Phagocytosis Escape by a Staphylococcus aureus Protein That Connects Complement and Coagulation Proteins at the Bacterial Surface

Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a ‘capsule’-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein

Host-pathogen Interaction at the Intestinal Mucosa Correlates With Zoonotic Potential of Streptococcus suis

Streptococcus suis has emerged as an important cause of bacterial meningitis in adults. The ingestion of undercooked pork is a risk factor for human S. suis serotype 2 (SS2) infection. Here we provide experimental evidence indicating that the gastrointestinal tract is an entry site of SS2 infection. We developed a noninvasive in vivo model to study oral SS2 infection in piglets. We compared in vitro interaction of S. suis with human and porcine intestinal epithelial cells (IEC). Two out of 15 piglets showed clinical symptoms compatible with S. suis infection 24-48 hours after ingestion of SS2. SS2 was detected in mesenteric lymph nodes of 40% of challenged piglets. SS2 strains isolated from patients showed significantly higher adhesion to human IEC compared to invasive strains isolated from pigs. In contrast, invasive SS9 strains showed significantly higher adhesion to porcine IEC. Translocation across human IEC, which occurred predominately via a paracellular route, was significantly associated with clonal complex 1, the predominant zoonotic genotype. Adhesion and translocation were dependent on capsular polysaccharide production. SS2 should be considered a food-borne pathogen. S. suis interaction with human and pig IEC correlates with S. suis serotype and genotype, which can explain the zoonotic potential of SS

The staphylococcal toxin Panton-Valentine Leukocidin targets human C5a receptors.

International audiencePanton-Valentine Leukocidin (PVL) is a staphylococcal bicomponent pore-forming toxin linked to severe invasive infections. Target-cell and species specificity of PVL are poorly understood, and the mechanism of action of this toxin in Staphylococcus aureus virulence is controversial. Here, we identify the human complement receptors C5aR and C5L2 as host targets of PVL, mediating both toxin binding and cytotoxicity. Expression and interspecies variations of the C5aR determine cell and species specificity of PVL. The C5aR binding PVL component, LukS-PV, is a potent inhibitor of C5a-induced immune cell activation. These findings provide insight into leukocidin function and staphylococcal virulence and offer directions for future investigations into individual susceptibility to severe staphylococcal disease

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