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 [1895][1896][1897][1898][1899][1900][1901][1902][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.

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.Lactococcus lactis is a gram-positive lactic acid bacterium that is widely used in the production of fermented food products, and as such, it is considered a food-grade microorganism. Experimental data and genomic analyses indicate that only a few proteins are naturally secreted in L. lactis (4,32,38), and a plasmid-free strain does not produce the extracytoplasmic protease PrtP (13). These features have drawn the attention of researchers to the potential use of L. lactis for secretion of proteins of biotechnological interest. Thus, L. lactis has been extensively engineered for production and export of heterologous proteins with high added value, such as antigens or enzymes (2, 6, 8-12, 20, 22, 23, 31, 35). For this purpose, several genetic tools have been developed for L. lactis, and the potential of this organism as a prokaryotic host for heterologous protein production has been confirmed (7,9,23,40).Systems that allow controlled levels of expression of foreign proteins in L. lactis may offer certain advantages over constitutive systems (8). The nisin-controlled expression (NICE) system (7, 18), based on a combination of the P nisA promoter and the nisRK regulatory genes, has proven to be highly versatile (8,17,18) and has already been used to express different heterologous proteins (2,6,10,11,35).Protein export to the cell surface or into the medium is a preferred means of protein expression for several biotechnological applications (9, 23). However, poor expression and proteolytic degradation of heterologous proteins are limiting factors for stable protein production in bacteria. In Escherichia coli and Bacillus subtilis, several exported proteases that are associated with turnover of both natural and foreign proteins have been described (15,25,26,30,37). In contrast to E. coli and B. subtilis, L. lactis has a unique extracellular housekeeping protease, HtrA (high-temperature requirement), as demonstrated by construction of an L. lactis htrA-IL1403 mutant strain (previously designated htrA [33...

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 g 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.The gastrointestinal tract is constantly exposed to substantial amounts of food and bacterial components. In the healthy gut, the immune system is able to create a balance between mucosal immunity and systemic tolerance. In food allergy, this balance is impaired and oral tolerance to dietary antigen is not achieved or maintained (14,19,29). Cow's milk allergy is an important problem in infants because it affects 1.9 to 2.8% of infants in the first 2 years of life in various countries of northern Europe (17, 18). Beta-lactoglobulin (BLG) is the most abundant protein of the whey fraction of milk and is regarded as a dominant allergen together with casein (37). In our laboratory, we use BLG as a model protein to evaluate and modulate the immune response to a food allergen in mice, and we have produced both anti-BLG monoclonal antibodies (MAb) (25) and recombinant BLG in Escherichia coli (8). Moreover, the BLG structure is well documented (27); it is a 162-aminoacid globular protein which contains two intramolecular disulfide bonds. In this study, we produced BLG in Lactococcus lactis, a food-grade bacterium, and used these recombinant strains to measure a potential specific immune response after intranasal or oral administration in mice.L. lactis is a gram-positive lactic acid bacterium that is nonpathogenic, noninvasive, and noncolonizing and that has GRAS ("generally regar...

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.Rotavirus is the major etiologic agent of severe diarrhea in infants and young children around the world (6). Rotavirus infects mature villus enterocytes in the small intestine. Nonstructural protein 4 (NSP4), encoded by gene 10, has been shown to be an intracellular receptor for double-layered particles. Purified NSP4 and a 22-amino-acid peptide (amino acids 114 to 135) were both capable of inducing dose-related diarrhea after intraperitoneal or intraduodenal administration to 6-to 10-day-old mice (1). NSP4 is thought to act as an enterotoxin which triggers chloride secretion by a calcium-dependent signal transduction pathway. To study the biological properties of NSP4, it is necessary to produce large quantities of NSP4 protein. Previously, Estes et al. produced and purified fulllength NSP4 from Spodoptera frugiperda 9 cells infected with a recombinant baculovirus expressing rotavirus gene 10 of strain SA11 (22). Newton et al. (12) produced part of NSP4 (amino acids 86 to 175) as a fusion with a 36-kDa domain of glutathione S-transferase.Developing efficient gene expression and protein secretion systems in nonpathogenic gram-positive lactic acid bacteria is an original approach for producing proteins of therapeutic interest (15, 25) and a new strategy for rotavirus vaccination. These lactic acid bacteria possess many properties which make them good candidates for oral vaccination purposes; e.g., they have generally regarded as safe status or adjuvant properties (15). They have already been used to produce several bacterial antigens and interleukins (19). Some viral antigens or parts of viral antigens have been produced in lactic acid bacteria; antigen M6-gp41E has been produced in Lactobacillus plantarum (5), a fragment of the human immunodeficiency virus type 1 envelope protein has been produced in Streptococcus gordonii (16), 250 amino acids of rotavirus protein VP7 have been produced in L. plantarum, an epitope of foot-and-mouth disease virus protein VP1 (amin...

A new controlled production system to target heterologous proteins to cytoplasm or extracellular medium is described for Lactococcus lactis NCDO2118. It is based on the use of a xylose-inducible lactococcal promoter, P(xylT). The capacities of this system to produce cytoplasmic and secreted proteins were tested using the Staphylococcus aureus nuclease gene (nuc) fused or not to the lactococcal Usp45 signal peptide. Xylose-inducible nuc expression is tightly controlled and resulted in high-level and long-term protein production, and correct targeting either to the cytoplasm or to the extracellular medium. Furthermore, this expression system is versatile and can be switched on or off easily by adding either xylose or glucose, respectively. These results confirm the potential of this expression system as an alternative and useful tool for the production of proteins of interest in L. lactis.

A system for controlled targeting of heterologous protein was developed in the food-grade bacterium Lactococcus lactis. It is composed of the L. lactis strain NZ9000 and of two broad host range expression vectors pCYT:Nuc and pSEC:Nuc for, respectively, cytoplasmic and secreted staphylococcal nuclease (Nuc) nisin-inducible production. The level of intracellular production of Nuc measured with pCYT:Nuc (3 mg l 31 ) is significantly lower than the one obtained with pSEC:Nuc (V20 mg l 31 ). The secretion efficiency (SE) of Nuc is estimated to be V70%, corresponding to V15 mg of secreted Nuc l 31 . Furthermore, we established that Nuc production continued in L. lactis 10 h after a 1-h nisin-pulse induction. This system was then used for intra-and extracellular production of a protein of therapeutical interest in L. lactis, the ovine interferon-omega (IFN-g). The SE and the quantity of secreted active IFN-g were evaluated respectively to be V70% and V1 mg l 31 (Vtwo-fold higher than the cytoplasmic form). ß

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 (ϳ40%). 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.Lactococcus lactis is widely used in the food industry and is considered a good candidate for production of heterologous proteins for developing nutraceuticals or new live vaccine strategies. Numerous genetic tools have been developed for gene expression and protein secretion in L. lactis (14), and many heterologous proteins have already been produced in L. lactis, including bacterial and viral antigens (3, 39, 48) and enzymes and cytokines (2, 43). Recently, efficient food-grade production systems were developed in L. lactis (19,42). However, so far there have been few analyses of lactococcal host factors involved in heterologous protein production and secretion machinery.Factors that affect secretion efficiency (secreted NucT versus total NucT) include elements of the secretion machinery itself, as well as factors involved in protein stability or degradation and folding. Furthermore, conditions that alter the microenvironment at the cell surface may affect protein folding and release into the culture medium (29,45). For the L. lactis secretion machinery, knowledge of the lactococcal genetic apparatus required for secretion is essentially limited to genome sequence information derived from L. lactis strain IL1403 (7). Several sec genes were revealed by sequence homology (7). Other putative and/or unidentified lactococcal genes may also be involved in the secretion process. It is notable that SecDF, which is involved in the late steps of translocation, is absent in L. lactis. Also, L. lactis has only a single signal peptidase for nonlipoproteins, compared to six such enzymes for the grampositive paradigm organism Bacillus subtilis (46). The unique signal peptidase and the absence of SecDF are potential bottlenecks when an L. lactis strain is engineered for high-level secretion. L. lactis encodes a unique cell surface protease, HtrA. An htrA mutant stabilizes production of several heterologous exported proteins or...

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 ß-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 (~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 ~8 µg/ml (~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.