Figure 1.
Cytotoxic effects of various B. cereus strains on intestinal epithelial cells (IEC).
Ptk6 cells were treated with 14 different B. cereus strains and two isogenic mutants, morphological changes were monitored over time using light microscopy. A. 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 plcR deletion mutant. Intact monolayer (green). B. Representative images of Ptk6 cell monolayers. Bar, 20 µm.
Figure 2.
Identification of cytotoxic protein from B. cereus supernatant.
A. Bacterial supernatant of B. cereus NVH 0075-95 ΔnheBC 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. Gel filtration fractions 12–14 transferred cytotoxicity to Ptk6 cells and contained two distinct proteins migrating at 34 kDa and 25 kDa (red asterisks). C. Comparing total extracellular proteins of WT and mutant B. cereus strains, two potential cytotoxic proteins (red asterisks) were absent in the supernatant of avirulent ΔplcR strain.
Figure 3.
Growth behavior and colony phenotypes of parental and mutant B. cereus strains.
B. cereus NVH 0075-95 (WT, •), isogenic nheBC (ΔnheBC, ×), sph (Δsph, ♦) and nheBC sph (ΔnheBCΔsph, <$>\raster="rg1"<$>) null mutants. Strains were cultivated in LB at 37°C and growth was monitored by measuring optical density at 600 nm (OD600) (A). Error bars represent standard errors derived from n = 3 independent experiments. Colony morphology on (B) Columbia Blood agar (5% sheep blood, Oxoid) and (C) MYP agar (mannitol egg yolk polymyxin agar, Oxoid) was used for specific detection of beta-hemolytic (visible as cleared zones around colonies) and PC-PLC enzyme activity (lecithin precipitation zone), respectively.
Figure 4.
Genomic organization of the chromosomal plc – sph gene cluster.
The operon comprises phosphatidylcholine-specific phospholipase C (plc) and sphingomyelinase (sph). Pplc-sph refers to the putative promoter region of the plc-sph operon. Three black bars in the promoter region represent the identified −35, −10 boxes and the ribosome-binding site. Pplc (white thick arrow) designates a 538-bp fragment spanning the operon promoter region Pplc-sph used for successful complementation, while the inter-genic region is covered by Psph (499bp; white thick arrow). The position of inplc_for and insph_rev primer binding for co-transcription analysis are indicated by two small black arrows. *putative promoter region directly upstream of sph start codon (Psph).
Figure 5.
Effect of sph deletion and complementation in trans on synthesis of SMase enzyme by B. cereus NHV 0075-95 and deduced mutant strains.
SMase activity was normalized to the protein concentration of prepared supernatant samples. Sph deletion completely abolished SMase activity, which could be restored by in trans expression of sph driven from the operon promoter region Pplc at different growth phases; OD600 = 4, black bar and OD600 = 7, grey bar. Data represent mean values ± SEM (n ≥3).
Figure 6.
Sph deletion effected B. cereus virulence in vitro.
A. Cytotoxic effect of sterile B. cereus 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. Cytotoxic effects of B. cereus supernatant on IEC were analyzed using flow cytometry. Ptk6 cells were treated with various dilutions of bacterial supernatant of B. cereus NVH 0075-95 WT (black line), ΔnheBC mutant (grey line), Δsph mutant (black dashed line) and ΔnheBCΔsph 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 Δsph mutant was strongly reduced at a dilution of 1∶8 compared to WT (a, P<0.05). Sph deletion in addition to Nhe inactivation significantly reduced cytotoxicity compared to Nhe inactivation alone (b, P<0.05). Data plotted represent mean values ± SEM (n = 3). C. 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 (Δsph supernatant, black line) compared to supernatant without Nhe (ΔnheBCΔsph supernatant, grey line) (*P<0.05). Addition of anti-NheB (1E11) antibody (10 µg/well) neutralizes Nhe activity (Δsph supernatant+α-NheB, black dotted line). Cytotoxicity is expressed in % of PI positive cells and data represent mean values ± SEM (n ≥3). D. CAMP-like test on sheep blood agar demonstrated complementation of extracellular hemolytic activity between B. cereus NVH 0075-95 Δsph and ΔnheBC. Beta-hemolytic activity appeared as cleared zone around the colonies.
Figure 7.
Sph deletion strongly reduced the pathogenicity of B. cereus NVH 0075-95 in a Galleria mellonella in vivo model.
A. Larvae were infected by intrahemocoelic injection of 105 CFU of vegetative B. cereus NVH 0075-95 (WT), the isogenic nheBC mutant (ΔnheBC), the isogenic sph null mutant (Δsph), the nheBC sph mutant strain (ΔnheBCΔsph) or its complemented strain (ΔnheBCΔsph comPplc) as described in Materials and Methods. Larvae infected with the non-insecticidal E. coli strain DH10B served as control group. G. mellonella 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 B. cereus WT (P<0.001). B. Survival and multiplication of B. cereus WT and isogenic mutant strains in G. mellonella after intrahemocoelic injection of 105 vegetative cells. Bacterial growth in two independent infection assays was monitored at indicated time points after infection (t = 0) by counting individual homogenates of five larvae per condition. CFUs recovered from dead larvae are indicated as encircled data points. Paired Student’s t-test was used to determine statistical differences between bacterial cell counts of the five treatment groups. In living or dead Galleria larvae cell counts of the sph mutant strains did not differ significantly from WT and ΔnheBC mutant.