Notably, 0.5 mM was the effective concentration of manganese used by Mukhopadhyay and Linstedt [14] in their study of Stx1 trafficking in HeLa cells. Figure 3D shows that CuSO4, like zinc, significantly reduced Stx2 translocation. This was a surprise because of the lack of protection by CuSO4 on TER. Nickel chloride also had no protective effect on TER and none on Stx2 translocation at 0.1 to 0.5 mM (data not shown). Figure 3 Effect of metals other than zinc on oxidant-induced changes in TER and on Stx2 translocation. As in Figure 2, the “standard” concentration of hypoxanthine
was 400 μM if not otherwise stated and the “standard” amount of XO was 1 U/mL. NSC23766 supplier Panel A, lack of protection by FeSO4 and MnCl2 on oxidant-induced ∆ TER. Panel B, lack of protection by FeSO4 on oxidant-induced Stx2 translocation. Panel C, lack of protection by MnCl2 on oxidant-induced Stx2 translocation. Panel D, protection by CuSO4 against oxidant-induced Stx2 movement across the monolayer. To summarize Figures 1, 2 and 3, zinc increased the TER in undamaged cells, and protected intestinal monolayers against the drop in TER induced by
DMSO, by hydrogen peroxide, and that induced by XO plus hypoxanthine. Zinc also protected against oxidant-induced translocation of Stx2 across the monolayers Emricasan in vitro at 0.1 to 0.3 mM concentration. These protective effects of zinc are AP26113 ic50 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 translocation across 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 Rebamipide 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 LEE-encoded 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 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.