The group A streptococcus (GAS) is an important human pathogen that causes a variety of diseases ranging from superficial infections of the throat and skin to invasive infections with high degrees of morbidity and mortality. Our long-term objective is to develop strategies to prevent or reduce the morbidity and mortality caused by GAS. M-related proteins (Mrp) are among a variety of virulence factors that contribute to the pathogenesis of these infections. A clear understanding of the mechanisms whereby Mrp contributes to the pathogenesis of streptococcal infections will help in devising methods to thwart these mechanisms. Infections due to GAS are primarily limited to humans and most animals are naturally resistant to GAS. The factors that contribute to this restricted host specificity are not well understood. The hypothesis of this proposal is that the ability of Mrp to preferentially bind human IgG contributes to host specificity of GAS and to the virulence of GAS.
The specific aims are:
Specific Aim 1. To determine the host specificity in the binding of IgG to Mrp. To determine the extent to which the binding of IgG is species-dependent, purified IgG from various animals will be commercially obtained and tested for direct binding to Mrp and for the ability to block the binding of human IgG to Mrp and to GAS. The binding affinities of Mrp for the various animal IgG will be determined and compared to that of humans.
Specific Aim 2. To determine if the IgG-binding domain of Mrp confers resistance to phagocytosis in human and mouse blood. We have localized the IgG-binding domain of Mrp4 to it's A repeats and have engineered a recombinant protein, rMrp??, in which the A repeats have been deleted in-frame. This construct does not bind IgG but still binds fibrinogen. To determine if the IgG-binding is involved in resistance to phagocytosis, we will use this construct to engineer a mutant of GAS in which the A repeats of Mrp are deleted in-frame and test it for growth in whole human blood and in mouse blood supplemented with human IgG. We have purified a recombinant protein containing the A repeats and will test this to determine if it can promote phagocytosis in human blood by blocking the binding of IgG to Mrp on the surface of GAS.
Specific Aim 3. To determine if the ability of Mrp to selectively bind human IgG contributes to virulence and host specificity of GAS in a mouse model. To investigate the impact of human IgG binding to Mrp on pathogenesis of GAS infections, groups of mice will be injected IP with either human IgG, human serum, human plasma, bovine IgG (negative control), or bovine serum (negative control) and then challenged with wild type M 4 S. pyogenes (SP4), with the Mrp-negative mutant (MP4), or with the mutant expressing Mrp?A. A comparison of survival rates will determine if the binding of human IgG to Mrp contributes to virulence in a mouse model and to host specificity.
Streptococcus pyogenes is the ninth leading cause of mortality among all microbial pathogens worldwide. The proposed studies will increase our understanding of how interactions between M-related protein (Mrp) and IgG contribute to virulence and to the host-specificity of GAS. A clear understanding of the molecular nature of the interactions between Mrp and human IgG may lead to the development of strategies that interfere with this interaction and thereby prevent or reduce the morbidity and mortality associated with infections by S pyogenes.