Design and Biological Evaluation of the Long-Acting C5-Inhibited Ornithodoros moubata Complement Inhibitor (OmCI) Modified with Fatty Acid.

Disorder of complement response is a significant pathogenic factor causing some autoimmune and inflammation diseases. The Ornithodoros moubata Complement Inhibitor (OmCI), a small 17 kDa natural protein, was initially extracted from soft tick salivary glands. The protein was found binding to complement C5 specifically, inhibiting the activation of the complement pathway, which is a successful therapeutic basis of complement-mediated diseases. However, a short half-life due to rapid renal clearance is a common limitation of small proteins for clinical application. In this study, we extended the half-life of OmCI by modifying it with fatty acid, which was a method used to improve the pharmacokinetics of native peptides and proteins. Five OmCI mutants were initially designed, and single-site cysteine mutation was introduced to each of them. After purification, four OmCI mutants were obtained that showed similar in vitro biological activities. Three mutants of them were subsequently coupled with different fatty acids by nucleophilic substitution. In total, 15 modified derivatives were screened and tested for anticomplement activity in vitro. The results showed that coupling with fatty acid would not significantly affect their complement-inhibitory activity (CH50 and AH50). OmCIT90C-CM02 and OmCIT90C-CM05 were validated as the applicable OmCI bioconjugates for further pharmacokinetic assessments, and both showed improved plasma half-life in mice compared with unmodified OmCI (15.86, 17.96 vs 2.57 h). In summary, our data demonstrated that OmCI conjugated with fatty acid could be developed as the potential long-acting C5 complement inhibitor in the clinic.

Juniperus pingii var. wilsonii acidic polysaccharide: Extraction, characterization and anticomplement activity.

A water-soluble acidic polysaccharide, XB-PS3, was isolated from the twigs of Juniperus pingii var. Wilsonii with a molecular weight of 86.04 kDa. By means of monosaccharide composition analysis, methylation, 2D NMR spectroscopy and UPLC-MS analysis, we concluded that XB-PS3 had a backbone composed of →2,4)-α-Manp-(1→ and →4)-α-GalpA-(1→ (60 % esterified), with an araban branch attached to O-2 of →2,4)-α-Manp-(1→. The possible repeating units were further validated by oligosaccharide analysis and partial acid hydrolysis. XB-PS3 exhibited potent anticomplement activity with CH50 value of 117.23 ± 18.74 µg/mL and interacted with C3, C4, C5 and C9 in the complement activation cascade. However, the anticomplement activity was significantly weakened when the galacturonic acids were reduced (CH50: 268.55 ± 16.82 µg/mL) or the branches were removed by partial hydrolysis (CH50: 197.76 ± 21.81 µg/mL), indicating the important role of uronic acids and branch structure in the polysaccharide's anticomplement activity.

Anticomplement and antitussive activities of major compound extracted from Chimonanthus nitens Oliv. leaf.

Chimonanthus nitens Oliv. leaf (CNOL), as a traditional Chinese medicine, has been widely used for the treatment of influenza and colds over a long history. However, the mechanism of colds related to the effects of CNOL have been little studied. In this study, the anticomplement and antitussive activities of different polarity extracts of CNOL were evaluated. Ethyl acetate extract (EAE) among different extracts not only significantly decreased cough times by 21-58% (P < 0.01), but also had anticomplement effects demonstrated by the CH50 values of 0.100 mg/ml. A total of 28 constituents (10 coumarins, 13 flavonoids and five phenolics) were identified in EAE based on the ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry technique. Eight compounds in EAE were evaluated by an ammonia-induced cough model to reveal the antitussive mechanisms and classical anticomplement pathway. The results indicated that the antitussive effects of scopoletin, kaempferol-3-O-rutinoside and kaempferol may depend on central mechanisms and that flavonoids such as compounds of kaempferol-3-O-rutinoside and kaempferol have better anticomplementary activity than coumarins like compounds of scopolin, scopoletin and isofraxidin. Taken together, kaempferol-3-O-rutinoside and kaempferol could be important chemical markers in the present study that might be used to evaluate the quality and biological activity of CNOL.

Screening of anti-complement active ingredients from Eucommia ulmoides Oliv. branches and their metabolism in vivo based on UHPLC-Q-TOF/MS/MS.

Eucommia ulmoides Oliv. (E. ulmoides) is a kind of plant with high medicinal value, there are known as the "gold plants". Some components and contents of barks and branches from E. ulmoides are similar, the barks are mainly used as medicine, but the branches have not been systematically studied and were discarded. In this paper, five fractions extracted from E. ulmoides branches were detected by the classical anti-complement activity assay in vitro. The n-butanol fraction of E. ulmoides branches showed excellent anti-complement activities with a CH50 value of 0.016 ±â€¯0.0014 mg·mL-1. A total of 76 compounds were identified from the n-butanol fraction, including 9 alkaloids, 18 organic acids, 22 lignans, 15 phenylethanoid glycosides and 12 other compounds. To further prove the anti-complement activity of potential active compounds, those compounds detectable in rat plasma after oral administration were tested by classical anti-complement activity assays. Genipin and pinoresinol 4-O-glucopyranoside had a certain complement inhibitory activity in the 17 potential anti-complements, their CH50 values were 0.050 ±â€¯0.0038 and 0.022 ±â€¯0.0018 mg·mL-1. UHPLC-Q-TOF/MS/MS was developed to profile and characterize the metabolites of genipin and pinoresinol 4-O-glucopyranoside in rat plasma. Twenty-one and seventeen metabolites were found, respectively. In summary, this study reported important clues for the further pharmacological and clinical studies of E. ulmoides branches. Meanwhile, it provided a practical strategy for rapid screening and identifying of in vivo anti-complement in traditional Chinese medicine.

Genome mining of Streptomyces xinghaiensis NRRL B-24674T for the discovery of the gene cluster involved in anticomplement activities and detection of novel xiamycin analogs.

Marine actinobacterium Streptomyces xinghaiensis NRRL B-24674T has been characterized as a novel species, but thus far, its biosynthetic potential remains unexplored. In this study, the high-quality genome sequence of S. xinghaiensis NRRL B-24674T was obtained, and the production of anticomplement agents, xiamycin analogs, and siderophores was investigated by genome mining. Anticomplement compounds are valuable for combating numerous diseases caused by the abnormal activation of the human complement system. The biosynthetic gene cluster (BGC) nrps1 resembles that of complestatins, which are potent microbial-derived anticomplement agents. The identification of the nrps1 BGC revealed a core peptide that differed from that in complestatin; thus, we studied the anticomplement activity of this strain. The culture broth of S. xinghaiensis NRRL B-24674T displayed good anticomplement activity. Subsequently, the disruption of the genes in the nrps1 BGC resulted in the loss of anticomplement activity, confirming the involvement of this BGC in the biosynthesis of anticomplement agents. In addition, the mining of the BGC tep5, which resembles that of the antiviral pentacyclic indolosesquiterpene xiamycin, resulted in the discovery of nine xiamycin analogs, including three novel compounds. In addition to the BGCs responsible for desferrioxamine B, neomycin, ectoine, and carotenoid, 18 BGCs present in the genome are predicted to be novel. The results of this study unveil the potential of S. xinghaiensis as a producer of novel anticomplement agents and provide a basis for further exploration of the biosynthetic potential of S. xinghaiensis NRRL B-24674T for the discovery of novel bioactive compounds by genome mining.

Chemical characterization and complement modulating activities of an arabinogalactan-protein-rich fraction from an aqueous extract of avocado leaves.

The aim of this study was to chemically characterize an arabinogalactan-protein-rich fraction (FRAGP) obtained from an aqueous extract of avocado leaves and investigate its effects on the classical pathway of the complement system. The FRAGP contained 4.6% ±â€¯1.8%, 22.5% ±â€¯4.9%, and 76.7% ±â€¯8.8% of total protein, arabinogalactan-protein, and carbohydrates, respectively. Arabinose and galactose were the main monosaccharide constituents. FT-IR and NMR data, together with linkage analyses, indicated the presence of a structure that included a (1 → 3)-linked ß-D-Galp main chain, mainly substituted at O-6 by Gal and Ara residues, which was characteristic of a type II arabinogalactan. The effect of FRAGP on the classical pathway of complement system was examined by a hemolytic fixation test and comparing with heparin, which was used as a control for inhibition. With pre-incubation, the IC50 of FRAGP was 1.90 ±â€¯1.1 µg/mL, which was similar to that of heparin (IC50 = 2.90 ±â€¯0.3 µg/mL). Without pre-incubation, the IC50 values were 18.6 ±â€¯3.7 and 8.0 ±â€¯4.1 µg/mL for FRAGP and heparin, respectively. Collectively, these results suggested that FRAGP has an inhibitory effect on the classical pathway of the complement system.

Anticomplement compounds from Polygonum chinense.

Five new compounds including two phenyldilactones (1, 2), two coumarins (3, 4) and a dimer of N-E-feruloyl tyramine (5) together with twenty-three known compounds (6-28) were isolated from a medicinal plant Polygonum chinense. The structures of the new compounds were established by detailed spectral analysis. The absolute configurations of 1 and 5 were elucidated by Mosher's method, Mo2(OAc)4-induced electronic circular dichroism (ECD) data, and ECD calculation. All the compounds were found to show potent anticomplement activity with CH50 and AP50 values ranging from 0.18 to 1.45 mM, and 0.26 to 2.80 mM, respectively. Phenyldilactones and phenylpropionic tyramines were firstly reported as anticomplement agents. The targets of compounds 1, 3, 5 and 10 in complement activation cascade were identified as well.

Identification of a staphylococcal complement inhibitor with broad host specificity in equid Staphylococcus aureus strains.

Staphylococcus aureus is a versatile pathogen capable of causing a broad range of diseases in many different hosts. S. aureus can adapt to its host through modification of its genome (e.g. by acquisition and exchange of mobile genetic elements that encode host-specific virulence factors). Recently, the prophage φSaeq1 was discovered in S. aureus strains from six different clonal lineages almost exclusively isolated from equids. Within this phage, we discovered a novel variant of staphylococcal complement inhibitor (SCIN), a secreted protein that interferes with activation of the human complement system, an important line of host defense. We here show that this equine variant of SCIN, eqSCIN, is a potent blocker of equine complement system activation and subsequent phagocytosis of bacteria by phagocytes. Mechanistic studies indicate that eqSCIN blocks equine complement activation by specific inhibition of the C3 convertase enzyme (C3bBb). Whereas SCIN-A from human S. aureus isolates exclusively inhibits human complement, eqSCIN represents the first animal-adapted SCIN variant that functions in a broader range of hosts (horses, humans, and pigs). Binding analyses suggest that the human-specific activity of SCIN-A is related to amino acid differences on both sides of the SCIN-C3b interface. These data suggest that modification of this phage-encoded complement inhibitor plays a role in the host adaptation of S. aureus and are important to understand how this pathogen transfers between different hosts.

Functional assignment for essential hypothetical proteins of Staphylococcus aureus N315.

Staphylococcus aureus, the causative agent of nosocomial infections worldwide, has acquired resistance to almost all antibiotics stressing the need to develop novel drugs against this pathogen. In S. aureus N315, 302 genes have been identified as essential genes, indispensable for growth and survival of the pathogen. The functions of 40 proteins encoded by S. aureus essential genes were found to be hypothetical and thus referred as essential hypothetical proteins (EHPs). The present study aims to carry out functional characterization of EHPs using bioinformatics tools/databases, whose performance was assessed by Receiver operating characteristic curve analysis. Evaluation of physicochemical parameters, homology search against known proteins, domain analysis, subcellular localization analysis and virulence prediction assisted us to characterize EHPs. Functional assignment for 35 EHPs was made with high confidence. They belong to different functional classes like enzymes, binding proteins, miscellaneous proteins, helicases, transporters and virulence factors. Around 35% of EHPs were from hydrolases family. A group of EHPs (32.5%) were predicted as virulence factors. Of 35, 19 essential pathogen-specific proteins were considered as probable drug targets. Two targets were found to be druggable and others were novel targets. Outcome of the study could aid to identify novel drugs for better treatment of S. aureus infections.

Anti-complementary constituents of Anchusa italica.

Activity-guided fractionation for complement inhibitors led to the isolation of 24 known compounds from Anchusa italica. Chemical types include eight megastigmane compounds, five triterpenoid compounds, five lignan compounds, three flavonoid compounds, two alkaloid compounds and one phenthyl alcohol compound. Among which, a lignan (medioresinol), an alkaloid (5-hydroxypyrrolidin-2-one) and a flavonoid (5-hydroxyl-3', 4', 6, 7-tetramethoxy flavone) exhibited better anticomplementary effects with CH50 values ranging from 0.07 to 0.82 mM, which are plausible candidates for developing potent anticomplementary agents.

Anticomplement triterpenoids from the roots of Ilex asprella.

Five new (1-5) and twenty-eight known (6-33) triterpenoids were isolated from the roots of Ilex asprella. The structures of the new compounds were elucidated by the detailed spectral analysis. The ursane and oleanane triterpenoids were found to show anticomplement activity with some structure-activity relationships. Several triterpenoids (1-3, 6-7) exhibited potent anticomplement activity with the CH50 and AP50 values of 0.058-0.131mg/mL and 0.080-0.444mg/mL, respectively. It was found that caffeoyl group could enhance activity remarkably, followed by coumaroyl and feruloyl group. The 28-carboxyl group was also important to anticomplement activity for the triterpenoids. However, the triterpenoids with lactone ring (4, 9-14) exhibited weak activity and triterpenoid glycosides (5, 23-33) showed no inhibition. The targets of several bioactive triterpenoids in complement activation cascade were identified as well.

Structure analysis of a heteropolysaccharide from Taraxacum mongolicum Hand.-Mazz. and anticomplementary activity of its sulfated derivatives.

A homogenous water-soluble polysaccharide, DPSW-A, with a deduced chemical structure was extracted from the herb Taraxacum mongolicum Hand.-Mazz. Moreover, 80.813-kDa DPSW-A is composed of three types of monosaccharide, namely rhamnose, arabinose, and galactose, at a molar ratio of 1.0:10.7:11.9. The main chain of DPSW-A contains Terminal-Galp, 1,3-Galp, 1,6-Galp, 1,3,6-Galp, and 1,2,4-Rhap; the branched chain contains Terminal-Araf, 1,5-Araf, and 1,3,5-Araf. The sulfated derivatives prepared from DPSW-A showed inhibitory effects on complement activation through the classical pathway (CH50: Sul-DPSW-A, 3.94±0.43µg/mL; heparin, 104.40±3.82µg/mL) and alternative pathway (AP50: Sul-DPSW-A, 42.76±0.46µg/mL; heparin, 43.42±0.22µg/mL). Mechanism studies indicated that Sul-DPSW-A inhibited complement activation by blocking C1q, C1r, C1s, and C9, but not C2, C3, C4, and C5. In addition, Sul-DPSW-A displayed limited anticoagulant effects. These results suggest that Sul-DPSW-A prepared from DPSW-A is valuable for treating diseases caused by excessive complement system activation.

One new cycloartane triterpene glycoside from Beesia calthaefolia.

One new cycloartane triterpene glycoside (1) was isolated from the whole plant of Beesia calthaefolia. Its structure was elucidated on the basis of extensive spectroscopic data analysis. Its inhibitory effect was measured by the classical pathway of the complement system, and compared with those of known related cycloartane glycosides 2 and 3, previously isolated by us from the same plant. Compounds 1 and 2 exhibited inhibitory activity of complement system with IC50 of 395.3 and 214 µM, respectively. The results suggested that OH at C-12, C-18 and C-15 along with the polarity could affect the inhibitory activity.

Creating functional sophistication from simple protein building blocks, exemplified by factor H and the regulators of complement activation.

Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a ß-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins.

Homogalacturonans from preinfused green tea: structural characterization and anticomplementary activity of their sulfated derivatives.

Two homogeneous water-soluble polysaccharides (TPSR4-2B and TPSR4-2C) were obtained from preinfused green tea. Their average molecular weights were estimated to be 41 kDa and 28 kDa, respectively. A combination of composition, methylation, and configuration analysis, as well as NMR spectroscopy, indicated that both TPSR4-2B and TPSR4-2C were poly-(1-4)-α-d-galactopyranosyluronic acid in which 30.5 ± 0.3% and 28.3 ± 0.5%, respectively, of uronic acid existed as methyl ester. Two sulfated derivatives (Sul-R4-2B and Sul-R4-2C) from TPSR4-2B and TPSR4-2C were prepared after sulfation with a 2:1 chlorosulfonic acid-pyridine ratio. The anticomplementary assay showed that Sul-R4-2B and Sul-R4-2C demonstrated a stronger inhibitory effect on the complement activation through the classic pathway, compared to that of heparin. Preliminary mechanism studies by using complement component depleted-sera indicated that both Sul-R4-2B and Sul-R4-2C selectively interact with C1q, C1r, C1s, C2, C5, and C9 but not with C3 and C4. The relationship between DS and the anticomplementary activity of sulfated derivatives of homogalacturonans showed that low sulfated derivatives of homogalacturonans also exhibited potent anticomplementary effect, which might greatly reduce the side effects related to heparin and oversulfated chondroitin sulfate, such as anticoagulant activity and allergic-type reaction. These results suggested that sulfated derivatives of homogalacturonans might be promising drug candidates for therapeutic complement inhibition.

Antioxidant and anticomplement functions of flavonoids extracted from Penthorum chinense Pursh.

Penthorum chinense Pursh is rich in flavonoids, which have strong antioxidant and anticomplement activities. In order to optimize their extraction conditions, various extraction parameters were chosen to identify their effects on flavonoids extraction. Single factor and Box-Behnken experimental designs consisting of 24 experimental runs and five replicates at zero point were applied to obtain the optimal extraction yield. The optimization conditions for flavonoids extraction were determined as follows: ethanol concentration 60.89%, extraction time 68.15 min, temperature 52.89 °C and liquid/solid ratio 19.70 : 1. The corresponding flavonoids content was 7.19%. The regression equation was found to fit well with the actual situation. Furthermore, the antioxidant activity (the free radical scavenging ability and ferric reducing/antioxidant power) and anticomplement ability of the flavonoids from P. chinense were determined. Results showed that the flavonoids of P. chinense displayed significant antioxidant and anticomplement activities. Its antioxidant activity can compete with ascorbic acid (Vc), whereas its anticomplement activity (IC50 = 111.6 µg ml(-1)) surpassed the effect of heparin (IC50 = 399.7 µg ml(-1)) which was used as the positive control, suggesting that P. chinense flavonoids and their related products could potentially be used as a promising natural agent in the treatment of humoral effector inflammation.

Staphylococcal Ecb protein and host complement regulator factor H enhance functions of each other in bacterial immune evasion.

Staphylococcus aureus is a major human pathogen causing more than a tenth of all septicemia cases and often superficial and deep infections in various tissues. One of the immune evasion strategies of S. aureus is to secrete proteins that bind to the central complement opsonin C3b. One of these, extracellular complement binding protein (Ecb), is known to interfere directly with functions of C3b. Because C3b is also the target of the physiological plasma complement regulator, factor H (FH), we studied the effect of Ecb on the complement regulatory functions of FH. We show that Ecb enhances acquisition of FH from serum onto staphylococcal surfaces. Ecb and FH enhance mutual binding to C3b and also the function of each other in downregulating complement activation. Both Ecb and the C-terminal domains 19-20 of FH bind to the C3d part of C3b. We show that the mutual enhancing effect of Ecb and FH on binding to C3b depends on binding of the FH domain 19 to the C3d part of C3b next to the binding site of Ecb on C3d. Our results show that Ecb, FH, and C3b form a tripartite complex. Upon exposure of serum-sensitive Haemophilus influenzae to human serum, Ecb protected the bacteria, and this effect was enhanced by the addition of the C-terminal domains 19-20 of FH. This finding indicates that the tripartite complex formation could give additional protection to bacteria and that S. aureus is thereby able to use host FH and bacterial Ecb in a concerted action to eliminate C3b at the site of infection.

Protostane and fusidane triterpenes: a mini-review.

Protostane triterpenes belong to a group of tetracyclic triterpene that exhibit unique structural characteristics. Their natural distribution is primarily limited to the genus Alisma of the Alismataceae family, but they have also been occasionally found in other plant genera such as Lobelia, Garcinia, and Leucas. To date, there are 59 known protostane structures. Many of them have been reported to possess biological properties such as improving lipotropism, hepatoprotection, anti-viral activity against hepatitis B and HIV-I virus, anti-cancer activity, as well as reversal of multidrug resistance in cancer cells. On the other hand, fusidanes are fungal products characterized by 29-nor protostane structures. They possess antibiotic properties against staphylococci, including the methicillin-resistant Staphylococcus aureus (MRSA). Fusidic acid is a representative member which has found clinical applications. This review covers plant sources of the protostanes, their structure elucidation, characteristic structural and spectral properties, as well as biological activities. The fungal sources, structural features, biological activities of fusidanes are also covered in this review. Additionally, the biogenesis of these two types of triterpenes is discussed and a refined pathway is proposed.

A novel peptide can mimic extracellular fibrinogen-binding protein to block the activation of complement system.

Extracellular fibrinogen-binding protein (Efb) of Staphylococcus aureus (S. aureus) is a bi-functional protein, which can specifically bind fibrinogen with its N terminus and inhibit deposition of C3b on the surface of S. aureus with its C terminus. Here, we screened the epitopes of Efb using phage display. Four peptides with consensus motif were screened. This consensus motif was identical to C terminus (161-164) of Efb. In the further investigation, it was found the synthesized peptide EC1 (154-165aa of Efb) could specifically bind C3/C3b and subsequently to block the activation of complement. Meanwhile, EC1 could inhibit the interaction between Efb and C3/C3b. Moreover, the interaction between the mutant protein of EmC1 (Efb without EC1) and C3 was decreased. And, the effect on the complement system of the mutant protein was dramatically declined compared with Efb. Our finding suggested that the peptide EC1 could mimic Efb to block complement system activation via binding C3.

A structurally dynamic N-terminal helix is a key functional determinant in staphylococcal complement inhibitor (SCIN) proteins.

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.