Influence of Lif, the lysostaphin immunity factor, on acceptors of surface proteins and cell wall sorting efficiency in Staphylococcus carnosus

Abstract

Proteins harboring a C-terminal cell wall sorting signal are covalently linked to pentaglycine acceptors within the staphylococcal peptidoglycan. This pentaglycine was modified when the lysostaphin immunity factor (Lif) of Staphylococcus simulans was expressed in Staphylococcus carnosus, likely by the exchange of two glycine residues for serine residues. A reporter protein was efficiently linked to the modified acceptor, indicating that the sorting reaction is not strictly dependent on the wild-type structures of the acceptors.

FIG. 1

Structures of reporter proteins and the staphylococcal peptidoglycan. (A) Schematic diagrams showing the domains of the S. hyicus lipase and proLipFnBPB, a reporter enzyme for cell wall anchoring, consisting of the cleavable signal peptide (SP), the propeptide (Pro), and the catalytic domain (Lipase) of S. hyicus lipase fused to the C-terminal region of the S. aureus fibronectin binding protein B (FnBPB′). FnBPB′ comprises the complete cell wall-spanning region and the cell wall sorting signal of the binding protein. (B) Structure of staphylococcal peptidoglycan with a C-terminally processed surface protein attached to the lysostaphin-sensitive wild-type pentaglycine acceptor of a branched anchor peptide. Cleavage sites of cell wall lytic enzymes used in this study are indicated (modified after references and 18).

FIG. 2

Lysostaphin sensitivity of branched anchor peptides solubilized with muramidase Ch from the cell wall of S. carnosus. Cells synthesizing proLipFnBPB encoded on plasmid pTX30 in the presence (+) or absence (−) of Lif (pCXlif) were washed, trichloroacetic acid-precipitated, and digested with muramidase Ch. Subsequently, the solubilized hybrid proteins were incubated with (+) or without (−) lysostaphin prior to Tricine–SDS-PAGE (10% acrylamide) and immunoblotting with prolipase-specific antiserum as described previously (15). The molecular masses of standard proteins (in kDa) are indicated on the left.

FIG. 3

Lysostaphin sensitivity of proLipFnBPB naturally released by S. carnosus into the culture supernatant during growth. Culture supernatants derived from clones synthesizing proLipFnBPB (pTX30) in the presence (+) or absence (−) of Lif (pCXlif) were collected and incubated with (+) or without (−) lysostaphin. As a reference, proLipFnBPB released with lysostaphin from the cell wall of S. carnosus harboring only pTX30 was included. Proteins were separated by Tricine–SDS-PAGE (10% acrylamide) and immunoblotted with prolipase-specific antiserum. The molecular masses of standard proteins (in kDa) are indicated on the left.