Rheumatic fever-associated Streptococcus pyogenes isolates aggregate collagen

Abstract

Acute rheumatic fever is a serious autoimmune sequel of Streptococcus pyogenes infection. This study shows that serotype M3 and M18 S. pyogenes isolated during outbreaks of rheumatic fever have the unique capability to bind and aggregate human basement membrane collagen type IV. M3 protein is identified as collagen-binding factor of M3 streptococci, whereas M18 isolates bind collagen through a hyaluronic acid capsule, revealing a novel function for M3 protein and capsule. Following in vivo mouse passage, conversion of a nonencapsulated and collagen-binding negative M1 S. pyogenes into an encapsulated, collagen-binding strain further supports the crucial role of capsule in mediating collagen binding. Collagen binding represents a novel colonization mechanism, as it is demonstrated that S. pyogenes bind to collagen matrix in vitro and in vivo. Moreover, immunization of mice with purified recombinant M3 protein led to the generation of anti-collagen type IV antibodies. Finally, sera from acute rheumatic fever patients had significantly increased titers of anti-collagen type IV antibodies as compared with healthy controls. These findings may suggest a link between the potential of rheumatogenic S. pyogenes isolates to bind collagen, and the presence of collagen-reactive autoantibodies in the serum of rheumatic fever patients, which may form a basis for post-streptococcal rheumatic disease. These anti-collagen antibodies may form a basis for poststreptococcal rheumatic disease.

Figure 1

Collagen matrix association of GAS. FESEM shows a thick CIV network on the surface of a collagen-binding M3 strain 86764 (18) isolated from a rheumatic fever patient (a) that is absent in strains that do not bind CIV (A40; b). Transmission electron microscopy of collagen-binding serotype M3 GAS 86764 shows collagen deposition on the surface using immunogold labeling (c, arrowheads). Asterisks indicate aggregated CIV on the streptococcal surface. Bars, 1 μm (a and b) and 0.25 μm (c).

Figure 2

CIV binding of M3 protein. (a and b) Ligand overlay assay of GST and GST-fused recombinant M proteins with soluble radiolabeled CIV. (a) Spot membrane; (b) Western blot. Molecular masses (kDa) of M3 and GST are indicated. (c) Model of M3 protein and the CIV-binding subfragments.

Figure 3

Visualization of streptococcal capsule and binding of CIV gold complexes to M18 GAS. M18 strain 87282 S. pyogenes associated with ARF exhibit a thick capsule (a), and loss of capsule after hyaluronidase treatment (d). Low-voltage FESEM (1.5 kV) reveals binding of CIV gold complexes on M18 capsular structures (b and c), and highly reduced binding of CIV gold complexes on hyaluronidase-treated bacteria (e). Bars, 1 μm (a and d), 0.5 μm (b and e), and 0.2 μm (c).

Figure 4

Capsule expression and collagen-binding activity of a mouse-passaged serotype M1 S. pyogenes. Transmission electron microscopic images show M1 S. pyogenes strain KTL3 grown directly in medium (a) or cultivated from blood of infected mice (b). (c) A highly encapsulated mouse-passaged M18 strain 87282. (d) Collagen-binding activities are shown for M18 and M1 isolates grown in medium (light gray bars) or cultivated from blood of infected mice (dark gray bars). Collagen binding is expressed as a ratio of bound radiolabeled CIV to streptococci. Bars, 0.25 μm (a–c).

Figure 5

S. pyogenes colonizes collagen in vitro and in vivo. FESEM analysis shows S. pyogenes colonizing collagen type I bundles (a) and fibers (b) in vitro. For in vivo localization, mice were infected subcutaneously with M3, M18, and M1 S. pyogenes. Infected skin was analyzed by FESEM analysis, showing S. pyogenes aggregated on and attached to collagen fibers (c and d). Analysis of skin sections by transmission electron microscopy shows intimate in vivo binding of streptococci to collagen. Serotypes shown are M3 (a, b, and h), M18 (c and g), and M1 (d–f). Bars, 10 μm (a), 2 μm (b), 2.5 μm (c), and 0.5 μm (d–h).

Figure 6

CIV-specific serum responses. (a) Serum Ig response in M3 protein–immunized mice. Reactivity was determined for preimmune serum pool (PI), serum pool of PBS-immunized (PBS) or M3 protein–immunized (M3) mice, and M3 serum pool after absorbing M3-reactive antibodies (M3-absorbed). (b) CIV-specific serum responses in patients and controls. Sera were from healthy controls (H, n = 27), pharyngitis patients (P, n = 9), or ARF patients (ARF, n = 5). Antigens were streptolysin O (SLO), GAS polysaccharide (GAS), human heart myosin (MYO), and CIV. Bars represent mean IgG titers of three experiments ± SD. (c) Anticollagen titer of individual patient sera. Titers are shown for each individual serum collected from ARF patients (triangles), pharyngitis patients (squares), or healthy individuals (circles).