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P Langella

Publications and source records attributed to P Langella.

At least 19 recordsLinked to original sources
PubMed2006

Constitutive delivery of bovine beta-lactoglobulin to the digestive tracts of gnotobiotic mice by engineered Lactobacillus casei.

The gut microbiota is critical for maturation of the immune system. Recent evidence suggests that early establishment of lactobacilli in the intestinal microbiota, during neonatal colonization or by probiotic supplementation, could prevent the development of allergic disorders. Postnatal maturation of the gut immune system with allergen-producing lactobacilli colonizing the digestive tract could then affect the development of further allergic sensitization. In this paper, we describe construction of a recombinant Lactobacillus casei strain that can constitutively deliver bovine beta-lactoglobulin (BLG), a major cow's milk allergen, to the guts of gnotobiotic mice. The blg gene was inserted into the L. casei chromosome downstream of an endogenous promoter. BLG production was improved by fusing the propeptide LEISSTCDA (LEISS) to the BLG mature moiety. This led to a 10-fold increase in LEISS-BLG production compared to the production obtained without the propeptide and also led to enhanced secretion corresponding to 5% of the total production. After inoculation into germfree C3H/HeN mice, the genetic stability of the recombinant strain and in vivo BLG production were confirmed for at least 10 weeks. BLG stimulation of spleen cells from mice monoassociated with the BLG-producing lactobacilli induced secretion of the Th1 cytokine gamma interferon and, to a lesser extent, the Th2 cytokine interleukin-5. No BLG-specific immunoglobulin G1 (IgG1), IgG2a, or IgA was detected in sera or in fecal samples. These results suggest that gut colonization with allergen-producing lactobacilli could provide a useful model for studying the modulation of allergic disorders.

Animals↗
PubMed2006

Construction and characterization of a Lactococcus lactis strain deficient in intracellular ClpP and extracellular HtrA proteases.

A Lactococcus lactis strain deficient in both its major proteases, intracellular (ClpP) and extracellular (HtrA), was constructed and characterized. This strain, hereafter called clpP-htrA, could be obtained only by conjugation between a clpP donor strain and an htrA recipient strain in the NZ9000 context, allowing heterologous gene expression under the control of the NICE (nisin-controlled expression) system. The clpP-htrA double mutant showed both higher stress tolerance (e.g. high temperature and ethanol resistance) and higher viability than single clpP or htrA mutant strains. In addition, the secretion rate of two heterologous proteins (staphylococcal nuclease Nuc and Nuc-E7) was also higher in clpP-htrA than in the wild-type strain. This strain should be a useful host for high-level production and quality of stable heterologous proteins.

Conjugation, Genetic↗
PubMed2006

Complementation of the Lactococcus lactis secretion machinery with Bacillus subtilis SecDF improves secretion of staphylococcal nuclease.

Unlike Bacillus subtilis and Escherichia coli, the gram-positive lactic acid bacterium Lactococcus lactis does not possess the SecDF protein, a component of the secretion (Sec) machinery involved in late secretion stages and required for the high-capacity protein secretion in B. subtilis. In this study, we complemented the L. lactis Sec machinery with SecDF from B. subtilis and evaluated the effect on the secretion of two forms of staphylococcal nuclease, NucB and NucT, which are efficiently and poorly secreted, respectively. The B. subtilis SecDF-encoding gene was tested in L. lactis at different levels. Increased quantities of the precursor and mature forms were observed only at low levels of SecDF and at high NucT production levels. This SecDF secretion enhancement was observed at the optimal growth temperature (30 degrees C) and was even greater at 15 degrees C. Furthermore, the introduction of B. subtilis SecDF into L. lactis was shown to have a positive effect on a secreted form of Brucella abortus L7/L12 antigen.

Bacillus subtilis↗
PubMed2005

Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis.

Lactococcus lactis, the model lactic acid bacterium, is a good candidate for heterologous protein production in both foodstuffs and the digestive tract. We attempted to produce Streptomyces tendae antifungal protein 1 (Afp1) in L. lactis with the objective of constructing a strain able to limit fungal growth. Since Afp1 activity requires disulfide bond (DSB) formation and since intracellular redox conditions are reportedly unfavorable for DSB formation in prokaryotes, Afp1 was produced as a secreted form. An inducible expression-secretion system was used to drive Afp1 secretion by L. lactis; Afp1 was fused or not with LEISSTCDA, a synthetic propeptide (LEISS) that has been described to be a secretion enhancer. Production of Afp1 alone was not achieved, but production of LEISS-Afp1 was confirmed by Western blot and immunodetection with anti-Afp1 antibodies. This protein (molecular mass: 9.8 kDa) is the smallest non-bacteriocin heterologous protein ever reported to be secreted in L. lactis via the Sec-dependent pathway. However, no anti-fungal activity was detected, even in concentrated samples of induced supernatant. This could be due to a too low secretion yield of Afp1 in L. lactis, to the absence of DSB formation, or to an improper DSB formation involving the additional cysteine residue included in LEISS propeptide. This raises questions about size limits, conformation problems, and protein secretion yields in L. lactis.

Antifungal Agents↗
PubMed2005

Improvement of bovine beta-lactoglobulin production and secretion by Lactococcus lactis.

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine beta-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (approximately 10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at approximately 8 microg/ml (approximately 2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Animals↗
PubMed2005

High-level resistance to oxidative stress in Lactococcus lactis conferred by Bacillus subtilis catalase KatE.

Lactococcus lactis, a lactic acid bacterium widely used for food fermentations, is often exposed to damaging stress conditions. In particular, oxidative stress leads to DNA, protein and membrane damages that can be lethal. As L. lactis has no catalase, the impact of production of the Bacillus subtilis haem catalase KatE on its oxidative stress resistance was tested. This cytoplasmic catalase was engineered for extracellular expression in L. lactis with an optimization strategy based on fusion to the nisin-inducible promoter and a lactococcal signal peptide (SP(Usp45)). The production of KatE by L. lactis conferred an 800-fold increase in survival after 1 h exposure to 4 mM hydrogen peroxide, and a 160-fold greater survival in long-term (3 days) survival of aerated cultures in a cydA mutant, which is unable to respire. The presence of KatE protected DNA from oxidative damage and limited its degradation after long-term aeration in a cydA/recA mutant, defective in DNA repair. L. lactis is thus able to produce active catalase that can provide efficient antioxidant activity.

Bacillus subtilis↗
PubMed2005

Oral administration of recombinant Lactococcus lactis expressing bovine beta-lactoglobulin partially prevents mice from sensitization.

BACKGROUND: The use of probiotics such as Lactococcus lactis and other lactic acid bacteria (LAB) has been proposed for the management of food allergy. However, no experimental study has clearly demonstrated any preventive or therapeutic inhibition of an allergen-specific IgE response. OBJECTIVE: We aimed to study the immunomodulatory effect of recombinant L. lactis expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, in a validated mouse model of allergy. METHODS: Six-week-old female Balb/c mice received five repeated doses of BLG, of L. lactis plus BLG, or of recombinant L. lactis by gavage. Different recombinant strains were inoculated, which corresponded to BLG doses ranging from 4 to 70 microg/mice. Mice were then sensitized by intra-peritoneal injection of BLG emulsified in incomplete Freund's adjuvant to induce high IgE concentrations. RESULTS: Pre-treatment with natural L. lactis plus BLG allowed induction of BLG-specific T-helper type 1 (Th1) response, and abrogated the oral tolerance induced by BLG alone, demonstrating the adjuvant effect of this non-colonizing LAB. Moreover, pre-treatment with some of the recombinant strains favoured the development of a Th1 response inhibiting the Th2 one: it induced a significant decrease of specific IgE response, and an intense increase of specific IgG2a and IFN-gamma productions. The most efficient strains that inhibited the IgE response were those producing the highest amounts of the BLG protein. CONCLUSION: Oral administration of some recombinant L. lactis was demonstrated to induce a specific Th1 response down-regulating a further Th2 one. Prophylaxis protocols will thus be evaluated using the most efficient strains.

Administration, Oral↗
PubMed2004

A food-grade delivery system for Lactococcus lactis and evaluation of inducible gene expression.

The genetic improvement of Lactococcus lactis is a matter of biotechnological interest in the food industry and in the pharmaceutical and medical fields. However, to construct a food-grade delivery system, both the presence of antibiotic markers or plasmid sequences should be avoided and the maintenance and expression of the cloned gene should be guaranteed. The objective of this work was to produce crossover mutants of L. lactis with a reporter gene under the control of an inducible promoter in order to evaluate the level of gene expression. We utilized a nuclease gene of Staphylococcus aureus as a reporter gene, P(nisA) as the nisin-inducible promoter, a non-essential gene involved in histidine biosynthesis of L. lactis as the site for homologous recombination, and pRV300 as a suicide vector for the genomic integration in L. lactis NZ9000. Single- and double-crossover mutants were identified by genotype and phenotype. Relative to episomal transformants of L. lactis, the level of expression of the heterologous protein after nisin induction was similar in the crossover mutants, suggesting that a single copy of the heterologous gene can be used to produce the protein of interest.

Bacterial Proteins↗
PubMed2004

Influence of lipoteichoic acid D-alanylation on protein secretion in Lactococcus lactis as revealed by random mutagenesis.

Lactococcus lactis, a food-grade nonpathogenic lactic acid bacterium, is a good candidate for the production of heterologous proteins of therapeutic interest. We examined host factors that affect secretion of heterologous proteins in L. lactis. Random insertional mutagenesis was performed with L. lactis strain MG1363 carrying a staphylococcal nuclease (Nuc) reporter cassette in its chromosome. This cassette encodes a fusion protein between the signal peptide of the Usp45 lactococcal protein and the mature moiety of a truncated form of Nuc (NucT). The Nuc secretion efficiency (secreted NucT versus total NucT) from this construct is low in L. lactis (approximately 40%). Twenty mutants affected in NucT production and/or in secretion capacity were selected and identified. In these mutants, several independent insertions mapped in the dltA gene (involved in D-alanine transfer in lipoteichoic acids) and resulted in a NucT secretion defect. Characterization of the dltA mutant phenotype with respect to NucT secretion revealed that it is involved in a late secretion stage by causing mature NucT entrapment at the cell surface.

Alanine↗
PubMed2002

Production of human papillomavirus type 16 E7 protein in Lactococcus lactis.

The E7 protein of human papillomavirus type 16 was produced in Lactococcus lactis. Secretion allowed higher production yields than cytoplasmic production. In stationary phase, amounts of cytoplasmic E7 were reduced, while amounts of secreted E7 increased, suggesting a phase-dependent intracellular proteolysis. Fusion of E7 to the staphylococcal nuclease, a stable protein, resulted in a highly stable cytoplasmic protein. This work provides new candidates for development of viral screening systems and for oral vaccine against cervical cancer.

Cloning, Molecular↗
PubMed2002

Controlled production of stable heterologous proteins in Lactococcus lactis.

The use of Lactococcus lactis (the most extensively characterized lactic acid bacterium) as a delivery organism for heterologous proteins is, in some cases, limited by low production levels and poor-quality products due to surface proteolysis. In this study, we combined in one L. lactis strain use of the nisin-inducible promoter P(nisA) and inactivation of the extracellular housekeeping protease HtrA. The ability of the mutant strain, designated htrA-NZ9000, to produce high levels of stable proteins was confirmed by using the staphylococcal nuclease (Nuc) and the following four heterologous proteins fused or not fused to Nuc that were initially unstable in wild-type L. lactis strains: (i) Staphylococcus hyicus lipase, (ii) the bovine rotavirus antigen nonstructural protein 4, (iii) human papillomavirus antigen E7, and (iv) Brucella abortus antigen L7/L12. In all cases, protein degradation was significantly lower in strain htrA-NZ9000, demonstrating the usefulness of this strain for stable heterologous protein production.

Antigens, Bacterial↗
PubMed2001

Identification of Mur, an atypical peptidoglycan hydrolase derived from Leuconostoc citreum.

A gene encoding a protein homologous to known bacterial N-acetyl-muramidases has been cloned from Leuconostoc citreum by a PCR-based approach. The encoded protein, Mur, consists of 209 amino acid residues with a calculated molecular mass of 23,821 Da including a 31-amino-acid putative signal peptide. In contrast to most of the other known peptidoglycan hydrolases, L. citreum Mur protein does not contain amino acid repeats involved in cell wall binding. The purified L. citreum Mur protein was shown to exhibit peptidoglycan-hydrolyzing activity by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An active chimeric protein was constructed by fusion of L. citreum Mur to the C-terminal repeat-containing domain (cA) of AcmA, the major autolysin of Lactococcus lactis. Expression of the Mur-cA fusion protein was able to complement an acmA mutation in L. lactis; normal cell separation after cell division was restored by Mur-cA expression.

Amino Acid Sequence↗
PubMed2001

Bovine rotavirus nonstructural protein 4 produced by Lactococcus lactis is antigenic and immunogenic.

Rotavirus nonstructural protein 4 (NSP4) can induce diarrhea in mice. To get insight into the biological effects of NSP4, production of large quantities of this protein is necessary. We first tried to produce the protein in Escherichia coli, but the nsp4 gene proved to be unstable. The capacity of the generally regarded as safe organism Lactococcus lactis to produce NSP4 either intra- or extracellularly was then investigated by using the nisin-controlled expression system. Production of recombinant NSP4 (rNSP4) was observed in L. lactis for both locations. In spite of a very low secretion efficiency, the highest level of production was obtained with the fusion between a lactococcal signal peptide and rNSP4. Cultures of the rNSP4-secreting strain were injected into rabbits, and a specific immune response was elicited. The anti-rNSP4 antibodies produced in these rabbits recognized NSP4 in MA104 cells infected by rotavirus. We showed that L. lactis is able to produce antigenic and immunogenic rNSP4 and thus is a good organism for producing viral antigens.

Animals↗
PubMed2001

Signal peptide and propeptide optimization for heterologous protein secretion in Lactococcus lactis.

Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SP(Nuc)) by that of L. lactis protein Usp45 (SP(Usp)) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SP(Usp) than when directed by SP(Nuc). This SP(Usp) effect on Nuc secretion is not due to a better antifolding activity, since SP(Usp):Nuc precursor proteins display enzymatic activity in vitro, while SP(Nuc):Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SP(Usp) and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.

Amino Acid Sequence↗
PubMed2001

Induction of mucosal immune response after intranasal or oral inoculation of mice with Lactococcus lactis producing bovine beta-lactoglobulin.

The bovine beta-lactoglobulin (BLG) is a major cow's milk allergen. Here, we evaluated the immune response against BLG induced in mice, using the organism Lactococcus lactis, which has GRAS ("generally regarded as safe") status, as a delivery vehicle. The cDNA of the blg gene, encoding BLG, was expressed and engineered for either intra- or extracellular expression in L. lactis. Using a constitutive promoter, the yield of intracellular recombinant BLG (rBLG) was about 20 ng per ml of culture. To increase the quantity of rBLG, the nisin-inducible expression system was used to produce rBLG in the cytoplasmic and extracellular locations. Although the majority of rBLG remained in the cytoplasm, the highest yield (2 microg per ml of culture) was obtained with a secreting strain that encodes a fusion between a lactococcal signal peptide and rBLG. Whatever the expression system, the rBLG is produced mostly in a soluble, intracellular, and denatured form. The BLG-producing strains were then administered either orally or intranasally to mice, and the immune response to BLG was examined. Specific anti-BLG immunoglobulin A (IgA) antibodies were detected 3 weeks after the immunization protocol in the feces of mice immunized with the secreting lactococcal strain. Specific anti-BLG IgA detected in mice immunized with lactococci was higher than that obtained in mice immunized with the same quantity of pure BLG. No specific anti-BLG IgE, IgA, IgG1, or IgG2a was detected in sera of mice. These recombinant lactococcal strains constitute good vehicles to induce a mucosal immune response to a model allergen and to better understand the mechanism of allergy induced by BLG.

Administration, Intranasal↗
PubMed1999

Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system.

Lactic acid bacteria (LAB) are Gram-positive bacteria and are generally regarded as safe (GRAS) organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc) was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE) of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV) epitopeprotein fusion (BCV-Nuc). BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

Antigens↗
PubMed1998

A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis.

Lactococcus lactis, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins. We examined the secretion efficiency and capacity of L. lactis by using the Staphylococcus aureus nuclease (Nuc) as a heterologous model protein. When expressed in L. lactis from an efficient lactococcal promoter and its native signal peptide, only approximately 60% of total Nuc was present in a secreted form at approximately 5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency was reduced further to approximately 30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). We identified a modification which improved secretion efficiency of both native Nuc and NucT. A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site. In the case of Nuc, secretion efficiency was increased to approximately 80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to approximately 20 mg per liter). The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90%. Similarly, the secretion efficiency of a third protein, the alpha-amylase of Bacillus stearothermophilus, was also improved by LEISSTCDA. These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in L. lactis.

Amino Acid Sequence↗