pathogens in addition to the microbicidal effect could enhance the therapeutic success rate. To address particularly the first issue, we decided to investigate in the present study in detail the influence of NCT, NVC-422 and NVC-612 on a clinically important secreted bacterial toxin, which causes granulocyte invasion into the tissue. We chose Shiga toxin 2 (Stx2), which is produced by enterohemorrhagic Escherichia coli (EHEC) [20]. Stx2 consists of an enzymatically active A subunit (32 kDa) and a non- covalently linked B subunit pentamer (7,7 kDa for each monomer) responsible for interaction with glycolipid receptors on target eukaryotic cells. The A subunit possesses N-glycosidase activity and cleaves a single adenine residue from 28S ribosomal RNA. This depurination results in inhibition of protein synthesis and target cell death. The amino acid sequence of Stxs has already been determined [21], which was important for the performance of our present study. EHEC are the major cause of hemolytic-uremic syndrome (HUS) in childhood. HUS is characterized by a clinical triad of microvascular glomerular thrombosis, consumptive thrombocyto- penia and microangiopathic hemolytic anemia. The thrombotic microangiopathy is especially severe in the kidney and can lead to acute renal failure. This is connected with activation of the complement system [22], with infiltration of neutrophilic granu- locytes in the glomeruli [23,24] and with leukocytosis in peripheral blood [25]. It may be hypothesized that one effect of leukocytes is inactivation of the causative agent of microangiopathy and inflammation, i.e. Shiga toxin. Long-lived oxidants could be involved in this process. The aim of this study was to investigate for the first time the impact of NCT on a virulence factor from the molecular process to the functional and biological consequences. Stx2 was used as a model for an important secreted toxin, which causes infiltration of NCT-producing leukocytes. Results Inhibition of Growth of EHEC and of Stx2 Production in the Presence of Sublethal Concentrations of NCT The CFU counts of EHEC 178 in the presence of 1.65 mM, 2.2 mM and 2.75 mM NCT in EHEC Direct Medium are shown in Figure 1A and the Stx2 production in Figure 1B (Figure S1A and B for EHEC 22). Bacterial growth from the starting point of 6.5 log10 was necessary for detection of Stx2. NCT (1.65 mM) had no influence on both bacterial growth and toxin production. For 2.2 mM and 2.75 mM, a growth inhibition of 2 and 4 h was observed, respectively. The course of Stx2 levels was similar, and the toxin production was blocked for 2 and 4 h by 2.2 mM and 2.75 mM NCT. Very similar results were found for 1.65–2.75 mM NVC-612 (data not shown). Compared to NCT and NVC-612, NVC-422 exhibited stronger activity (Figure 1C and D and Figure S1C and D for EHEC 22). Already 1.65 mM NVC-422 led to a reduction of the bacterial count by approximately 1.5 log10 and completely abrogated Stx2 production. A lag of regrowth and inhibition of Stx2 for 4 h was caused by 1.1 mM NVC-422, while 0.55 mM had no effect on both parameters. This can most likely be attributed to the higher oxidation capacity because of the two chlorine atoms in the molecule. The tests with EHEC 22 confirmed all these results with a lag time of 2–4 h for the same concentrations of test substances and for both parameters (Figure S1A–D). N-Chlorotaurine Inacitvates Shiga Toxin Inhibition of the Cytotoxic Effect of Stx2 against Vero Cells by NCT To investigate the capability of NCT and its analogs to impact the biological function of Stx2, the supernatant of EHEC cultures was treated for 30 min with the oxidants and added to Vero cells. Cells were monitored on the typical cytopathic effect by light microscopy for 3 days (Figure 2). When 55 mM (final concentration) NCT was added to the supernatant, only few dead cells (,5%) were seen as in controls treated with RPMI + FCS without any additives. A cytopathic effect typical for Stx was completely absent. With 5.5 mM NCT, some cells (approximately 20% of the value of the positive control) showing the Stx-specific cytopathic effect could be seen, although the total number of cytopathic cells was not significantly higher compared to the untreated control (Figure 2A). The results with NVC-422 and NVC-612 were very similar (Figure 2B and 2C). LDH assay (Figure 2D) and trypan blue exclusion tests (data not shown) confirmed these findings. A kinetic experiment disclosed that the inhibition of the Stx effect by NCT occurred already after 1 min (Figure 2E). The results were the same for both EHEC strains (data not shown for strain no. 22). In contrast to growth inhibition of EHEC (see above), NVC-422 offered the same activity than the other two test compounds. Taurine and dimethyltaurine (55 mM each) added to the supernatant of EHEC cultures did not decrease the number of cells showing a cytopathic effect and therefore did not influence the activity of Stx2 (data not shown). Consumption of Oxidation Capacity by EHEC Medium To estimate the maximum oxidation capacity available for inactivation of Stx2 in the previous tests, we measured the chlorine consumption of the test oxidants by EHEC Direct Medium using redox-iodometry. Results are depicted in Figure 3A and B. Over a period of 30 min, the oxidation capacity of 55 mM NCT and NVC-612 declined to 64%, of 5.5 mM to 45%, of 2.75 mM to 25.5% and of 1.375 mM to 12% of its basic value. The oxidation capacity of 55 mM NVC-422 decreased to 85%, of 5.5 mM to 72%, of 2.75 mM to 53.5% and of 1.375 mM to 38%. NVC-422 showed approximately 25% less chlorine consumption in EHEC Direct Medium compared to NCT and NVC-612. Binding of Stx2 on GEnC Cells (FACS Analysis) After treatment with 55 mM NCT for 30 min, purified Stx2 lost its ability to bind to cell surfaces. Extracellular accumulation of Stx2 was completely absent on cell surfaces when incubated with NCT-treated Stx2 compared to the untreated control (Figure 4A). Penetration of Stx2 into GEnC Cells (Confocal Laser Scanning Microscopy) Confocal laser scanning microscopy revealed no binding or uptake of Stx2 treated with NCT. However, the accumulation and uptake of Stx2 treated with PBS was clearly shown (Figure 4B). Structural Changes of Stx2 by NCT Evaluated by SDS- PAGE Coomassie stained gels as shown in Figure 5 revealed the known separation of the purified Stx2 in A and B subunits (lanes 10 mg Stx). Treatment with NCT for 30 min revealed a slight shift of the A subunit band and the B subunit band (Figure 5A). The impact on subunit A1 (to be seen in the reducing buffer) was even more pronounced. In total, six marked new bands occurred with the NCT treated Stx2 in non- PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e47105