N-Chlorotaurine Inacitvates Shiga Toxin Figure 1. Inhibition of growth of EHEC 178 and of Stx2 production in the presence of sublethal concentrations of NCT and NVC- 422. (A) CFU counts of EHEC in Direct Medium, to which NCT was added to final concentrations of 0 mM (control), 1.65 mM, 2.2 mM and 2.75 mM at 37uC. (B) Stx2 produced by EHEC under the same conditions as in (A), measured by ELISA, related to the 6 h value of the control without NCT. (C) CFU counts of EHEC in Direct Medium, to which NVC-422 was added to final concentrations of 0 mM (control), 0.55 mM, 1.1 mM and 1.65 mM at 37uC. (D) Stx2 produced by EHEC under the same conditions as in (C), measured by ELISA, related to the 6 h value of the control without NVC-422. Mean values 6 SD from three independent experiments are shown in (A–D). *P,0.05; **P,0.01. doi:10.1371/journal.pone.0047105.g001 reducing buffer (designated a–f in Figure 5), and six in reducing buffer, which indicate fundamental molecular changes of the toxin. Bands a, b, c, d, and e from the sample dissolved in non- reducing buffer were cut out for mass spectrometry analysis to detect also changes that might have been reversed by mercaptoethanol of the reducing buffer. For improved separa- tion of the low molecular weight range, a tricine gel with non- reducing buffer was performed with even higher Stx concen- tration (25 mg) for the test lane (Figure 5B). Indeed, a clear shift and separation of subunit B could be observed. Bands a and b were evaluated by mass spectrometry. Structural Changes of Stx2 by NCT Evaluated by Mass Spectrometry We were able to show the molecular sites of oxidative attack of subunits A and B of Stx2. Mass spectrometry detected mono- and di-oxidation of methionine. This leads to the formation of sulfoxides (mono-oxidation) and sulfones (di-oxidation). Cysteine showed not only mono- and di-oxidation but also tri-oxidation. In PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e47105