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

BclA inhibited antibody responses against spores.

<p>(<b>A</b>) C57BL/6 mice were i.n. inoculated with ~1×10⁸ spores of 7702, Δ<i>bclA</i> or vehicle control once and blood collected at 2 weeks post inoculation (2wk), or inoculated again with the same spores and dose at 2 weeks and blood collected at 4 weeks after the initial inoculation (4wk). Anti-spore antibodies in the serum were detected using ELISA. Data shown were combined from at least three independent experiments. The mouse number for the various groups is as follows: control, n = 8; 2wk experiment, n = 30 and 29 for 7702 and Δ<i>bclA</i>, respectively; 4wk experiment, n = 30 and 28 for for 7702 and Δ<i>bclA</i>, respectively. (<b>B</b>) C3^-/- mice were i.n. inoculated with vehicle control, or ~5×10⁵ spores of 7702 or Δ<i>bclA</i> and blood collected at 2 weeks post inoculation. Anti-spore antibodies in the serum were detected using ELISA. Data shown were combined from at least three independent experiments, with n = 10, 24 and 21 for control, 7702 and Δ<i>bclA</i>, respectively. *, <i>p</i> < 0.05; **, <i>p <</i> 0.01; ****, <i>p</i> < 0.0001; <i>t</i> test.</p

BclA-mediated CFH binding promoted degradation of C3b on the spore surface and downregulated further C3 activation.

<p>(<b>A</b>) and (<b>B</b>) Spores were incubated with purified human C3b, CFH and CFI. C3 fragments deposited on the spore surface were detected using anti-C3 polyclonal antibodies (<b>A</b>). The image shown is representative of at least three independent experiments. The ratio of iC3b/C3b was determined by quantifying the density of the corresponding bands in western blots using Image J (<b>B</b>). The β chain represents C3b + iC3b, and the α” chain represents iC3b. Results were combined from three independent experiments. (<b>C</b>) and (<b>D</b>) Rate of iC3b deposition on spores. Spores were incubated with 10% NHS for the indicated time and subjected to flow cytometry analysis using iC3b-specific antibody. The results shown are mean fluorescence intensity normalized to that at 10 minutes, respectively, and combined from at least three independent experiments (<b>C</b>). The rate of iC3b deposition (ΔiC3b/Δt) was calculated by linear regression analysis of the normalized data (GraphPad Prism 6) (<b>D</b>). (<b>E</b>) Determination of C3a concentration. GVB⁰ buffer containing 20% NHS (no antibody), 20% NHS pre-treated with the CFH functional blocking antibody OX24 (240 nM final conc.) or mouse IgG1 control (240 nM) was incubated with no spore, 7702, Δ<i>bclA</i> or Δ<i>bclA</i>/BclA spores at 37°C for 30 min and centrifuged to remove the spores. C3a concentrations in the supernatants were determined using the Human C3a ELISA kit (BD OptEIA^™) and normalized to the respective no spore control. Data was combined from four experiments, each with duplicate wells. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001; <i>t</i> test.</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

BclA impaired protective immunity against lethal <i>B</i>. <i>anthracis</i> challenges.

<p>C57BL/6 mice were i.n. inoculated with a sub-lethal dose of ~1×10⁸ spores of 7702, Δ<i>bclA</i> or vehicle control once (<b>A</b>) or twice (<b>B</b>). Mice were then challenged with ~1×10¹⁰ 7702 spores by i.p. injection 15 days after the last i.n. inoculation and monitored for survival. Data shown in (<b>A</b>) were combined from two independent experiments, with n = 7, 8, and 10 for ctrl-7702, 7702–7702, and Δ<i>bclA</i>-7702, respectively. Data shown in (<b>B</b>) were combined from two independent experiments, with n = 6, 8 and 12 for ctrl-7702, 7702–7702, and Δ<i>bclA</i>-7702, respectively. Log-rank test was used to statistical comparison of the survival curves.</p

BclA significantly promoted spore persistence in the mouse lungs.

<p>Mice were i.n. inoculated with sub-lethal doses of various spores. Lungs were collected, homogenized and either dilution plated to determine the total viable bacterial counts, or heated at 68°C and dilution plated to determine the spore counts. (<b>A</b>). C57BL/6 mice were i.n. inoculated with ~1×10⁸ spores of 7702, Δ<i>bclA</i> or Δ<i>bclA</i>/BclA per mouse. Lungs were collected 2 weeks post inoculation. Data shown were combined from at least two independent experiments (7702, n = 12; Δ<i>bclA</i>, n = 7; Δ<i>bclA</i>/BclA, n = 4). (<b>B</b>) Balb/c mice were i.n. inoculated with ~ 1.5 ×10⁷ spores per mouse of <i>B</i>. <i>subitilis</i> containing pDG1662 vector (n = 20) or pDG1662-BclA (n = 20). Lungs were harvested at one week post inoculation. Data shown were combined from two independent experiments. (<b>C</b>). C3^-/- mice were more susceptible to <i>B</i>. <i>anthracis</i> than C57BL/6. Therefore, a sub-lethal dose of ~ 5×10⁵ spores/mouse was used for i.n. inoculation of C3^-/- mice. Lungs were collected at 2 weeks post inoculation. Data shown were combined from at least two independent experiments (7702, n = 14; Δ<i>bclA</i>, n = 12). (<b>D—G</b>) Mice were i.p. inoculated with lethal doses of 7702 or Δ<i>bclA</i> spores. (<b>D</b>) C57BL/6 mice were inoculated with ~1×10⁸ spores/mouse of 7702 (n = 10) or Δ<i>bclA</i> (n = 10) and survival monitored. Data shown were combined from two independent experiments. (<b>E</b>) C3^-/- mice were inoculated with ~5×10⁶ spores/mouse of 7702 (n = 11) or Δ<i>bclA</i> (n = 11). Data shown were combined from two independent experiments. (<b>F</b>) Bacterial burden in the lungs and spleen of C57BL/6 mice inoculated with ~1×10⁸ spores of 7702 (n = 10) or Δ<i>bclA</i> (n = 10) at 48 hours post inoculation. Data shown were combined from two independent experiments. (<b>G</b>) Bacterial burden in the lungs and spleen of C3^-/- mice inoculated with ~5×10⁶ spores of 7702 (n = 6) or Δ<i>bclA</i> (n = 4) at 48 hours post inoculation. Data shown were combined from two independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ****, <i>p</i> < 0.0001; <i>t</i> test. Analysis of survival curves was done using Log-rank test.</p