Characterization of PAK-P3 and P3-CHA bacteriophages.

<p>(<b>A</b>, <b>C</b>) Electron micrographs of PAK-P3 (<b>A</b>) and P3-CHA (<b>C</b>). Scale bar: 100 nm. (<b>B</b>) SDS-PAGE of PAK-P3 and P3-CHA proteins; only the most abundant proteins give visible signals (MW: molecular weight markers, the arrow points to the major capsid proteins). (<b>D</b>) Clustal alignment of the three major capsid proteins of PAK-P1, PAK-P3 and P3-CHA bacteriophages with their closest homologs in the database with known function (the major capsid protein of Felix 01 bacteriophage; NP_944891). Major capsid proteins of PAK-P3 and P3-CHA are 100% identical.</p

Bacteriophage P3-CHA cure and prevent lung infections caused by a clinical <i>P. aeruginosa</i> strain.

<p>(<b>A–B</b>) Survival curves of mice infected with the CHA strain and treated or pre-treated with P3-CHA bacteriophage. (<b>A</b>) PBS (♦), 3×10⁶ (•) and 3×10⁷ (□) pfu of bacteriophage were given intranasally 2 h after bacteria (3×10⁶ cfu) were administered. This curative treatment appears to be dose dependent (P<0.0001 for both bacteriophage doses compared to PBS and P<0.01 between 3×10⁶ and 3×10⁷ bacteriophage doses). (<b>B</b>) Four days before infection with 3×10⁶ bacteria, mice were given either 3×10⁷ (•), or 3×10⁸ (□) pfu of P3-CHA or 3×10⁸ pfu of heat-inactivated P3-CHA solution (♦). These survival curves indicate that the preventative treatment is dose dependent (P<0.0005 and P<0.0001 for 3×10⁷ and 3×10⁸ bacteriophage doses respectively compared to heat-inactivated bacteriophage solution and P<0.0005 between 3×10⁷ and 3×10⁸ bacteriophage doses). (<b>C–D</b>) 20 h after infection with strain CHA, mice were euthanized and BAL fluids were assayed for bacteria (<b>C</b>) and bacteriophages (<b>D</b>). In the curative treatment protocols, mice were treated with PBS or bacteriophage 2 h post infection (phage 2 h pi). In the preventative treatment protocols, mice were intranasally administered bacteriophage solution (4d phage) or heat-inactivated bacteriophage solution (4d phage 80°C) four days before infection. (<b>C</b>) Bacterial counts were significantly lower in the BAL fluids from mice that had received either curative or preventative bacteriophage treatment than the respective control treatment (* P<0.05, and ** P<0.01). (<b>D</b>) Bacteriophage counts were significantly lower in the BAL fluids from mice that had received bacteriophage treatment than the non-infected animals (** P<0.01) or the non-infected animals pre-treated four days earlier with the bacteriophage P3-CHA (* P<0.05).</p

Histological and immunohistochemical analyses of lung sections from mice treated or pre-treated with bacteriophage P3-CHA.

<p>(<b>A–D</b>; scale bar 200 µm) Thin sections of lungs obtained after 20 h from, (<b>A</b>) mice infected with the CHA strain, (<b>B</b>) infected mice treated with bacteriophage P3-CHA, (<b>C</b>) mice pre-treated with bacteriophage P3-CHA four days before infection, and (<b>D</b>) uninfected mice were stained with hematoxylin-eosin and Gram. Histological analyses included the determination of a lesion severity score (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016963#s4" target="_blank">materials and methods</a>) to allow comparison (<b>M</b>). This score was significantly lower for mice given curative or preventive bacteriophage treatments than for untreated mice (* P<0.05). (<b>E–L</b>) Immunohistochemistry was performed with anti-<i>Pseudomonas</i> antibodies on sections obtained from the same samples as above (<b>E–H</b>; scale bar 100 µm and <b>I–L</b>; scale bar 50 µm). In lung sections from infected mice, bacteria are both intracellular and extracellular (<b>E</b> and <b>I</b>) whereas no signal can be seen in lung sections from uninfected mice (<b>H</b> and <b>L</b>). The signal was lower following either curative (<b>F</b> and <b>J</b>) or preventive (<b>G</b> and <b>K</b>) bacteriophage treatments and bacteria were only observed in macrophage cells.</p

Persistence of P3-CHA bacteriophage in lungs of uninfected mice.

<p>Three groups of five mice were given 3×10⁸ pfu of bacteriophage P3-CHA, administered intranasally. The number of bacteriophages in BAL fluids was determined 1, 4 and 5 days post-administration (n = 5).</p

Efficacy of plating of PAK-P3 and P3-CHA bacteriophages on CF <i>P. aeruginosa</i> strains.

<p>Efficacies of PAK-P3 and P3-CHA bacteriophages on their reference strains, respectively. PAK and CHA strains were fixed at 100%. Results are expressed as relative percentage to these references.</p

Survival curve of infected mice treated with bacteriophage PAK-P3 compared to P3-CHA.

<p>Mice were infected intranasally with 3×10⁶ cfu (CHA strain) and 2 h later were treated with either PBS (♦) or 3×10⁷ pfu of bacteriophage PAK-P3 (•), or 3×10⁷ pfu of bacteriophage P3-CHA (□) also administered intranasally (P<0.005 for both P3-CHA and PAK-P3 bacteriophage doses compared to PBS and P<0.05 between P3-CHA and PAK-P3 bacteriophage doses).</p

Induction of apoptosis mediated by (r)GAPDH.

<p>A) 5×10⁵ RAW264.7 murine macrophages were left untreated (Medium) or treated for 24 h with 1 µM staurosporin or 50 µg/mL rGAPDH, or 200 µL of culture supernatant from GBS, <i>S. pyogenes</i>, <i>L. lactis</i> or <i>S. aureus</i> left untreated (unt) or immunoprecipitated with rabbit anti-GAPDH IgG pAb (ipGAPDH), with rabbit anti-SodA IgG pAb (ipSodA) or with a normal rabbit IgG (control IgG). B) 5×10⁵ Bone marrow-derived macrophages from C57BL/6 mice were left untreated (Medium) or treated for 24 h with 1 µM staurosporin, or rGAPDH at the indicated concentrations. After treatment, the macrophages were fixed and stained for TUNEL using the Promega DeadEnd™ Fluorometric TUNEL kit. Samples were acquired and the % of cells that incorporated fluorescein-dUTP (TUNEL positive cells) was quantified. C) For IF, 1×10⁵ C57BL/6 bone marrow-derived macrophages were left untreated or treated for 24 h with 50 µg/mL rGAPDH. After that period, cells were fixed and stained as mentioned in B) and visualized in a fluorescence microscope. Scale bars, 15 µm. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029963#s2" target="_blank">Results</a> shown are representative of two independent experiments. ns, non-significant; *, P<0.05; **, P<0.01.</p

Reassociation capacity of rGAPDH.

<p>A) ELISA plates were coated with 6.5×10⁷ CFU/well of heat inactivated NEM316 overnight at 4°C. They were incubated with 100 µL/well of different amounts of rGAPDH (0, 10 or 50 µg/mL) or rGAPDH 50 µg/mL with 75 µg of rabbit anti-rGAPDH IgG pAb (50+αGAPDH). rGAPDH was detected using a HRP coupled mouse pentaHis antibody. After revelation, the OD₄₅₀ was registered using a microplate reader. B) 200 µg of rGAPDH (+rGAPDH) or PBS (−rGAPDH) was added to an exponentially grown culture of several GBS strains belonging to different serotypes (Ia, Ib, II, III, IV, V, VI, and VII), <i>S. pyogenes</i>, <i>L. lactis</i>, <i>S. aureus</i>, and <i>E. coli</i>. After incubation, total proteins extracts were subjected to SDS-PAGE followed by transfer to nitrocellulose membrane (rGAPDH: 10 ng; Total proteins +/− rGAPDH: 8 µg). This membrane was incubated with an HRP-coupled mouse pentaHis antibody or rabbit anti-SodA antibody (loading control) followed by HRP-conjugated goat anti-rabbit secondary antibody. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029963#s2" target="_blank">Results</a> shown are representative of two independent experiments. **, P<0.01; ***, P<0.001.</p

Interaction of rGAPDH with pilus structural subunits.

<p>ELISA plates were coated with 10 µg/mL of PilA, PilB, PilC, S10 domain of HvgA (HvgA-S10) or rGAPDH proteins. These plates were incubated with 100 µL/well of different amounts of rGAPDH (0, 6.25, 12.5, 25, 50 or 100 µg/mL). GAPDH was detected using a rabbit anti-rGAPDH IgG pAb. After the revelation, the OD₄₅₀ was registered using a microplate reader. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029963#s2" target="_blank">Results</a> shown are representative of two independent experiments.</p

Evaluation of surface-bound and extracellular GAPDH levels in GBS strain NEM316 (WT) and pilus mutant strains.

<p>A) Immunoelectron microscopy (IEM) picture of the surface-bound GAPDH in NEM316. Scale bar, 100 nm. B) Fluorescence-activated cell sorter analysis (FACS) of WT, PilB⁻ and PilA/C⁻ incubated with a polyclonal rabbit anti-GAPDH IgG (grey filled histogram for WT or black line histogram for PilB⁻ and PilA/C⁻ strains) or normal rabbit IgG (control IgG-grey dotted histogram). Mean Fluorescence Intensity (MFI) in lower panel is reported relative to control IgG. C) Western Blot detection of GAPDH in culture supernatants (SN) of exponentially growing WT, PilB⁻ and PilA/C⁻ strains. 25 µL of each concentrated supernatant, 10 ng of rGAPDH or 2 µg of WT total proteins were loaded in the gel. After transfer to a membrane, proteins were detected using a polyclonal rabbit anti-rGAPDH IgG antibody, rabbit anti-SodA antibody and rabbit anti-CAMP antibody followed by HRP-conjugated goat anti-rabbit antibody. D) Quantifications of GAPDH relative to the loading control protein (CAMP) in the supernatant of WT, PilB⁻ and PilA/C⁻ strains, were performed with ImageJ software. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029963#s2" target="_blank">Results</a> shown are representative of three independent experiments. *, P<0.05.</p