Sequence alignment of TIR domain proteins.

<p>Sequence alignment of the TIR domains from BaTdp (BA_4098, residues 126–266), BwTdp from <i>Bacillus weihenstephanensis</i> (KEZ82977.1, residues 132–256), TcpB from <i>Brucella melitensis</i> (NP5404591.1, residues 116–250), TcpC from <i>Escherichia coli</i> CFT073 (NP_754290.1, residues 169–280), TcpF from <i>Enterococcus faecalis</i> (CCO72761.1, residues 3–160), TirS from <i>Staphylococcus aureus</i> (WP_000114516.1, residues 142–246), SaTlp1 from <i>S</i>. <i>aureus</i> (CAQ50581.1, residues 202–308), YpTdp from <i>Yersinia pestis</i> (NP_ 669733.1, residues 139–240), and human proteins MyD88 (AAH13589.1, residues 146–296), TLR2 (AAH33756.1, residues 641–784) and TLR4 (NP_003257.1, residues 621–781). The Box 1 and Box 2 regions are indicated by red boxes. The conserved glycine residue in the box 2 region is marked with an asterisk. Structural features indicated are based on the TcpB structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158575#pone.0158575.ref019" target="_blank">19</a>]. The alignment was generated using MAFFT v.7220 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158575#pone.0158575.ref035" target="_blank">35</a>] and visualised in Jalview [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158575#pone.0158575.ref036" target="_blank">36</a>].</p

Colonisation of lungs by <i>B</i>. <i>anthracis</i> STI or <i>B</i>. <i>anthracis</i> STI Δ<i>BaTdp</i> during murine model of infection.

<p>Eight groups of 6 6–8 week old A/J female mice were infected via the intra-nasal route with either <i>B</i>. <i>anthracis</i> STI or <i>B</i>. <i>anthracis</i> STI Δ<i>BaTdp</i>. At 3, 4, 5 or 6 days post-infection, lungs were removed from mice and bacterial numbers enumerated. Open symbols indicate mice that died during the experiment. The bar represents the median bacterial load for each group. WT = Wild Type <i>B</i>. <i>anthracis</i> STI, MT = Mutant <i>B</i>. <i>anthracis</i> STI Δ<i>BaTdp</i>. Data points show the bacterial burden per lung. Significance markers indicate the output of Bonferroni’s post-tests where P < 0.01. Bacterial burden data from dead mice was not included in the analysis.</p

Expression of BaTdp increases lipidation of cellular LC3.

<p>(A) HEK293T cells were transfected with plasmid encoding either BaTdp (wild type) or BaTdp (G164A) protein. After 24 or 48 hours post-transfection, cells were lysed in PBS containing 1% SDS and lysates were resolved by SDS-PAGE and analysed by Western blot using an anti-LC3B primary antibody or anti-GAPDH antibody for loading control. Result shown is representative of three independent experiments. Figures show cropped lanes from non-adjacent lanes of the same blot. Images have been cropped to only show the regions of interest. (Uncropped versions of blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158575#pone.0158575.s001" target="_blank">S1 Fig</a>) (B) Western blots were analysed by densitometry using ImageJ software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158575#pone.0158575.ref043" target="_blank">43</a>]. For each sample, the LC3B-II value was normalised against corresponding GAPDH value. Bars represent mean values of three independent experiments. <i>Error bars</i>, SD of triplicates. *P < 0.05 (by two-tailed Student’s t-test).</p

<i>In vitro</i> signalling assays indicate that BaTdp does not affect TLR signalling.

<p>(A) HEK-TLR4 cells were transfected with increasing amounts of vector DNA containing the gene encoding BaTdp (wildtype) or the BaTdp (G164A). 24 hours post-transfection, cells were challenged with LPS (0.1 μg/mL) and the production of inflammatory cytokines, TNFα and IL-8, was assessed by ELISA 24 hours later. Bars represent mean values of three independent experiments. <i>Error bars</i>, SD of triplicates. (B) HEK-TLR2 cells were transfected with plasmid encoding for BaTdp or BaTdp (G164A) and challenged with LTA (1 μg/mL) 24 hours post-transfection, followed by assessment of IL-8 content in the culture supernatant by ELISA after an additional 24 hours. Bars represent mean values of three independent experiments. <i>Error bars</i>, SD of triplicates. (C) HEK-TLR4 cells were co-transfected with plasmids encoding FLAG-tagged MyD88 and GFP-tagged BaTdp. 24 hours post transfection, after being stained with mitotracker dye to stain cellular mitochondria, cells were fixed, permeabilised and stained with an anti-FLAG antibody and visualised with a red fluorescent secondary antibody. All scale bars, 10 μm. Original magnification ×100.</p