Hung Ton-That

Sortase-catalysed anchoring of surface proteins to the cell wall of Staphylococcus aureus

Many surface proteins of Gram‐positive bacteria are anchored to the cell wall envelope by a transpeptidation mechanism, requiring a C‐terminal sorting signal with a conserved LPXTG motif. Sortase, a membrane protein of Staphylococcus aureus, cleaves polypeptides between the threonine and the glycine of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl‐group of threonine and the amino‐group of peptidoglycan cross‐bridges. S. aureus mutants lacking the srtA gene fail to anchor and display some surface proteins and are impaired in the ability to cause animal infections. Sortase acts on surface proteins that are initiated into the secretion (Sec) pathway and have their signal peptide removed by signal peptidase. The S. aureus genome encodes two sets of sortase and secretion genes. It is conceivable that S. aureus has evolved more than one pathway for the transport of 20 surface proteins to the cell wall envelope.

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

Surface proteins of Gram-positive bacteria play important roles during the pathogenesis of human infections and require sortase for anchoring to the cell-wall envelope. Sortase cleaves surface proteins at the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine (T) and the amino group of cell-wall crossbridges. The NMR structure of sortase reveals a unique β-barrel structure, in which the active-site sulfhydryl of cysteine-184 is poised for ionization by histidine-120, presumably enabling the resultant thiolate to attack the LPXTG peptide. Calcium binding near the active site stimulates catalysis, possibly by altering the conformation of a surface loop that recognizes newly translocated polypeptides. The structure suggests a mechanistic relationship to the papain/cathepsin proteases and should facilitate the design of new antiinfective agents.

Multiple Enzymatic Activities of the Murein Hydrolase from Staphylococcal Phage φ11 IDENTIFICATION OF A d-ALANYL-GLYCINE ENDOPEPTIDASE ACTIVITY

Bacteriophage muralytic enzymes degrade the cell wall envelope of staphylococci to release phage particles from the bacterial cytoplasm. Murein hydrolases of staphylococcal phages φ11, 80α, 187, Twort, and φPVL harbor a central domain that displays sequence homology to knownN-acetylmuramyl-l-alanyl amidases; however, their precise cleavage sites on the staphylococcal peptidoglycan have thus far not been determined. Here we examined the properties of the φ11 enzyme to hydrolyze either the staphylococcal cell wall or purified cell wall anchor structures attached to surface protein. Our results show that the φ11 enzyme has d-alanyl-glycyl endopeptidase as well asN-acetylmuramyl-l-alanyl amidase activity. Analysis of a deletion mutant lacking the amidase-homologous sequence, φ11(Δ181–381), revealed that the d-alanyl-glycyl endopeptidase activity is contained within the N-terminal 180 amino acid residues of the polypeptide chain. Sequences similar to this N-terminal domain are found in the murein hydrolases of staphylococcal phages but not in those of phages that infect other Gram-positive bacteria such as Listeria or Bacillus.

Anchor structure of staphylococcal surface proteins. IV. Inhibitors of the cell wall sorting reaction.

Surface proteins of Staphylococcus aureus are covalently linked to the bacterial cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. Cleavage between the threonine and the glycine of the LPXTG motif liberates the carboxyl of threonine to form an amide bond with the amino of the pentaglycine cross-bridge in the staphylococcal peptidoglycan. We asked whether antibiotic cell wall synthesis inhibitors interfere with the anchoring of surface proteins. Penicillin G, a transpeptidation inhibitor, had no effect on surface protein anchoring, whereas vancomycin and moenomycin, inhibitors of cell wall polymerization into peptidoglycan strands, slowed the sorting reaction. Cleavage of surface protein precursors did not require a mature assembled cell wall and was observed in staphylococcal protoplasts. A search for chemical inhibitors of the sorting reaction identified methanethiosulfonates andp-hydroxymercuribenzoic acid. Thus, sortase, the enzyme proposed to cleave surface proteins at the LPXTG motif, appears to be a sulfhydryl-containing enzyme that utilizes peptidoglycan precursors but not an assembled cell wall as a substrate for the anchoring of surface protein.

Anchor Structure of Staphylococcal Surface Proteins A BRANCHED PEPTIDE THAT LINKS THE CARBOXYL TERMINUS OF PROTEINS TO THE CELL WALL

Surface proteins of Staphylococcus aureus are anchored to the cell wall by a mechanism requiring a COOH-terminal sorting signal. Previous work demonstrated that the sorting signal is cleaved at the conserved LPXTG motif and that the carboxyl of threonine (T) is linked to the staphylococcal cell wall. By employing different cell wall lytic enzymes, surface proteins were released from the staphylococcal peptidoglycan and their COOH-terminal anchor structure was revealed by a combination of mass spectrometry and chemical analysis. The results demonstrate that surface proteins are linked to a branched peptide (NH2-Ala-γ-Gln-Lys-(NH2-Gly5)-Ala-COOH) by an amide bond between the carboxyl of threonine and the amino of the pentaglycine cross-bridge that is attached to the ε-amino of lysyl. This branched anchor peptide is amide-linked to the carboxyl ofN-acetylmuramic acid, thereby tethering the COOH-terminal end of surface proteins to the staphylococcal peptidoglycan.

Anchor structure of staphylococcal surface proteins: III. Role of the femA, femB, and femX factors in anchoring surface proteins to the bacterial cell wall

Surface proteins of Staphylococcus aureus are covalently linked to the bacterial cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. Cleavage between the threonine and the glycine of the LPXTG motif liberates the carboxyl of threonine to form an amide bond with the pentaglycyl cross-bridge in the staphylococcal peptidoglycan. Here, we asked whether altered peptidoglycan cross-bridges interfere with the sorting reaction and investigated surface protein anchoring in staphylococcalfem mutants. S. aureus strains carrying mutations in the femA, femB, femAB, or the femAX genes synthesize altered cross-bridges, and each of these strains displayed decreased sorting activity. Characterization of cell wall anchor structures purified from thefem mutants revealed that surface proteins were linked to cross-bridges containing one, three, or five glycyl residues, but not to the ε-amino of lysyl in muropeptides without glycine. When tested in a femAB strain synthesizing cross-bridges with mono-, tri-, and pentaglycyl as well as tetraglycyl-monoseryl, surface proteins were found anchored mostly to the five-residue cross-bridges (pentaglycyl or tetraglycyl-monoseryl). Thus, although wild-type peptidoglycan appears to be the preferred substrate for the sorting reaction, altered cell wall cross-bridges can be linked to the COOH-terminal end of surface proteins.

Anchor structure of staphylococcal surface proteins: II. COOH-terminal structure of muramidase and amidase-solubilized surface protein

Surface proteins of the Gram-positive organismStaphylococcus aureus are anchored to the bacterial cell wall by a transpeptidation mechanism during which the polypeptide is cleaved between the threonine (T) and the glycine (G) of the LPXTG motif. The carboxyl of threonine is subsequently amide linked to the amino of the pentaglycyl cross-bridge within the staphylococcal peptidoglycan. Previous work examined the anchor structure of surface proteins solubilized from the peptidoglycan by treatment with lysostaphin or φ11 hydrolase and identified COOH-terminally linked triglycyl orl-Ala-d-iGln-l-Lys(Gly5)-d-Ala and MurNAc-[l-Ala-d-iGln-l-Lys(Gly5)-d-Ala](β1–4)-GlcNAc, respectively. Here, we report the anchor structure of surface proteins solubilized with N-acetylmuramidase andN-acetylmuramyl-l-alanine amidase.N-Acetylmuramidase-released surface protein was linked to MurNAc-[l-Ala-d-iGln-l-Lys(Gly5)-d-Ala](β1–4)-GlcNAc, whereas N-acetylmuramyl-l-alanine amidase treatment of the cell wall solubilized surface proteins linked tol-Ala-d-iGln-l-Lys(Gly5)-d-Ala. Most, but not all, anchor structures were cross-linked to other cell wall subunits, in which the d-alanyl at position four was amide linked to the pentaglycyl of a neighboring wall peptide.