Crucial Role of the CB3-Region of Collagen IV in PARF-Induced Acute Rheumatic Fever

Acute rheumatic fever (ARF) and rheumatic heart disease are serious autoimmune sequelae to infections with Streptococcus pyogenes. Streptococcal M-proteins have been implicated in ARF pathogenesis. Their interaction with collagen type IV (CIV) is a triggering step that induces generation of collagen-specific auto-antibodies. Electron microscopy of the protein complex between M-protein type 3 (M3-protein) and CIV identified two prominent binding sites of which one is situated in the CB3-region of CIV. In a radioactive binding assay, M3-protein expressing S. pyogenes and S. gordonii bound the CB3-fragment. Detailed analysis of the interactions by surface plasmon resonance measurements and site directed mutagenesis revealed high affinity interactions with dissociation constants in the nanomolar range that depend on the recently described collagen binding motif of streptococcal M-proteins. Because of its role in the induction of disease-related collagen autoimmunity the motif is referred to as “peptide associated with rheumatic fever” (PARF). Both, sera of mice immunized with M3-protein as well as sera from patients with ARF contained anti-CB3 auto-antibodies, indicating their contribution to ARF pathogenesis. The identification of the CB3-region as a binding partner for PARF directs the further approaches to understand the unusual autoimmune pathogenesis of PARF-dependent ARF and forms a molecular basis for a diagnostic test that detects rheumatogenic streptococci

Identification of a streptococcal octapeptide motif involved in acute rheumatic fever.

Acute rheumatic fever is a serious autoimmune sequela of pharyngitis caused by certain group A streptococci. One mechanism applied by streptococcal strains capable of causing acute rheumatic fever is formation of an autoantigenic complex with human collagen IV. In some geographic regions with a high incidence of acute rheumatic fever pharyngeal carriage of group C and group G streptococci prevails. Examination of such strains revealed the presence of M-like surface proteins that bind human collagen. Using a peptide array and recombinant proteins with targeted amino acid substitutions, we could demonstrate that formation of collagen complexes during streptococcal infections depends on an octapeptide motif, which is present in collagen binding M and M-like proteins of different beta-hemolytic streptococcal species. Mice immunized with streptococcal proteins that contain the collagen binding octapeptide motif developed high serum titers of anti-collagen antibodies. In sera of rheumatic fever patients such a collagen autoimmune response was accompanied by specific reactivity against the collagen-binding proteins, linking the observed effect to clinical cases. Taken together, the data demonstrate that the identified octapeptide motif through its action on collagen plays a crucial role in the pathogenesis of rheumatic fever. Eradication of streptococci that express proteins with the collagen binding motif appears advisable for controlling rheumatic fever

Biological functions of GCS3, a novel plasminogen-binding protein of Streptococcus dysgalactiae ssp. equisimilis

Increasing awareness of the relevance of Streptococcus dysgalactiae ssp. equisimilis as a human pathogen motivates the analysis of its pathomechanisms. One of the mechanisms that increases infectivity and dissemination of several streptococcal species is the recruitment and subsequent activation of host plasminogen on the streptococcal surface. This study identified GCS3 as a novel plasminogen-binding M protein of S. dysgalactiae ssp. equisimilis and revealed a difference in the mode of binding as compared to the plasminogen-binding protein PAM of S. pyogenes. In contrast to PAM, GCS3 did not bind to the kringle 1-3 region of plasminogen. Despite this difference, GCS3 exerts the same function of recruiting plasminogen to the streptococcal surface, which can be activated by streptokinase and host plasminogen activators to serve as a spreading factor. Moreover, we demonstrate a role of GCS3 in plasminogen-dependent streptococcal adherence to human pharyngeal cells (cell line Detroit 562) that indicates an additional function of the protein as an adhesin in the oral cavity

Differences in the aromatic domain of homologous streptococcal fibronectin-binding proteins trigger different cell invasion mechanisms and survival rates.

Group A streptococci (GAS, Streptococcus pyogenes) and Group G streptococci (GGS, Streptococcus dysgalactiae ssp. equisimilis) adhere to and invade host cells by binding to fibronectin. The fibronectin-binding protein SfbI from GAS acts as an invasin by using a caveolae-mediated mechanism. In the present study we have identified a fibronectin-binding protein, GfbA, from GGS, which functions as an adhesin and invasin. Although there is a high degree of similarity in the C-terminal sequence of SfbI and GfbA, the invasion mechanisms are different. Unlike caveolae-mediated invasion by SfbI-expressing GAS, the GfbA-expressing GGS isolate trigger cytoskeleton rearrangements. Heterologous expression of GfbA on the surface of a commensal Streptococcus gordonii and purified recombinant protein also triggered actin rearrangements. Expression of a truncated GfbA (lacking the aromatic domain) and chimeric GfbA/SfbI protein (replacing the aromatic domain of SfbI with the GfbA aromatic domain) on S. gordonii or recombinant proteins alone showed that the aromatic domain of GfbA is responsible for different invasion mechanisms. This is the first evidence for a biological function of the aromatic domain of fibronectin-binding proteins. Furthermore, we show that streptococci invading via cytoskeleton rearrangements and intracellular trafficking along the classical endocytic pathway are less persistence than streptococci entering via caveolae

Binding of PARF mutants to CB3.

<p>(<i>A</i>) Dot blot experiment with ¹²⁵I-CB3 as the soluble ligand. Dots 1–9 are the immobilized mutants of M protein FOG (for description see table in <i>B</i>); dot 10 is recombinant wild type protein. Recombinant M3- (+) and M18-proteins (−) were used as positive and negative control, respectively. The table in <i>B</i> allows to compare the experiment with previous data on full length collagen IV<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004666#pone.0004666-Dinkla1" target="_blank">[5]</a>.</p

Rheumatic fever–associated Streptococcus pyogenes isolates aggregate collagen

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

Electron microscopy of M3-CIV complexes.

<p>Micrographies (<i>left panel</i>) and corresponding cartoons (<i>right panel</i>) show complexes that consist of M3-protein and CIV. Block arrows highlight M3-protein (depicted in gray in the cartoons), both free and bound to CIV (depicted in black in the cartoons). Arrow heads point out the 7S regions of CIV, while line arrows points out the globular heads of the CIV molecules. The bars represent 100 nm in the representative overviev (<i>A</i>) and 50 nm in the panel of selected complexes (<i>B</i>).</p

CB3 binding by M3-protein.

<p>(<i>A</i>) Interaction with ¹²⁵I-CB3 was measured in a pull-down assay with, <i>M3-GST</i> (<i>black bar</i>), or <i>M18-GST</i> (<i>grey bar</i>) protein coupled to glutathion sepharose beads. A control sample with glutathion separose alone (−) was included (<i>white bar</i>). (<i>B</i>) Binding of ¹²⁵I-CIV (black bars) or ¹²⁵I-CB3 (<i>grey bars</i>) to <i>S. gordonii</i> that heterologously expressed M3 protein on its surface (<i>SGO M3</i>) and that of the wild type control (<i>SGO</i>) was expressed as a percentage of total radioactivity input. Error bars in <i>A</i> and <i>B</i> represent the standard deviation of the triplicate measurements. (<i>C</i> and <i>D</i>) Surface plasmon resonance measurements of the interaction between M3-protein and immobilized CIV (<i>C</i>) or CB3 (<i>D</i>), respectively. Injection of M3-protein at different concentrations (a: 50 µg/ml, b: 25 µg/ml, c: 12.5 µg/ml, d: 6.25 µg/ml) started at t = 0 s and stopped at t = 120 s. The response is expressed in response units (RU).</p