Formation of small transmembrane pores: An intermediate stage on the way to Bacillus cereus non-hemolytic enterotoxin (Nhe) full pores in the absence of NheA

• Published in: Biochemical and biophysical research communications 2016-01, Vol.469 (3), p.613-618
• Main Authors: Zhu, Kui, Didier, Andrea, Dietrich, Richard, Heilkenbrinker, Uta, Waltenberger, Eva, Jessberger, Nadja, Märtlbauer, Erwin, Benz, Roland
• Format: Article
• Language: English
• Subjects: Animals; Bacillus cereus; Bacillus cereus - physiology; Bacterial Proteins - metabolism; Bacterial Toxins - metabolism; Cell Membrane Permeability - physiology; Cercopithecus aethiops; Cytotoxicity; Enterotoxins - metabolism; Lipid-bilayer membrane; Membrane Fluidity - physiology; Non-hemolytic enterotoxin; Pore-forming toxins; Vero Cells
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2015.11.126

The non-hemolytic enterotoxin (Nhe) of Bacillus cereus is a three-partite toxin formed of the components NheA, -B and –C. Pore formation and subsequent lysis of target cells caused by Nhe is an orchestrated process comprising three steps: (i) formation of NheB/C oligomers in solution, (ii) attachment of the oligomers to the cell membrane, (iii) binding of NheA to the oligomers. The present study aimed to characterize the properties of the NheB/C complex and the fate of the target cell upon binding. An enzyme immunoassay allowing kinetic measurements and surface plasmon resonance revealed the fast and high affinity formation of the NheB/C oligomers. The benefit of these complexes is a more stable cell binding as well as stronger and earlier cytotoxic effect. High molecular mass hetero-oligomers (620 kDa) probably consisting of one NheC and up to 15 NheB were detected by size-exclusion chromatography and on native PAGE immunoblots. Due to the NheBC application the morphology and membrane permeability of Vero cells is partly disturbed. Formation of stable transmembrane channels with a conductance of about 870 pS and a diameter of about 2 nm due to the application of NheBC could be demonstrated in lipid bilayer experiments. Thus, the NheBC complex itself has a tendency to increase the membrane permeability prior to the emergence of full pores containing also NheA. •Bacillus cereus NheBC complexes show high affinity binding (approx. KD 4.8 × 10−10 M).•NheBC complexe size is approx. 620 kDa.•The complex triggers stronger binding and earlier pore formation on Vero cells.•Lipid bilayer channels show a conductivity of approx. 870 pS and a diameter of 2 nm.