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. 2014 Dec 15;193(12):6161-6171.
doi: 10.4049/jimmunol.1401600. Epub 2014 Nov 7.

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

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

Jordan L Woehl et al. J Immunol. .
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Abstract

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.

Figures

Figure 1
Figure 1. Eap Inhibits Complement Activation via the Classical and Lectin Pathways
The effect of Eap on distinct routes of complement activation was assessed via ELISA-based methods. (a) The effect of 1 μM Eap, EapH1, or EapH2 on CP (left) and LP-mediated (right) complement activation was measured across a dilution series of NHS. Activation was detected as C5b-9 deposition on an ELISA plate surface. Legend is inset. (b) The effect of 1 μM Eap, EapH1, or EapH2 on CP and LP-mediated complement activation in 1% (v/v) NHS was measured. Activation was detected as C3b deposition on an ELISA plate surface. (c) The same experiment as in panel b, except that activation was detected as C4b deposition on an ELISA plate surface. Error bars represent the mean ± standard deviation of three independent experiments. Measures of statistical significance in panels b and c were determined by an unpaired t-test of each experimental series versus buffer control. **, p≤0.01; ***, p≤0.001; ns, not significant.
Figure 2
Figure 2. Eap Inhibits Opsonization, Phagocytosis, and Killing of Staphylococcus aureus
The impact of recombinant Eap on complement deposition and phagocytosis of S. aureus Newman strains was assessed using flow cytometry. (a) C3b deposition on the surface of S. aureus Newman WT or Δeap in the presence of 1 μM Eap or a buffer control. Legend is inset. (b) Phagocytosis of S. aureus Newman WT or Δeap in the presence of 1 μM Eap or a buffer control. Legend is inset in the adjacent panel. (c) Extent of phagocytosis of S. aureus Newman Δeap using 1% (v/v) NHS in the presence of 1 μM Eap, EapH1, or EapH2, or a buffer control. (d) Neutrophil-mediated killing of S. aureus Newman Δeap opsonized in the presence of 1 μM Eap or a buffer control. Error bars represent the mean ± standard deviation of three independent experiments and at least two different donors. Legend is inset. Measures of statistical significance were determined by an unpaired t-test of each experimental series versus the corresponding buffer control for each strain and serum concentration as appropriate. *, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001; ns, not significant.
Figure 3
Figure 3. Eap Forms a Nanomolar Affinity Complex with Complement Component C4b
(a) Analysis of the C4b/Eap complex by analytical gel-filtration chromatography. Chromatograms for C4b and C4b/Eap are shown in the left panel, while Coomassie-stained SDS-PAGE analysis of the peak fractions from each injection is shown in the right panel. Control lanes are designated as: C, C4b; E, Eap; and M, molecular weight marker. (b) The ability of untagged Eap, EapH1, and EapH2 to compete the AlphaScreen signal generated by myc-Eap and C4b-biotin was assessed over a logarithmic dilution series. While three independent trials were carried out, the data presented here are from single representative titrations. The smooth line indicates the outcome of fitting all points to a dose-response curve when competition was observed. Legend is inset. (c) Binding of Eap, EapH1, and EapH2 to an oriented C4b-biosensor surface. The peak signals achieved following injection stop for samples at 1 and 10 μM, done in triplicate, were normalized to the molecular weight of their respective analyte proteins. Note that error bars are shown, but represent comparatively small variations due to the high precision of the assay system.
Figure 4
Figure 4. The Third Domain of Eap is Necessary for C4b Binding and Inhibition of the CP/LP
(a) Diagram of full-length and domain-deleted forms of Eap used to map functional sites within the Eap protein. (b) The ability of untagged Eap, Eap12, Eap23, Eap34, and an equimolar mixture of individual Eap domains (Eap MIX) to compete the AlphaScreen signal generated by myc-Eap and C4b-biotin was assessed over a logarithmic dilution series. While three independent trials were carried out, the data presented here are from a single representative titrations. The smooth line indicates the outcome of fitting all points to a dose-response curve when competition was observed. Legend is inset. (c) The effect of including 1 μM Eap, or various truncations thereof, on C5b-9 deposition on ELISA plates coated with either CP- (left panel) or LP-specific (right panel) activators. 1% (v/v) NHS was used as a source of complement components. Error bars represent the mean ± standard deviation of three independent experiments. Measures of statistical significance were determined by one-way ANOVA for the various Eap truncations versus buffer control alone. *, p≤0.05; **, p≤0.01; ****, p≤0.0001; ns, not significant.
Figure 5
Figure 5. Eap Binding Inhibits the Interaction of Complement Component C2 with C4b
(a) The ability of recombinant human C2, C2b, and C4BP to compete the AlphaScreen signal generated by myc-Eap and C4b-biotin was assessed over a logarithmic dilution series. While three independent trials were carried out, the data presented here are from a single representative titration. The smooth line indicates the outcome of fitting all points to a dose-response curve. (b) Representative data from an SPR competition experiment where the effect of 1 μM Eap on the interaction of 200 nM C2 with a C4b-biotin surface was examined. A legend showing the identity of each sensorgram is inset. The residual C2 binding in the presence of Eap is shown as a dashed line, while the sensorgram for the same concentration of C2 in the absence of any Eap is shown as the darkest black line. (c) Residual C2 binding in the presence of various concentrations of Eap fit to a dose-response curve (IC50 = 50 nM). (d) Identical experiment to panel a, with the exception that the ability of recombinant human C2, C4BP, and Eap to compete the AlphaScreen signal generated by myc-Eap34 and C4b-biotin was assessed. (e) Identical experiment to panel b, with the exception that Eap34 was used as the competitor instead of Eap. The residual C2 binding in the presence of Eap34 is shown as a dashed line, while the sensorgram for the same concentration of C2 is shown as the darkest black line. (f) Residual C2 binding in the presence of various concentrations of Eap34 fit to a dose-response curve (IC50 = 870 nM).
Figure 6
Figure 6. Proposed Mechanism for Eap-mediated Inhibition of the CP/LP C3 and Its Similarities to the S. aureus AP Inhibitor, Efb-C
The overall structural similarities between C4b and C3b are represented by the similar shapes of their cartoon representations. The shaded green rectangle represents the macroglobulin-like core, the orange square the C345C domain, the small pink rectangle the CUB domain, and filled circle the thioester-containing domain (i.e. C4d and C3d). The thin blue rectangle represents the γ-chain unique to C4/b. The inhibitor Eap is shown in the left panel with two domains filled in yellow to represent the domains 3 and 4 ‘active site’, as described in Figs. 4 and 5. The inhibitor Efb-C is shown as a blue cylinder in the right panel. Efb-C binding to the C3d domain (45) and stabilization of an open, inactive conformation of C3b that is unable to bind FB (10) is depicted by reorientation of the CUB-TED region relative to the macroglobulin-like core of C3b.

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