Representative images of immunofluorescently stained lung sections from mice challenged with <i>B. anthracis</i> spores.

<p>Mice were infected i.n. with ∼10⁸ spores/mouse. Lungs were harvested at 2 and 4 weeks, fixed, sectioned and stained with anti-BclA antibodies and secondary antibodies conjugated to Alexa Fluor 594 (red), Alexa Fluor 488-conjugated phalliodin (green) and DAPI (blue), as described in the Materials and Methods section. Representative images are shown to indicate spore association with the small airway epithelium (<b>A</b> and <b>B</b>) and the alveolar epithelium (<b>C</b>–<b>E</b>). Arrows indicate spores. The areas around those spores were enlarged and shown in boxed insets. Scale bars represent 10 µm.</p

<i>B. anthracis</i> spores persisted in the lung significantly better than spores of <i>B. subtilis</i>.

<p>Mice were inoculated i.n. with 7702 spores (<i>B. ant</i>), PY79 spores (<i>B. sub</i>), or overnight cultures of HB101 (<i>E. coli</i>) at a dose of ∼1.3×10⁷ cfu/mouse. Lungs were harvested at 2 (<b>A</b>) and 4 (<b>B</b>) weeks post-inoculation, homogenized, and plated for total viable bacteria (closed circles) or heat-resistant dormant spores (open circles). The results were combined from at least 2 independent experiments. **, <i>p</i><0.01; ***, <i>p</i><0.001; compared to respective total bacteria and spore titers in <i>B. anthracis</i> infected lungs.</p

H&E stained lung sections of mice challenged with <i>B. anthracis</i> spores.

<p>Lungs from infected and control mice were collected at 2, 4, and 8 weeks post-inoculation, fixed and subjected to H&E staining. Representative images displaying the alveoli and the airway from mice infected for 2 (<b>A</b>–<b>C</b>), 4 (<b>D</b>–<b>F</b>), and 8 weeks (<b>G</b>–<b>I</b>), and uninfected control mice (<b>J</b>–<b>L</b>) are shown. Scale bars represent 20 µm.</p

The majority of persisting spores associated tightly with the lung tissue.

<p>Mice were inoculated i.n. with ∼1.3×10⁷ spores/mouse (<b>A</b> and <b>B</b>) and ∼1.1×10⁸ spores/mouse (<b>C</b> and <b>D</b>). Lungs were lavaged with sterile PBS and collected. Total bacteria (closed circles) and spore (open circles) titers in the lung tissues and BAL fluids at 2 (<b>A</b> and <b>C</b>) and 4 (<b>B</b> and <b>D</b>) weeks were determined. The results were combined from at least two independent experiments. *, <i>p</i><0.05; ***, <i>p</i><0.001; compared to respective total bacteria and spore titers in the lung tissue.</p

Confocal analysis of immunofluorescently stained lung sections.

<p>Lung sections were stained as described in the Materials and Methods section and analyzed by confocal microscopy. Representative images are shown. <b>A</b>, an image showing a spore (red) surrounded by F-actin (green). The projections show all planar views, including <i>xy</i> (center panel), <i>xz</i> (right panel) and <i>yz</i> (top panel)-stacks. <b>B</b>, the section was stained with wheat germ agglutinin to outline the plasma membrane (red) and anti-BclA antibodies to detect spores (green). Scale bars represent 10 µm.</p

Bacterial and spore burden in the lungs of mice at 2, 4, and 8 weeks post-intranasal inoculation.

<p>Mice were inoculated i.n. with ∼1.1×10⁸ or ∼1.3×10⁷ spores/mouse. Lungs were harvested at 2, 4, and 8 weeks, homogenized, and dilution plated with or without heat treatment. The results were combined from at least two independent experiments. Closed circles represent total viable bacteria and open circles heat-resistant dormant spores. *, <i>p</i><0.05; **, <i>p</i><0.01; compared to respective total bacteria and spore titers at 2 weeks.</p

Bacteria and spore burden in the lungs of mice.

a<p>Average retained dose was determined by harvesting the lungs 1 hour after intranasal inoculation and dilution plating the lung homogenates.</p>b<p>The amounts of total bacteria and spores in the lung as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066177#pone-0066177-g001" target="_blank">Figure 1</a> legend are expressed as the mean±SEM at 2, 4, or 8 weeks post-inoculation. Results are combined from two independent experiments.</p>c<p>Statistical analysis was performed using the two-tailed Student's t-test. *, <i>p</i><0.05; **, <i>p</i><0.01; compared to respective total bacteria and spore titers at 2 weeks.</p>d<p>Not determined.</p

Bacterial and spore burden in various organs at 2 and 4 weeks post-inoculation.

<p>Mice were inoculated i.n. with ∼1.3×10⁷ spores/mouse. Bacterial burden in the lung, spleen, kidney, trachea, and nasopharynx (NP) at 2 (<b>A</b>) and 4 (<b>B</b>) weeks was determined as described in the Materials and Methods section. The results were combined from at least 2 independent experiments. Closed circles represent total viable bacteria and open circles heat-resistant dormant spores. **, <i>p</i><0.01; ***, <i>p</i><0.001; compared to respective total bacteria and spore titers in the lung.</p

BclA-mediated CFH recruitment inhibited downstream complement activation <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Complement hemolytic assay. Spores were incubated with 20% NHS and centrifuged. The supernatants (1:10 diluted) were used to perform complement hemolytic assays using opsonized sheep erythrocytes (EA-SRBC). Data shown was from at least three independent experiments. (<b>B</b>) Determination of C5a levels in human serum incubated with the different spores. GVB⁰ buffer containing 20% NHS was pre-treated with buffer only (no antibody), OX24, or control IgG1, followed by incubation with 7702, Δ<i>bclA</i> or Δ<i>bclA</i>/BclA spores. C5a levels in the supernatants were measured using the Human Complement Component C5a DuoSet. Data shown was combined from two independent experiments, each with duplicate wells. (<b>C</b>) Determination of C5a levels in mouse BAL fluid. C57BL/6 were i.n. inoculated with 7702 (n = 8), Δ<i>bclA</i> (n = 8), Δ<i>bclA</i>/BclA (n = 8) spores or PBS (n = 6). BAL fluids were collected 6 hours later and C5a level in the supernatant determined using the Mouse Complement Component C5a DuoSet. Data shown were combined from two independent experiments, each with duplicate wells. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01. ****, <i>p</i> < 0.0001, <i>t</i> test.</p

<i>B</i>. <i>anthracis</i> spore surface protein BclA mediated CFH binding to spores.

<p>Spores were incubated with purified human CFH in PBS buffer containing D-alanine. Spore-bound CFH was determined by flow cytometry (<b>A</b>), solid phase binding assay (<b>B</b>) and Western blot (<b>C</b>). Flow cytometry results were combined from at least three independent experiments. Solid phase binding assay results were combined from two independent experiments, each with duplicate wells. Western blots shown were representative of at least three independently performed experiments. (<b>D</b>) Recombinant BclA protein (rBclA) bound to immobilized human CFH in a concentration-dependent manner. Results were combined from three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001; <i>t</i> test.</p