Monday, June 5, 2023
6/5/2023
PROJECT ABSTRACT The innate immune system is best known as an essential defense against invading microbes. In humans, this response requires an immediate and concerted action by both humoral and cellular components, which are represented by the complement system and neutrophils, respectively. Efficient killing of microbes by this so- called “complement/neutrophil axis” is predicated upon a highly orchestrated and stepwise series of molecular recognition events and biochemical transformations, which at their most fundamental level involve enzymes. As a consequence of host/pathogen co-evolution, the Gram-positive bacterium Staphylococcus aureus has developed a powerful array of small protein inhibitors that block many of the central enzymatic players of the innate immune response. In this regard, we identified three secreted staphylococcal proteins, called Eap, EapH1, and EapH2 (denoted “EAP proteins”), which potently inhibit three different proteases known as Neutrophil Serine Proteases (NSPs) that are critical components of the neutrophil's anti-bacterial arsenal. In addition to this, Eap itself also inhibits assembly of a multi-subunit protease system that is required for function of the classical and lectin complement pathways. Separately, we also identified a new staphylococcal protein, called “SPIN”, that is a potent inhibitor of the HOCl-generating myeloperoxidase (MPO) found in neutrophils. Collectively, these S. aureus proteins interfere with bacterial killing in both in vitro systems and animal models. While our initial studies on EAP proteins and SPIN have provided important information on the structure, function, and mechanism of these novel enzyme inhibitors, many significant questions still remain. In this project, we will employ a combination of structural, biochemical, functional, and informatics approaches to address these issues. In the first series of investigations, we will determine how the individual repeating domains of S. aureus Eap inhibit NSPs. This will provide a means for comparative analysis to the Eap homologs, EapH1 and EapH2, which are more extensively characterized. We will also work to define the structural determinants within Eap that allow this protein, but not EapH1 or EapH2, to inhibit the complement system in addition to NSPs. In the second series of investigations, we will explore the structural transitions that allow SPIN to adopt an inhibitory conformation upon binding to MPO. We will also define the structural determinants within SPIN proteins that provide an exquisite level of selectivity for MPO when compared to closely related heme peroxidases. In our final series of studies, we will leverage our extensive structural and functional data on SPINs and staphylococcal complement inhibitors toward establishing a paradigm for understanding the physical basis for host species specificity of virulence proteins. By completing this research plan, we will further our understanding of two novel classes of enzyme inhibitors that function at the host/pathogen interface, and lay the basic science foundation for future development of anti-bacterial and anti-inflammatory therapies arising from the information we uncover.
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