Constitutive Delivery of Bovine β-Lactoglobulin to the Digestive Tracts of Gnotobiotic Mice by EngineeredLactobacillus casei

Abstract

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 ␤-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.Food allergy affects 1 to 2% of adults and 5 to 8% of children in Western countries (25). With an incidence of 1.9 to 2.8%, cow's milk allergy is the most common allergy in early infancy (13). Patients may be sensitized to various proteins, mainly ␤-lactoglobulin (BLG) and caseins (39). Food allergy generally corresponds to an inappropriate immune response characterized by disruption of the Th1/Th2 balance toward a Th2 profile that results in the production of immunoglobulins E (IgE) specific for food antigens. Th2 cells produce interleukin-4 (IL-4), IL-5, and IL-13, whereas the Th1 response is characterized by gamma interferon (IFN-␥) and IL-12 synthesis. The Th1 and Th2 responses inhibit each other's development and function via the cytokines produced (26).Development of allergy is multifactorial, and it includes genetic factors and also different environmental factors, such as lifestyle and the intestinal microbiota. The intestinal microbiota seems to be critical because of its role in the postnatal maturation of the immune system. At birth, the digestive tract is sterile and the neonatal immune response is characterized by a polarized Th2 cytokine profile. During postnatal gut colonization, the gut immune system is exposed to a wide range of bacterial antigens, which apparently play a major role in driving the initial Th2-skewed immune response toward a more finely...

Induction of Immune Responses in Mice after Oral Immunization with RecombinantLactobacillus caseiStrains Expressing EnterotoxigenicEscherichia coliF41 Fimbrial Protein

“…The recombinant live oral vaccines expressing pathogen-derived antigens on the surfaces of bacteria can be an alternative method for providing protection because ETEC replicate mainly in the villi of the small intestines. Mucosal immunization offers a number of advantages over other routes of antigen delivery, including convenience, cost effectiveness, and the induction of both local and systemic immune responses (7,9,10,12,23,41). The goal is to provide the first line of defense by effectively eliminating pathogens at the mucosal surface.…”

“…For mucosal immunization, lactic acid bacteria (LAB) are more attractive as delivery vehicles than other live vaccine vectors (e.g., Shigella, Salmonella, and Listeria spp.) (1,3,20,21) because LAB are considered safe, they exhibit adjuvant properties, and they are weakly immunogenic (7,9,10,12,23,41). In addition, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular (18).…”

Efficient production and secretion of bovine β-lactoglobulin by Lactobacillus casei

“…While in L. lactis the optimal secretion was obtained with the LEISS-BLG form, the highest level of secretion in L. casei was reached with the LEISS-Nuc-BLG construct. Use of the two-plasmid NICE system resulted also in a 500-fold increase in BLG production compared to the level obtained with recombinant L. casei strains possessing one copy of the blg gene into their chromosome [14]. However, Western blot analysis of Nuc production (Fig.…”

“…We previously described the construction of L. casei strains carrying a chromosomal BLG expression cassette inserted downstream an endogenous constitutive promoter [14]. Such chromosomal insertions led to BLG yields reaching ~2 μg/L of culture.…”

Secretion of Biologically Active Heterologous Oxalate Decarboxylase (OxdC) inLactobacillus plantarumWCFS1 Using Homologous Signal Peptides

“…Genetically modified LAB such as Lactococcus lactis have been used for delivery of interleukin-2, -6, and -10 to combat murine colitis [15, 16]. Lactobacillus casei has been used for the delivery of β -lactoglobulin in the digestive tract of mice [17]. Similarly, modified Lactobacillus was shown to protect against dental caries in rat [18].…”