DataSheet_1_Antibodies to coagulase of Staphylococcus aureus crossreact to Efb and reveal different binding of shared fibrinogen binding repeats.pdf

Staphylococcus aureus pathology is caused by a plethora of virulence factors able to combat multiple host defence mechanisms. Fibrinogen (Fg), a critical component in the host coagulation cascade, plays an important role in the pathogenesis of this bacterium, as it is the target of numerous staphylococcal virulence proteins. Amongst its secreted virulence factors, coagulase (Coa) and Extracellular fibrinogen-binding protein (Efb) share common Fg binding motives and have been described to form a Fg shield around staphylococcal cells, thereby allowing efficient bacterial spreading, phagocytosis escape and evasion of host immune system responses. Targeting these proteins with monoclonal antibodies thus represents a new therapeutic option against S. aureus. To this end, here we report the selection and characterization of fully human, sequence-defined, monoclonal antibodies selected against the C-terminal of coagulase. Given the functional homology between Coa and Efb, we also investigated if the generated antibodies bound the two virulence factors. Thirteen unique antibodies were isolated from naïve antibodies gene libraries by antibody phage display. As anticipated, most of the selected antibodies showed cross-recognition of these two proteins and among them, four were able to block the interaction between Coa/Efb and Fg. Furthermore, our monoclonal antibodies could interact with the two main Fg binding repeats present at the C-terminal of Coa and distinguish them, suggesting the presence of two functionally different Fg-binding epitopes.</p

Phagocytosis inhibition by Efb is independent of complement inhibition.

<p>A. Phagocytosis of fluorescently labeled <i>S. epidermidis</i> and <i>E. coli</i> by purified human neutrophils in the presence of human plasma (5%) and Efb. B. Immunoblot detecting surface-bound C3b after incubation of <i>S. aureus</i> with 5% human plasma in the presence of 5 mM EDTA or 0.5 µM Efb. Blot is a representative of 3 independent experiments. C. Alternative pathway hemolysis of rabbit erythrocytes in 5% human plasma and Efb (mutants) (1 µM). Bars are the mean ± se of three independent experiments. **<i>P</i><0.005 for Efb versus buffer (two-tailed Student's <i>t</i>-test). D. Phagocytosis with a washing step. Fluorescent <i>S. aureus</i> was first incubated with 5% serum to deposit complement. Bacteria were washed and subsequently mixed with neutrophils and Fg in the presence or absence of Efb (0.5 µM). Graph is a representative of three independent experiments.</p

Full-length Efb inhibits phagocytosis of <i>S. aureus</i> in human plasma.

<p>A. Phagocytosis of fluorescently labeled <i>S. aureus</i> by purified human neutrophils in the presence of human serum or plasma and Efb (0.5 µM). B. Histology image of human neutrophils incubated with <i>S. aureus</i> and 2.5% plasma in the presence or absence of Efb (0.5 µM). Cells were stained using Diff-Quick. C. Dose-dependent phagocytosis inhibition by Efb in the presence of 2.5% human plasma. IC₅₀ was calculated using non-linear regression analysis, R² = 0.95. D–F. Phagocytosis in the presence of 5% human serum supplemented with either full-length human Fg (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003816#ppat-1003816-g001" target="_blank">Fig. 1D</a>), the D domain of human Fg (1 µM or 86 µg/ml) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003816#ppat-1003816-g001" target="_blank">Fig. 1E</a>) or mouse Fg (WT or lacking the Mac-1 binding site) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003816#ppat-1003816-g001" target="_blank">Fig. 1F</a>). A, C–F are mean ± se of three independent experiments. B is a representative image. *<i>P</i><0.05, **<i>P</i><0.005 for Efb versus buffer (two-tailed Student's <i>t</i>-test).</p

Simultaneous binding to Fg and C3 is essential for phagocytosis inhibition by Efb.

<p>A. Schematic overview of Efb mutants generated in this study. Efb is depicted in its secreted form (30–165) lacking the signal peptide (1–29). Bounding boxes indicate Fg- and C3-binding domains. The N-terminus of Efb (light grey, 9 kD) harbors two Fg binding sites named Fg1 (residues 30–67) and Fg2 (residues 68–98). The C-terminus of Efb (dark grey, 7 kD) harbors the C3 binding site (residues R131 and N138). EfbΔFg1 has deletion of residues 30–45, resulting in non-functional binding Fg1; whereas EfbΔFg2 has deletion of residues 68–76, resulting in non-functional binding Fg2. B–C. Phagocytosis of fluorescent <i>S. aureus</i> by human neutrophils in the presence of 5% human plasma and Efb fragments (B) or Efb mutants (C) (all at 1 µM). B,C are mean ± se of three independent experiments. **<i>P</i><0.005 for Efb versus buffer (two-tailed Student's <i>t</i>-test).</p

Purified Efb blocks phagocytosis <i>ex vivo</i> and <i>in vivo</i>.

<p>A. <i>Ex vivo</i> phagocytosis of fluorescent <i>S. aureus</i> incubated with 50% human whole blood and Efb (1 µM). Neutrophils were gated based on forward and side scatter properties. B. <i>In vivo</i> phagocytosis of fluorescent <i>S. aureus</i> by human neutrophils in the mouse peritoneum. Neutrophils were attracted to the peritoneal cavity using carrageenan (i.p.) and subsequently challenged with 10⁸ heat-inactivated fluorescent <i>S. aureus</i> and Efb (1 µM) for 1 hour. The peritoneal lavage was collected and neutrophil phagocytosis was analyzed by flow cytometry. Neutrophils were gated based on Gr-1 expression. The mouse experiments were carried out three times. In each experiment, we used 3 mice per group and the cells of these 3 mice were pooled for phagocytosis analysis. C. Representative histograms of B. A,B are mean ± se of three independent experiments. *<i>P</i><0.05, **<i>P</i><0.005 for Efb versus buffer (two-tailed Student's <i>t</i>-test).</p

Efb attracts Fg to the bacterial surface.

<p>A. ELISA showing that Efb can bind Fg and C3b at the same time. C3b-coated microtiter wells were incubated with Efb (mutants) and, after washing, incubated with 50 nM Fg that was detected with a peroxidase-conjugated anti-Fg antibody (Abcam). Graph is a representative of two independent experiments performed in duplicate. B. Binding of Alexa488-labeled Fg (60 µg/ml) to serum-opsonized <i>S. aureus</i> in the presence of Efb (mutants) (0.5 µM). Graph represents mean ± se of three independent experiments. *<i>P</i><0.05, **<i>P</i><0.005 for Efb versus buffer (two-tailed Student's <i>t</i>-test). N.S. is not significant. C. Confocal analysis of samples generated in B (representative images). D. TEM pictures of <i>S. aureus</i> incubated with 5% human plasma in the absence or presence of Efb (0.5 µM). Three representative images are shown.</p

Efb prevents recognition of opsonic C3b and IgG.

<p>A–B. Flow cytometry assay detecting binding of soluble CR1 (A) or anti-IgG antibody (B) to pre-opsonized <i>S. aureus</i> in the presence of buffer, Efb (0.5 µM) and/or Fg (200 µg/ml). C. Efb inhibits phagocytosis of encapsulated <i>S. aureus</i> by human neutrophils. FITC-labeled <i>S. aureus</i> strain Reynolds (high capsule CP5 expressing strain) was incubated with human plasma and/or Efb (0.5 µM) in the presence (dotted line) or absence (solid line) of polyclonal rabbit anti-CP5 antibody. All figures represent the mean ± se of three separate experiments. *<i>P</i><0.05, **<i>P</i><0.005 for Efb+Fg versus buffer (A,B) or Efb versus buffer (for dotted lines) (two-tailed Student's <i>t</i>-test).</p

Endogenously produced Efb blocks phagocytosis via complex formation.

<p>A. <i>Left</i>. Immunoblot detecting Efb in 4 h and 20 h culture supernatants of <i>S. aureus</i> Newman; fixed concentrations of His-tagged Efb were loaded as controls. <i>Right</i>. Immunoblot of 4 h culture supernatants of <i>S. aureus</i> Newman (WT), an isogenic Efb deletion mutant (ΔEfb) and its complemented strain (ΔEfb+pEfb). Blots were developed using polyclonal sheep anti-Efb and Peroxidase-labeled donkey anti-sheep antibodies. Blot is a representative of two independent experiments. B. Flow cytometry analysis of the binding of Alexa488-labeled Fg to pre-opsonized <i>S. aureus</i> in the presence of 4 h culture supernatants (2-fold diluted) or purified Efb (250 nM). C. <i>In vitro</i> phagocytosis of fluorescently labeled <i>S. aureus</i> by purified human neutrophils. Pre-opsonized <i>S. aureus</i> was first incubated with 4 h culture supernatants (2-fold diluted) or purified Efb (250 nM) and subsequently mixed with Fg and neutrophils. D. <i>In vivo</i> phagocytosis of GFP-expressing wild-type or Efb-deficient <i>S. aureus</i> strains by neutrophils in the mouse peritoneal cavity. Neutrophils were attracted to the peritoneal cavity using carrageenan (i.p.) and subsequently injected with 300 µl of GFP-expressing wild-type (SA WT) or Efb-deficient (SAΔEfb) <i>S. aureus</i> strains during the exponential phase of growth. The peritoneal lavage was collected 1 h thereafter and neutrophil phagocytosis was analyzed by flow cytometry. Neutrophils were gated based on Gr-1 expression. Graphs in B–D represent mean ± se of three independent experiments. *<i>P</i><0.05, **<i>P</i><0.005 for WT versus Buffer or ΔEfb (two-tailed Student's <i>t</i>-test).</p