EPEC bacteria were grown in DMEM tissue culture medium in the absence and presence of 0.3 mM zinc acetate. In the absence of zinc, the envelope of the bacteria appeared intact

(Figures 4A-C). However, after growth in DMEM in the presence of zinc the outer membrane of the bacteria appeared compromised, and we observed what appeared to be multiple membrane blebs on individual bacteria (Figures 4D,E). Furthermore, we also observed bacteria with irregularly shaped inner membranes (Figure 4F). These data provided direct evidence that zinc damages the EPEC envelope. Figure 4 The effects of zinc stress on INCB28060 concentration the EPEC envelope imaged by transmission electron microscopy. After 10-hour growth in DMEM medium, cultures were grown for an additional 5 hours in the absence (A,B) and presence (D,E) of 0.3 mM zinc acetate. EPEC bacteria were pelleted, the medium discarded, and bacteria then were resuspended in 0.1 M MgSO4. Samples were placed on carbon formvar grids, stained with 1.3% uranyl acetate and viewed

by transmission electron microscopy. The same procedure was repeated with 1-hour growth in DMEM medium, followed by an additional 5-hours of growth in the absence (C) and presence (F) of 0.3 mM zinc acetate. Arrow points to outer membrane blebs in (D). (A,D) Bars 1.0 μm; (B-C,D-F) Bars 0.1 μm. Chemical disruption of the EPEC envelope diminishes type III secretion Zinc stimulates the expression of rpoE (Figure 3) and physically damages the EPEC envelope Thymidylate synthase (Figure 4).

These data demonstrated that, as for laboratory strains of E. coli, zinc causes envelope stress in EPEC. Along with P505-15 down-regulation of LEE genes encoding type III secretion system components envelope stress could, at least in part, explain why zinc reduces diarrhoea in a rabbit illeal loop model of infection [11]. To test this hypothesis we monitored proteins secreted from EPEC grown in DMEM in the presence of ammonium metavanadate (NH4VO3). Ammonium metavanadate causes envelope stress and specifically stimulates the rpoE regulon [24, 34]. Thus our prediction was that this chemical, in a manner similar to zinc, would diminish protein secretion via the type III secretion system of EPEC strain E2348/69. To test this prediction strain E2348/69 was grown in DMEM overnight, in static cultures in the presence of increasing concentrations of NH4VO3. Bacteria were pelleted, and secreted proteins were harvested from the supernatant by GF120918 datasheet TCA-precipitation. To control for proteins being released from the bacteria independently from the type III secretion system, we also harvested supernatant proteins from the strain CVD452, deleted for escN, encoding the ATPase [26]. We monitored secretion in the presence of zinc because protein secretion was previously shown to be diminished in the presence of this metal ion [11].