RESUMO
In this study, Lactococcus lactis was engineered to express mutated internalin A on its surface and to secrete large amounts of listeriolysin O (LLO) in order to improve its potential as a vehicle for DNA vaccination. Western blotting experiments demonstrated that the bacterium expressed LLO in both the cytoplasmic and extracellular compartments, with higher quantities found in the culture supernatants. A hemolytic assay showed that the recombinant strain secreted 250 ng active LLO/mg total protein. This mInlA/LLO-producing strain of L. lactis may be used as an alternative tool in DNA vaccination against a number of infectious diseases or in cancer therapy.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/imunologia , Lactococcus lactis/genética , Listeria monocytogenes/imunologia , Mutação , Proteínas Recombinantes , Vacinas Bacterianas , Membrana Celular/metabolismo , Expressão Gênica , Hemólise , Lactococcus lactis/metabolismo , VacinaçãoRESUMO
Allergic diseases affect up to 30% of the western population, and their prevalence is increasing. Probiotics are able to modulate the mucosal immune response, and clinical trials demonstrated that specific strains, especially lactic acid bacteria (LAB) ones, reduce allergic symptoms. Moreover, the use of recombinant probiotics has been evaluated as possible strategies for the immunotherapy of allergic diseases. The production and delivery of allergens by recombinant LAB in concert with their ability to induce a Th1-type immune response have been shown to be a promising mucosal vaccination strategy in mouse model. The aim of this article is to review the applications of probiotics in allergy immunotherapy with a special focus on recombinant LAB delivering proteins or DNA.
Assuntos
Hipersensibilidade/terapia , Imunoterapia , Probióticos/uso terapêutico , Alérgenos/genética , Alérgenos/imunologia , Animais , Bifidobacterium/genética , DNA/administração & dosagem , Humanos , Hipersensibilidade/imunologia , Imunidade nas Mucosas , Lactobacillus/genética , Camundongos , Hipersensibilidade a Leite/terapia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologiaRESUMO
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.
Assuntos
Lactococcus lactis/metabolismo , Lactoglobulinas/biossíntese , Nuclease do Micrococo/metabolismo , Oligopeptídeos/metabolismo , Animais , Bovinos , Modelos Animais de Doenças , Lactococcus lactis/imunologia , Lactoglobulinas/imunologia , Camundongos , Nuclease do Micrococo/imunologia , Hipersensibilidade a Leite/imunologia , Oligopeptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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 æg/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.