Zinc also protected

monolayers from damage induced by hydrogen peroxide, an oxidant host defense that is released in LY2874455 manufacturer response to EPEC and STEC infection [22, 23]. We also examined if zinc and other metals had any effect of the translocation of Stx across T84 monolayers and found that it reduced toxin translocation as well. We also reexamined the ability of zinc to inhibit Stx production from STEC bacteria and correlated it with zinc’s ability to block the onset of the SOS bacterial stress response, as measured by recA expression, an early and quantifiable marker of the Geneticin solubility dmso SOS response. While other metals occasionally mimicked zinc’s effects in one particular attribute or another, zinc was unique in its ability to simultaneously Selleckchem Quisinostat exert protective effects

on host tissues while also inhibiting multiple bacterial pathways associated with STEC virulence such as the recA/SOS response, EHEC secreted proteins (Esps), the adhesins intimin and Tir, and Stx production. No other metal tested showed the same broad combination of beneficial effects as did zinc. Methods Bacterial strains used Bacterial strains used are listed in Table  1. Bacteria were grown overnight in LB broth at 37°C with 300 rpm shaking, then subcultured into the medium for the expression studies, usually DMEM medium or minimal medium. In this report, when bacteria were subcultured in “DMEM” this refers to DMEM/F12 Buspirone HCl medium supplemented with 18 mM NaHCO3 and 25 mM HEPES, pH 7.4, but without serum or antibiotics. Table 1 Bacterial strains used Strain name Pathotype/serotype Comment Reference Popeye-1 STEC; O157:H7 stx2; stx2c United States 2006 spinach-associated outbreak strain. [12] EDL933 STEC; O157:H7 stx1; stx2 [23] TSA14 STEC O126:H11 stx1 [23] JLM281 recA-lacZ reporter strain derived from laboratory strain MC4100 recA is used as a measure of the SOS response to DNA damage

in E. coli [24] JLM165 LEE4-lacZ reporter strain LEE4 encodes the EPEC and EHEC secreted proteins (Esps) [25] KMTIR3 LEE5-lacZ reporter LEE5 encodes Tir and intimin [26] mCAMP bla-lacZ reporter β-lactamase [25] MG1655 Used as susceptible host strain for bacteriophage plaque assays.   [27] Assays using T84 cells grown in polarized monolayers in Transwell inserts T84 cells were grown to confluency over 7 to 10 days on 12 mm Transwell inserts (Corning Life Sciences, Lowell, MA) in T84 medium with 8% fetal bovine serum and antibiotics as described. The Transwells were of 0.4 μm pore size polycarbonate plastic, and were not coated with collagen or other proteins. Trans-epithelial electrical resistance (TER) was measured using an Evom2 meter (World Precision Instruments, Tampa, FL) and the STX2 chopstick electrode. (It is mere coincidence that the electrode has a name similar to the toxin we were studying.