A novel sortase, SrtC2, from Streptococcus pyogenes anchors a surface protein containing a QVPTGV motif to the cell wall

Abstract

The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.

FIG. 1.

FCT regions of different GAS strains. (A) Genomic organization of the FCT regions from different GAS strains. In M1 strains, srtC1 is located in a potential operon with genes encoding a putative signal peptidase (sipA1) and three potential sortase substrate proteins, cpa, SPy128, and SPy130. A similar gene arrangement is seen in other strains. A divergently transcribed gene encoding a potential regulator of this operon (rofA and nra) is located upstream of cpa. Homologous genes are shaded alike. Potential sortase recognition motifs are given in parentheses after gene names. The sequences for M1 (13), M3 (2), M5 (genome sequence at http://www.sanger.ac.uk, GenBank accession no. NC_002958), M6 (3), M12 (3), M18 (49), and M49 (3, 42) have been published. The star denotes that the srtB gene from M12 contains an internal stop codon (codon 102). (B) Multiple sequence alignment of SrtC amino acid sequences from the above strains. Alignment was performed with ClustalW software.

FIG. 2.

Anchoring of Orf100_HA to the cell surface requires SrtC2. (A) Regions of the srtC2 operon cloned into pNZ276 under the P_lac promoter. Plasmid pJRS1316 contains sipA2 and orf100_HA, while pJRS1317 contains sipA2, orf100_HA, and srtC2. (B) Whole-cell immunoblot analysis for surface localization of Orf100_HA in JRS4 strains harboring pNZ276, pJRS1316, and pJRS1317. Strains were grown overnight and resuspended in saline at an optical density at 600 nm of 2.0. Serial twofold dilutions were spotted onto a nitrocellulose membrane and reacted with monoclonal antibody HA-7. (C) Western immunoblot of cell wall and culture supernatant extracts of JRS4 containing pJRS1316 or pJRS1317 reacted with HA-7 monoclonal antibody. Cell wall extracts were prepared with phage lysin (14) and immunoprecipitated prior to SDS-PAGE. Proteins were separated by SDS-PAGE on 4 to 12% gradient gels, transferred to nitrocellulose, and detected with HA-7 monoclonal antibody. The sizes of molecular mass standards (in kilodaltons) are indicated to the left. The heavy (H) and light (L) chains of the antibody used for immunoprecipitation are also indicated.

FIG. 3.

Role of QVPTG motif in SrtC2-dependent anchoring of Orf100_HA to the cell surface. Strains JRS4 and JRS758 were transformed with pJRS1329, pJRS1330, pJRS1317, and pJRS1316 and assayed for the presence of Orf100_HA as described for Fig. 2. (A) Whole-cell immunoblot analysis. (B) Western immunoblot of cell wall extracts. (C) Western immunoblot of culture supernatant fractions. Lanes: 1, JRS4/pJRS1329; 2, JRS758/pJRS1329; 3, JRS4/pJRS1330; 4, JRS758/pJRS1330; 5, JRS4/pJRS1317; 6, JRS758/pJRS1317; 7, JRS4/pJRS1316; 8, JRS758/pJRS1316. The sizes of molecular mass standards (in kilodaltons) are indicated to the left.

FIG. 4.

Orf100_HA containing an LPSTGE motif is processed by SrtA. Cell wall and culture supernatant fractions from strains JRS4/pJRS1330 and JRS758/pJRS1330 were separated by SDS-12% PAGE and assayed for the presence of Orf100_HA by Western immunoblot. The sizes of molecular mass standards (in kilodaltons) are indicated to the left.

FIG. 5.

Effect of SrtC2-dependent anchoring of Orf100_HA on the surface display of M6 protein. Whole-cell immunoblots of JRS4 strains containing plasmids pNZ276, pJRS1316 (srtC2 mutant), and pJRS1317 (srtC2⁺) and strain JRS758 (srtA mutant) were prepared as described for Fig. 2 and reacted with monoclonal antibody 10B6.

FIG. 6.

Fates of the different Orf100 proteins investigated in this study. (A) Orf100 is synthesized in the cytoplasm and transported to the cytoplasmic membrane, where the signal peptide (Sig) is removed by a mechanism that may require SipA2. (B) Orf100 is tethered to the membrane by its C-terminal hydrophobic region and charged tail. (C) SrtC2 recognizes and cleaves the QVPTG motif. (D) SrtC2 links the cleaved protein to the cell wall precursor. (E) Same as steps A and B. (F) SrtA recognizes and cleaves the LPSTGE motif, but other inherent properties of Orf100_HA-LPSTGE prevent it from being linked to the cell wall precursor. (G) Instead, this protein is released into the culture supernatant. This figure was adapted from that of Navarre and Schneewind (34) and modified to include the results from this study.