E. Zinc also protected against oxidant-induced translocation of Stx2 across the monolayers at 0.1 to 0.3 mM concentration. These protective effects of zinc are attributable to actions of zinc on the host tissues, not on bacteria. None of the four other metals tested (iron, manganese, copper, or nickel) protected against oxidant-induced decrease in TER, but copper was still able to reduce Stx2 translocation0.0.Crane et al. BMC Microbiology 2014, 14:145 http://www.biomedcentral.com/1471-2180/14/Page 8 ofacross monolayers (Figure 3D). Our results did not support the idea, advanced by Mukhopadhyay and Linstedt, that manganese was the metal with the greatest promise for protection against STEC infection in the clinical setting [14]. Zinc still seemed to be a candidate for such studies, but to address this more fully we compared zinc and other metals for their ability to block bacterial signaling and stress-response pathways associated with virulence. Stx production and release in STEC bacteria is strongly regulated by the SOS stress response system in E. coli [18,38]. In contrast, Stx production is quite insensitive to commonly mentioned signaling pathways such as quorum sensing, and to transcription factors such as the LEEencoded regulator (Ler) and Plasmid-encoded regulator (Per) [25,39-41]. This is not surprising since stx1 and stx2 are encoded on phages similar to phage lambda, and these phage genes are strongly activated by the DNA PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28045099 damage triggered by certain antibiotics [18], hydrogen peroxide [22,42], or ultraviolet light. An early, reliable, and quantifiable marker of the SOS response is the expression of recA [43,44]. We hypothesized that zinc’s ability to inhibit Stx production arises from its ability to inhibit the SOS response and recA. To test this, we measured recA expression using a recA-lacZ reporter gene construct using the Miller assay method and compared those results with metals ability to inhibit Stx production. Figure 4A shows that zinc inhibits ciprofloxacin-induced Stx2 production strongly and in a dose-dependent manner. In contrast, MnCl2 had no such ability to inhibit either ciprofloxacin-induced Stx2 production (Figure 4B) or basal (non-antibiotic treated) Stx release [12]. Figure 4C shows that recA expression increased in reporter strain JLM281 when hypoxanthine is added in the presence of the order Stattic enzyme XO, but not in the absence of XO. Hydrogen peroxide itself showed a recA activation curve with a similar shape (Figure 4D). Zinc acetate inhibited ciprofloxacininduced recA expression (Figure 4E) as well as hydrogenperoxide induced recA expression (data not shown). Zinc acetate was more efficacious and more potent in inhibition of ciprofloxacin-induced recA expression that MnCl2 or NiCl2 (Figure 4F) and more than FeSO4, CuSO4, or gallium nitrate (Figure 4G). Gallium was tested because of its position next to zinc on the Periodic Table and because others had reported it had anti-virulence activity [45]. Figure 4H shows that zinc acetate was more potent than zinc oxide nanoparticles, CoCl2, or bismuth subcitrate in inhibition of recA induced by ciprofloxacin. Bismuth was tested because of its long use as a treatment for infectious diarrhea [46,47], and zinc oxide nanoparticles were reported to have activity against Campylobacter jejuni [48]. In summary, zinc acetate was more potent and more effective in inhibiting ciprofloxacin-induced recA than any other metal shown in Figure 4. Zinc also blocked recA induced by mitomy.
