The identity of EspB was confirmed by an in-gel tryptic digest followed by mass spectrometry (data not shown). Increasing concentrations of NH4VO3 caused diminished protein PX-478 cost see more secretion in a concentration dependent manner, such that at 10 mM of this chemical secretion of EspB was diminished by more than 70%. Because NH4VO3 stresses the bacterial envelope, specifically targeting the RpoE stress pathway, we concluded
that stress to the EPEC envelope caused decreased protein secretion via the type III secretion system. Figure 5 Zinc and ammonium metavanadate both inhibit protein secretion from EPEC. Cultures of EPEC strain E2348/69 were grown statically overnight in DMEM with varied concentrations of zinc acetate or ammonium metavanadate to an OD600 of 0.8 – 1.0. A culture of an EPEC strain deficient in type III secretion (ΔescN) was included as a control. Cells were removed by centrifugation, then proteins in the culture medium were precipitated with 25% find more trichloroacetic acid and visualized with SDS-PAGE. The volume of supernatant precipitated was chosen such that volume (ml)×culture
OD600 = 6.0. Zinc precipitates phosphate from the tissue culture medium DMEM Through the course of growing EPEC cultures in DMEM we observed that, not the doubling time, but rather the growth yield was modestly diminished in the presence of Rebamipide zinc acetate (data not shown). In addition, CFU/ml values after overnight growth in DMEM were ∼1.0 x 109 versus 5.0 x 108
in the absence and presence of 0.3 mM zinc. As phosphate is present in DMEM at 1 mM concentration, zinc phosphate is insoluble in solution, and we observed a small amount of white precipitate in DMEM in the presence of zinc acetate (data not shown), we hypothesized that the addition of zinc removed phosphate from this tissue culture medium. Indeed we observed that after the addition of millimolar concentrations of zinc, the concentration of soluble phosphate diminished in a linear fashion in DMEM (Figure 6). Therefore we concluded that zinc removed the essential element phosphorous from solution, and was the most likely explanation for the modestly diminished EPEC growth yield in the presence of zinc. Figure 6 Effect of added zinc on soluble phosphate remaining in DMEM. Zinc acetate was added to DMEM and incubated at 37°C. Remaining soluble phosphate was quantitated with a Mol-Bio Green assay described in Methods. Discussion In this report we begin to elucidate the molecular mechanisms by which zinc diminishes EPEC virulence. Though previous data had indicated that zinc reduces LEE gene expression, in a Ler-dependent manner , as a negative control in this report we also observed that zinc reduced expression of the bla gene, encoding β-lactamase.