Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria

Abstract

The M6 protein from Streptococcus pyogenes is the best-characterized member of a family of cell envelopeassociated proteins. Based on the observation that the C-terminal sorting signals of these proteins can drive cell wall anchoring of heterologous unanchored proteins, we have cloned and expressed the emm6 structural gene for the M6 protein in various lactic acid bacteria (LAB). The emm6 gene was successfully expressed from lactococcal promoters in several Lactococcus lactis strains, an animal-colonizing Lactobacillus fermentum strain, Lactobacillus sake, and Streptococcus salivarius subsp. thermophilus. The M6 protein was efficiently anchored to the cell wall in all strains tested. In lactobacilli, essentially all detectable M6 protein was cell wall associated. These results suggest the feasibility of using the C-terminal anchor moiety of M6 for protein surface display in LAB.Surface presentation of heterologous molecules in grampositive bacteria is of increasing interest for applications in various fields of biotechnology. Major achievements concerning the surface display of heterologous antigens (25, 33), immunoglobulins (19), and enzymes (40) were recently reported. The absence of an outer membrane in gram-positive bacteria makes them particularly attractive for use in the exposure of bioactive molecules to the extracellular compartment.Lactic acid bacteria (LAB) constitute a family of gram-positive bacteria which are extensively used in the fermentation of raw agricultural products and in the manufacture of a wide variety of food products (5). The burst of information concerning LAB genetics and metabolism, as well as the development of expression and secretion tools, has opened the door for new (non)alimentary applications of these bacteria, such as those described above (8,29). The use of LAB as in vivo delivery vectors for biologically active molecules (e.g., antigens, enzymes, or biological peptides) is made attractive by their nonpathogenicity and ability to survive passage along an oral route down to the intestine. The fulfillment of this project necessitates a delivery system comprising a vehicule (in this case, a LAB species) and a system to present molecules at the cell surface. To do this, we examined the cell wall-anchoring potential of the M6 protein of Streptococcus pyogenes. M6 (49 kDa) is among the best characterized of the cell wall-anchored proteins and has already been successfully used to drive cell wall anchoring of recombinant fusion proteins to the surface of Streptococcus gordonii (25). More than 60 cell wall-anchored proteins have been identified in gram-positive organisms, and 2 such proteins, a cell wall proteinase and a clumping factor, were characterized in the model LAB, Lactococcus lactis (11,17,22). All these proteins share a rather similar C-terminal anchoring tail of about 35 amino acids, suggesting that the anchoring mechanism is conserved in gram-positive organisms. The anchoring structure includes an LPXTG motif followed by a stretch of about 23 hydrophobic amino acids a...

Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

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SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion

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Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4

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