, 2010). Figure 1a shows that the addition of 40 μL (1 : 50) of the supernatant of MHI 1672 did not induce propidium fluorescence unless supplemented with recombinant NheC. This increase in fluorescence could be prevented including the monoclonal antibody Mab 1E11 (against NheB) in the bathing solution, excluding the

possibility that NheC alone can induce propidium uptake in Vero cells (Fig. 1a). To examine the effect of DDM on propidium uptake induced by the intact Nhe complex, we pre-incubated B. cereus NVH 75/95 supernatant with DDM at its critical micelle concentration (CMC = 0.2 mM) AC220 nmr prior to mixing with the cell suspensions at 1 : 80 dilution. This abolished the propidium uptake in Vero cells (Fig. 1b) but not when NVH 75/95 supernatant was incubated with water (solvent for DDM) or with DDM at 0.05 mM, i.e. less than the CMC (Fig. 1b). The same results were observed using human intestinal HT29 epithelial cells (data not shown). Similar inhibition of propidium uptake was obtained when NVH 75/95 supernatant was pre-incubated with the CMC of beta-octyl glucoside (20 mM, data not shown). These

data are consistent with one or more of the three Nhe components interacting with the micelles, thereby preventing pore formation when subsequently exposed to the Vero and HT29 cells. Using the NheC-deficient B. cereus MHI 1672 culture supernatant, we examined the effect of DDM on NheC. Pre-incubation of purified NheC with 0.2 mM DDM did not inhibit its ability to restore propidium uptake in Vero cells when added to the culture supernatant of B. cereus MHI 1672 (Fig. 1c). Similar results were obtained in HT-29 Selleckchem Lapatinib cells (data not shown). ANS has been widely used to monitor the changes in protein conformation via an increase in fluorescence

upon binding to exposed hydrophobic regions of proteins (Slavík, 1982). Figure 2a shows the increase in ANS fluorescence intensity and blue shift of the wavelength maximum following pre-incubation of NheB with DDM micelles compared with pre-incubation find more with water. DDM (0.2 mM) did not induce any changes in the ANS fluorescence with NheA (Fig. 2b). NheC exhibited a blue shift in the wavelength maximum but no increase in fluorescence intensity (Fig. 2c). Thus, of the three Nhe proteins, DDM induced the conformational changes in NheB as observed with other pore forming toxins (Sangha et al., 1999). To indicate the location of the conformational changes in NheB induced by DDM, we sought changes in the intrinsic tryptophan fluorescence. NheB contains three tryptophan residues, all located in the alpha helical bundles of the protein. No significant changes in the emission maximum of NheB fluorescence were observed with (333–334 nm) and without treatment with DDM (334 nm; Supporting Information, Fig. S1). The emission wavelength maximum shifted to 354 nm after denaturation with 8 M urea.