Abstract
The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) binds to fibronectin via protein F. In this study, we have investigated the binding properties of protein F to various multimeric tissue forms of fibronectin that appear on cell surfaces and in the extracellular matrix. We show that binding of S. pyogenes through protein F is more efficient to an in vitro-derived polymerized form of fibronectin (superfibronectin) than to soluble fibronectin immobilized in a solid phase. In addition, Chinese hamster ovary cells overexpressing the alpha5beta1 integrin produced an increased amount of a fibronectin matrix and consequently bound a higher number of S. pyogenes cells. Inhibition and direct binding assays using purified proteins demonstrated that binding to a fibronectin matrix involved both domains of protein F (UR and RD2) that have previously been implicated in interactions with fibronectin. Using intact S. pyogenes bacteria in which various domains of protein F were expressed as hybrids with the surface-exposed region of an unrelated protein, we revealed that, in contrast to the predominantly UR-mediated binding to soluble fibronectin, the maximal binding to the fibronectin matrix required RD2 in addition to UR. Since in some infections S. pyogenes may initially encounter a matrix form of fibronectin, these results suggest that UR and RD2 may be important for the initiation of streptococcal infectious processes.
Highlights
The adherence of pathogenic bacteria to host tissues is an important prerequisite for bacterial colonization and subsequent development of disease [1]
We show that binding of S. pyogenes through protein F is more efficient to an in vitro-derived polymerized form of fibronectin than to soluble fibronectin immobilized in a solid phase
Using intact S. pyogenes bacteria in which various domains of protein F were expressed as hybrids with the surface-exposed region of an unrelated protein, we revealed that, in contrast to the predominantly UR-mediated binding to soluble fibronectin, the maximal binding to the fibronectin matrix required RD2 in addition to UR
Summary
Bacterial Strains and Growth Conditions—Escherichia coli strains SG13009(pREP4) (Qiagen Inc.) and MV1184 (Takara Shuzo) were used for expression of fusion proteins. Purified protein containing various concentrations of either UR, RD2, or UR plus five RD2 repeats was added to superfibronectin-coated wells for 1 h at room temperature. 100 l of the purified fusion protein containing either UR, RD2, or UR plus five repeats of RD2 at 10 g/ml was added to superfibronectin- or the III1-C-coated well at various concentrations and incubated for 2 h at room temperature. After washing the wells three times with PBS/T, the amount of each purified protein bound to superfibronectin or to the III1-C fragment was measured by ELISA using a monoclonal RGS1⁄7His antibody (Qiagen Inc.) and an alkaline phosphate-conjugated anti-mouse IgG antiserum (Sigma).
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