Reactivity of monoclonal antiserum EAII-6G6-2-3, specific for <i>Ba</i> neutral cell wall polysaccharide [19], with HF-SCWPs from <i>Bc</i> group strains.

<p>Meso Scale Discovery (MSD) multi-array high bind 96 well plates were coated with a fixed concentration of HF-SCWP antigen (2 μg/ml) from different <i>Bacillus</i> species and probed with the monoclonal antibody in serial two fold dilutions. Bound antibody was detected by using 2.5 μg/ml of sulfo-tagged goat anti-mouse IgM detection antibody. Data points are the average of three independent experiments. Error bars represent one standard error. Reactivity reported as effective concentration (EC₅₀) titer in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183115#pone.0183115.t004" target="_blank">Table 4</a></b>.</p

<i>Bacillus anthracis</i> and <i>B</i>. <i>cereus</i> strains used in this study.

<p><i>Bacillus anthracis</i> and <i>B</i>. <i>cereus</i> strains used in this study.</p

Percent and type of structural substituent at the 3-position of backbone ManNAc residues in HF-SCWPs from examined infection-associated <i>B</i>. <i>cereus</i> strains.

<p>Percent and type of structural substituent at the 3-position of backbone ManNAc residues in HF-SCWPs from examined infection-associated <i>B</i>. <i>cereus</i> strains.</p

600-MHz ¹H and ¹³C NMR parameters observed for the HF-released secondary cell wall polysaccharide released by HF treatment of great ape isolate <i>B</i>. <i>cereus</i> strain CA cell walls^a.

<p>600-MHz ¹H and ¹³C NMR parameters observed for the HF-released secondary cell wall polysaccharide released by HF treatment of great ape isolate <i>B</i>. <i>cereus</i> strain CA cell walls<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183115#t002fn003" target="_blank">^a</a>.</p

Expanded region of proton NMR spectra identifying the locations and area ratios of the Gal substituents on HF-SCWPs from <i>B</i>. <i>cereus</i> strains.

<p><b>(A)</b> human isolate <i>B</i>. <i>cereus</i> G9241; <b>(B)</b> great ape isolate <i>B</i>. <i>cereus</i> CA (Cameroon); <b>(C)</b> great ape isolate <i>Bc</i> CI (Côte d’Ivoire). The α-anomeric signal originates from the free reducing end of all these polysaccharides, and co-migrates with a new residue (<b>K</b>) in the <i>Bc</i> CA and <i>Bc</i> CI SCWPs. This reducing end (α-GlcNAc residue) was removed by borodeuteride reduction for subsequent experiments, to facilitate integration and characterization of the residue <b>K</b> system. The percentage of Gal (<b>G</b>) and Gal-Gal disaccharide (<b>J</b>+<b>K</b>) substitution at ManNAc residues (<b>B</b>+<b>B′</b>+<b>B″</b>) is estimated by examination of the signal areas for these residues. The residue <b>G</b> spin system undergoes a shift at several positions, and is designated residue <b>J</b>, when it is substituted by residue <b>K</b> (refer to <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183115#pone.0183115.t002" target="_blank">Table 2</a></b>).</p

Immunochemical analysis of HF-SCWP with monoclonal and polyclonal antisera.

<p>Immunochemical analysis of HF-SCWP with monoclonal and polyclonal antisera.</p

Proton NMR spectra comparing the <i>Bc</i> Cameroon HF-SCWP before and after reduction of the reducing end with borodeuteride.

<p>The effect of borodeuteride reduction on the signal at (<b>K</b> + α) is demonstrated: <b>(A)</b> native <i>Bc</i> CA HF-SCWP before reduction; <b>(B)</b> the <i>Bc</i> CA HF-SCWP after reduction of the reducing end (red-HF-SCWP). The contribution from the α-reducing end anomeric signal to residue <b>K</b> signal area was almost completely eliminated by 1 h treatment, allowing unambiguous assignment of residue <b>K</b> system and area measurements. Following reduction, the <b>K</b>/<b>J</b> ratio appears to approach 1:1, reflecting the presence of the <b>K</b>α(1→3)<b>J</b>α(1→ disaccharide. Considerable overlap of the <b>K</b> and <b>J</b> anomeric signals appears to result in some inaccuracy in the integration, as the HSQC spectra demonstrate that both α- and β- reducing end anomeric protons were absent following borodeuteride reduction (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183115#pone.0183115.s004" target="_blank">S4 Fig</a></b>).</p

Virulence of the <i>Bacillus cereus</i> bv <i>anthracis</i> and CA derivative strains by the subcutaneous route in guinea pigs.

<p>Guinea pigs were inoculated with graded spore inocula of each strain by subcutaneous route in the flank (four animals per dose). The presence of pBCXO1 and PBCXO2 and the gene inactivated on pBCXO1 is specified where applicable. Results are expressed as mean lethal dose (LD50) and mean time to death in days (MTD, mean ± SD). Each experiment was performed at least twice.</p><p>Virulence of the <i>Bacillus cereus</i> bv <i>anthracis</i> and CA derivative strains by the subcutaneous route in guinea pigs.</p

Oligonucleotides for expression analysis used in this study.

<p>Oligonucleotides for expression analysis used in this study.</p

Local role of the HA capsule and <i>in vivo</i> dissemination of the <i>B</i>. <i>cereus</i> bv <i>anthracis</i> CA strain during intranasal infection in mice.

<p>Mice were inoculated intranasally with spores of <b>(A)</b> CARP-<i>lux</i> (13 mice, inoculum 1 x 10⁸), <b>(B)</b> CAR-<i>lux</i> (16 mice, inoculum 1 x 10⁶; all mice died), or <b>(C)</b> CAP-<i>lux</i> (14 mice, inoculum 1 x 10⁶; all mice died) strains and bioluminescence was analysed at the indicated times after infection as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003455#pntd.0003455.g005" target="_blank">Fig. 5</a> (D: days). (<b>Ab-e)</b> Histological characterisation of the infected brain tissue shown in <b>Aa</b>, bottom panel; <b>(Ab)</b> Diffuse inflammatory lesion centred on leptomeninges (LM), multifocally extending to the brain parenchyma (star); <b>(Ac)</b> high density of bacteria in the leptomeninges and Virchow-Robin spaces; <b>(Ad)</b> inflammatory infiltrates consisting of neutrophils, haemorrhages and oedema provoking a marked distension of leptomeninges and <b>(Ae),</b> at higher magnification, extending to the cerebral parenchyma with the presence of bacteria in the neuropil (arrowhead) highly suggestive of a rupture of the blood-brain barrier. <b>(Ab & d)</b>: HE staining; <b>(Ac & e)</b>: Gram staining.</p