Staphylococcus aureus Sortase, an Enzyme that Anchors Surface Proteins to the Cell Wall

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Science  30 Jul 1999:
Vol. 285, Issue 5428, pp. 760-763
DOI: 10.1126/science.285.5428.760


Surface proteins of Gram-positive bacteria are linked to the bacterial cell wall by a mechanism that involves cleavage of a conserved Leu-Pro-X-Thr-Gly (LPXTG) motif and that occurs during assembly of the peptidoglycan cell wall. A Staphylococcus aureus mutant defective in the anchoring of surface proteins was isolated and shown to carry a mutation in the srtA gene. Overexpression of srtA increased the rate of surface protein anchoring, and homologs of srtA were found in other pathogenic Gram-positive bacteria. The protein specified bysrtA, sortase, may be a useful target for the development of new antimicrobial drugs.

Hospital isolates ofStaphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis have become resistant to most, if not all, known therapeutic regimens (1). Many antibiotics, including penicillin and its derivatives, target the transpeptidation reaction of bacterial cell wall synthesis, which cross-links peptidoglycan strands (2). To search for other cell wall synthesis reactions that may serve as targets for antimicrobial therapy, we have focused on the anchoring of surface proteins to the peptidoglycan of Gram-positive bacteria.

Surface proteins not only promote interaction between the invading pathogen and animal tissues, but also provide ingenious strategies for bacterial escape from the host's immune response (3). In the case of S. aureus protein A, immunoglobulins are captured on the microbial surface and camouflage bacteria during the invasion of host tissues (4). Protein A is cleaved by a transpeptidase, sortase, between the threonine and the glycine of a conserved LPXTG motif (5). The carboxyl group of threonine is amide-linked to the amino group of the pentaglycine cross-bridge, thereby tethering the COOH-terminal end of protein A to the bacterial cell wall (6). This reaction, called cell wall sorting, is strikingly similar to the penicillin-sensitive transpeptidation reaction, and is likely to occur in most Gram-positive bacteria (7).

To identify cell wall sorting mutants, we mutagenized S. aureus strain OS2 with nitrosoguanidine (8). Temperature-sensitive (ts) mutants were identified and 1000 were transformed with pSEB-SPA490-524, a plasmid encoding a reporter protein that allows measurement of surface protein anchoring (9). The SEB-SPA490-524 precursor (P1) is exported from the cytoplasm, and its NH2-terminal leader peptide is removed to generate the P2 intermediate (Fig. 1A) (10). P2 is cleaved by sortase at the LPXTG motif to generate the mature, surface-anchored protein (M). After labeling with [35S]Met for 5 min, the reporter protein in strain OS2 was distributed as follows: P1 (5%), P2 (19%), and M (76%) (Fig. 1B) (11). We used this assay to screen 1000 ts mutants and identified two strains in which there was aberrant accumulation of P2 (47% in SM317 and 26% in SM329) (Fig. 1B). Pulse-chase analysis (12) revealed that in strain OS2, P2 was cleaved and anchored within 2 min, whereas in strain SM317 these events required more than 10 min (Fig. 1C). In strain SM329, P2 processing required 3 min, suggesting a mild defect in cell wall sorting (13).

Figure 1

Isolation of a staphylococcal mutant defective in cell wall sorting of surface proteins. (A) Primary structure of the surface protein precursor SEB-SPA490-524, a fusion between enterotoxin B (SEB) and COOH-terminal protein A (SPA) sequences. P1 is directed across the cytoplasmic membrane by an NH2-terminal leader peptide, and is then cleaved to generate P2. P2 bears a COOH-terminal sorting signal that includes an LPXTG motif, a hydrophobic domain (black bar), and a positively charged tail (boxed +). The sorting signal of P2 is cleaved at the LPXTG motif, and the mature protein (M) is linked to the cell wall. (B)Staphylococcus aureus ts mutants were screened for the accumulation of P2 by labeling with [35S]Met. SM317 and SM329 accumulate more P2 than does the wild-type (WT) strain OS2. (C) Pulse-chase analysis of SEB-SPA490-524 anchoring in wild-type and mutant strains.

Previous work showed that mutations in S. aureus fem genes slow the anchoring of surface proteins to the cell wall (14). These genes are thought to specify enzymes that catalyze the addition of glycines to the ɛ-amino of lysine within the peptidoglycan precursor lipid II (15). To examine whether the SM317 and SM329 mutants were defective in cell wall synthesis, we tested their sensitivity to lysostaphin, an enzyme that cuts the pentaglycine cross-bridges of the staphylococcal cell wall (16). In contrast to fem mutants, which are resistant to lysostaphin (17), strains SM317 and SM329 were sensitive at concentrations that also inhibited growth of wild-type staphylococci, indicating that their sorting defects are not caused by a mutationally altered cell wall cross-bridge (18). To measure cell wall synthesis, we grew the wild-type and mutant strains in minimal medium containing [3H]Lys or [3H]Leu. Because lysine (but not leucine) is a component of the cell wall, the ratio of [3H]Lys:[3H]Leu incorporation into acid-precipitable and protease-resistant murein polymer is a measure of cell wall synthesis (19). Wild-type S. aureusdisplayed a ratio of 30, and the inhibition of cell wall synthesis by vancomycin reduced this ratio to 1.5. Strains SM317 and SM329 displayed ratios of 18 and 19, respectively, which indicates that the accumulation of P2 in strain SM317 is not caused by a defect in cell wall synthesis.

To determine the cell wall anchor structure of surface proteins in strain SM317, we introduced into cells a plasmid (pHTT4) specifying the reporter protein SEB-MH6-CWS (Fig. 2A) (6). The cell wall was purified and digested with mutanolysin, which hydrolyzes the glycan strands (6). Mutanolysin-released surface protein was purified by chromatography on nickel-nitrilotriacetic acid (Ni-NTA) and cleaved at Met with cyanogen bromide. COOH-terminal peptides bearing cell wall anchor structures were purified by a second affinity chromatography step and analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) (Fig. 2). A series of ion signals with regularly spaced mass increments was revealed. These measurements are consistent with increasing numbers of peptidoglycan subunits linked to the COOH-terminal threonine of surface protein (20). If surface protein is tethered to cross-linked peptidoglycan of strain SM317, digestion of muramidase-solubilized anchor peptides with φ11 hydrolase should produce anchor peptide linked to murein tetrapeptide and disaccharide-tetrapeptide (21) (Fig. 2). This was tested, and the doubly digested anchor peptides generated the predicted ion signals (Fig. 2C). Thus, surface proteins of S. aureus SM317 are tethered to cross-linked peptidoglycan in a manner that is indistinguishable from the anchor structure of polypeptides in wild-type staphylococci. These results suggest that the accumulation of P2 in strain SM317 is caused by a defect in sortase.

Figure 2

Structure of surface protein anchor in strain SM317. (A) Primary structure of SEB-MH6-CWS and its linkage to the cell wall. The glycan strands of the staphylococcal cell wall consist of a repeating disaccharideN-acetylmuramic acid-(β1-4)-N-acetylglucosamine (MN-GN) in which the lactyl of muramic acid is linked to the wall peptide (l-Ala-d-iGln-l-Lys-d-Ala). Wall peptides are linked to the pentaglycine cross-bridge, which tethers the ɛ-amino of l-Lys to the carboxyl of d-Ala. The cell wall can be cut at specific sites with the enzymes muramidase (solid arrow) and φ11 hydrolase (open arrows). (B) SEB-MH6-CWS was solubilized by digesting the staphylococcal cell wall with muramidase (mutanolysin) and purified by affinity chromatography on Ni-NTA resin. The polypeptide was cleaved with cyanogen bromide (CnBr), and COOH-terminal anchor peptides were purified by another affinity chromatography step. The structure of the mutanolysin-released anchor peptides of strain SM317 was analyzed by MALDI-MS (6). (C) Muramidase-released anchor peptides were digested with φ11 hydrolase and analyzed by MALDI-MS. The observed ions represent anchor peptide linked to cell wall tetrapeptide (m/z 2236) and murein-disaccharide tetrapeptide (m/z2715 and 2757).

We reasoned that overexpression of sortase from a multicopy plasmid should reduce the concentration of P2 in both wild-type and mutant S. aureus. A plasmid library of 2000 random DNA fragments from S. aureus OS2 was screened for sequences that reduce the accumulation of P2 in strain SM317 and two plasmids, pGL1631 and pGL1834, were identified (Fig. 3) (22). Transformation with pGL1834 reduced the P2 concentration in strain SM317 by 35%, in strain SM329 by 14%, and in wild-type S. aureus OS2 by 9%. All three strains showed a rapid increase in P2 processing (Fig. 3C). We mapped the critical S. aureus sequences in pGL1631 and pGL1834 to a gene that we named srtA (surface protein sorting A) (18, 23).

Figure 3

Overexpression of srtA reduces P2 accumulation in wild-type S. aureus strain OS2 and in two mutant strains, SM317 and SM329. (A) Transformants of a multicopy plasmid library in strain SM317 were screened by labeling with [35S]Met for a decrease in the accumulation of P2. Plasmid pGL1834 contains the srtA gene cloned into pC194-mcs. (B) The srtA gene of strain SM317 (pGL1898) or of strain OS2 (pGL1897) was transformed into SM317 and analyzed for P2 processing. (C) All three strains were transformed with pGL1834 and subjected to pulse-chase analysis (12).

The srtA gene specifies a protein of 206 amino acids with a potential NH2-terminal signal peptide/membrane anchor sequence and a presumed active-site cysteine at position 184, consistent with the observation that the cell wall sorting reaction is sensitive to reagents that modify sulfhydryl groups (Fig. 4) (10). Database searches revealed that srtA homologs are present in Actinomyces naeslundii, Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Streptococcus mutans, Streptococcus pneumoniae, andStreptococcus pyogenes. All srtA homologs displayed absolute conservation of the cysteine-encoding codon 184 (18).

Figure 4

Deduced amino acid sequence of thesrtA gene (25). The NH2-terminal hydrophobic membrane anchor sequence is boxed. The single Cys is shaded. The srtA gene of strain SM317 carries two mutations, one in codon 35 replacing Asp (GAT) with Gly (GGT), and another in codon 180 replacing Thr (ACA) with Ala (AGA). The altered amino acids are indicated in bold. The DNA sequence of plasmid pGL1834 has been submitted to GenBank (accession number AF162687).

To examine whether the defect in cell wall sorting of S. aureus SM317 is caused by a srtA mutation, we amplified the genes from S. aureus OS2 and SM317 by PCR, and cloned them into a multicopy vector, which was then transformed into S. aureus SM317 (18, 23). The wild-type (OS2)srtA gene reduced the accumulation of P2 by 32%, whereas the mutant had no effect (Fig. 3B). DNA sequence analysis revealed mutations in codons 35 and 180 in the SM317 srtA gene. Interestingly, multicopy expression of wild-type srtA(pGL1894) did not fully complement the ts growth phenotype of SM317. The transformed mutant grew at 42°C but slower than the wild type, suggesting that the conditional lethal phenotype of S. aureus SM317 is not caused solely by the mutations insrtA.

Together, our data reveal that srtA encodes sortase, the transpeptidase that anchors surface proteins to the bacterial cell wall (24). In principle, purified SrtA protein can be used to screen for compounds that inhibit cell wall sorting, a strategy that may lead to new therapies for human infections caused by Gram-positive bacteria.

  • * To whom correspondence should be addressed. E-mail: olafs{at}


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