Anchor structure of cell wall surface proteins in Listeria monocytogenes

Many surface proteins of Gram-positive bacteria are anchored to the cell wa ll by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. In Staphylococcus aureus, surface proteins are cleaved betwee n the threonine and the glycine of the LPXTG motif. The carboxyl of threoni ne is subsequently amide linked to the amino group of the pentaglycine cell wall crossbridge. Here we investigated the anchor structure of surface pro teins in Listeria monocytogenes. A methionine and six histidines (MH6) were inserted upstream of the LPXTG motif of internalin A (InlA), a cell-wall-a nchored surface protein of L. monocytogenes. The engineered protein InlA-MH 6-Cws was found anchored in the bacterial cell wall. After peptidoglycan di gestion with phage endolysin, InlA-MH6-Cws was purified by affinity chromat ography. COOH-terminal peptides of InlA-MH6-Cws were obtained by cyanogen b romide cleavage followed by purification on a nickel-nitriloacetic acid col umn. Analysis of COOH-terminal peptides with Edman degradation and mass spe ctrometry revealed an amide linkage between the threonine of the cleaved LP XTG motif and the amino group of the nz-diaminopimelic acid crossbridge wit hin the listerial peptidoglycan. These results reveal that the cell wall an choring of surface proteins in Gram-positive bacteria such as S. aureus and L. monocytogenes occurs by a universal mechanism.