Kasra X. Ramyar

Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

Significance Neutrophils are among the first immune cells to migrate to the site of infection and clear invading bacteria. They store large amounts of neutrophil serine proteases (NSPs) that play key roles in immune defense. Unfortunately, NSPs also contribute to tissue destruction in a variety of inflammatory disorders. In this study we discover that the pathogenic bacterium Staphylococcus aureus secretes a family of highly potent and specific NSP inhibitors that promote the pathogenicity of this bacterium in vivo. From crystallography experiments, we conclude that these proteins constitute a unique class of NSP inhibitors, which can be used to design novel treatment strategies against excessive NSP activity. Furthermore, this study significantly increases our understanding of the complex nature of S. aureus infections. Neutrophils are indispensable for clearing infections with the prominent human pathogen Staphylococcus aureus. Here, we report that S. aureus secretes a family of proteins that potently inhibits the activity of neutrophil serine proteases (NSPs): neutrophil elastase (NE), proteinase 3, and cathepsin G. The NSPs, but not related serine proteases, are specifically blocked by the extracellular adherence protein (Eap) and the functionally orphan Eap homologs EapH1 and EapH2, with inhibitory-constant values in the low-nanomolar range. Eap proteins are together essential for NSP inhibition by S. aureus in vitro and promote staphylococcal infection in vivo. The crystal structure of the EapH1/NE complex showed that Eap molecules constitute a unique class of noncovalent protease inhibitors that occlude the catalytic cleft of NSPs. These findings increase our insights into the complex pathogenesis of S. aureus infections and create opportunities to design novel treatment strategies for inflammatory conditions related to excessive NSP activity.

Molecular Basis for Complement Recognition and Inhibition Determined by Crystallographic Studies of the Staphylococcal Complement Inhibitor (SCIN) Bound to C3c and C3b.

The human complement system plays an essential role in innate and adaptive immunity by marking and eliminating microbial intruders. Activation of complement on foreign surfaces results in proteolytic cleavage of complement component 3 (C3) into the potent opsonin C3b, which triggers a variety of immune responses and participates in a self-amplification loop mediated by a multi-protein assembly known as the C3 convertase. The human pathogen Staphylococcus aureus has evolved a sophisticated and potent complement evasion strategy, which is predicated upon an arsenal of potent inhibitory proteins. One of these, the staphylococcal complement inhibitor (SCIN), acts at the level of the C3 convertase (C3bBb) and impairs downstream complement function by trapping the convertase in a stable but inactive state. Previously, we have shown that SCIN binds C3b directly and competitively inhibits binding of human factor H and, to a lesser degree, that of factor B to C3b. Here, we report the co-crystal structures of SCIN bound to C3b and C3c at 7.5 and 3.5 A limiting resolution, respectively, and show that SCIN binds a critical functional area on C3b. Most significantly, the SCIN binding site sterically occludes the binding sites of both factor H and factor B. Our results give insight into SCIN binding to activated derivatives of C3, explain how SCIN can recognize C3b in the absence of other complement components, and provide a structural basis for the competitive C3b-binding properties of SCIN. In the future, this may suggest templates for the design of novel complement inhibitors based upon the SCIN structure.

The Extracellular Adherence Protein from Staphylococcus aureus Inhibits the Classical and Lectin Pathways of Complement by Blocking Formation of the C3 Proconvertase

The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. Although the majority of staphylococcal complement inhibitors act on the alternative pathway to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical pathway (CP) and lectin pathway (LP). We screened a collection of recombinant, secreted staphylococcal proteins to determine whether S. aureus produces other molecules that inhibit the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 proconvertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion.

Advances in Understanding the Structure, Function, and Mechanism of the SCIN and Efb Families of Staphylococcal Immune Evasion Proteins

Our understanding of both the nature and diversity of Staphylococcal immune evasion proteins has increased tremendously throughout the last several years. Among this group of molecules, members of the SCIN and Efb families of complement inhibitors have been the subject of particularly intense study. This work has demonstrated that both types of proteins exert their primary function by inhibiting C3 convertases, which lie at the heart of the complement-mediated immune response. Despite this similarity, however, significant differences in structure/function relationships and mechanisms of action exist between these bacterial proteins. Furthermore, divergent secondary effects on host immune responses have also been described for these two protein families. This chapter summarizes recent advances toward understanding the structure, function, and mechanism of the SCIN and Efb families, and suggests potential directions for the field over the coming years.

Diversity in the C3b contact residues and tertiary structures of the staphylococcal complement inhibitor (SCIN) protein family.

Background: Staphylococcus aureus has evolved a web of mechanisms to disrupt the human complement system. Results: We report the structures of two staphylococcal complement inhibitor proteins, SCIN-B and SCIN-D. Conclusion: We have identified differences in C3b recognition within active SCIN proteins and suggest a physical basis for lack of C3b binding by SCIN-D. Significance: This analysis may inform future design of complement-targeted therapeutics. To survive in immune-competent hosts, the pathogen Staphylococcus aureus expresses and secretes a sophisticated array of proteins that inhibit the complement system. Among these are the staphylococcal complement inhibitors (SCIN), which are composed of three active proteins (SCIN-A, -B, and -C) and one purportedly inactive member (SCIN-D or ORF-D). Because previous work has focused almost exclusively on SCIN-A, we sought to provide initial structure/function information on additional SCIN proteins. To this end we determined crystal structures of an active, N-terminal truncation mutant of SCIN-B (denoted SCIN-B18–85) both free and bound to the C3c fragment of complement component C3 at 1.5 and 3.4 Å resolution, respectively. Comparison of the C3c/SCIN-B18–85 structure with that of C3c/SCIN-A revealed that both proteins target the same functional hotspot on the C3b/C3c surface yet harbor diversity in both the type of residues and interactions formed at their C3b/C3c interfaces. Most importantly, these structures allowed identification of Arg44 and Tyr51 as residues key for SCIN-B binding to C3b and subsequent inhibition of the AP C3 convertase. In addition, we also solved several crystal structures of SCIN-D to 1.3 Å limiting resolution. This revealed an unexpected structural deviation in the N-terminal α helix relative to SCIN-A and SCIN-B. Comparative analysis of both electrostatic potentials and surface complementarity suggest a physical explanation for the inability of SCIN-D to bind C3b/C3c. Together, these studies provide a more thorough understanding of immune evasion by S. aureus and enhance potential use of SCIN proteins as templates for design of complement targeted therapeutics.

Biochemical and structural basis for inhibition of Enterococcus faecalis hydroxymethylglutaryl-CoA synthase, mvaS, by hymeglusin.

Hymeglusin (1233A, F244, L-659-699) is established as a specific β-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme-inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS-hymeglusin cocrystal structure (1.95 Å) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.

A Structurally Dynamic N-terminal Helix Is a Key Functional Determinant in Staphylococcal Complement Inhibitor (SCIN) Proteins

Background: SCIN proteins from S. aureus mediate immune evasion by binding to C3b. Results: A structurally dynamic region exists within N termini of SCIN proteins and affects their C3b binding and inhibitory properties. Conclusion: The N terminus is a critical functional determinant in multiple SCINs. Significance: This work provides a new model for understanding SCIN structure and function. Complement is a network of interacting circulatory and cell surface proteins that recognizes, marks, and facilitates clearance of microbial invaders. To evade complement attack, the pathogenic organism Staphylococcus aureus expresses a number of secreted proteins that interfere with activation and regulation of the complement cascade. Staphylococcal complement inhibitors (SCINs) are one important class of these immunomodulators and consist of three active members (SCIN-A/-B/-C). SCINs inhibit a critical enzymatic complex, the alternative pathway C3 convertase, by targeting a functional “hot spot” on the central opsonin of complement, C3b. Although N-terminal truncation mutants of SCINs retain complement inhibitory properties, they are significantly weaker binders of C3b. To provide a structural basis for this observation, we undertook a series of crystallographic and NMR dynamics studies on full-length SCINs. This work reveals that N-terminal SCIN domains are characterized by a conformationally dynamic helical motif. C3b binding and functional experiments further demonstrate that this sequence-divergent N-terminal region of SCINs is both functionally important and context-dependent. Finally, surface plasmon resonance data provide evidence for the formation of inhibitor·enzyme·substrate complexes ((SCIN·C3bBb)·C3). Similar to the (SCIN·C3bBb)2 pseudodimeric complexes, ((SCIN·C3bBb)·C3) interferes with the interaction of complement receptors and C3b. This activity provides an additional mechanism by which SCIN couples convertase inhibition to direct blocking of phagocytosis. Together, these data suggest that tethering multi-host protein complexes by small modular bacterial inhibitors may be a global strategy of immune evasion used by S. aureus. The work presented here provides detailed structure-activity relationships and improves our understanding of how S. aureus circumvents human innate immunity.

Immune evasion by a staphylococcal inhibitor of myeloperoxidase

Significance Staphylococcus aureus secretes numerous proteins to evade our innate immune system, for example to evade opsonization and phagocytosis by neutrophils. Here we describe the discovery that S. aureus has evolved a protein, called SPIN, that specifically binds and inhibits the human myeloperoxidase enzyme (MPO). MPO is located inside the granules of neutrophils and is important in the oxidative burst against pathogens. We identify the molecular mode of action of SPIN inhibiting MPO, illustrate this with the cocrystal structure, and show that SPIN is important for bacterial survival by MPO-dependent killing. Our study shows that S. aureus fights back after it is engulfed by neutrophils, which will help our understanding of the complex nature of S. aureus infections. Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein “staphylococcal peroxidase inhibitor” (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing.

Crystallization of human complement component C3b in the presence of a staphylococcal complement-inhibitor protein (SCIN).

Staphylococcus aureus secretes a number of small proteins that effectively attenuate the human innate immune response. Among these, the staphylococcal complement-inhibitor protein (SCIN) disrupts the function of the complement component 3 (C3) convertase that is initiated through either the classical or the alternative pathway and thereby prevents amplification of the complement response on the bacterial surface. Recent studies have shown that SCIN may affect the activities of the C3 convertase by binding in an equimolar fashion to C3b, which is itself an integral although non-enzymatic component of the convertase. In order to better understand the nature of the C3b-SCIN interaction, the hanging-drop vapor-diffusion technique was used to crystallize human C3b in the presence of a recombinant form of SCIN. These crystals diffracted synchrotron X-rays to approximately 6 A Bragg spacing and grew in a primitive tetragonal space group (P4(1)2(1)2 or P4(3)2(1)2; unit-cell parameters a = b = 128.03, c = 468.59 A). Cell-content analysis of these crystals was consistent with the presence of either two 1:1 complexes or a single 2:2 assembly in the asymmetric unit, both of which correspond to a solvent content of 51.9%. By making use of these crystals, solution of the C3b-SCIN structure should further our understanding of complement inhibition and immune evasion by this pathogen.

MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne's Disease

ABSTRACT The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johnes disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johnes disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis.