The M6 protein of Streptococcus pyogenes and its potential as a tool to anchor biologically active molecules at the surface of lactic acid bacteria.

Abstract

Much attention is currently devoted to the development of tools enabling the presentation of proteins at the surface of Gram-positive bacteria. Successful anchoring of antigens (9), immunoglobulins (4) and enzymes (15) in various hosts including Staphylococcus carnosus (4, 15), Staphylococcus xylosus (5) and Streptococcus gordoni (9) was recently reported. The carrier proteins used in those studies were portions of cell-wall anchored protein A of Staphylococcus aureus (4, 5, 15) or the M6 protein of Streptococcus pyogenes (9). We believe that this kind of system can offer exciting applications in various fields of biotechnology using lactic acid bacteria (LAB) as a vector. LAB are present in large numbers (ca. 109/g) in fermented foods such as cheese and yogurt and some are able to survive the oral route down to the intestine. Some, such as Lactobacillus fermentum are also part of the flora which colonizes the gastrointestinal tract of man and animals. LAB are therefore interesting candidates whose ability to deliver specific biological activities along the gastrointestinal tract should be tested. The carrier molecule chosen is the M6 protein. The M6 molecule is one of the best characterized cell-wall anchored proteins among a family of about 60 such molecules described in Gram-positive bacteria. These proteins all harbour a rather similar C-terminal tail of ca. 32 aminoacids involved in the anchoring process. This anchoring signal is composed of a LPXTG motif followed by a hydrophobic domain and a charged C-terminus. A recent model of anchoring suggests that the surface proteins are first exported across the membrane by a Sec-dependent process. The C-terminal hydrophobic tail acts as a stop transfer sequence within the membrane and positions the LPXTG motif so that it can be cleaved by a so-called sortase machinery (12). To examine whether this anchoring process is efficient in LAB, we present here the cloning and expression in LAB of the emm6 gene for the M6 protein and we report the levels of M6 anchored to and displayed on the cell-wall. We also analyzed the ability of the M6 leader peptide and cell-wall anchor to respectively export and anchor a heterologous enzyme.