Julia A. Sharp

Staphylococcus aureus surface protein SdrE binds complement regulator factor H as an immune evasion tactic.

Similar to other highly successful invasive bacterial pathogens, Staphylococcus aureus recruits the complement regulatory protein factor H (fH) to its surface to inhibit the alternative pathway of complement. Here, we report the identification of the surface-associated protein SdrE as a fH-binding protein using purified fH overlay of S. aureus fractionated cell wall proteins and fH cross-linking to S. aureus followed by mass spectrometry. Studies using recombinant SdrE revealed that rSdrE bound significant fH whether from serum or as a purified form, in both a time- and dose-dependent manner. Furthermore, rSdrE-bound fH exhibited cofactor functionality for factor I (fI)-mediated cleavage of C3b to iC3b which correlated positively with increasing amounts of fH. Expression of SdrE on the surface of the surrogate bacterium Lactococcus lactis enhanced recruitment of fH which resulted in increased iC3b generation. Moreover, surface expression of SdrE led to a reduction in C3-fragment deposition, less C5a generation, and reduced killing by polymorphonuclear cells. Thus, we report the first identification of a S. aureus protein associated with the staphylococcal surface that binds factor H as an immune evasion mechanism.

Peptide Inhibitor of Complement C1, a Novel Suppressor of Classical Pathway Activation: Mechanistic Studies and Clinical Potential

The classical pathway of complement plays multiple physiological roles including modulating immunological effectors initiated by adaptive immune responses and an essential homeostatic role in the clearance of damaged self-antigens. However, dysregulated classical pathway activation is associated with antibody-initiated, inflammatory diseases processes like cold agglutinin disease, acute intravascular hemolytic transfusion reaction (AIHTR), and acute/hyperacute transplantation rejection. To date, only one putative classical pathway inhibitor, C1 esterase inhibitor (C1-INH), is currently commercially available and its only approved indication is for replacement treatment in hereditary angioedema, which is predominantly a kinin pathway disease. Given the variety of disease conditions in which the classical pathway is implicated, development of therapeutics that specifically inhibits complement initiation represents a major unmet medical need. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement. In vitro studies have demonstrated that these peptide inhibitors of complement C1 (PIC1) bind to the collagen-like region of the initiator molecule of the classical pathway, C1q. PIC1 binding to C1q blocks activation of the associated serine proteases (C1s–C1r–C1r–C1s) and subsequent downstream complement activation. Rational design optimization of PIC1 has resulted in the generation of a highly potent derivative of 15 amino acids. PIC1 inhibits classical pathway mediated complement activation in ABO incompatibility in vitro and inhibiting classical pathway activation in vivo in rats. This review will focus on the pre-clinical development of PIC1 and discuss its potential as a therapeutic in antibody-mediated classical pathway disease, specifically AIHTR.

Peptide Inhibitor of Complement C1 (PIC1) Rapidly Inhibits Complement Activation after Intravascular Injection in Rats

The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1). In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases.

Disruption of the alternative pathway convertase occurs at the staphylococcal surface via the acquisition of factor H by Staphylococcus aureus

Staphylococcus aureus is a significant human pathogen that causes skin-structure, invasive, and hospital-associated infections worldwide. The complement system is vital to innate defense against many bacterial infections. As shown with other pathogens, mechanisms for circumventing complement attack may include recruitment of the complement regulatory protein factor H (fH). In the present study, we show that S. aureus binds fH in a dose-dependent and time-dependent manner. Interestingly, this interaction does not require complement activation nor C3-fragment presence and occurs efficiently in the absence of other serum components suggesting a mechanism other than bridging between intermediary molecules. However, fH binding is greater when incubated with normal human serum compared to heat-inactivated serum, which suggests that complement activation may enhance fH binding. S. aureus-bound fH was found to inhibit the alternative pathway through disruption of the alternative pathway C3 convertase as shown by an increase in Bb release and a decrease in total C3-fragment deposition. Furthermore, S. aureus-bound fH retains cofactor activity for factor-I mediated cleavage of C3b. These studies show that the acquisition of fH to the S. aureus surface inhibits complement-mediated opsonization via disruption of the alternative pathway convertase; thus, we report an immune-evasion mechanism not previously described for S. aureus.

Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis

Prevention of acute hemolytic transfusion reactions is a worldwide concern. The objective of this study was to develop a simple rat model of complement‐mediated acute intravascular hemolysis.

Peptide Inhibitor of Complement C1 (PIC1) Inhibits Growth of Pathogenic Bacteria

Peptide Inhibitor of Complement C1 (PIC1) is a family of 15 amino acid peptides that inhibit complement activation via the classical and lectin pathways and inhibit myeloperoxidase. PIC1 peptides were originally derived from a region of limited homology with defensin human neutrophil peptide 1 (HNP-1). Despite having undergone extensive rearrangements of amino acid sequence subsequently, PIC1 peptides retain the defensin-like characteristics of being cysteine rich and amphiphilic. To date, defensin-like antimicrobial activity for PIC1 has not been explored. Here we report the antimicrobial activity of PIC1 for multiple pathogenic bacteria tested in minimum inhibitory concentration (MIC)-type assays. PIC1variant PA-dPEG24 was found to have antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Neisseria meningitidis, Neisseria gonorrhoeae, Gardnerella vaginalis, and Prevotella bivia. Confocal microscopy demonstrated PIC1 localized to the surface of P. aeruginosa and S. aureus consistent with the defensins. Testing PIC1 variants with amino acid substitutions revealed differences in complement inhibition and antimicrobial effects suggesting these occur via independent mechanisms. PIC1 inhibited P. aeruginosa growth in normal human serum suggesting the antimicrobial effect was dominant versus the survival benefit resulting from complement inhibition. In summary, these experiments demonstrate that PIC1 peptides have broad antimicrobial activity against pathogenic bacteria similar to defensins.