N-chlorotaurine, a long-lived oxidant produced by human leukocytes, inactivates Shiga toxin of enterohemorrhagic Escherichia coli

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.

Figure 1. Inhibition of growth of EHEC 178 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 37°C. (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 37°C. (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 ± SD from three independent experiments are shown in (A–D). *P<0.05; **P<0.01.

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 ± SD from three independent experiments are shown in (A–E). *P<0.05; **P<0.01.

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 ± SD from three independent experiments are shown.

Figure 4. Binding to and penetration of purified Stx2 into human kidney glomerular endothelial cells (GEnC) was inhibited by NCT.

(A) FACS analysis of binding of Stx2 to GEnC cells. Purified Stx2 was incubated with 55 mM NCT (1% w/v) in PBS at 37°C for 30 min. Controls with Stx2 in PBS or without Stx2 were performed in parallel. Cells were incubated for 4 h at 37°C. 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 ± 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 Oyster®-488. Subsequently, labeled Stx2 was incubated in 55 mM NCT (1% w/v) or PBS (control) for 30 min at 37°C. 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, 6000× magnification, scale bar 10 µm). By contrast, using NCT-treated Stx2, no fluorescence occurred, indicating the absence of penetration.

Figure 5. Impact of NCT on purified Stx2 demonstrated in gel electrophoresis.

(A) Purified Stx2 was treated with 55 mM NCT for 30 min at RT. Then, an aliquot was used for SDS-PAGE. Reducing buffer contained 4.5% v/v mercaptoethanol. A gel containing bands of aliquots containing 10 or 15 µg Stx2 is shown (one representative of three independent experiments). Bands a–f were subjected to in-gel digestion and mass spectrometry. (B) Purified Stx2 was treated with 55 mM NCT for 30 min at RT and subjected to a tricine gel for improved separation of the low molecular weight bands. Note the separation of the B subunit in bands a and b, which were subjected to in-gel digestion and mass spectrometry.

Figure 6. Oxidation of thio groups and aromatic groups of Stx2 detected with mass spectrometry.

Bands a-f from the gels, as depicted for one representative in Figure 5, were analyzed with mass spectrometry after in-gel digestion. Similar results gained from three independent experiments are shown. Positions and occurrence in bands are demonstrated. Amino acids 1–22 (subunit A) and 1–19 (subunit B), respectively, comprise the signal peptides. (A) Oxidation of all methionines and cysteines of subunit A occurred. Positions of oxidized amino acids in the sequence are shown and their mono- to tri-oxidation in relation to the bands excised from the gels. (B) Chlorination of phenylalanine, tyrosine, and histidine of subunit A. (C) Oxidation of methionine and chlorination of tyrosine of subunit B.

Figure 7. Survey of the impacts of NCT on Stx2.

The found effects of NCT on Stx2 are summarized in this figure.