Molecular genetic analysis of a group A Streptococcus operon encoding serum opacity factor and a novel fibronectin-binding protein, SfbX

Abstract

The group A Streptococcus (GAS) sof gene encodes the serum opacity factor protein, which is capable of opacifying mammalian sera and binding at least two host proteins, fibronectin and fibrinogen. The sof gene exists in approximately 50% of clinical isolates, and there is a classical association of so-called nephritogenic strains with the opacity factor-positive phenotype. In both a type emm49 strain and a type emm12 strain, the sequences upstream of the 5' end of sof and downstream of the putative terminator were determined to be nearly identical to a region in the M type 1 genome approximately 10 kb upstream of the emm1 gene. This close genetic linkage is likely reflected in the strict correlation of opacity factor phenotype with specific emm genotypes. A new fibronectin-binding protein gene, sfbX, was discovered immediately downstream of sof in emm12 and emm49 strains and in several other sof-positive strains. The sof and sfbX genes were found to be expressed on the same transcription unit, which was correlated with the putative promoter and rho-independant terminator sequences that flank these two genes. The sfbX genes from different emm types are predicted to encode approximately 650-residue surface-bound proteins sharing 89 to 92% sequence identity. SfbX residues approximately 1 to 480 are not highly similar to those of other known proteins, with the closest match being the Staphylococcus aureus coagulase protein. The remaining portions of these proteins (residues 481 to 650) contain four putative fibronectin-binding repeats highly similar to those of other streptococcal fibronectin-binding proteins and a potential LP(X)SG cell wall anchor motif. Targeted in-frame allelic-exchange mutagenesis, complementation, and heterologous-expression studies found that serum opacification is encoded by sof alone and that sfbX encodes a fibronectin-binding function. A recombinant SfbX protein was found to bind immobilized fibronectin and to partially inhibit GAS adherence to fibronectin. The sfbX gene was found to be present only in sof-positive strains, and together these genes could influence the spectrum of tissues colonized by sof-positive GAS.

FIG. 1.

Chromosomal location of the GAS emm49 serum opacity factor locus (sof49-sfbX49) with respect to the published emm1 genome sequence.

FIG. 2.

RT-PCR analysis demonstrates that the sof49 and sfbX49 genes are transcribed on the same mRNA and thus represent an operon. Asterisks indicate stop codons. The two underlined sequences between the sof49 and sfbX structural genes indicate an inverted repeat. A putative transcriptional terminator immediately downstream of sfbX is underlined, with the two inverted repeats boxed.

FIG. 3.

(a) Structural comparison of the predicted Sof49 and SfbX49 proteins. S.S., signal sequence; Fn, fibronectin. (b) High degree of homology between Sof and SfbX in the terminal fibronectin-binding repeats (underlined), cell wall anchor (box), and membrane-spanning domain. (c) Alignment of two SfbX49 putative fibronectin-binding repeat regions with similar repeats from other gram-positive bacterium fibronectin-binding proteins. The published sequences for Sof2 (10), PrtF1 and PrtF2 (43), and Sfbi (48) are from Streptococcus pyogenes; FnbpA (44) is from Staphylococcus aureus; and FnbA (28) is from Streptococcus dysgalactiae.

FIG. 4.

Contribution of the Sof49 and SfbX49 gene products to opacification of horse serum. Precise in-frame allelic-exchange mutagenesis, complementation analysis, and heterologous-expression studies indicate that sof alone encodes the opacity factor phenotype.

FIG. 5.

(a) Contribution of the Sof49 and SfbX49 gene products to the fibronectin (Fn)-binding ability of log-phase GAS assessed by using isogenic allelic-exchange knockout mutants. (b) Complementation of the sof and sfbX knockout mutants by returning the single gene on a plasmid vector. Wild-type (w.t.) and mutant strains transformed with the vector alone (pDCerm) served as controls. (c) Ability of the cloned GAS sof49 and sfbX49 genes to confer fibronectin-binding activity to L. lactis. ∗, P < 0.05; ∗∗, P < 0.0001. The bacterial inoculum was 10⁶ CFU in 100 μl for all experiments.

FIG. 6.

(a) Western blot confirmation of recombinant SfbX protein after affinity column purification. (b) Dose-dependent binding of recombinant SfbX protein to immobilized fibronectin. (c) Dose-dependent competitive inhibition of GAS fibronectin-binding by recombinant SfbX.