image_1.PDF

<p>The complement system is an ancient part of the innate immune system important for both tissue homeostasis and host defense. However, bacteria like Staphylococcus aureus (SA) possess elaborative mechanisms for evading both the complement system and other parts of the immune system. One of these evasive mechanisms—important in causing chronic and therapy resistant infections—is the intracellular persistence in non-immune cells. The objective of our study was to investigate whether persistent intracellular SA infection of epidermal keratinocytes resulted in complement activation. Using fluorescence microscopy, we found that persistent SA, surviving intracellularly in keratinocytes, caused activation of the complement system with formation of the terminal complement complex (TCC) at the cell surface. Skin samples from atopic dermatitis patients analyzed by bacterial culture and microscopy, demonstrated that SA colonization was associated with the presence of intracellular bacteria and deposition of the TCC in epidermis in vivo. Complement activation on keratinocytes with persistent intracellular bacteria was found with sera deficient/depleted of the complement components C1q, Mannan-binding lectin, or complement factor B, demonstrating involvement of more than one complement activation pathway. Viable bacterial counts showed that complement activation at the cell surface initiated cellular responses that significantly reduced the intracellular bacterial burden. The use of an inhibitor of the extracellular signal-regulated kinase (ERK) abrogated the complement-induced reduction in intracellular bacterial load. These data bridge the roles of the complement system in tissue homeostasis and innate immunity and illustrate a novel mechanism by which the complement system combats persistent intracellular bacteria in epithelial cells.</p

EDC34 inhibits contact activation at negatively charged surfaces.

<p>(<b>A</b>) Effect of EDC34 on the contact system was assessed by measuring the aPTT in human citrate plasma. The control peptide DAA14 was used at 50 µM. (<b>B</b>) Measurement of effects of EDC34 (50 µM) and DAA14 (50 µM) on the extrinsic (PT) and common pathway (TCT) of coagulation. (<b>C</b>) Measurement of aPTT, PT and TCT clotting times of mouse citrate plasma in absence (Control) or presence of 50 µM EDC34. (<b>D</b>) Plasma kallikrein activity induced by negatively charged kaolin in human citrate plasma in absence (Control) or presence of EDC34 (50 µM) or DAA14 (50 µM), was determined by a chromogenic substrate assay. Data are presented as mean±SEM relative to control without peptides (n = 4; One-Way ANOVA Bonferroni's Multiple Comparison Test). (<b>E</b>) Plasma kallikrein activity in human citrate plasma induced by the indicated bacteria in absence (Control) or presence of the peptides EDC34 (50 µM) or DAA14 (50 µM) was measured by a chromogenic substrate assay. Data are presented as mean±SEM relative to control without peptides (n = 5). (<b>F</b>) Western blot analysis of HK and HK degradation products: 1, No peptide; 2, EDC34; 3, DAA14. (<b>G</b>) Bradykinin release in human citrate plasma incubated with kaolin in absence (Control) or presence of EDC34 (50 µM) or DAA14 (50 µM) (n = 3, mean±SEM is presented; One-Way ANOVA Bonferroni's Multiple Comparison Test).</p

Antimicrobial activities of EDC34.

<p>(<b>A</b>) Antibacterial effects of EDC34 at the indicated concentrations were studied in viable count assays, using the bacteria <i>E. coli</i> ATCC 25922 (complement sensitive) and <i>E. coli</i> O18:K1 (complement-resistant). Analyses were performed in 10 mM Tris, 0.15 M NaCl, pH 7.4 (Buffer) or buffer containing 20% citrate plasma (CP) or heat-inactivated citrate plasma (HCP). Mean with SD is shown (n = 3). (<b>B</b>) Antimicrobial and hemolytic effects of EDC34 against the indicated bacteria in whole blood (n = 3, mean±SEM is presented). (<b>C</b>) Antimicrobial effects of EDC34 and DAC31, a mouse derived TFPI-2 C-terminal peptide against <i>E. coli</i> in human or mouse citrate plasma (n = 3, mean±SEM is presented).</p

EDC34 enhances the binding of complement proteins to bacteria.

<p>(<b>A</b>) <i>E. coli</i> ATCC 25922 and <i>P. aeruginosa</i> PA01 bacteria were incubated 30–60 min with citrate plasma alone (Control) or supplemented with EDC34 (at 3 µM) at 37°C. The bacterial cells and supernatants were collected and proteins were detected by immunoblotting with antibodies recognizing C1q or C5b-9. CP, citrate plasma; S, supernatant or unbound bacteria; P, pellet or material bound to bacterial cells. (<b>B</b>), as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003803#ppat-1003803-g002" target="_blank">figure 2A</a>, but antibodies against C3a were used. (<b>C</b>) Quantification of deposition of complement components on bacteria by flow cytometry, <i>left panel</i>, comparison of the mean proportion of bacteria positive for C1q/C3a binding in citrate plasma and in plasma supplemented with EDC34. <i>Right panel</i>, comparative degree of C1q and C3a binding to <i>E. coli</i> and <i>P. aeruginosa</i> strains, expressed as means of the fluorescence index (<i>FI</i>; proportion of bacteria positive for C1q/C3a multiplied by the mean intensity of C1q/C3a binding). (n = 4, mean±SEM is presented; Two-Way ANOVA Bonferroni's Multiple Comparison Test). (<b>D</b>) TFPI-2 and C3a binding to bacteria in fibrin slough collected from an infected chronic wound were visualized by using gold-labeled antibodies of different sizes, specific for the C-terminus of TFPI-2 (20 nm) and C3a (10 nm), respectively. Insert shows a higher magnification.</p

EDC34 is antimicrobial and anti-coagulative in an <i>E. coli</i> infection model <i>in vivo</i>.

<p>BALB/c mice were injected (i. p.) with <i>E. coli</i> DH5-α (2×10⁸ cfu) and treated with EDC34 (0.5 mg) as indicated in <b>A</b> and <b>B</b>. (<b>A</b>) Survival is presented (<i>E. coli</i> infection; n = 13, treatment with EDC34 i.p., or sc; n = 8, Log-rank Mantel-Cox test) and (<b>B</b>) weight was determined daily. As only one animal survived in the untreated group (after day 2), the weight curve for untreated mice is not shown (EDC34-ip. 0 h vs. EDC34-sc. 1 h (***); EDC34-ip. 0 h vs. EDC34-ip. 1 h (*); and EDC34-sc. 1 h vs. EDC34-ip. 1 h (ns), mean±SEM is presented; Two-Way ANOVA Bonferroni's Multiple Comparison Test). (<b>C–G</b>) BALB/c mice were injected (i. p.) with <i>E. coli</i> DH5-α (1.5×10⁸ cfu) and immediately treated with EDC34 (0.5 mg, i. p.). The following parameters were determined: (<b>C</b>) Cfu in peritoneal wash fluid (n = 15/group, mean±SEM is presented; Two-Way ANOVA Bonferroni's Multiple Comparison Test), (<b>D</b>) cfu in blood and indicated organs (n = 9/group, mean±SEM is presented; Two-Way ANOVA Bonferroni's Multiple Comparison Test), (<b>E</b>) aPTT and PT in citrate plasma (n = 9/group, mean±SEM is presented; Two-Way ANOVA Bonferroni's Multiple Comparison Test), (<b>F</b>) TAT complexes (8 h post-infection, n = 8/group, mean±SEM is presented; One-Way ANOVA Bonferroni's Multiple Comparison Test), and (<b>G</b>) cytokines (Control, 0 h; n = 7, <i>E. coli</i> and EDC34 treatment; n = 9/group, mean±SEM is presented; Two-Way ANOVA Bonferroni's Multiple Comparison Test).</p

Activities of the C-terminal TFPI-2 peptide (EDC34).

<p>(<b>A</b>) Schematic representation of TFPI-2. The EDC34 peptide sequence is indicated. (<b>B</b>) Migration of EDC34 on 16.5% SDS-PAGE. The arrow indicates the position of the neutrophil elastase generated C-terminal peptide, whereas the asterisks indicate the positions of C-terminal fragments found in human fibrin slough. (<b>C</b>) LPS-binding activity of EDC34. Peptides were applied to a nitrocellulose membrane followed by incubation in PBS (containing 2% bovine serum albumin) with iodinated (¹²⁵I)-LPS. Similar to LL-37, the EDC34 peptide bound LPS. The binding was inhibited by heparin (6 mg/ml). (<b>D</b>) Interaction of EDC34 with bacterial surfaces. The indicated bacteria (1–2×10⁹ cfu/ml, 50 µl) were incubated with 3 µM TAMRA-labeled EDC34 in 450 µl of 10 mM Tris, pH 7.4, 0.15 M NaCl (Buffer), or 450 µl of human citrate-plasma (Plasma), and samples were analyzed by FACS. (<b>E</b>) Permeabilizing effects of peptides on <i>E. coli</i>. Bacteria were incubated with 30 µM of EDC34 or LL-37, and permeabilization was assessed using the impermeant probe FITC. (<b>F</b>) Electron microscopy analysis. <i>P. aeruginosa</i> and <i>S. aureus</i> bacteria were incubated for 2 h at 37°C with 30 µM of EDC34 and LL-37 and visualized by negative staining. Control; buffer control.</p

Peptide effects on liposomes and biophysical studies.

<p>(<b>A</b>) The membrane permeabilizing effect of EDC34 and LL-37 was recorded by measuring fluorescence release of carboxyfluorescein from PA (negatively charged) liposomes. The experiments were performed in 10 mM Tris buffer. Values represents mean of triplicate samples. (<b>B</b>) Helical content of EDC34 and LL-37 peptides in presence of negatively charged liposomes (PA). The structure of EDC34 was largely unaffected by the addition of liposomes. (<b>C</b>) CD spectra of EDC34 and LL-37 in Tris buffer and in presence of LPS. For control, CD spectra for buffer and LPS alone are also presented.</p

Identification and expression of TFPI-2 in human skin and wounds.

<p>(<b>A</b>) Immunohistochemical identification of TFPI-2 in normal skin, acute skin wound and in chronic venous leg ulcer tissue (chronic wound). Skin biopsies were taken from normal skin (n = 3), acute wounds (n = 3) and from the wound edges of patients with chronic venous ulcers (n = 3). Representative sections are shown. Scale bar is 100 µm. (<b>B</b>) TFPI-2 expression levels in wounds <i>in vivo</i> and <i>ex vivo</i>. Expression levels of TFPI-2, as determined by array data, in wounded and non-wounded skin are presented (n = 3).</p

Activities on eukaryotic cells. Hemolytic effects of the indicated peptides.

<p>The cells were incubated with different concentrations of the peptides, 2% Triton X-100 (Sigma-Aldrich) served as positive control. The absorbance of hemoglobin release was measured at λ 540 nm and is expressed as % of Triton X-100 induced hemolysis (note the scale of the y-axis). Effects of LL-37 are shown for comparison.</p

Antimicrobial activities of EDC34.

<p>(<b>A</b>) Antimicrobial activity of EDC34 (at 100 µM in RDA) was tested against the indicated microbes. For determination of effects, <i>E. coli</i> ATCC 25922, <i>P. aeruginosa</i> ATCC 27853, <i>S. aureus</i> ATCC 29213 or <i>B. subtilis</i> ATCC 6633 isolates (4×10⁶ cfu) or C. <i>albicans</i> ATCC 90028 and <i>C. parapsilosis</i> ATCC 90018 (1×10⁵ cfu) were inoculated in 0.1% TSB agarose gel. Each 4 mm-diameter well was loaded with 6 µl of peptide. The zones of clearance correspond to the inhibitory effect of each peptide after incubation at 37°C for 18–24 h. LL-37 (at 100 µM) was used for control (mean values are presented, n = 3). (<b>B</b>) Antibacterial effects of EDC34 and LL-37 against the indicated bacterial strains in viable count assays. 2×10⁶ cfu/ml bacteria were incubated in 50 µl with peptides at the indicated concentrations in 10 m.</p