Fibronectin Receptors from Gram-Positive Bacteria: Comparison of Active Sites

Abstract

Many parasitic bacteria express fibronectin binding proteins that are located on the cell surface. These proteins may act as adhesins and mediate the adherence of the microorganisms to fibronectin-containing host tissues. The ligand binding sites in the fibronectin receptor proteins from Gram-positive bacteria are composed of unique 37-48 amino acid long motifs that are repeated 3-4 times. We have now expressed the ligand binding sites of fibronectin receptors from Staphylococcus aureus, Streptococcus dysgalactiae (two receptors), and Streptococcus pyogenes as recombinant proteins. The purified recombinant proteins have the expected molecular weights as indicated by electrospray mass spectroscopy although they migrate abnormally on SDS-PAGE. Each recombinant protein effectively inhibited the binding of 125I-labeled intact fibronectin or the N-terminal fibronectin domain to Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus pyogenes. The relative inhibitory potency of the different recombinant proteins was similar for all target bacteria and is reflected in their relative affinities for fibronectin. Synthetic peptides corresponding to the repeat units of the ligand binding site of the fibronectin receptor proteins were shown to inhibit the binding of the N-terminal fibronectin fragment to Streptococcus pyogenes cells. Together with amino acid sequence comparison, these data demonstrate that the repeat motif of the fibronectin receptor of Streptococcus pyogenes conforms to the consensus sequence previously reported for the Staphylococcus aureus receptor and to one of the Streptococcus dysgalactiae receptors (McGavin et al., 1993).

High Affinity Streptococcal Binding to Human Fibronectin Requires Specific Recognition of Sequential F1 Modules

“…In addition, sequence comparisons could not always predict or explain the binding properties of apparently homologous repeats. The cross-reactivity observed in inhibition studies with FnBRs from different species was also difficult to interpret (5,35,41).…”

“…4B). 5 F1 shows that these modules share 51 and 45% sequence identity, respectively. This seems to be reflected in a certain degree of homology between F1-binding motifs in FnBRs.…”

Characterization of the Interaction Between the Staphylococcus Aureus Clumping Factor (ClfA) and Fibrinogen

“…Adherence was determined as a function of the absorbance at 495 nm as quantitated using a plate reader (Thermo Max, Molecular Devices). To determine the inhibition of bacterial attachment by purified recombinant proteins, varying concentrations of Clf33 or rFnBD-D ( [25], a recombinant truncated form of the fibronectin-binding MSCRAMM FnBPA of S. aureus that expresses fibronectin-binding activity) were incubated for 30 min in the fibrinogen-coated wells after the blocking step. The bacterial suspension was then added to the wells that contained the potential inhibitors.…”

“…The prepared blood was incubated with either a range from 0.5 pM to 4.04 pM Clf33, 0.35 yM and 1.5 pM rFnBD-A ( [25], a recombinant truncated form of the fibronectin-binding MSCRAMM FnBPA of Streptococcus dysgalactiae) or an equal volume of buffer A at 37°C for 5 min. The fluorescent dye, mepacrine (quinacrine dihydrochloride; Sigma), was added at a final concentration of 10 pM in order to visualize the platelets [33].…”

Interactions of the streptococcal C5a peptidase with human fibronectin

“…Hydrogen bonding [13], electrostatic interactions [14], specific interactions between surface proteins and extracellular matrix proteins [11,15,16], and shear forces [17] all have been shown to mediate bacterial adhesion. GBS will only adhere to adsorbed Fn, not soluble Fn1, 18,19; this behavior helps GBS evade the host immune system since soluble Fn acts as an opsonin [20].…”