Differential recognition of surface proteins in Streptococcus pyogenes by two sortase gene homologs

Abstract

The interaction of Streptococcus pyogenes (group A streptococcus [GAS]) with its human host requires several surface proteins. In this study, we isolated mutations in a gene required for the surface localization of protein F by transposon mutagenesis of the M6 strain JRS4. This gene (srtA) encodes a protein homologous to Staphylococcus aureus sortase, which covalently links proteins containing an LPXTG motif to the cell wall. The GAS srtA mutant was defective in anchoring the LPXTG-containing proteins M6, protein F, ScpA, and GRAB to the cell surface. This phenotype was complemented when a wild-type srtA gene was provided in trans. The surface localization of T6, however, was unaffected by the srtA mutation. The M1 genome sequence contains a second open reading frame with a motif characteristic of sortase proteins. Inactivation of this gene (designated srtB) in strain JRS4 affected the surface localization of T6 but not M6, protein F, ScpA, or GRAB. This phenotype was complemented by srtB in trans. An srtA probe hybridized with DNA from all GAS strains tested (M types 1, 3, 4, 5, 6, 18, 22, and 50 and nontypeable strain 64/14) and from streptococcal groups C and G, while srtB hybridized with DNA from only a few GAS strains. We conclude that srtA and srtB encode sortase enzymes required for anchoring different subsets of proteins to the cell wall. It seems likely that the multiple sortase homologs in the genomes of other gram-positive bacteria have a similar substrate-specific role.

FIG. 1.

Construction of srtB-1 and srtB-2 alleles. (A) Internal fragments of srtB were amplified by PCR and cloned into pZErO-2.1 (pJRS779) and pUCSpec (pJRS789). Numbers above the srtB open reading frame and the fragments cloned into pJRS779 and pJRS789 refer to the locations of encoded amino acid residues of the JRS4 srtB gene. (B) Integration of pJRS779 and pJRS789 into the chromosome of JRS4 results in one copy of srtB (srtB 5′) encoding a protein truncated after amino acid 256 (pJRS779) or 206 (pJRS789) and a second copy (srtB 3′) lacking a promoter and the first 55 (pJRS779) or 59 (pJRS789) amino acids. The signal sequence of SrtB is indicated by a striped box. Open and solid arrows represent srtB and aad9, respectively. The srtB promoter is indicated with a bent arrow preceding a region of unknown length. The internal region of srtB cloned into pJRS779 and pJRS789 is indicated by a striped box.

FIG. 2.

Multiple sequence alignment of SrtA with homologous proteins identified by Blast search. The alignment was performed using ClustalW. The GenBank accession numbers for the sequences are as follows: S. pyogenes (Spyog) SrtA, AAK34025; S. gordonii (Sgord) SrtA, AAG41778; L. lactis (Llact) hypO, AAK05211; B. licheniformis (Blich) Amyl, AAA73122; S. aureus (Saure) SrtA, AAD48437; and A. naeslundii (Anaes) fimbria-associated protein (fimassoc), AAC13546 (truncated after amino acid 251).

FIG. 3.

Effect of the srtA mutation on surface anchoring of LPXTG proteins by GAS. (A) Dot blot analysis for surface localization of M6, protein F, ScpA, GRAB, and T6 proteins in JRS4 and srtA derivatives. Each strain was grown overnight in THY broth, and cell density was standardized to an optical density at 600 nm of 2.0. Individual spots represent 10-μl aliquots of twofold serial dilutions of each strain. (B) Western immunoblot analysis of JRS4 and srtA derivatives with monoclonal antibody 10B6. CW, cell wall fraction; S, culture supernatant fraction. Proteins were separated by SDS-PAGE on 4 to 12% gradient gels, transferred to nitrocellulose, and detected with 10B6 as described in Materials and Methods. The sizes of molecular mass standards are indicated to the left (in kilodaltons). The arrow to the right represents the position of mature M6 protein.

FIG. 4.

Multiple sequence alignment of SrtB using ClustalW with homologous proteins identified by Blast search. The GenBank accession numbers for the sequences are as follows: S. aureus (Saure) SrtA, AAD48437; S. pyogenes (Spyog) SrtA, AAK34025; and S. pyogenes SrtB, AAK33243.

FIG. 5.

Dot blot analysis for the surface localization of M6, protein F, ScpA, GRAB, and T6 proteins in JRS4 and srtB derivatives. Dot blots were prepared as described for Fig. 3.

FIG. 6.

Southern hybridization analysis for the presence of srtA (A) and srtB (B) genes in strains of GAS and other gram-positive bacteria. DNA was digested with HindIII and separated by 0.8% agarose gel electrophoresis. Following transfer to a nylon membrane, srtA- and srtB-homologous sequences were detected by hybridization with an internal fragment labeled with [α-³²P]dCTP. Gp, group; NT, nontypeable.

FIG. 7.

Alignment of the C-terminal amino acid sequences of GAS proteins containing an LPXTG motif. Alignments were performed using ClustalW. Spy designations are annotations from the genome sequence of the M1 GAS strain SF370 (16). The sequence of the corresponding region of the M6 protein analyzed in this study is identical to the M1 sequence (23). References for the sequences are as follows: SPy130, -416, -747, -843, -872, -1494, and -1972 (57); Scl2 (35, 52, 69); GRAB (54); Scl1 (34, 53); Fba (64); ScpA (7); M1 (20). Protein F (61) and T6 (57) sequences are from the M6 strain JRS4 and are not present in the M1 genome.