(Project Description) The human body relies on neutrophils to provide a sterilizing innate immune response against bacterial pathogens. Although neutrophils circulate inside the blood in a quiescent state, they are rapidly activated in reponse to a number of biochemical patterns which signify either that potential pathogens are present or that cellular damage has occured. Activation of neutrophils results in remarkable changes in their morphology, and triggers mobilization and secretion of their cytosolic granules. It is these granules which contain critical components of the neutrophil?s anti-bacterial arsenal. Two of the most abudant components of these granules are the enzyme myeloperoxidase (MPO), which converts hydrogen peroxide into cytotoxic hypohalous acids, and a series of chymotrypsin-like serine proteases (NSPs), which can directly attack the pathogen cell by cleaving proteins that are either exposed on its surface or secreted into the environment. Together, the combined action of MPO and NSPs form the foundation of neutrophil-mediated innate defense against invading bacteria. As a consequence of host/pathogen co-evolution, the Gram-positive bacterium Staphylococcus aureus has developed a powerful array of small protein inhibitors that effectively block many of the critical components of the human innate immune response. In this regard, we recently identified three secreted staphylococcal proteins, called Eap, EapH1, and EapH2 (denoted ?EAP proteins?), which potently inhibit NSPs, as well as a novel staphylococccal inhibitior of MPO, called SPIN. Through collaborative efforts, we have established that both EAP proteins and SPIN are required for maximal S. aureus virulence in animal infection models. In this project, we will use a synergistic series of crystallographic and solution NMR methods, physical biochemistry approaches, and activity assays to provide detailed structure/function information on these novel staphylococcal inhibitors of neutrophil granule enzymes. We will accomplish this overall goal through two concurrent Specific Aims. In the first Aim, we will investigate the structural basis for the selectivity of EAP domains toward NSPs, examine whether changes in protein dynamics influence EAP/NSP interactions, and define a structure/activity relationship for NSP inhibition by EAP domain proteins. In the second Aim, we will determine the structural basis for SPIN/MPO binding, examine whether SPIN undergoes changes in conformation upon interaction with MPO, and define the biochemical determinants which mediate MPO inhibition by SPIN. Finally, since NSPs and MPO are known to play signifcant roles in damaging host cells and tissues in a number of human inflammatory diseases, we will explore whether synthetic peptides based upon the structures of EAP proteins and SPIN bound to their targets can mimic the therapeutically-valuable activities of these staphylococcal immune evasion proteins. By completing this research plan, we will lay the basic science foundation for future development of anti-bacterial and anti-inflammatory therapies based upon the information that we uncover here.
(Public Health Relevance Statement) In this project, we will investigate two newly discovered families of Staphylococcus aureus proteins that act as potent inhibitors of key enzyme systems required for the anti-bacterial activities of neutrophils. The first family of inhibitors, called EAP proteins, targets a series immune proteases found exclusively in human neutrophils, while the second inhibitor, called SPIN, blocks the myeloperoxidase which generates cytotoxic hypohalous acids from hydrogen peroxide. Since neutrophils are the primary type of white blood cell responsible for our body?s innate response against S. aureus as well as a key driver of inflammatory processes in human diseases like COPD/emphysema, rheumatoid arthritis, and atherosclerosis, understanding how S. aureus blocks neutrophil functions may reveal new approaches to preventing and/or treating staphylococcal infections and provide us with a basic science foundation to support future development of novel anti-inflammatory inhibitory therapeutics.
