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PubMed2005

Lactococcin MMT24, a novel two-peptide bacteriocin produced by Lactococcus lactis isolated from rigouta cheese.

Lactococcin MMT24 is a novel bacteriocin produced by Lactococcus lactis MMT24, a strain isolated from a Tunisian traditional cheese. The bacteriocin shows a narrow antimicrobial activity against closely related lactic acid bacteria. Lactococcin MMT24 is heat resistant, remains active after incubation at pH 3 to 10, lyophilization, long-term storage at -20 degrees C and is sensitive to treatment with proteolytic enzymes. The mode of action of lactococcin MMT24 was identified as bactericidal. Purification of the active compound showed that lactococcin MMT24 consists of two distinct peptides, named pepalpha and pepbeta, whose complementary action is necessary for full antibacterial activity. Optimal antibacterial activity was obtained when the complementary peptides pepalpha and pepbetawere present in equal amounts. Mass spectrometry analysis showed masses of 3765.33 Da and 3255.26 Da for pepalpha and pepbeta, respectively. These molecular masses do not correspond to those of so far described bacteriocins. Addition of 50 nmol l(-1) of lactococcin MMT24 to cells of L. lactis ssp. cremoris ATCC11603 induced increase in the concentration of K+ in supernatant indicating a massive leakage of this ion from the cells. This release was most likely caused by pores formation by the pepalphaand pepbeta peptides in the target bacterial membrane.

Anti-Bacterial Agents↗
PubMed2006

Combined high pressure and temperature induced lethal and sublethal injury of Lactococcus lactis--application of multivariate statistical analysis.

It was the aim of this work to determine the combined effects of pressure, temperature, and co-solutes on Lactococcus lactis, and to detect correlations between culture-dependent and culture-independent methods for assessment of cellular viability and sublethal injury. Therefore, the pressure induced inactivation of L. lactis MG 1363 was investigated in buffer and in buffer with 1.5 M sucrose or 4 M NaCl at a pressure range of 0.1 to 500 MPa and a temperature range of 5 to 50 degrees C. The inactivation was characterised by viable cell counts, stress resistant cell counts, membrane integrity, metabolic activity, and the activity of the multi-drug-resistance transport enzyme LmrP. L. lactis was most resistant to pressure application at 20-30 degrees C. Sucrose protected towards inactivation at any temperature, NaCl provided protection at high temperatures only. By using Principal Component Analysis, correlations were detected between viable cell counts and metabolic activity as well as stress resistant cell counts and LmrP activity. In conclusion, the pressure-inactivation of L. lactis is strongly temperature dependent, baroprotection by sucrose occurs at any temperature but the baroprotective effects of NaCl is temperature dependent. Further on, a combination of two experimental methods fully describe lethal and sublethal injury of pressure treated cells. These simplification of data acquisition and model development facilitates the establishment of pressure processes in food technology.

Bacterial Proteins↗
PubMed2006

Cell membrane damage induced by lacticin 3147 enhances aldehyde formation in Lactococcus lactis IFPL730.

Amino acid catabolism is mainly initiated in Lactococcus lactis by a transamination reaction that leads to the formation of alpha-keto acids. In addition, a novel alpha-keto acid decarboxylase enzyme, rare in lactic acid bacteria, responsible for the conversion of alpha-keto acids into aldehydes has been reported in L. lactis IFPL730. The effect of lacticin 3147-induced cell damage on both amino acid transamination and alpha-keto acid decarboxylation by L. lactis IFPL730 leading to the formation of aldehydes from amino acids was investigated. Cell membrane permeabilization induced by lacticin 3147 facilitated the diffusion of amino acids into the cells and thus, enhanced amino acid transamination and formation of alpha-keto acids. However, alpha-keto acid decarboxylation was not affected by cell membrane permeabilization since decarboxylation of alpha-keto acids in both control and lacticin 3147-treated cells were similar, suggesting that these substrates could freely diffuse inside the cells. Nevertheless, the formation of 2-methylbutyraldehyde from isoleucine was enhanced in lacticin 3147-treated cells. The increase in alpha-keto acids formation rate by L. lactis IFPL730 due to lacticin 3147-induced cell damage, led to a concomitant increase in the subsequent decarboxylation reaction that complete the metabolic pathway to aldehyde production from amino acids. The present study points out to the use of the food grade lacticin 3147 along with L. lactis IFPL730 as a valuable tool in the development of cheese flavour.

Aldehydes↗
PubMed2006

Oral delivery of beta-lactamase by Lactococcus lactis subsp. lactis transformed with Plasmid ss80.

The objective was to use normal flora to deliver protein/peptide drugs orally. A probiotic bacterium, Lactococcus lactis subsp. lactis (L. lactis) transformed with Plasmid ss80, which made it able to synthesize and secrete beta-lactamase, a 29 kDa protein, was used as the delivery system for beta-lactamase. Oral absorption of beta-lactamase in rats when delivered by this L. lactis system was investigated. The oral bioavailability of beta-lactamase delivered by 3x10(7) of the L. lactis was equivalent to 209 mU of i.v. dose, and the estimated relative bioavailability was 16.7%. When delivered by beta-lactamase free solution form, the relative oral bioavailability was 4.7%, which increased to 6.0% when co-administered with 3x10(7) of the untransformed L. lactis. The results demonstrated that the L. lactis significantly increased the beta-lactamase oral bioavailability by 2-3-folds (p<0.01), the mean residence time (MRT) by 3-4 times (p<0.01), and the mean absorption time (MAT) by 6-14 times (p<0.01), as compared to the free solution form with/without the untransformed L. lactis. In conclusion, the L. lactis is more efficient in delivering beta-lactamase orally compared with the free solution form. It also provides a sustained delivery mechanism for beta-lactamase. Gene-transformed normal flora may be used as an efficient and sustained delivery system for protein drugs through oral route.

Administration, Oral↗
PubMed2006

Delivery of TEM beta-lactamase by gene-transformed Lactococcus lactis subsp. lactis through cervical cell monolayer.

Lactococcus lactis subsp. lactis transformed with Plasmid ss80 (encoding the production and secretion of TEM beta-lactamase) was used for the delivery of beta-lactamase through the C-33A (cervix cell) monolayer. The viability of the cell monolayers co-cultured with L. lactis was examined by the trypan blue exclusion method. The integrity of the monolayers was monitored by measuring the transport of mannitol and propranolol as well as the transepithelial electrical resistance. The transport rate of beta-lactamase through C-33A monolayer was increased by four- and nine-folds (p < 0.05) at the first hour by the transformed L. lactis compared to the free solution with or without presence of the untransformed L. lactis, respectively. This increase was gradually diminished after the 1st hour: it became 30 and 50% (p < 0.05) at 10 h. The presence of the untransformed L. lactis with free solution delivery also increased the transport rate by 100% at 1 h (p < 0.05) and 15% at 10h (p>0.05). The increase in transport rate by the transformed L. lactis is most probably due to the concentrate of beta-lactamase on C-33A monolayer. When co-cultured with the L. lactis, the C-33A cell viability and the monolayer TEER remained steady for 10 h. The presence of L. lactis did not change the transport of propranolol and mannitol through the monolayers. In conclusion, the transformed L. lactis significantly (p < 0.05) increased the transport of beta-lactamase through the cervical monolayers, indicating probiotic bacteria delivery may be a promising approach for protein delivery through the vagina.

Bacterial Adhesion↗
PubMed2005

Immunomodulatory effects of IL-12 secreted by Lactococcus lactis on Th1/Th2 balance in ovalbumin (OVA)-induced asthma model mice.

Asthma is a chronic lung disease characterized by allergen-induced airway inflammation and orchestrated by Th2 cells. Interleukin-12, a Th1-promoting cytokine, is capable of inhibit the Th2-driven allergen-induced airway changes and therefore considered as an attractive molecule to treat asthma. Recent epidemiological and clinical studies suggest a possible role of Lactococcus lactis in the prevention of allergic diseases. In this study, we evaluated the immunomodulatory effects of live L. lactis secreting a biologically active form of IL-12 (LL-IL12) in a mouse model of ovalbumin (OVA)-induced asthma. Intranasal mice administration with LL-IL12 resulted in a shift Th2 to Th1 with elevated IFN-gamma and decreased IL-4 levels. In addition, a profound decrease in airway hyper-responsiveness and pulmonary inflammation was also observed in mice administered with LL-IL12. These promising preclinical results suggest the feasibility of this approach to be used in the treatment of asthma.

Adjuvants, Immunologic↗
PubMed2005

Production of xylitol from D-xylose by recombinant Lactococcus lactis.

The D-xylose reductase from Pichia stipitis CBS 5773 and the xylose transporter from Lactobacillus brevis ATCC 8287 were expressed in active form in Lactococcus lactis NZ9800. Xylitol production was investigated using non-growing recombinant cells in high cell-density under microaerobic conditions in the presence of xylose and glucose. Besides xylose, the recombinant strain with xylose reductase activity reduced l-arabinose and D-ribose in significant extent to the corresponding pentitols. The ratio of xylitol produced per glucose consumed was almost 10-fold higher under glucose limitation than the ratio in the presence of excess initial glucose. The co-expression of the xylose transporter with the xylose reductase did not increase the efficiency of xylitol production appreciably when compared to the strain in which only the xylose reductase gene was expressed. A fed-batch experiment with high initial xylose concentration (160 gl(-1)) under glucose limitation was carried out using the strain co-expressing xylose reductase and xylose transporter genes. The xylitol yield from xylose was 1.0 mol mol(-1) and the ratio of xylitol produced per glucose consumed was 2.5 mol mol(-1). The volumetric productivity was 2.72 gl(-1)h(-1) at 20 h. Of the xylose initially present, 34% was consumed. Analysis of the fermentation metabolites revealed a shift from homolactic to mixed acid fermentation at early stages of the experiment.

Aldehyde Reductase↗
PubMed2005

In vitro expression of the restriction endonucleases LlaMI and ScrFI isolated from Lactococcus lactis M19 and UC503.

A new restriction endonuclease LlaMI has been characterized in Lactococcus lactis subsp. cremoris M19. LlaMI recognizes the sequence 5'-CCNGG-3' and cuts after the second cytosine. This restriction endonuclease is related to commercially available ScrFI but not identical to it. Comparative analysis of the predicted amino acid sequences of LlaMI and ScrFI indicates five non-conservative amino acid changes between these two restriction enzymes. These two enzymes were expressed in vitro as histidine-tagged fusion proteins. LlaMI was shown to be more sensitive to high salt concentration than ScrFI. Southern blotting and hybridization analysis indicate that the gene for LlaMI R/M system is chromosomally encoded.

Amino Acid Substitution↗
PubMed2005

Internalin-expressing Lactococcus lactis is able to invade small intestine of guinea pigs and deliver DNA into mammalian epithelial cells.

The use of the food-grade bacterium Lactococcus lactis as antigen delivery vehicle at the mucosal level is an attractive vaccination strategy intensively explored during the last decade. In this study, we developed L. lactis strains which could be used as a DNA delivery vector to combine both advantages of mucosal delivery and of DNA vaccination. To render lactococci capable of invading epithelial cells, the Listeria monocytogenes inlA gene was cloned and expressed in L. lactis under transcriptional control of the native promoter. Western blot and immunofluorescence assays revealed that recombinant lactococci efficiently displayed the cell wall anchored form of InlA. We demonstrated that this expression promotes internalization of L. lactis inlA+ into the human epithelial cell line Caco-2. Gentamicin assay showed that invasiveness of L. lactis in these cells is approximately 100-fold higher for L. lactis inlA+ than for wild type (wt) L. lactis strains. Moreover, we showed that L. lactis inlA+ is able to enter intestinal cells in vivo, after oral inoculation of guinea pigs. After internalization, L. lactis inlA+ was able to deliver a functional eukaryotic gfp gene into epithelial Caco-2 cells; GFP was detected in 1% of internalized cells. The L. lactis inlA+ strain will be a useful bacterial vector for the development of new live oral DNA vaccines. It also constitutes an interesting new model to study the role of internalin in bacterial localization in the animal host.

Animals↗
PubMed2006

Addition of glucose enables determination of luciferase activity in carbon-starved, stationary phase Lactococcus lactis cells.

We describe a simple method for measuring luciferase activity in the stationary phase of Lactococcus lactis. Due to large fluctuations in the energy and redox pools of stationary phase bacterial cells, measurement of luciferase activities does not yield reliable results. Upon addition of relatively small amounts of glucose, the pools are restored and measurement of luciferase becomes possible. Since luciferase activities are easily measured, our method allows to apply this simple analytical tool in stationary phase cells.

Glucose↗
PubMed2006

Expression, purification, and characterization of arginine deiminase from Lactococcus lactis ssp. lactis ATCC 7962 in Escherichia coli BL21.

The arcA gene that encodes arginine deiminase (ADI, EC 3.5.3.6)--a key enzyme of the ADI pathway--was cloned from Lactococcus lactis ssp. lactis ATCC 7962. The deduced amino acid sequence of the arcA gene showed high homology with the arcA gene from Lactobacillus plantarum (99%) and from Lactobacillus sakei (60%), respectively. The arcA gene from Lc. lactis spp. lactis ATCC 7962 was expressed in soluble fraction of recombinant Escherichia coli BL21. ADI produced from Lc. lactis spp. lactis ATCC 7962 (LADI) in E. coli BL21 (DE3) was purified using sequential Q-Sepharose anion exchange and Sephacryl S-200 gel filtration column chromatography. The final yield of LADI in the purification procedure was 63.5%, and the specific activity was 140.27 U/mg. The presence of purified LADI was confirmed by N-terminal sequencing and determination of the molecular mass. The LADI had a molecular mass of about 140 kDa, and comprised a homotrimer of 46 kDa in the native condition. LADI exhibited only 35% amino acid sequence homology with ADI from Mycoplasma arginini. However, LADI shared a similar three dimensional structure. The K(M) and V(max) values for arginine were 8.67+/-0.045 mM (mean+/-SD) and 344.83+/-1.79 micromol/min/mg, respectively, and the optimum temperature and pH for the production of LADI were 60 degrees C and 7.2.

Amino Acid Sequence↗
PubMed2004

General and specialized vectors derived from pBM02, a new rolling circle replicating plasmid of Lactococcus lactis.

This paper reports the construction of several general cloning vectors and a specialized depurative vector based on a new lactococcal plasmid that replicates by the rolling circle mechanism [pBM02; Plasmid 49 (2003) 118]. Most vectors are shuttle vectors for Escherichia coli-Lactococcus lactis and carry replicons of both ColE1 and pBM02 plasmids (ColE1 is used even though the pBM02 replicon is fully active in both Gram-positive and Gram-negative organisms). Segregational and structural studies indicated that the new vectors were stable enough for the majority of applications. Further, since the basic replicon is compatible with plasmid derivatives of pWV01 and pSH71, they can be maintained in the same cell with members of the two largest vector series for L. lactis and other lactic acid bacteria, the pGK, and the pNZ series.

Chromosome Mapping↗
PubMed2004

Development of an inducible system to control and easily monitor gene expression in Lactococcus lactis.

This report describes the implementation and use of a maltose-inducible system for regulated gene expression in Lactococcus lactis. The system was established using Green Fluorescent Protein as reporter. The transcription of a gene of interest from the inducible promoter of pLS1RGFP plasmid vector can be easily monitored by fluorescence spectroscopy and microscopy. As an example, the lactococcal ribonuclease III was overproduced in an active form.

Blotting, Western↗
PubMed2006

Construction of transposition insertion libraries and specific gene inactivation in the pathogen Lactococcus garvieae.

This paper reports the development of genetic tools in Lactococcus garvieae, an important Gram-positive bacterial pathogen affecting both fish and mammals. The vector pGKV210, a broad host range vector, was introduced by electroporation into L. garvieae UNIUD074. The maximal frequency obtained was 3.2 x 10(5) transformants/mug of DNA. Moreover, this effect is highly reproducible and appears to be constant, since all L. garvieae strains tested were transformed. Once the optimal transformation procedure was established, it was used to generate isogenic and transposition mutants. Insertional mutagenesis of the L. garvieae SA9H10L gene, similar to a Streptococcus pyogenes gene encoding the M protein (emm64), was carried out using the conditional replication plasmid pORI19. Transposition mutagenesis using the streptococcal temperature-sensitive suicide vector pTV408 to deliver Tn917 into the chromosome of L. garvieae was also achieved at a frequency of ca. 10(-4). Transposon flanking DNA sequences were obtained by plasmid rescue in Escherichia coli and their sequencing analysis demonstrated that the transposon was inserted at different chromosomal loci. Tn917 also made it possible to select a mutant in the operon involved in mannitol fermentation in this microorganism. The results obtained in the present study lay the foundation for future research on the virulence mechanisms of L. garvieae.

Chromosomes, Bacterial↗
PubMed2005

Unity in organisation and regulation of catabolic operons in Lactobacillus plantarum, Lactococcus lactis and Listeria monocytogenes.

Global regulatory circuits together with more specific local regulators play a notable role when cells are adapting to environmental changes. Lactococcus lactis is a lactic acid bacterium abundant in nature fermenting most mono- and disaccharides. Comparative genomics analysis of the operons encoding the proteins and enzymes crucial for catabolism of lactose, maltose and threhalose revealed an obvious unity in operon organisation . The local regulator of each operon was located in a divergent transcriptional direction to the rest of the operon including the transport protein-encoding genes. Furthermore, in all three operons a catabolite responsive element (CRE) site was detected inbetween the gene encoding the local regulator and one of the genes encoding a sugar transport protein. It is evident that regardless of type of transport system and catabolic enzymes acting upon lactose, maltose and trehalose, respectively, Lc. lactis shows unity in both operon organisation and regulation of these catabolic operons. This knowledge was further extended to other catabolic operons in Lc. lactis and the two related bacteria Lactobacillus plantarum and Listeria monocytogenes. Thirty-nine catabolic operons responsible for degradation of sugars and sugar alcohols in Lc. lactis, Lb. plantarum and L. monocytogenes were investigated and the majority of those possessed the same organisation as the lactose, maltose and trehalose operons of Lc. lactis. Though, the frequency of CRE sites and their location varied among the bacteria. Both Lc. lactis and Lb. plantarum showed CRE sites in direct proximity to genes coding for proteins responsible for sugar uptake. However, in L. monocytogenes CRE sites were not frequently found and not in the vicinity of genes encoding transport proteins, suggesting a more local mode of regulation of the catabolic operons found and/or the use of inducer control in this bacterium.

Bacterial Proteins↗
PubMed2004

A Plasmodium falciparum GLURP-MSP3 chimeric protein; expression in Lactococcus lactis, immunogenicity and induction of biologically active antibodies.

Plasmodium falciparum malaria is a major cause of morbidity and mortality worldwide. To evaluate the efficacy of a possible vaccine antigen against P. falciparum infection, a fusion protein, derived from P. falciparum Glutamate-rich protein (GLURP) genetically coupled to P. falciparum Merozoite surface protein 3 (MSP3) was produced in Lactococcus lactis as a secreted recombinant GLURP-MSP3 fusion protein. The hybrid protein was purified to homogeneity by ion exchange and hydrophobic-interaction chromatography and its composition was verified by MALDI MS, SDS/PAGE and Western blotting with antibodies against antigenic components of GLURP and MSP3. Mice immunized with the hybrid protein produced higher levels of both GLURP- and MSP3-specific antibodies than mice immunized with either GLURP, MSP3 or a mix of both. The protective potential of the hybrid protein was also demonstrated by in vitro parasite-growth inhibition of mouse anti-GLURP-MSP3 IgG antibodies in a monocyte-dependent manner. These results indicate that the GLURP-MSP3 hybrid could be a valuable strategy for future P. falciparum vaccine development.

Animals↗
PubMed2006

Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations.

A putative protective protein from Plasmodium falciparum merozoites, MSA2, was expressed in two different ways on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. The first display format exploits an LPXTG-type anchoring motif of the lactococcal proteinase PrtP to covalently anchor MSA2 to the genetically modified producer cells. In a second display format, MSA2 was fused to the peptidoglycan-binding domain (Protein Anchor) of the lactococcal cell wall hydrolase AcmA and was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells, termed Gram-positive enhancer matrix (GEM) particles. The L. lactis recombinants carrying covalently bound MSA2 were used to immunise rabbits through nasal and oral routes. The highest levels of IgG antibodies reacting with near-native MSA2 on merozoites was elicited by oral administration. Intestinal antibodies to MSA2 were produced only after oral immunisation. MSA2-specific T(h)-cell activation could be demonstrated. Based on these results, the immunogenicity in oral immunisations of MSA2, bound non-covalently to non-genetically modified L. lactis GEM particles, was compared with MSA2 that was bound covalently to genetically modified L. lactis. These two forms elicited similar titres of serum antibodies. The results illustrate the potential of using non-genetically modified L. lactis as a safe vaccine delivery vehicle to elicit systemic antibodies, thereby avoiding the dissemination of recombinant DNA into the environment.

Administration, Intranasal↗