Concerted Action of Sphingomyelinase and Non-Hemolytic Enterotoxin in Pathogenic <i>Bacillus cereus</i>

<div><p><i>Bacillus cereus</i> causes food poisoning and serious non-gastrointestinal-tract infections. Non-hemolytic enterotoxin (Nhe), which is present in most <i>B. cereus</i> strains, is considered to be one of the main virulence factors. However, a <i>B. cereus</i> Δ<i>nheBC</i> mutant strain lacking Nhe is still cytotoxic to intestinal epithelial cells. In a screen for additional cytotoxic factors using an <i>in vitro</i> model for polarized colon epithelial cells we identified <i>B. cereus</i> sphingomyelinase (SMase) as a strong inducer of epithelial cell death. Using single and double deletion mutants of <i>sph</i>, the gene encoding for SMase, and <i>nheBC</i> in <i>B. cereus</i> we demonstrated that SMase is an important factor for <i>B. cereus</i> cytotoxicity <i>in vitro</i> and pathogenicity <i>in vivo.</i> SMase substantially complemented Nhe induced cytotoxicity <i>in vitro</i>. In addition, SMase but not Nhe contributed significantly to the mortality rate of larvae <i>in vivo</i> in the insect model <i>Galleria mellonella</i>. Our study suggests that the role of <i>B. cereus</i> SMase as a secreted virulence factor for <i>in vivo</i> pathogenesis has been underestimated and that Nhe and SMase complement each other significantly to cause full <i>B. cereus</i> virulence hence disease formation.</p></div

Identification of cytotoxic protein from <i>B. cereus</i> supernatant.

<p><b>A.</b> Bacterial supernatant of <i>B. cereus</i> NVH 0075-95 Δ<i>nheBC</i> was separated on a Superdex-75 10/300 GL gel filtration column. Chromatogram of fractionated bacterial proteins is shown (fraction1–24). Protein fractions were tested on Ptk6 cells for cytotoxicity as described in Experimental procedures. Protein fractions obtained from gel filtration were analyzed by SDS-PAGE. <b>B.</b> Gel filtration fractions 12–14 transferred cytotoxicity to Ptk6 cells and contained two distinct proteins migrating at 34 kDa and 25 kDa (red asterisks). <b>C.</b> Comparing total extracellular proteins of WT and mutant <i>B. cereus</i> strains, two potential cytotoxic proteins (red asterisks) were absent in the supernatant of avirulent Δ<i>plcR</i> strain.</p

Cytotoxic effects of various <i>B. cereus</i> strains on intestinal epithelial cells (IEC).

<p>Ptk6 cells were treated with 14 different <i>B. cereus</i> strains and two isogenic mutants, morphological changes were monitored over time using light microscopy. <b>A.</b> At an MOI of 1 all strains tested caused epithelial cell rounding (yellow) and detachment (red) within 2–4 h after infection except for the <i>plcR</i> deletion mutant. Intact monolayer (green). <b>B.</b> Representative images of Ptk6 cell monolayers. Bar, 20 µm.</p

<i>Sph</i> deletion effected <i>B. cereus</i> virulence <i>in vitro</i>.

<p><b>A</b>. Cytotoxic effect of sterile <i>B. cereus</i> supernatants (1∶4 diluted) on IEC. Intact monolayer (green), cell rounding ≤50% (yellow), cell rounding >50% (orange) and 95–100% cell detachment (red) are indicated. <b>B</b>. Cytotoxic effects of <i>B. cereus</i> supernatant on IEC were analyzed using flow cytometry. Ptk6 cells were treated with various dilutions of bacterial supernatant of <i>B. cereus</i> NVH 0075-95 WT (black line), Δ<i>nheBC</i> mutant (grey line), Δ<i>sph</i> mutant (black dashed line) and Δ<i>nheBC</i>Δ<i>sph</i> mutant (grey dashed line). Samples were stained with Propidium iodide (PI) for dead epithelial cells and cytotoxicity is expressed in % of PI positive cells as determined by flow cytometric analysis. Cytotoxicity of the Δ<i>sph</i> mutant was strongly reduced at a dilution of 1∶8 compared to WT (a, <i>P</i><0.05). <i>Sph</i> deletion in addition to Nhe inactivation significantly reduced cytotoxicity compared to Nhe inactivation alone (b, <i>P</i><0.05). Data plotted represent mean values ± SEM (n = 3). <b>C</b>. Cooperative cytotoxic interaction of SMase and Nhe. Addition of various concentrations of recombinant SMase (0.05, 0.1 and 0.2 U/ml) to diluted (1∶16) bacterial supernatants caused significantly higher cytotoxicity against Ptk6 cells when subtoxic Nhe concentrations were present (Δ<i>sph</i> supernatant, black line) compared to supernatant without Nhe (Δ<i>nheBC</i>Δ<i>sph</i> supernatant, grey line) (*<i>P</i><0.05). Addition of anti-NheB (1E11) antibody (10 µg/well) neutralizes Nhe activity (Δ<i>sph</i> supernatant+α-NheB, black dotted line). Cytotoxicity is expressed in % of PI positive cells and data represent mean values ± SEM (n ≥3). <b>D</b>. CAMP-like test on sheep blood agar demonstrated complementation of extracellular hemolytic activity between <i>B. cereus</i> NVH 0075-95 Δ<i>sph and</i> Δ<i>nheBC</i>. Beta-hemolytic activity appeared as cleared zone around the colonies.</p

<i>Sph</i> deletion strongly reduced the pathogenicity of <i>B. cereus</i> NVH 0075-95 in a <i>Galleria mellonella in vivo</i> model.

<p><b>A.</b> Larvae were infected by intrahemocoelic injection of 10⁵ CFU of vegetative <i>B. cereus</i> NVH 0075-95 (WT), the isogenic <i>nheBC</i> mutant (Δ<i>nheBC</i>), the isogenic <i>sph</i> null mutant (Δ<i>sph</i>), the <i>nheBC sph</i> mutant strain (Δ<i>nheBC</i>Δ<i>sph</i>) or its complemented strain (Δ<i>nheBC</i>Δ<i>sph</i> comP<i>plc</i>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061404#s2" target="_blank">Materials and Methods</a>. Larvae infected with the non-insecticidal <i>E. coli</i> strain DH10B served as control group. <i>G. mellonella</i> survival data are plotted as Kaplan-Meier plots. Data are retrieved from two independent infection experiments with a total of 60 larvae per condition. Both experiments showed very similar results. Statistical significance was determined using log-rank analysis. An asterisk indicates treatment groups with a survival distribution function statistically different from <i>B. cereus</i> WT (<i>P</i><0.001). <b>B.</b> Survival and multiplication of <i>B. cereus</i> WT and isogenic mutant strains in <i>G. mellonella</i> after intrahemocoelic injection of 10⁵ vegetative cells. Bacterial growth in two independent infection assays was monitored at indicated time points after infection (t<i> = </i>0) by counting individual homogenates of five larvae per condition. CFUs recovered from dead larvae are indicated as encircled data points. Paired Student’s <i>t</i>-test was used to determine statistical differences between bacterial cell counts of the five treatment groups. In living or dead <i>Galleria</i> larvae cell counts of the <i>sph</i> mutant strains did not differ significantly from WT and Δ<i>nheBC</i> mutant.</p

FTIR spectra of <i>L. monocytogenes</i> re-isolated from challenged mice.

<p>(A) Average FTIR spectra derived from 129/Sv (dotted), Tyk2−/− (dashed) and C57BL/6 (solid) isolates are shown from the whole spectral range (B) Subtraction spectra were generated from second derivative, vector-normalized, average FTIR spectra of the three different mouse genotypes. Spectra from C57BL/6 mice were subtracted from 129/Sv isolates (black), spectra from C57BL/6 mice were subtracted from Tyk2−/− (red) and spectra from Tyk2−/− were subtracted from 129/Sv isolates (blue). Most pronounced differences were observed in the spectral range of 1700–1500 cm⁻¹ (protein region) and can be assigned to amide I (1670–1625 cm⁻¹) and amide II (1560–1530 cm⁻¹).</p

Cereulide synthetase gene cluster from emetic : Structure and location on a mega virulence plasmid related to toxin plasmid pXO1-3

<p><b>Copyright information:</b></p><p>Taken from "Cereulide synthetase gene cluster from emetic : Structure and location on a mega virulence plasmid related to toxin plasmid pXO1"</p><p>BMC Microbiology 2006;6():20-20.</p><p>Published online 2 Mar 2006</p><p>PMCID:PMC1459170.</p><p>Copyright © 2006 Ehling-Schulz et al; licensee BioMed Central Ltd.</p>(for details on probes see Supplemental Materials Table S1). Hybridization with both probes revealed a single band for emetic strains which has the same size as the pBc10987 plasmid from ATCC 10987 (lane 2)

ANOVA-pairwise comparison to assess significant differentiation among the three genotypes (<i>p</i>-values).

<p>ANOVA-pairwise comparison to assess significant differentiation among the three genotypes (<i>p</i>-values).</p

Cereulide synthetase gene cluster from emetic : Structure and location on a mega virulence plasmid related to toxin plasmid pXO1-2

<p><b>Copyright information:</b></p><p>Taken from "Cereulide synthetase gene cluster from emetic : Structure and location on a mega virulence plasmid related to toxin plasmid pXO1"</p><p>BMC Microbiology 2006;6():20-20.</p><p>Published online 2 Mar 2006</p><p>PMCID:PMC1459170.</p><p>Copyright © 2006 Ehling-Schulz et al; licensee BioMed Central Ltd.</p>ted. Residues identical to amino acids from Ces core motifs are printed in boldface type. from CesA2 (D-Leu) and CesB1 (L--Val) were aligned to short chain dehydrogenases (SDR) and ketoreductases (KR); partial sequences including putative NADPH binding sites (solid bar) and the catalytic residues of SDRs/KR (printed in boldface type). Numbers between hyphens in the first lines refer to residues not shown before residues depicted in the second lines. JamL: KR from Jamaicamides synthetase (GenBank accession no. ) of ; EryA: KR from erythronolide synthetase of (GenBank accession no. ). GlcDh: Glucose 1-dehydrogenase from (Swissprot accession no. P39482)

Cereulide synthetase gene cluster from emetic : Structure and location on a mega virulence plasmid related to toxin plasmid pXO1-0

<p><b>Copyright information:</b></p><p>Taken from "Cereulide synthetase gene cluster from emetic : Structure and location on a mega virulence plasmid related to toxin plasmid pXO1"</p><p>BMC Microbiology 2006;6():20-20.</p><p>Published online 2 Mar 2006</p><p>PMCID:PMC1459170.</p><p>Copyright © 2006 Ehling-Schulz et al; licensee BioMed Central Ltd.</p>regions showing homologies to toxin plasmids from group members are printed as hatched boxes. For details on CDS designation see Table 2. The bars refer to probes used to test the conservation of genes in the group (see Table 3) Inset: Structure of cereulide according to [13]