SclA, a novel collagen-like surface protein of Streptococcus pyogenes

Abstract

Surface proteins of Streptococcus pyogenes are important virulence factors. Here we describe a novel collagen-like surface protein, designated SclA (streptococcal collagen-like surface protein). The sclA gene was identified in silico using the Streptococcal Genome Sequencing Project with the recently identified protein GRAB as the probe. SclA has a signal sequence and a cell wall attachment region containing the prototypic LPXTGX motif. The surface-exposed part of SclA contains a unique NH(2)-terminal domain of 73 amino acids, followed by a collagen-like region. The sclA gene was found to be positively regulated by Mga, a transcriptional activator of several S. pyogenes virulence determinants. A mutant lacking cell wall-associated SclA was constructed and was found to be as effective as wild-type bacteria in platelet aggregation, survival in fresh human blood, and adherence to pharyngeal cells. The sclA gene was found in all 12 S. pyogenes strains that were investigated using PCR. Sequence analysis revealed that the signal sequence and the cell wall attachment region are highly conserved. The collagen-like domain is variable in its NH(2)-terminal region and has conserved repeated domains in its COOH-terminal part. SclA proteins from most strains have additional proline-rich repeats spacing the collagen-like domain and the cell wall attachment sequence. The unique NH(2)-terminal region is hypervariable, but computer predictions indicate a common secondary structure, with two alpha helices connected by a loop region. Immune selection may explain the hypervariability in the NH(2)-terminal region, whereas the preserved secondary structure implies that this region has a common function. These features and the Mga regulation are shared with the M protein of S. pyogenes. Moreover, as with the gene encoding the M protein, phylogenetic analysis indicates that horizontal gene transfer has contributed to the evolution of sclA.

FIG. 1

Schematic representation of the SclA protein from strain SF370. A putative signal sequence (Ss) is followed by an A domain and a CLR in gray. The CLR contains two partially overlapping repeats, denoted by 1 and 2. In the COOH-terminal part is a putative cell wall-spanning domain (W), including the typical LPXTGX motif and a hydrophobic membrane-spanning domain (M).

FIG. 2

(A) Total RNA was obtained from the AP1 strain at early logarithmic growth phase (EL), late logarithmic growth phase (LL), and early stationary phase (ES). RNA was subjected to Northern blotting using a probe hybridizing with sclA. A part of the gel was stained with ethidium bromide to visualize rRNA bands, used as molecular weight markers. (B) Total RNA from AP1, BMJ71, and BMJ71pMGA.1 was prepared from bacteria in early logarithmic growth phase and subjected to Northern blotting using the same probe as for panel A. (C) Representation of the putative Mga-binding element located at −107 to −147 bp from the start codon of sclA. Putative −35 and −10 boxes are indicated.

FIG. 3

The SclA protein was expressed as a GST fusion. GST and GST-SclA samples were separated by SDS-PAGE (12% acrylamide; reducing conditions), and two identical gels were run. One gel was stained with Coomassie brilliant blue (STAIN), and the other was subjected to Western blotting using an antiserum to GST-SclA (BLOT).

FIG. 4

(A) Insertion-duplication mutagenesis was used to delete the part of sclA encoding the membrane-spanning M domain and the COOH-terminal part of the W domain in strain AP1 to generate the SclA⁻ strain. (B) Growth media from AP1 and SclA⁻ were TCA precipitated, and proteins were subjected to SDS-PAGE (12% acrylamide gels; reducing conditions). One gel was stained with Coomassie brilliant blue (STAIN), and two replicas were subjected to Western blotting (BLOT) using SclA antiserum or preimmune serum (dilution, 1:1,000), respectively.

FIG. 5

(A) Schematic comparison of predicted mature SclA proteins from 10 S. pyogenes strains. Amino acid positions are indicated. The unique A domain is followed by the CLR, depicted in light gray. Four types of repeats, denoted by 1, 2, 3, and 4, are present in the CLRs. A region of >95% identity is represented by the dark gray shade. The type 5 repeat is a proline-rich repeat of 21 aa, while the 5* repeats are variants of type 5 with 2 to 5 extra amino acids. In the COOH-terminal part is the conserved wall-spanning region (W) containing the LPATGE motif. (B) Schematic depiction of the two alpha helices in the A domain from the SclA protein of 10 different M serotypes. Amino acid numbers are given on top of the box. The black color indicates that 9 or 10 of the A domains had a helical prediction in the area, the dark gray that 6 to 8 A domains were predicted to be helical, the light gray that 3 to 5 were predicted to be helical, and the white that 0 to 2 were predicted to be helical. Only regions where both predictions indicated alpha helices were regarded as helical.