N-Chlorotaurine Inacitvates Shiga Toxin Figure 2. NCT, NVC-422, and NVC-612 inhibit the cytopathic effect of Stx2 in Vero cells. Supernatants of EHEC 178 cultures were treated with 1.38–55 mM of oxidants for 30 min (A–D), diluted 100-fold in RPMI + 10% v/v FCS, and added to Vero cell cultures, which were monitored for the typical cytopathic effect. (A–C) Numbers of cytopathic cells per visual field at a 100x magnification after incubation with EHEC supernatant for 72 h. Comparison of RPMI + FCS without Stx (negative control), supernatant of the non-EHEC strain ATCC 11229 (shown in A), untreated supernatant (positive control), and supernatant treated with NCT (A), NVC-422 (B), or NVC-612 (C). (D) Quantification of cell death by LDH assay after incubation with EHEC supernatant. Values were related to untreated supernatant. An additional separate positive control was performed with 1% Igepal. (E) Supernatants of EHEC 178 cultures were treated with 55 mM NCT for 1–30 min, followed by Vero cell assay and evaluation as in A–C. Mean values 6 SD from three independent experiments are shown in (A–E). *P,0.05; **P,0.01. doi:10.1371/journal.pone.0047105.g002 that case sulfenic acid (mono-oxidation), sulfinic acid (di-oxidation) and sulfonic acid (tri-oxidation) were generated. Remarkably, all cysteines and methionines in the subunit A were oxidized. Furthermore, chlorination of the aromatic amino acids phenylal- anine, tyrosine, and histidine was found. The exact positions of the oxidized and chlorinated amino acids are illustrated in Figure 6. Notably, in bands a and b from the tricine gel (Figure 5B), we found not only the predominant sequences of subunit B, but also fragments of subunit A (data not shown in Figure 6), indicating oxidative fragmentation of this subunit. Discussion As early as 1918, hypochlorous acid and chloramine T have been shown to remove the lethal effect of Bacillus welchii toxin on pigeons after an incubation time of 5 min [26]. Decades later, the active chlorine compounds NaOCl and monochloramine were shown in few studies to inactivate aflatoxins, endotoxins and ricin, mainly in connection with their use as water disinfectants [27,28], and recently as irrigation solutions for root canals in odontology [29]. The finding that mainly NCT, but also other chloramines are produced by leukocytes as long-lived oxidants with antiinflamma- tory and antimicrobial properties, implicated their importance for the human defense system [1,4,5,30]. Rapid loss of virulence of pathogens and first studies on inactivation or downregulation of the virulence factors gliotoxin and aspartyl proteinases in the presence of NCT [9,17–19] prompted us to investigate in detail the impact of this substance against a model toxin. In this study we have shown that the even more stable dimethylated analogs NVC- 422 and NVC-612 have similar properties as NCT, indicating importance not only for innate immunity, but also for the application of these substances as topical antiinfectives in medicine. The first step of the investigations, incubation of EHEC in NCT, revealed that inhibition of bacterial growth and Stx2 concentration in the supernatant are tightly connected. Interest- ingly, this was slightly different with Candida spp. in a previous study, where a decrease of aspartyl proteinases was found by NCT concentrations too low to inhibit fungal growth [18]. Therefore, the minimal NCT-concentration to inhibit virulence factors in relation to the minimal inhibitory concentration seems to be specific for each factor. It is known that the first attack of NCT leads to chlorination of the surface of pathogens (‘‘chlorine cover’’, [16]). This impact is sufficient to induce a loss of virulence, as demonstrated in the mouse peritonitis model for S. aureus and S. pyogenes, but not a loss of viability of the bacteria [9,17,18]. Therefore, it is near ad hand that virulence factors on the surface of pathogens and secreted ones will be oxidized and may be inactivated. This is confirmed by the direct inactivation of Stx2 by NCT in the present study and of gliotoxin in a recent one [19]. For killing of bacteria and fungi, penetration of NCT is necessary [9,16,31], and it is logical that in this case also virulence factors will be inactivated keeping in mind the multiple sites of attack in E. coli found by proteomics [31]. However, at sublethal concentra- tions of NCT (see Figure 1) it remains unclear if minimal amounts of active chlorine can penetrate bacteria or if signal transduction plays a role. Additionally, it is an open question whether toxin production or release or both are inhibited under these conditions. Also the inhibition of bacterial growth by NCT per se may contribute significantly to the absence of toxin in the supernatant. The direct chemical impact of NCT on the toxin could be clearly proved by SDS-PAGE and mass spectrometry. N-chloro amino acids are known to react with thio groups (sulfhydryls and thioethers), aromatic C-H compounds, and amino groups [11]. Oxidation capacity is lost by the first two kinds of reaction, while the latter one leads to transfer of the active chlorine to the reaction Figure 3. Decrease of oxidation capacity of NCT and NVC-422 in EHEC Direct Medium. Stock solutions of NCT (A) or NVC-422 (B) were 10-fold diluted in this medium to final concentrations of 55 mM, 5.5 mM, 2.75 mM and 1.375 mM. The oxidation capacity was measured with redox potentiometry after indicated time-points. Results with NVC- 612 resembled those of NCT. Mean values 6 SD from three independent experiments are shown. doi:10.1371/journal.pone.0047105.g003 PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e47105