Surface expression of SdrE increases cleavage of C3b and reduces total C3-fragment deposition.

<p>A. <i>L. lactis</i> were incubated with (+) or without (−) 10% HI-serum to bind serum fH, washed thoroughly, then resuspended with fI and C3b (0.5 µg each) for 2.5 hr, 37°C; supernatants were assessed for C3b cleavage via Western blot. B, C and D: <i>L. lactis</i> were incubated with NHS; deposited C3-fragments were stripped with 25 mM methylamine and quantitated by ELISA. B, All pathways, 15-minute incubation, various serum concentrations (<i>p</i><0.001, as a group). C, All pathways, 10% NHS, varied incubation times (<i>p</i><0.0001, as a group). D, Alternative pathway only (Mg-EGTA-GVBS), 10% NHS, various incubation times (<i>p</i><0.0001, as a group). Data represent at least 3 independent experiments ± SEM.</p

fH binding to recombinant proteins.

<p>rSdrE, rClfA and BSA (10 µg/mL) were adsorbed to a microtitre plate and assessed for fH binding using heat-inactivated serum (HI-serum): A, Serum proteins bound to immobilized rSdrE, rClfA, and BSA in microtiter wells were extracted with 2% SDS and analyzed by anti-fH Western blot. B, fH binding via Western blot, as described in (A) quantitated using optical densitometry; *<i>p</i> = 0.03. C, Modified ELISA using 15% HI-serum and various time points; <i>p</i><0.001 as a group. D, Modified ELISA using various concentrations of HI-serum, 15-minute incubation; <i>p</i><0.001 as a group. Data represent the mean of at least three independent experiments ± SEM.</p

Mass spectrometry of fH-binding protein detected via fH cross-linking identifying SdrE peptides.

<p>Mass spectrometry of fH-binding protein detected via fH cross-linking identifying SdrE peptides.</p

Factor H binds to <i>S. aureus</i> cell wall proteins.

<p>A, Far-western analysis (purified fH overlay) of <i>S. aureus</i> cell wall proteins fractionated via ionic exchange chromatography; 9, 10, and 11 represent fraction numbers. B, Sypro-ruby stained SDS-PAGE gel, complimentary to (A). C, Peptide map of Serine-aspartate repeat-containing protein E (SdrE) with peptides identified by LC-ESI-MS/MS shown in bold and underlined. D, Clumping Factor A (ClfA) peptide map with peptides identified by LC-ESI-MS/MS shown in bold and underlined.</p

Factor H cross-linked to <i>S. aureus</i> cell wall protein/s.

<p>A, Anti-fH Western blot of purified fH cross-linked to <i>S. aureus</i> cell wall protein/s indicated by the asterisk (*); XL: cross-linker, BS³. B, LC-ESI-MS/MS mass spectra map of <i>S. aureus</i> protein SdrE found cross-linked to fH. Matched peptides are shown in bold and underlined.</p

SdrE expression reduces <i>L. lactis</i> killing by PMNs.

<p><i>L. lactis</i> were incubated with 10% NHS with or without PMNs and tumbled at 37°C. Samples were taken at various time points, diluted in sterile water, and plated (<i>p</i> = 0.0016, as a group). Data represent the mean of at least 4 independent experiments ± SEM.</p

Purified fH binding to recombinant proteins.

<p>A, A representative fH overlay dot blot using a purified fH dilution series as a quantitation control (right column, <u>fH</u>). rSdrE, rClfA and BSA (5 µg) were adsorbed to a PDVF membrane (left column), blocked, then overlaid with purified fH (20 µg in 10 ml block buffer); BSA was used as a control. fH binding was determined via optical densitometry using the purified fH dilution series as a standard curve. B, Quantitative fH binding via dot blot, as described in (A), *<i>p</i><0.01; n = 4. C, Modified ELISA: rSdrE, rClfA and BSA were adsorbed to a microtiter plate and incubated with various amounts of purified fH for 1 hr; data represent the mean of at least three independent experiments ± SEM.</p

Serum fH binds to SdrE-expressing <i>L. lactis</i> in a dose-dependent manner.

<p>A, <i>L. lactis</i> isogenic mutants were immobilized to a PVDF membrane and overlaid with 5% HI-serum; fH binding was quantitated using a serum fH standard curve and optical densitometry; *<i>p</i> = 0.02. B, <i>L. lactis</i> isogenic mutants were incubated with various concentrations of HI-serum for 1 hr; bound proteins were extracted with 2% SDS and analyzed by fH ELISA using chicken antibodies specific for human fH; <i>p</i> = 0.005, as a group. C, <i>L. lactis</i> were incubated with 20% HI-serum; bound proteins were extracted with 2% SDS and analyzed by anti-fH Western blot. Data represent the mean of at least three independent experiments ± SEM.</p

PA-dPEG24 inhibits complement activation <i>in vivo</i>.

<p>(A) Rats were IV administered PA-dPEG24 at 10 (n = 2) or 20mg/mL (n = 2) in 0.9% NaCl or received a vehicle control (n = 2) or were sham treated (n = 2). B, Rats were IV administered PA-dPEG24 at 10mg/mL (n = 4), 20mg/mL (n = 4) or 30mg/mL (n = 4) in 10mM Na₂HPO₄, 0.9% NaCl or received a vehicle control (n = 3). At the indicated time points post-administration, blood was collected and plasma isolated. Plasma was tested in hemolytic assays with human AB erythrocytes. Error bars represent the SEM.</p

Linear mode MALDI-TOF mass spectrometry analysis of PA-dPEG24 oligomeric state.

<p>PA-dPEG24 in acidified water was analyzed by linear mode mass spectrometry. The spectrum shows two peaks for PA-dPEG24 with a monomer peak at 2778 and a dimer form at 5553.</p