Design of a protein-targeting system for lactic acid bacteria

We designed an expression and export system that enabled the targeting of a reporter protein (the staphylococcal nuclease Nuc) to specific locations i n Lactococcus lactis cells, i.e., cytoplasm, cell wall, or medium. Optimiza tion of protein secretion and of protein cell wall anchoring was performed with L. lactis cells by modifying the signals located at the N and C termin i, respectively, of the reporter protein. Efficient translocation of precur sor (similar to 95%) is obtained using the signal peptide from the lactococ cal Usp45 protein and provided that the mature protein is fused to overall anionic amino acids at its N terminus; those residues prevented interaction s of Nuc with the cell envelope. Nuc could be covalently anchored to the pe ptidoglycan by using the cell wall anchor motif of the Streptococcus pyogen es M6 protein. However, the anchoring step proved to not be totally efficie nt in L. lactis, as considerable amounts of protein remained membrane assoc iated. Our results may suggest that the defect is due to limiting sortase i n the cell. The optimized expression and export vectors also allowed secret ion and cell wall anchoring of Nuc in food-fermenting and commensal strains of Lactobacillus. In all strains tested, both secreted and cell wall-ancho red Nuc was enzymatically active, suggesting proper enzyme folding in the d ifferent locations. These results provide the first report of a targeting s ystem in lactic acid bacteria in which the final location of a protein is c ontrolled and biological activity is maintained.