Opacification Domain of Serum Opacity Factor Inhibits Beta-Hemolysis and Contributes to Virulence of Streptococcus pyogenes

Abstract

Serum opacity factor (SOF) is a cell surface virulence factor made by the human pathogen Streptococcus pyogenes. We found that S. pyogenes strains with naturally occurring truncation mutations in the sof gene have markedly enhanced beta-hemolysis. Moreover, deletion of the sof gene in a SOF-positive parental strain resulted in significantly increased beta-hemolysis. Together, these observations suggest that SOF is an inhibitor of beta-hemolysis. SOF has two major functional domains, including an opacification domain and a fibronectin-binding domain. Using a SOF-positive serotype M89 S. pyogenes parental strain and a panel of isogenic mutant derivative strains, we evaluated the relative contribution of each SOF functional domain to beta-hemolysis inhibition and bacterial virulence. We found that the opacification domain, rather than the fibronectin-binding domain, is essential for SOF-mediated beta-hemolysis inhibition. The opacification domain, but not the fibronectin-binding domain of SOF, also contributed significantly to virulence in mouse models of bacteremia and necrotizing myositis. Inasmuch as the opacification domain of SOF is known to interact avidly with host high-density lipoprotein (HDL), we speculate that SOF-HDL interaction is an important process underlying SOF-mediated beta-hemolysis inhibition and SOF-mediated virulence. IMPORTANCEStreptococcus pyogenes is a major human pathogen causing more than 700 million infections annually. As a successful pathogen, S. pyogenes produces many virulence factors that facilitate colonization, proliferation, dissemination, and tissue damage. Serum opacity factor (SOF), an extracellular protein, is one of the virulence factors made by S. pyogenes. The underlying mechanism of how SOF contributes to virulence is not fully understood. SOF has two major features: (i) it opacifies host serum by interacting with high-density lipoprotein, and (ii) it inhibits beta-hemolysis on blood agar. In this study, we demonstrate that the domain of SOF essential for opacifying serum is also essential for SOF-mediated beta-hemolysis inhibition and SOF-mediated virulence. Our results shed new light on the molecular mechanisms of SOF-host interaction.

Keywords: Streptococcus pyogenes; beta-hemolysis; high-density lipoprotein; serum opacity factor; virulence.

FIG 1

Serotype M89 S. pyogenes strains with naturally occurring truncation mutations in sof have increased beta-hemolysis and abolished serum opacity activity. (A) Schematic depiction of the sof gene of three M89 strains. The 5′ end of the sof gene encodes a secretion signal sequence that targets proteins across the membrane. The 3′ end of the sof gene encodes a cell wall-anchoring domain with an LPXXG anchoring motif (4). Sites with naturally occurring nucleotide (nt) insertions (ins) or deletions (del) are labeled with vertical lines. (B) Colony morphology and beta-hemolysis of strains after overnight incubation at 37°C (top) and serum opacity activity of the three M89 strains (bottom). Strain MGAS27556 is a wild-type control M89 strain.

FIG 2

Serotype M28 S. pyogenes strains with naturally occurring truncation mutations in sof have increased beta-hemolysis and abolished serum opacity activity. (A) Schematic depiction of the sof gene of three M28 strains. Sites with nucleotide (nt) insertions (ins) or deletions (del) are labeled with vertical lines. (B) Colony morphology and beta-hemolysis (top) and serum opacity activity (bottom) of the three M28 strains. Strain MGAS29482 is a wild-type control M28 strain.

FIG 3

Characteristics of serotype M89 reference strain MGAS27556 and its isogenic mutant derivatives with different deletions in sof. (A) Schematic depiction of the sof gene in strain MGAS27556 and the three isogenic mutant strains. (B and C) Serum opacity activity (B) and fibronectin-binding activity (C) of assayed strains. (D) Colony morphology and beta-hemolysis of assayed strains. (E and F) Quantitation of beta-hemolysis (E) and SLO hemolytic activity (F) of assayed strains. Data are expressed as mean ± standard deviation (B, C, E, and F). n = 4 (B), 7 (C), 20 (E), and 3 (F). *, P < 0.05 versus the wild-type strain MGAS27556 using one-way analysis of variance and Dunnett’s posttest.

FIG 4

Strain virulence in mouse invasive infection models. (A) Ability of wild-type and isogenic mutant strains to cause near-mortality in a mouse model of bacteremia. IP, intraperitoneal. (B) Ability of wild-type and mutant strains to cause near-mortality in a mouse model of necrotizing myositis. IM, intramuscular. Near-mortality is expressed as Kaplan-Meier survival curves. Statistical differences are determined using the log rank test. *, P < 0.05 relative to wild-type strain MGAS27556. (C) Histologic analysis of infected limb tissue. Limbs were visually inspected at 3 days postinoculation, and microscopic examination was performed at 1 day postinoculation (hematoxylin and eosin stain; original magnification, ×4). Boxes indicate that MGAS27556 and Δsof-FD strains cause very large lesions (upper panel) with extensive destruction of the fascia, muscle, and soft tissue (lower panel). Arrows indicate that Δsof-OD and Δsof strains cause comparably smaller lesions that are restricted to the fascial plane. All isogenic mutant strains were derived from wild-type parental strain MGAS27556 (serotype M89).