ABSTRACT
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.
Subject(s)
Lactococcus lactis/metabolism , Bacterial Proteins , Carrier Proteins , Antifungal Agents/isolation & purification , Antifungal Agents/metabolism , Antifungal Agents/pharmacology , Aspergillus fumigatus/drug effects , Blotting, Western , Microbial Sensitivity Tests , Paecilomyces/drug effects , Plasmids/genetics , Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Carrier Proteins/genetics , Carrier Proteins/pharmacology , Trichophyton/drug effectsABSTRACT
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