methods without a correction for PCR efficiency (ratio of 92-144) and with results based on conventional culture method (ratio of 34). Overall, this study demonstrates that q-PCR associated with a relative quantification analysis is a powerful tool for the monitoring of microorganisms in tissue. It could be used as an alternative to standard curve approach especially for the investigation of microbial load in vaccine models.

Animals↗
PubMed2006

Development of lactococcal GEM-based pneumococcal vaccines.

We report the development of a novel protein-based nasal vaccine against Streptococcus pneumoniae, in which three pneumococcal proteins were displayed on the surface of a non-recombinant, killed Lactococcus lactis-derived delivery system, called Gram-positive Enhancer Matrix (GEM). The GEM particles induced the production of the proinflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) by macrophages as well as the maturation of dendritic cells. The pneumococcal proteins IgA1 protease (IgA1p), putative proteinase maturation protein A (PpmA) and streptococcal lipoprotein A (SlrA) were anchored in trans to the surface of the GEM particles after recombinant production of the antigens in L. lactis as hybrids with a lactococcal cell wall binding domain, named Protein Anchor domain (PA). Intranasal immunisation with the SlrA-IgA1p or trivalent vaccine combinations without additional adjuvants showed significant protection against fatal pneumococcal pneumonia in mice. The GEM-based trivalent vaccine is a potential pneumococcal vaccine candidate that is expected to be easy to administer, safe and affordable to produce.

Administration, Intranasal↗