Protein H, an antiphagocytic surface protein in Streptococcus pyogenes.

Surface-associated M protein is a major virulence factor in Streptococcus pyogenes which confers bacterial resistance to phagocytosis. However, many S. pyogenes strains also express additional structurally related so-called M-like proteins. The strain studied here is of the clinically important M1 serotype and expresses two structurally related surface proteins, the M1 protein and protein H. Mutants were generated that expressed only one or none of these proteins at the bacterial surface. For survival in human blood either protein H or M1 protein was sufficient, whereas the double mutant was rapidly killed. The protein-binding properties of protein H, M1 protein, and the mutants suggest that bacterial binding of immunoglobulin G and factor H or factor H-like protein 1, which are regulatory proteins in the complement system, contribute to the antiphagocytic property.

Streptococcal protein H forms soluble complement-activating complexes with IgG, but inhibits complement activation by IgG-coated targets.

Protein H, a surface protein of Streptococcus pyogenes interacting with the constant Fc region of IgG, is known to be released from the streptococcal surface by a cysteine proteinase produced by the bacteria. Poststreptococcal glomerulonephritis and rheumatic fever are conditions in which immune complexes and autoimmune mechanisms have been suggested to play pathogenetic roles. The present study demonstrates that addition of protein H to human serum produces complement activation with dose-dependent cleavage of C3. The activation was IgG-dependent and the result of complexes formed between IgG and protein H. These complexes were size heterogeneous with molecular masses of 400 kDa to 1.4 MDa. Using complement-depleted serum reconstituted with complement proteins, the activation by protein H was found to be dependent of the classical, but independent of the alternative pathway of complement. In contrast to results of experiments based on soluble protein H.IgG complexes, complement activation was inhibited by protein H when IgG was immobilized on a surface. The interaction between C1q and immunoglobulins represents the first step in the activation of the classical pathway, and protein H efficiently inhibited the binding of C1q to IgG immobilized on polyacrylamide beads. Protein H reduced C3 deposition on the IgG-coated beads and inhibited immune hemolysis of IgG-sensitized erythrocytes. Finally, significantly less C3 was deposited on the surface of protein H-expressing wild-type streptococci than on the surface of isogenic mutant bacteria devoid of protein H. The results demonstrate that protein H.IgG complexes released from the streptococcal surface can produce complement breakdown at the sites of infection, whereas complement activation on bacterial surfaces is inhibited. This should have important implications for host-parasite relationships. In addition, soluble protein H.IgG complexes might contribute to immunological complications of streptococcal infections.

Characterization of the binding properties of protein LG, an immunoglobulin-binding hybrid protein.

Protein LG is a 50-kDa hybrid molecule containing four Ig-light-chain-binding domains from protein L of Peptostreptococcus magnus and two IgG-Fe-binding repeats from streptococcal protein G. Here we analyse the binding of protein LG to Ig from several mammalian species. Protein LG was shown to bind human IgG of all subclasses and other Ig classes that carry kappa chains. The binding to human IgG was only marginally influenced by changes in temperature (4-37 degrees C) or salt concentration (0-1.6 M), and was stable over a wide pH range (pH 4-10). Protein LG bound to Ig from 11 of 12 mammalian species, including those of rabbit, mouse and rat. The affinity constants obtained for the interactions between protein LG and polyclonal IgG from rabbit (4.0 x 10(9) M-1), mouse (1.7 x 10(9) M-1) and rat (1.3 x 10(9) M-1) were similar to the value previously reported for the interaction between the hybrid protein and human polyclonal IgG (5.9 x 10(9) M-1). The interaction between protein LG and a mouse IgG mAb was not influenced by the presence of the specific protein antigen, nor was the binding of this antibody to its ligand affected by protein LG. Inhibition experiments demonstrated that the Ig-binding site of one of the fusion partners retained its ligand-binding capacity when the other component was occupied. Protein LG selectively absorbed 85-90% of the total Ig present in human and rabbit sera and 75-80% of the Ig in sera from mouse and rat. Human serum depleted of C1q, factor D and properdin and preabsorbed by protein LG could be used as a source for other complement factors. These data demonstrate that protein LG is a very versatile Ig-binding protein.

Biological properties of a Streptococcus pyogenes mutant generated by Tn916 insertion in mga.

The mga regulon of Streptococcus pyogenes contains genes which contribute to the pathogenicity and virulence of this significant human pathogen. Transposon insertional inactivation of the regulatory mga gene in a S. pyogenes strain of the clinically important M1 serotype, blocked the expression of four genes located downstream of mga. These genes encode the M1 protein, the IgG-binding protein H, protein SIC which is an extracellular inhibitor of complement, and the C5a peptidase which interferes with granulocyte migration. The wild-type strain is resistant to phagocytosis and adheres to human skin tissue sections; properties that were lost in the transposon mutant. Moreover, the mutant was less virulent to mice but more cytolytic to human lymphocytes, the latter due to an increased activity of streptolysin S, whereas the production of streptolysin O, another toxin of S. pyogenes, was not affected. The mga mutation was complemented in trans with an intact mga gene which restored the phenotype of the wild-type strain.

Protein LG: a hybrid molecule with unique immunoglobulin binding properties.

Immunoglobulin (Ig)-binding bacterial proteins have attracted theoretical interest for their role in molecular host-parasite interactions, and they are widely used as tools in immunology, biochemistry, medicine, and biotechnology. Protein L of the anaerobic bacterial species Peptostreptococcus magnus binds Ig light chains, whereas streptococcal protein G has affinity for the constant (Fc) region of IgG. In this report, Ig binding parts of protein L and protein G were combined to form a hybrid molecule, protein LG, which was found to bind a large majority of intact human Igs as well as Fc and Fab fragments, and Ig light chains. Binding to Ig was specific, and the affinity constants of the reactions between protein LG and human IgG, IgGFc fragments, and kappa light chains, determined by Scatchard plots, were 5.9 x 10(9), 2.2 x 10(9), and 2.0 x 10(9) M-1, respectively. The binding properties of protein LG were more complete as compared with previously described Ig-binding proteins when also tested against mouse and rat Igs. This hybrid protein thus represents a powerful tool for the binding, detection, and purification of antibodies and antibody fragments.