Effect of pyrophosphate (PPi), methylenediphosphonic acid (PcP) and imidodiphosphate (PnP) on <i>Nm</i> growth on iron-depleted medium.

<p>The experiment was repeated three times. Representative results are presented. <b>+++</b>: large colonies (1 to 1.5 mm diameter); −: no growth.</p><p>Effect of pyrophosphate (PPi), methylenediphosphonic acid (PcP) and imidodiphosphate (PnP) on <i>Nm</i> growth on iron-depleted medium.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Assay of the ability of desferal, pyrophosphate (PPi), methylenediphosphonic acid (PcP) and imidodiphosphate (PnP) to bind iron.

<p>150 nanomoles of desferal (♦), PPi (○), PcP (□), or PnP (X) were added to a 1 ml mix (1/4 V/3/4 V) of distilled water and CAS assay solution <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107612#pone.0107612-Schwyn1" target="_blank">[32]</a> at room temperature. Every 10 min for 60 min absorbance was measured at 630 nm. (▪): No agent added. The experiment was repeated three times. A representative result is presented.</p

Use of iron pyrophosphate (FePPi), FeNo3 and FeCl3 as iron sources by <i>Nm.</i>

<p>Experiments were repeated three times. Representative results are presented. <b>+++</b>: large colonies (1 to 1.5 mm diameter); <b>+</b>: small colonies (<0.5 mm diameter); −: no growth.</p><p>Use of iron pyrophosphate (FePPi), FeNo3 and FeCl3 as iron sources by <i>Nm.</i></p

<i>Nm</i> growth in the mouse model in the presence of human transferrin (Htf) or methylenediphosphonic acid (PcP).

<p>The tested strains were isolated on a GCB plate supplemented with S1 and S2 complements and grown for 18 h at 37°C in the presence of 5% CO₂. Bacteria were suspended in sterile physiological serum to obtain a cell density of 2.5×10⁶ bacteria/ml. When specified, 100 µl of the tested iron source were added to 400 µl of the bacterial suspension to obtain 0.05 mM for human transferrin and 5 mM for PcP. For the control experiment, 100 µl of physiological serum were added. For each experiment, the mixtures were injected intraperitoneally into five mice and bioluminescence was measured 30 min, and 360 min after injection, as described in Materials and methods. At t = 360 min, blood and peritoneal washes samples were taken, diluted in physiological serum and plated on GCB solid medium. After 18 h incubation at 37°C in the presence of 5% CO₂, the colonies were counted. Data represent the means ± SD from 3 independent experiments of groups of five mice per time point in each experiment. Student’s <i>t</i>-test results were included in the figure and in the table. CFU: colony-forming unit.</p

Primers used in this study.

<p>Primers used in this study.</p

TonB-independent use of transferrin as an iron source.

<p>The tested strains were isolated on a GCB plate supplemented with S1 and S2 complements and grown for 18 h at 37°C in the presence of 5% CO₂. GCB plates depleted for iron by addition of desferal were supplemented with human or bovine transferrin added at a 5 µM final concentration. When specified, PPi, PcP and PnP were added at a 1 mM final concentration. Bacteria were isolated on test plates and incubated for 18 h at 37°C in the presence of 5% CO₂. The experiment was repeated three times. Representative results are presented.</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

The <i>B</i>. <i>cereus</i> bv <i>anthracis</i> CA strain expresses a PDGA and a HA capsule, and toxins.

<p><b>(A)</b> Capsule expression in the CAP(Δ<i>pagA</i>), the CAR and the CAR-H(Δ<i>hasA</i>) strains in inducing conditions; the polyglutamate (PDGA) and hyaluronic acid (HA) capsule was visualised by immunofluorescence with a polyclonal anti-PDGA immune serum or by India ink staining; degradation of the HA capsule was achieved by incubation with hyaluronidase as described in the Materials and Methods section. (<b>B)</b> The production of toxin components PA and LF in overnight bacterial culture supernatants was determined by western blot with or without CO₂/bicarbonate as described in the Materials and Methods section.</p