N-Chlorotaurine, a Long-Lived Oxidant Produced by Human Leukocytes, Inactivates Shiga Toxin of Enterohemorrhagic Escherichia coli Christian Eitzinger1, Silvia Ehrlenbach1, Herbert Lindner2, Leopold Kremser2, Waldemar Gottardi1, Dmitri Debabov3, Mark Anderson 3 , Markus Nagl1*., Dorothea Orth1. 1 Department of Hygiene, Microbiology and Social Medicine, Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria, 2 Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria, 3 NovaBay Pharmaceuticals, Inc., Emeryville, California, United States of America Abstract N-chlorotaurine (NCT), the main representative of long-lived oxidants produced by granulocytes and monocytes, is known to exert broad-spectrum microbicidal activity. Here we show that NCT directly inactivates Shiga toxin 2 (Stx2), used as a model toxin secreted by enterohemorrhagic Escherichia coli (EHEC). Bacterial growth and Stx2 production were both inhibited by 2 mM NCT. The cytotoxic effect of Stx2 on Vero cells was removed by $5.5 mM NCT. Confocal microscopy and FACS analyses showed that the binding of Stx2 to human kidney glomerular endothelial cells was inhibited, and no NCT- treated Stx2 entered the cytosol. Mass spectrometry displayed oxidation of thio groups and aromatic amino acids of Stx2 by NCT. Therefore, long-lived oxidants may act as powerful tools of innate immunity against soluble virulence factors of pathogens. Moreover, inactivation of virulence factors may contribute to therapeutic success of NCT and novel analogs, which are in development as topical antiinfectives. Citation: Eitzinger C, Ehrlenbach S, Lindner H, Kremser L, Gottardi W, et al. (2012) N-Chlorotaurine, a Long-Lived Oxidant Produced by Human Leukocytes, Inactivates Shiga Toxin of Enterohemorrhagic Escherichia coli. PLoS ONE 7(11): e47105. doi:10.1371/journal.pone.0047105 Editor: Michael Hensel, University of Osnabrueck, Germany Received April 12, 2012; Accepted September 10, 2012; Published November 6, 2012 Copyright: 2012 Eitzinger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by NovaBay Pharmaceuticals, Inc., Emeryville, California, United States of America. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Dmitri Debabov and Mark Anderson are employed by and Markus Nagl was a consultant for NovaBay Pharmaceuticals, Inc., the funder of this study. In this study, pure NVC-612 (molecular weight 209.63 g/mol) and NVC-422 (molecular weight 244.07 g/mol) were from NovaBay Pharmaceuticals Inc. There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: m.nagl@i-med.ac.at . These authors contributed equally to this work. Introduction N-chlorotaurine (Cl-HN-CH2-CH2-SO3H, NCT), an N-chloro derivate of the amino acid taurine, is a long lived oxidant produced in high amounts by stimulated human granulocytes and monocytes [1–3]. It has been shown to downregulate in vitro proinflammatory chemokines, cytokines, and enzymes, such as TNFa, nitric oxide, and interleukins 1beta, 2, 6, 8, 12 [4–6]. Oxidation of IkBa and the following inhibition of activation of NF-kB is assumed as a major basic mechanism [7], as well as induction of hemoxygenase-1 [8]. Furthermore, NCT exerts broad-spectrum microbicidal activity down to micromolar concentrations, so that its contribution to inactivation of pathogens by phagocytes has been discussed [9,10]. The possibility to synthesize the substance chemically as crystalline sodium salt (Cl-HN-CH2-CH2-SO3-Na, [11]) paved the way for numerous studies investigating mainly the 55 mM (1% w/v) aqueous solution on its suitability as a topical antiinfective agent in human medicine (for review see [12]). One major advantage is the very good tolerability as a mild endogenous active chlorine compound, so that sensitive body regions and cavities can be irrigated [12]. These findings encouraged the development of analog chloramines with even higher solution stability, N- monochloro-dimethyltaurine (Cl-HN-CH2-(CH3)2-CH2-SO3-Na, NVC-612) and N,N-dichloro-dimethyltaurine (Cl2-N-CH2- (CH3)2-CH2-SO3-Na, NVC-422), which are promising new antiinfective agents [13–15]. Investigating the mechanisms of action of NCT, we found that chlorination of the surface of bacteria and fungi occurred within 1 min, before the killing started [16]. Importantly, incubation of bacteria for such sublethal times in NCT removed their virulence, as demonstrated for S. aureus and S. pyogenes in the mouse peritonitis model [9,17]. Moreover, secretory aspartyl proteinases of Candida albicans were found to be downregulated by sublethal concentra- tions of NCT [18]. Loss of virulence was connected with a lag of regrowth of pathogens, generally designated as postantibiotic effect [9,17,18]. Besides that, we hypothesized that not only the pathogens can be attacked by NCT, but also their virulence factors may be directly oxidized and inactivated. This concept was supported by the finding that gliotoxin of Aspergillus fumigatus is obviously inactivated by this chlorine compound [19]. The consequences would be at least dual: First, long-lived oxidants produced by granulocytes and monocytes may act as tools of innate immunity to inactivate secreted or surface-bound virulence factors. Second, upon clinical application of chloramines as antiinfective solutions, an impact on the metabolites of PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e47105