N-Chlorotaurine Inacitvates Shiga Toxin in PBS or without Stx2 were performed in parallel. Cells were incubated for 4 h at 37uC. Stx2 bound on the cell surface was detected using a mouse- anti-Stx2-antibody and a FITC-labeled polyclonal goat-anti-mouse secondary antibody. MFI mean fluorescence intensity. Mean values 6 SD from three independent experiments are shown. (B) Confocal laser scanning microscopy of penetration of Stx2 into GEnC cells. Labeling of Stx2 was done with OysterH-488. Subsequently, labeled Stx2 was incubated in 55 mM NCT (1% w/v) or PBS (control) for 30 min at 37uC. The sample was 100-fold diluted in the cell culture, and the fluorescence was monitored. The figure shows a fluorescent cell treated with Stx2 in PBS after 1h (one representative of three independent experiments, 60006 magnification, scale bar 10 mm). By contrast, using NCT-treated Stx2, no fluorescence occurred, indicating the absence of penetration. doi:10.1371/journal.pone.0047105.g004 partner (transchlorination, transhalogenation, [1,11]). In perfect accordance, we found oxidation of cysteines, methionines, and the aromatic amino acids histidine, phenylalanine, and tyrosine in the subunit A of Stx2. Transchlorination could not be detected in our setup, which is well explainable by the instability of the formed chloramines during the preparation process including gel electro- phoresis and in-gel digestion. As a consequence of these chemical reactions, gel electrophoresis with NCT-treated Stx2 revealed bands with lower, but also higher molecular weight (Figure 5). The latter phenomenon has recently been observed with the toxin ricin, lactate dehydrogenase, and lysozyme treated with hypo- chlorous acid or (to a lesser extent) with monochloramine and has been explained by formation of higher-order aggregates [27]. Moreover, ovalbumin-chlorination by hypochlorous acid has been found to promote formation of aggregates stabilized by non- covalent bonds [32]. Although a definite explanation has not yet been provided, it is clear that both fragmentation and aggregation of target proteins by NCT and other active chlorine compounds result from oxidation of the described groups. Cystine, tyrosine, and methionine are present in active sites of Stx2 [21]. Since we found oxidation of all cysteines and methionines and chlorination of tyrosine 99 in subunit A, inactivation of the toxin by NCT was a logic consequence and clearly proven in the Vero cell assays. Notably, cysteines 263 and 282 form the disulfide bond between subunit A1 and A2 [21], so that their oxidation will probably lead to conformational changes. It has to be taken into account that a part of the oxidation capacity is rapidly consumed by thio groups of the medium in which the toxin was dissolved (decline within 1 min, see Figure 3), and that this part relatively increases if the applied concentration of NCT is lowered. Therefore, the remaining free concentration of NCT to inactivate Stx2 was lower than the added one. From these tests can be deduced that approximately 1–3 mM NCT is the threshold of antitoxic activity. The consumption of the dichloro compound NVC-422 was approximately 25% lower than that of NCT and NVC-612. Although, in accordance to that, NVC-422 showed a higher antimicrobial activity, its minimal antitoxic concentration was similar. The reason for that is not known in detail, but the observation fits to previous data that dichloro representatives of active chlorine compounds are not in all cases more active than the monochloro ones [33]. A comparison between Vero cell assays and EHEC growth curves reveals that a higher concentration of NCT (about 3 mM) is needed for inactivation of Stx2 than for growth inhibition (about 2 mM). In subunit B of Stx2, we found oxidation of methionine and chlorination of tyrosine, too. This perfectly fits to FACS analysis and confocal microscopy, which demonstrated that NCT treated Stx2 loses its capability of binding and penetrating its target cell, which are functions of the subunit B. From these tests and from the impact found on subunit A, it can be deduced that the toxin is oxidized at multiple sites, including those decisive for its activity (Figure 7). Such a multi-target attack is typical for active chlorine compounds and obviously connected with the broad spectrum of antimicrobial activity and the absence of resistance development of pathogens [9,11,12,31]. One may consider also the other way around that NCT might oxidize the body cells and impact the Stx receptor CD77 (globotriaosylceramide), thus preventing penetration of the toxin. However, since NCT incubated with Stx was at least largely inactivated by dilution before addition to the kidney cells (Figure 4) and since NCT does not react with amides such as CD77 (as opposed to amines [11]), it must be assumed that indeed the impact on the toxin was causative for the found absence of penetration of NCT-treated toxin and not an impact on the receptor. Antitoxic activity of long-lived oxidants is one more aspect of their antimicrobial activity and might be of importance for both their role in innate immunity and their application for local treatment of infections. Regarding innate immunity, these compounds are created via myeloperoxidase at the sites of infection – as a mixture of different chloramines, from which NCT is a major component [1,2]. The question is, if the concentration of long-lived oxidants would be sufficient in vivo to inactivate microbes or their toxins. In the supernatant of stimulated granulocytes, up to approximately 100 mM have been found [1,2,9], and also within the phagocytic granules the concentrations of chlorine compounds are generally estimated as sufficient to inactivate or at least attack bacteria [34,35]. Moreover, in vivo, chloramine levels of up to 288696 mM were measured in sputum samples of cystic fibrosis patients [36]. Taking into account chlorine consumption in sputum, rather high levels can be conceived to be produced by leukocytes. It is also assumed that long-lived oxidants may accumulate during inflammation and persist for h in view of permanently arriving granulocytes [5]. Because of all these results, it may be conceived that chloramines are also involved in oxidation of products of pathogens produced in vivo. Neutrophilic infiltration has been found in glomeruli of patients with hemolytic uremic syndrome [23,24]. Generally, such infiltration connected with complement activation is thought to trigger the tissue destruction [37]. However, in view of our results, there might be also beneficial effects like inactivation of Stx, which would fit to the goal of leukocyte migration to remove a nox in the body. Upon application of NCT for topical treatment of infections, sufficient concentrations to inactivate bacterial toxins are achieved with high probability. In different body regions, for instance crural ulcers, the eye, the outer ear, the oral cavity, and the urinary tract, 55 mM (1% w/v) NCT has been well tolerated and effective in clinical trials [12]. This concentration does not only kill different kinds of microbes after incubation times of a few min to 4 h [12], but also removed the virulence of staphylococci and streptococci within 1 min proven in the mouse peritonitis model [9,17], and impaired secretory aspartyl proteinases of Candida albicans before viability decreased [18]. Since we clearly demonstrated multiple oxidation of the example virulence factor Stx2 and its rapid loss of function within 1–5 min by 55 mM NCT in addition to these findings, it is very probable that not only the microorganisms, but also their products are inactivated during topical treatment. The analogs NVC-612 and NVC-422 obviously have similar properties with the advantage of higher stability at room temperature. PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e47105