Streptococcal protein FOG, a novel matrix adhesin interacting with collagen I in vivo.

Group G streptococcus (GGS) is a human pathogen of emerging clinical significance. It causes skin and soft tissue infections, occasionally resulting in life-threatening conditions such as sepsis and necrotizing fasciitis. We recently identified FOG, a novel surface protein of GGS with fibrinogen binding and immune evasion properties. Here we investigated the role of FOG in streptococcal primary adhesion to host tissue. A FOG-expressing clinical isolate adhered more efficiently to human skin biopsies ex vivo and to the murine dermis in vivo than a FOG-deficient strain. Scanning and transmission electron microscopy of skin specimens exhibited that this property was assigned to the ability of FOG to interact with collagen I, a major interstitial component of the dermis. Overlay experiments with human skin extracts and radiolabeled FOG followed by matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis identified both the alpha1- and alpha2-chains of collagen I as targets for FOG. Transmission electron microscopy of the molecular complexes revealed thread-like FOG molecules binding via their NH2 termini to distinct sites on collagen I monomers and fibrils. The results demonstrate that FOG is important for GGS adhesion in vivo, implying a pathogenic role for this surface protein.

Activation of the complement system generates antibacterial peptides.

The complement system represents an evolutionary old and significant part of the innate immune system involved in protection against invading microorganisms. Here, we show that the anaphylatoxin C3a and its inactivated derivative C3a-desArg are antibacterial, demonstrating a previously unknown direct antimicrobial effect of complement activation. The C3a peptide, as well as functional epitopes in the sequence, efficiently killed the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and the Gram-positive Enterococcus faecalis. In mice, a C3a-derived peptide suppressed infection by Gram-positive Streptococcus pyogenes bacteria. Fluorescence and electron microscopy demonstrated that C3a binds to and induces breaks in bacterial membranes. C3a was also found to induce membrane leakage of liposomes. These findings provide an interesting link between the complement system and antimicrobial peptides, which are two important branches of innate immunity.

Characterization of a pseudoachondroplasia-associated mutation (His587-->Arg) in the C-terminal, collagen-binding domain of cartilage oligomeric matrix protein (COMP).

We have introduced a pseudoachondroplasia-associated mutation (His(587)-->Arg) into the C-terminal collagen-binding domain of COMP (cartilage oligomeric matrix protein) and recombinantly expressed the full-length protein as well as truncated fragments in HEK-293 cells. CD spectroscopy revealed only subtle differences in the overall secondary structure of full-length proteins. Interestingly, the mutant COMP did not aggregate in the presence of calcium, as does the wild-type protein. The binding site for collagens was recently mapped to amino acids 579-595 and it was assumed that the His(587)-->Arg mutation influences collagen binding. However full-length mutant COMP bound to collagens I, II and IX, and the binding was not significantly different from that of wild-type COMP. Also a COMP His(587)-->Arg fragment encompassing the calcium-binding repeats and the C-terminal collagen-binding domain bound collagens equally well as the corresponding wild-type protein. The recombinant fragments encompassing the C-terminal domain alone showed multiple bands following SDS/PAGE, although their theoretical molecular masses could be verified by MS. A temperature-induced conformational change was observed in CD spectroscopy, and negative-staining electron microscopy demonstrated that both wild-type and mutant proteins formed defined elongated aggregates after heating to 60 degrees C. Our results suggest that the His(587)-->Arg mutation is not itself deleterious to the structure and collagen-binding of COMP.

SC1/hevin. An extracellular calcium-modulated protein that binds collagen I.

SC1, a member of the BM-40 family of extracellular matrix proteins, was recombinantly expressed in a eukaryotic expression system. The full-length protein as well as truncated versions were purified to homogeneity under non-denaturing conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of full-length SC1 revealed a mass of 87.8 kDa of which 16.8 kDa is contributed by posttranslational modifications. In electron microscopy, after negative staining, SC1 was revealed as a globule attached to a thread-like structure. A calcium dependence of the SC1 conformation could be demonstrated by fluorescence spectroscopy. In the extracellular matrix of cultured osteosarcoma cells SC1 was found associated with collagen I-containing fibrils, and binding of SC1 to reconstituted collagen I fibrils could be demonstrated by immunogold labeling and electron microscopy. SC1 showed a broad expression in a variety of tissues.