material (Reprosil) was used. The gradient (solvent A: 0.1% v/v formic acid; solvent B: 0.1% v/v formic acid in 85% v/v acetonitrile) started at 4% B. The portion of solvent B was increased linearly from 4% to 50% during 50 min and from 50% to 100% during 5 min. A flowrate of 250 nL/min was applied. Database search was performed in Proteome Discoverer (version 1.2.0.208, ThermoScientific) using the Sequest search engine and a database consisting of 545 sequences to which the sequences of Shiga toxin 2, A and B subunit, were added. Variable modifications were oxidation and di-oxidation at Cys and Met, tri-oxidation at Cys, and chlorination at Phe, His, Trp, and Tyr. Specific cleavage sites for trypsin (KR) were selected with two missed cleavage sites allowed. Peptide tolerance was610 ppm and MS/MS tolerance was60.8 Da. The criteria for positive identi- fication of peptides were Xcorr .2.3 for doubly charged ions, and Xcorr .2.8 for triply charged ions. For MS analysis, the displayed sequence (Figure 6) and the corresponding numbers were taken from the up-to-date UniProt database (including the signal peptide) with the accession numbers Q8XBV2 for Stx2, subunit A, and Q8X531 for Stx2, subunit B. The signal peptide comprised amino acids 1–22 for subunit A and 1–19 for subunit B. Statistics Data are presented as mean values and standard deviations (SD). Student’s unpaired t test in case of two groups, or one-way ANOVA and Bonferroni’s and Dunnett’s multiple comparison test in case of more than two groups were used to test for a difference between the test and control group. P,0.05 was considered significant for all tests. Calculations were done with GraphPad Prism 5 software. References 1. Grisham MB, Jefferson MM, Melton DF, Thomas EL (1984) Chlorination of endogenous amines by isolated neutrophils. J Biol Chem 259: 10404–10413. 2. Weiss SJ, Klein R, Slivka A, Wei M (1982) Chlorination of taurine by human neutrophils. J Clin Investig 70: 598–607. 3. Weiss SJ (1989) Tissue destruction by neutrophils. N Engl J Med 320: 365–376. 4. Koprowski M, Marcinkiewicz J (2002) Taurine chloramine - its role in immunity and new perspectives for clinical use. Centr Eur J Immunol 27: 69–74. 5. Marcinkiewicz J (1997) Neutrophil chloramines: missing links between innate and acquired immunity. Immunol Today 18: 577–580. 6. Park E, Jia J, Quinn MR, Schuller-Levis G (2002) Taurine chloramine inhibits lymphocyte proliferation and decreases cytokine production in activated human leukocytes. Clin Immunol 102: 179–184. 7. Kanayama A, Inoue J, Sugita-Konishi Y, Shimizu M, Miyamoto Y (2002) Oxidation of Ikappa Balpha at methionine 45 is one cause of taurine chloramine-induced inhibition of NF-kappa B activation. J Biol Chem 277: 24049–24056. 8. Marcinkiewicz J, Walczewska M, Olszanecki R, Bobek M, Biedron R, et al. (2009) Taurine haloamines and heme oxygenase-1 cooperate in the regulation of inflammation and attenuation of oxidative stress. Adv Exp Med Biol 643: 439– 450. 9. Nagl M, Hess M, Pfaller K, Hengster P, Gottardi W (2000) Bactericidal activity of micromolar N-chlorotaurine - evidence for its antimicrobial function in the human defence system. Antimicrob Agents Chemother 44: 2507–2513. 10. Passo SA, Weiss SJ (1984) Oxidative mechanisms utilized by human neutrophils to destroy Escherichia coli. Blood 63: 1361–1368. 11. Gottardi W, Nagl M (2002) Chemical properties of N-chlorotaurine sodium, a key compound in the human defence system. Arch Pharm Pharm Med Chem 335: 411–421. 12. Gottardi W, Nagl M (2010) N-chlorotaurine, a natural antiseptic with outstanding tolerability. J Antimicrob Chemother 65: 399–409. 13. Iovino SM, Krantz KD, Blanco DM, Fernandez JA, Ocampo NF, et al. (2011) NVC-422 topical gel for the treatment of impetigo. Int J Clin Exp Pathol 4: 587– 595. 14. Wang L, Khosrovi B, Najafi R (2008) N-Chloro-2, 2-dimethyltaurines: a new class of remarkably stable N-chlorotaurines. Tetrahedron Lett 49: 2193–2195. 15. Wang L, Belisle B, Bassiri M, Xu P, Debabov D, et al. (2011) Chemical Characterization and Biological Properties of NVC-422, a Novel, Stable N- Chlorotaurine Analog. Antimicrob Agents Chemother 55: 2688–2692. N-Chlorotaurine Inacitvates Shiga Toxin Supporting Information Figure S1 Inhibition of growth of EHEC 22 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. (TIF) Acknowledgments We are grateful to Prof. Helge Karch, Institute for Hygiene and National Consulting Laboratory on Hemolytic Uremic Syndrome, University of Mu¨ nster, Germany, for providing purified Stx2. We thank Martin Hermann for his help in performance of confocal microscopy. We are grateful to Andrea Windisch for technical assistance. Author Contributions Conceived and designed the experiments: CE SE HL WG DD MA MN DO. Performed the experiments: CE SE LK. Analyzed the data: CE HL LK DD MN DO. Contributed reagents/materials/analysis tools: HL MN DO. Wrote the paper: CE SE MN DO. Critically revised the data: CE SE HL LK WG DD MA MN DO. Critically revised the manuscript: CE SE HL LK WG DD MA MN DO. Final approval of the manuscript: CE SE HL LK WG DD MA MN DO. 16. Gottardi W, Nagl M (2005) Chlorine covers on living bacteria: the initial step in antimicrobial action of active chlorine compounds. J Antimicrob Chemother 55: 475–482. 17. Nagl M, Hengster P, Semenitz E, Gottardi W (1999) The postantibiotic effect of N-chlorotaurine on Staphylococcus aureus. Application in the mouse peritonitis model. J Antimicrob Chemother 43: 805–809. 18. Nagl M, Gruber A, Fuchs A, Lell C, Lemberger EM, et al. (2002) Impact of N- chlorotaurine on viability and production of secreted aspartyl proteinases of Candida spp. Antimicrob Agents Chemother 46: 1996–1999. 19. Reeves EP, Nagl M, O’Keeffe J, Kelly J, Kavanagh K (2006) Effect of N- chlorotaurine on Aspergillus, with particular reference to destruction of secreted gliotoxin. J Med Microbiol 55: 913–918. 20. Orth D, W u¨rzner R (2010) Complement in typical hemolytic uremic syndrome. Semin Thromb Hemost 36: 620–624. 21. Fraser ME, Fujinaga M, Cherney MM, Melton-Celsa AR, Twiddy EM, et al. (2004) Structure of shiga toxin type 2 (Stx2) from Escherichia coli O157:H7. J Biol Chem 279: 27511–27517. 22. Orth D, Khan AB, Naim A, Grif K, Brockmeyer J, et al. (2009) Shiga toxin activates complement and binds factor H: evidence for an active role of complement in hemolytic uremic syndrome. J Immunol 182: 6394–6400. 23. Sauter KA, Melton-Celsa AR, Larkin K, Troxell ML, O’Brien AD, et al. (2008) Mouse model of hemolytic-uremic syndrome caused by endotoxin-free Shiga toxin 2 (Stx2) and protection from lethal outcome by anti-Stx2 antibody. Infect Immun 76: 4469–4478. 24. Inward CD, Howie AJ, Fitzpatrick MM, Rafaat F, Milford DV, et al. (1997) Renal histopathology in fatal cases of diarrhoea-associated haemolytic uraemic syndrome. British Association for Paediatric Nephrology. Pediatr Nephrol 11: 556–559. 25. Exeni RA, Fernandez GC, Palermo MS (2007) Role of polymorphonuclear leukocytes in the pathophysiology of typical hemolytic uremic syndrome. ScientificWorldJournal 7: 1155–1164. 26. Taylor HD, Austin JH (1918) The action of antiseptics on the toxin of Bacillus welchii: a preliminary note. J Exp Med 27: 375–381. 27. Cole KD, Gaigalas A, Almeida JL (2008) Process monitoring the inactivation of ricin and model proteins by disinfectants using fluorescence and biological activity. Biotechnol Prog 24: 784–791. 28. Yang CY (1972) Comparative studies on the detoxification of aflatoxins by sodium hypochlorite and commercial bleaches. Appl Microbiol 24: 885–890. PLOS ONE | www.plosone.org 12 November 2012 | Volume 7 | Issue 11 | e47105