RESUMO
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.
Assuntos
Lactococcus lactis/metabolismo , Proteínas de Bactérias , Proteínas de Transporte , Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Western Blotting , Testes de Sensibilidade Microbiana , Paecilomyces/efeitos dos fármacos , Plasmídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Trichophyton/efeitos dos fármacosRESUMO
Lactococcus lactis, the most extensively characterized lactic acid bacterium, is a mesophilic- and microaerophilic-fermenting microorganism widely used for the production of fermented food products. During industrial processes, L. lactis is often exposed to multiple environmental stresses (low and high temperature, low pH, high osmotic pressure, nutrient starvation and oxidation) that can cause loss or reduction of bacterial viability, reproducibility, as well as organoleptic and/or fermentative qualities. Among these stress factors, oxidation can be considered one of the most deleterious to the cell, causing cellular damage at both molecular and metabolic levels. During the last two decades, considerable efforts have been made to improve our knowledge of oxidative stress in L. lactis. Many genes involved with both oxidative stress resistance and control mechanisms have been identified; functionally they seem to overlap. The finding of new genes, and a better understanding of the molecular mechanisms of stress resistance in L. lactis and other lactic acid bacterium, will lead to the construction and isolation of stress-resistant strains. Such strains could be exploited for both traditional and probiotic uses
Assuntos
Estresse Oxidativo/fisiologia , Lactococcus lactis/metabolismo , Complexos Multienzimáticos/metabolismo , Estresse Oxidativo/genética , Genes Bacterianos/genética , Lactococcus lactis/genética , NADH NADPH Oxirredutases/metabolismo , Peroxidases/metabolismo , Recombinases Rec A/metabolismo , Sobrevivência Celular/genética , Superóxido Dismutase/metabolismoRESUMO
Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium). A promising application of L. lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines. The expression of heterologous proteins and antigens as well as the various delivery systems developed in L. lactis, and its use as an oral vaccine carrier are discussed
Assuntos
Animais , Vetores Genéticos , Lactococcus lactis/genética , Vacinas , Antígenos/genética , Antígenos/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Imunidade nas Mucosas , Lactococcus lactis/metabolismo , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Food-borne diseases are of major concern worldwide. To date, around 250 different food-borne diseases have been described, and bacteria are the causative agents of two thirds of food-borne disease outbreaks. Among the predominant bacteria involved in these diseases, Staphylococcus aureus is a leading cause of gastroenteritis resulting from the consumption of contaminated food. Staphylococcal food poisoning is due to the absorption of staphylococcal enterotoxins preformed in the food. Here, we briefly review the latest data on staphylococcal enterotoxins and some papers exemplifying the interactions between S. aureus and the food matrix; environmental factors affecting staphylococcal enterotoxin production are discussed