In the <i>irp2</i> knockout strain the production of yersiniabactin is blocked.

<p>Yersiniabactin production was tested using a GFP reporter assay with the knockout strain WA-CS <i>irp1</i>::Kan^r containing the pCJG3.3N plasmid as the reporter strain to detect yersiniabactin in supernatants. The number of bacteria versus the measured arbitrary amount of fluorescence from 50,000 counted bacteria is shown in panel A. A) Supernatants from the <i>irp2</i> knockout (E4Δ<i>irp2</i>) cultures (grey) induced a lower amount of GFP compared to supernatants from the wild-type (WT) cultures (white). This indicates that production of yersiniabactin was blocked in the <i>irp2</i> knockout. The experiments were performed in duplicate. B) The GFP reporter assay is dependent on yersiniabactin production, and production of yersiniabactin in the <i>irp2</i> knockout is blocked. Yersiniabactin production is only seen in the wild-type (WT) cells cultured in iron-depleted medium (NBD), while the <i>irp2</i> knockout (E4Δ<i>irp2</i>) strain cultured in NBD and iron-containing medium (NB), as well as the wild-type strain grown in NB, did not produce yersiniabactin. Three different experiments were performed, with each sample analyzed in duplicate. C) The expression of HMWP1 and HMWP2 is disrupted by the insertion of a kanamycin resistance gene into <i>irp2</i> using the Tagetron Knockout System. The <i>irp2</i> knockout (E4Δ<i>irp2</i>) strain was not able to produce HMWP1 and HMWP2 when cultured in NBD. M: marker; lane 1: wild-type strain E4; lane 2–5: <i>irp2</i> gene knockouts created using the wild-type strain E4; lane 6 wild-type EHOS strain 03-702; lane 7: wild-type EHOS strain 03-819 served as HPI-positive control because the HMWP2 of this strain was confirmed by Edman degradation.</p

ROS production by PMNs is reduced by yersiniabactin.

<p>To determine the effect of yersiniabactin on the ROS production of PMNs, PMNs were pre-incubated with yersiniabactin. PMNs were then activated by PMA, and the production of ROS was measured using luminol. A) Absolute ROS production measured in arbitrary fluorescence units (A.U.) of PMNs after pre-incubation with yersiniabactin or iron-saturated yersiniabactin or the controls (PMNs that were only pre-incubated in RPMI/HSA medium and PMNs that were pre-incubated with yersiniabactin but not stimulated with PMA). B) The yersiniabactin-mediated inhibition of ROS production by PMNs is concentration dependent. Red line: the concentration-dependent decrease in production of ROS following treatment with yersiniabactin. Blue line: no decrease in ROS production following treatment with different concentrations of iron-saturated yersiniabactin. Black dotted line: negative control (PMNs incubated with yersiniabactin, but not stimulated with PMA); ***<i>p</i> value<0.0005, **<i>p</i> value<0.005, *<i>p</i> value<0.05. C) Treatment of PMNs with unsaturated lactoferrin or holo-transferrin partly inhibits the yersiniabactin-mediated inhibition of ROS production. Black bars: relative ROS production following treatment with yersiniabactin; gray bars: relative ROS production following treatment with yersiniabactin and unsaturated lactoferrin; white bars: relative ROS production following treatment with yersiniabactin and holo-transferrin. ^aConcentration of the iron chelator. D) Treatment of PMNs with saturated lactoferrin or apo-transferrin partly inhibits the yersiniabactin-mediated inhibition of ROS production. Black bars: relative ROS production following treatment with yersiniabactin; gray bars: relative ROS production following treatment with yersiniabactin and saturated lactoferrin; white bars: relative ROS production following treatment with yersiniabactin and apo-transferrin. All experiments were replicated three times, and each sample was analyzed in duplicate.</p

Other iron chelators also reduce ROS production by PMNs.

<p>Along with yersiniabactin, aerobactin, deferoxamine, and deferiprone also reduce ROS production by PMNs. To determine the effect of other iron-binding molecules on ROS production by PMNs, PMNs were pre-incubated with aerobactin, deferoxamine, deferiprone, or yersiniabactin. The pre-treated PMNs were then activated using PMA, and the production of ROS was measured using luminol. Red line: the concentration-dependent decrease in ROS production following yersiniabactin treatment. Gray line: the decrease in ROS production following treatment with aerobactin. Yellow line: the concentration-dependent decrease in ROS production following deferiprone treatment. Green line: the concentration-dependent decrease in ROS production following deferoxamine treatment. The level of ROS produced by stimulated PMNs without additives was set as 100%. All experiments were replicated three times, and each sample was analyzed in duplicate.</p

Growth curves of the EHOS E4 isolate and its corresponding <i>irp2</i> knockout strain.

<p>The results are presented as the mean of three experiments performed in duplicate on different days. Cells were grown in: A) nutrient broth treated with 1% Chelex; B) nutrient broth treated with 1% Chelex and supplemented with 80% iron-saturated lactoferrin (∼6.2 µM, Fe-LF) or unsaturated lactoferrin (∼6.4 µM,0-LF); C) nutrient broth treated with 1% Chelex and supplemented with 17 µM holo-transferrin or 17 µM apo-transferrin; D) nutrient broth treated with 1% Chelex and supplemented with 10 µM or 30 µM hemin.</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

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

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