6 mM Zn 1:20 4-fold decrease + 10 ng/ml cipro 1:640   + 10 cipro + 0.6 mM Zn 1:160 4-fold decrease All source strains were grown for 5 hours, 4 hours after addition of ciprofloxacin and/or zinc. Zn, zinc acetate; cipro, ciprofloxacin, usually added at ~ 1/3 of the MIC. Stx is an important virulence factor in STEC, but it is not the only one. Therefore, we also tested whether operons in the locus for enterocyte

effacement (LEE) were activated by oxidant stress, and if so, whether, they were susceptible to inhibition by zinc. We used LEE4-lacZ and LEE5-lacZ reporter strains; LEE4 encodes the EPEC and EHEC secreted proteins (Esps), and LEE5 encodes the critical adhesins Tir and intimin, and the CesT chaperone. Figure  6 shows that, in the presence of XO, GF120918 hypoxanthine substrate does modestly activate expression of both LEE4 (Figure  6A) and LEE5 (Figure  6B). Figure  6C shows that H2O2 also induced LEE5

expression in a manner similar to that triggered hypoxanthine plus XO, and as previously shown for ciprofloxacin [24]. Figure  6D shows that zinc acetate inhibited LEE4 expression, but unfortunately manganese chloride showed no such ability. Figure  6 shows first that LEE operons may be up-regulated by oxidant stress, and second that the virulence-inhibiting abilities of zinc extend to factors other than Stx including critical adhesins and Type III secreted proteins encoded in the LEE. While Figures  1, 2 and 3 focused on the protective Casein kinase 1 effects of zinc and other metals on intestinal cells, Figures  4, 5 and 6 extend our previous understanding of zinc’s direct effects on bacteria [11, 12], showing zinc’s ability find more to inhibit the SOS response as measured by recA expression (Figure  4), a property

not matched by any other metal tested. The good correlation between zinc’s inhibition of recA expression (Figure  4), filamentation (Additional file 1: Figure S1), phage production, and zinc’s inhibition of Stx toxin protein (Figure  4A) and stx RNA [12] suggests that zinc’s ability to block recA activation is an important part of the mechanism of action of this metal in STEC and EPEC infection. Figure 6 Rabusertib cost effect of zinc and other metals on expression of LEE operons as measured in reporter strains. Reporter strains JLM165 (for LEE4, encoding the Esps) KMTIR3 (for LEE5, encoding Tir and intimin) and mCAMP (for beta-lactamase) were used to measure gene expression using the Miller assay. Panels A and B, expression of LEE4 and LEE5 were significantly increased in dose-dependent fashion by hypoxanthine in the presence of XO, compared to without added XO. Panel C, LEE5 expression was modestly but significantly increased in response to H2O2. Panel D, zinc acetate, but not MnCl2, inhibited induced LEE4 expression. *significant compared to “plus cipro, no-metal” condition. Panel E, lack of effect of zinc on expression of beta-lactamase in the bla-lacZ reporter strain in two different types of liquid media, minimal medium (MM) and DMEM.