Staphylococcal protein Ecb impairs complement receptor-1 mediated recognition of opsonized bacteria

<div><p><i>Staphyloccus aureus</i> is a major human pathogen leading frequently to sepsis and soft tissue infections with abscesses. Multiple virulence factors including several immune modulating molecules contribute to its survival in the host. When <i>S</i>. <i>aureus</i> invades the human body, one of the first line defenses is the complement system, which opsonizes the bacteria with C3b and attract neutrophils by release of chemotactic peptides. Neutrophils express Complement receptor-1 [CR1, CD35) that interacts with the C3b-opsonized particles and thereby plays an important role in pathogen recognition by phagocytic cells. In this study we observed that a fraction of <i>S</i>. <i>aureus</i> culture supernatant prevented binding of C3b to neutrophils. This fraction consisted of <i>S</i>. <i>aureus</i> leukocidins and Efb. The C-terminus of Efb is known to bind C3b and shares significant sequence homology to the extracellular complement binding protein [Ecb). Here we show that <i>S</i>. <i>aureus</i> Ecb displays various mechanisms to block bacterial recognition by neutrophils. The presence of Ecb blocked direct interaction between soluble CR1 and C3b and reduced the cofactor activity of CR1 in proteolytic inactivation of C3b. Furthermore, Ecb could dose-dependently prevent recognition of C3b by cell-bound CR1 that lead to impaired phagocytosis of NHS-opsonized <i>S</i>. <i>aureus</i>. Phagocytosis was furthermore reduced in the presence of soluble CR1 [sCR1). These data indicate that the staphylococcal protein Ecb prevents recognition of C3b opsonized bacteria by neutrophil CR1 leading to impaired killing by phagocytosis and thereby contribute to immune evasion of <i>S</i>. <i>aureus</i>.</p></div

Effect of secreted molecules from <i>S</i>. <i>aureus</i> on binding of C3b to neutrophils.

<p>Fractionated <i>S</i>. <i>aureus</i> culture supernatant was incubated with C3b and neutrophils and cell-bound C3b was detected with a FITC-conjugated anti-C3 antibody using flow cytometry. <i>A</i>, Separated fractions of undiluted, or two or five times diluted culture supernatant were compared. The black bar represents binding of C3b to neutrophils in the absence of culture supernatant. Fl, fluorescence intensity. <i>B</i>, Histograms showing the effect of dilutions of the fraction 15 on binding of C3b to neutrophils. <i>C</i>, Silver stained SDS-PAGE gel of the fractions 11–21 with molecular weight marker indicated on left and major bands in fractions 15–16 indicated on right.</p

Effect of Ecb on proteolytic cleavage of C3b by sCR1 and factor I.

<p>Proteolysis of ¹²⁵I-C3b was analyzed by the intensity of the α’-chain in autoradiography. The intensity in the absence of sCR1 (negative control, black bar), was set to 100% and cleavage in the presence of sCR1 but absence of Ecb was used as a positive control (dark grey bar). The lanes in the upper panel shows the α’-chain in the SDS-page gel. The x-axis shows the presence (+) or absence (-) of the protein and the concentrations (0.002–2.2 μM) of Ecb in the sample.</p

Effect of Ecb on binding of C3b to human red blood cells.

<p><i>A</i>, RBC were incubated with C3b with or without Ecb. After washings, C3b bound to RBC was detected with flow cytometry using an antibody recognizing the C3c fragment (FI, fluorescence intensity). The assay was performed three times with duplicates and mean SD values are shown. <i>B</i> is a representative histogram of RBC incubated with C3b in the presence of Ecb (0–1.5 and 0–1.8, respectively). Student’s two-tailed <i>t</i>-test was used to determine the statistical significances (* <i>p</i><0.05; ** <i>p</i><0.01).</p

Effect of Ecb and FH on binding of C3b to sCR1.

<p>¹²⁵I-labeled sCR1 was incubated in wells of microtiter plates coated with C3b in the presence of the indicated proteins followed by washings and detection of the bound labeled protein with a gamma counter. <i>A</i>, Binding of ¹²⁵I-sCR1 to C3b in the presence of Ecb, or BSA as a control. <i>B</i>, Binding of ¹²⁵I-sCR1 to C3b in the presence of FH, or its fragments FH1-4, FH5-7, FH19-20, or BSA. <i>C</i>, Binding of ¹²⁵I-sCR1 to C3b in the presence of Ecb alone or together with FH or BSA as a control. <i>D</i>, Concentration of the protein needed to inhibit 50% of ¹²⁵I-sCR1 bound to C3b (IC50). The assay in panel <i>A</i> was performed three times, assays <i>B</i>, <i>C</i>, and <i>D</i> were performed twice, all with triplicates. Results of representative experiments are shown with mean SD. Differences between means was calculated using One-way ANOVA with Bonferroni multiple comparison (* <i>p</i><0.05).</p

Neutrophil binding-deficient mutants of SElX retain mitogenic activity.

<p>Isolated human PBMC were stimulated with recombinant SElX and sialic acid-binding mutants. After 72 h incubation, proliferation was determined by analysing the incorporation of [³H] thymidine. Results shown are the means of triplicate measurement from 3 human donors ± standard deviation of the mean.</p

List of primers used in this study.

<p>List of primers used in this study.</p

SElX binds to multiple neutrophil surface glycoproteins in a sialic acid-dependent manner.

<p>(i) Neutrophil lysates were analysed for peptide enrichment following incubation with wild-type SElX (WT) and SElX EKQD-A. Data shown indicates the mean number of peptides detected from each enriched protein (± SD from three donors). * denotes significance difference (p-value <0.05) in peptide enrichment between the wild-type and mutant protein as determined by multiple t-test comparisons (one unpaired, two tailed test per protein), without assuming similar standard deviation. (ii) ELISAs were performed comparing the interaction between staphylococcal proteins and recombinant CD50. Two-way ANOVA statistical analyses were performed. **** denotes a significant difference between SElX and SElX EKQD-A.</p

SElX protein sequence encodes a conserved sialic acid-binding motif.

<p>(i) A conserved glycan binding motif (colored red) is present in the amino acid sequences of characterised SSl-proteins and SElX. (ii) The sialic acid-binding motif is conserved in all 17 alleles of SElX. (iii) Amino acid conservation across the sialic acid-binding region of 7 staphylococcal neutrophil binding proteins (SSl2, SSl3, SSl4 SSl5, SSl6, SSl11 and SElX). The probability of residues at each position is proportional to the size of the letters. Image generated using the weblogo 3.4 program (<a href="http://weblogo.threeplusone.com/" target="_blank">http://weblogo.threeplusone.com/</a>). Chemical property color scheme: green is polar, blue is basic, red is acidic, purple is neutral and black is hydrophobic. Sequences were obtained from previously published work [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006461#ppat.1006461.ref006" target="_blank">6</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006461#ppat.1006461.ref023" target="_blank">23</a>].</p

SElX does not contribute to <i>S. aureus</i> virulence in a mouse skin abscess infection model.

<p>(i) Lesion size of each mouse (n = 10) was measured every 24 h post inoculation (p.i.) for 6 d. Mean lesion size ± SEM is plotted for each of the 4 USA300 LAC mutant groups. (ii) Bacteria were recovered from excised skin lesions and enumerated by serial dilutions. CFU were normalised to the weight of tissue homogenised to give the bacterial load per mg of tissue. CFU per mg are displayed for each infected animal; the horizontal line indicates the mean CFU/mg of tissue and vertical bars show the SEM for each group (n = 10) (iii) Representative images from histological examinations of skin lesions 72 h and 144 h post inoculation. Mounted sections were stained with haematoxylin and eosin. Black arrows on each image indicate the surface of the epidermis.</p