Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

Abstract

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine ␤-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding fulllength PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB٢, PrtB٢-BLG, and PrtB٢-T6) or 807 (PrtB⌬) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB٢-BLG yielded up to 170 g per 10 9 CFU in the culture supernatant and 9 g per 10 9 CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue.An inappropriate immunological response to bovine milk proteins, including ␤-lactoglobulin (BLG), has been put forward as a key factor in cow's milk allergy (34). Studies have shown that a reequilibration of this response toward a state of specific oral tolerance can be achieved upon oral administration of partially hydrolyzed cow's milk formula or BLG peptides obtained by tryptic hydrolysis (8,22). Interestingly, the degradation of cow's milk proteins by lactic acid bacteria (LAB) used in food fermentation was also found to generate tolerogenic peptides, with a suppression of the specific lymphoproliferative response (32). In addition, certain LAB strains are more and more widely used as a vehicle for the delivery of heterologous proteins to the mucosal immune system (13,20). Therefore, it can be hypothesized that the introduction of LAB capable of expressing full-length or BLG fragments may prove them to be a useful tool to aid in host protection against allergic sensitization to this dietary antigen.Lactococcus lactis is a gram-positive, nonpathogenic bacterium that is widely used in industrial food fermentation. A noncolonizing bacterium, L. lactis has been shown to be quite resistant to gastric acidity when administered together with food, remaining metabolically active all the way through the digestive tract (3). Its use as an antigen delivery vehicle for mucosal immunization has been reported (1,4,26,31). Induction of oral tolerance might require a large amount of target antigen, in contrast to immune modulators such as cytokines (30,31). Therefo...

Characterization of aLactococcus lactisStrain That Secretes a Major Epitope of Bovine Beta-Lactoglobulin and Evaluation of Its Immunogenicity in Mice

“…lactis was already used to produce entire Blg protein, and recombinant strains were shown to be immunogenic after intranasal and oral administration in mice (7). More recently, Bernasconi et al (5) showed that the Lactobacillus bulgaricus exported protease PrtB could enhance the export of both entire Blg and a poorly antigenic Blg peptide in L. lactis. Here, we constructed an L. lactis strain producing Blg41-60::Nuc, a recombinant fusion protein between Blg41-60 and the mature part of the staphylococcal nuclease (Nuc) (26), by use of the nisin-inducible expression system (9).…”

Determination of the Domain of the Lactobacillus delbrueckii subsp. bulgaricus Cell Surface Proteinase PrtB Involved in Attachment to the Cell Wall after Heterologous Expression of the prtB Gene in Lactococcus lactis

“…Proteins of interest were already genetically inserted in the catalytic domain of the proteinase PrtB. This system was successfully used for the expression of different antigens, such as beta-lactoglobulin and immunoglobulin E mimotopes, as tools for the modulation of tolerance and allergy (1,32). …”

“…A fragment comprising amino acids 280 to 467 of L. bulgaricus mature cell wall proteinase was overexpressed in Escherichia coli and used for production of antiserum in rabbits (1).…”

Surface display of respiratory syncytial virus glycoproteins in Lactococcus lactis NZ9000

“…Appropriate combinations of promoter, signal sequence and anchoring signal sequences under suitably efficient promoters are required for satisfactory surface expression in the lactic acid bacteria (Bernasconi et al. 2002).…”