coli genes [36], including those associated with EPEC virulence [11, 15] (Figure 2). Consistent with this conclusion, we found no evidence for specific regulation by zinc interacting with Ler, or involvement of the major zinc homeostasis regulators Zur or ZntR. However,

toward the goal of using dietary supplements to diminish the severity of disease caused by EPEC, and the related EHEC, zinc clearly reduces the expression of BFP, LEE genes, including the LEE1 operon encoding Ler, and stx encoding the Shiga toxin [11, 15] (Figure 2). Looking Osimertinib mouse for a general stress pathway to explain the observed down regulation of EPEC virulence genes, we observed stimulation of rpoE expression in the presence of zinc selleck screening library (Figure 3). We concluded that zinc caused envelope stress to EPEC grown in defined DMEM. Consistent with our observation, rpoE and a number of rpoE-dependent genes including rpoH and htrA were stimulated in the E. coli K-12 strain W3110 grown in LB in the presence of zinc chloride [31]. However, it is not likely that the RpoE sigma factor controls expression of LEE genes because the promoters identified

for the LEE operons of EPEC were clearly RpoD-dependent, having consensus sequences highly similar to those of promoters transcribed by the σ 70-containing RNA polymerase holoenzyme [14]. Zinc causes envelope stress, in part, by compromising protein tertiary structure, complexing with the thiol side chain of cysteine residues and/or disrupting disulfide bonds. Predictably, extracellular zinc causes a transient induction of the genes necessary for cysteine biosynthesis, thought to mop up excess cytoplasmic zinc [31]. A brief, transitory increase in intracellular zinc concentration most likely occurs inside of the bacterium, particularly

for the strains containing mutations in either zur or zntR, upon addition selleck of 0.3 to 0.5 mM zinc acetate to the culture medium. However, evidence suggests that zinc is quickly complexed to cysteine because the cysteine biosynthetic genes are stimulated by zinc stress [31] and then intracellular zinc concentrations return to normal conditions where free zinc is in the femtomolar range, less than one zinc molecule per bacterium [18]. In EPEC, the type III secretion system is assembled through the envelope, spanning the inner and outer membranes, and beyond, in order to inject effector proteins into the host cell click here cytoplasm [12, 37, 38]. Thus one would predict that zinc adversely affects the assembly, and integrity of the injectosome once assembled, ultimately preventing protein secretion. Here we demonstrate that zinc physically alters the EPEC envelope (Figure 4) and that the envelope stressor NH4VO3, which modifies lipid A of the LPS [34] and specifically stimulates the RpoE regulon, inhibits type III protein secretion in a manner similar to that observed for zinc [11] (Figure 5).