The RpoE Stress Response Pathway Mediates Reduction of the Virulence of Enteropathogenic Escherichia coli by Zinc

Abstract

Zinc supplements are an effective clinical treatment for infantile diarrheal disease caused by enteric pathogens. Previous studies demonstrated that zinc acts on enteropathogenic Escherichia coli (EPEC) bacteria directly to suppress several virulence-related genes at a concentration that can be achieved by oral delivery of dietary zinc supplements. Our in vitro studies showed that a micromolar concentration of zinc induced the envelope stress response and suppressed virulence in EPEC, providing a possible mechanistic explanation for zinc's therapeutic action. In this report, we investigated the molecular and physiological changes in EPEC induced by zinc. We found that micromolar concentrations of zinc reduced the bacterial growth rate without affecting viability. We observed increased membrane permeability caused by zinc. Zinc upregulated the RpoE-dependent envelope stress response pathway and suppressed EPEC virulence gene expression. RpoE alone was sufficient to inhibit virulence factor expression and to attenuate attaching and effacing lesion formation on human host cells. By mutational analysis we demonstrate that the DNA-binding motif of RpoE is necessary for suppression of the LEE1, but not the LEE4, operon. Predictably, inhibition of the RpoE-mediated envelope stress response in combination with micromolar concentrations of zinc reduced EPEC viability. In conclusion, zinc induces the RpoE and stress response pathways in EPEC, and the alternate sigma factor RpoE downregulates EPEC LEE and non-LEE virulence genes by multiple mechanisms.

FIG 1

Zinc acetate reduced the EPEC growth rate but not viability. EPEC strain E2348/69 was grown in DMEM and incubated at 37°C with aeration for 90 min. Then zinc acetate was added to a final concentration of 0.3, 0.5, or 1.0 mM; 1% glacial acetic acid treatment was added as a vehicle control. The optical density at 600 nm was then recorded in the microplate reader for 160 min (A). The resulting cultures were serially diluted after 4 h of growth and plated on nonselective LB agar plates in triplicate. Viability of bacteria was determined by determining CFU counts per milliliter after overnight incubation at 37°C (B).

FIG 2

Zinc acetate increased membrane permeability. EPEC bacteria were grown in DMEM in the presence of a dilution of the vehicle, 10% glacial acetic acid, or 0.3 mM or 0.5 mM zinc acetate for 4 h. A group of vehicle-treated cells was collected and treated with 70% EtOH for 15 min as a positive control. Cells were harvested and stained as described in Materials and Methods. A minimum of seven fields of images were captured, with at least 8,000 cells counted by CellProfiler, version 2.0, for each treatment. The ratio of fluorescent cells, indicative of permeable membrane, to total cells was computed, and statistically significant differences are indicated by asterisks (two-tailed t test; *, P < 0.05).

FIG 3

Zinc acetate activated the envelope stress response and suppressed LEE virulence. EPEC bacteria were grown in DMEM at 37°C with aeration for 2 h. Treatment with 0.3 mM zinc acetate or vehicle was added in the beginning or in the last 10 min of incubation. RNA was extracted and reverse transcribed into cDNA as described in the text. REST 2009 (Roche) software was used to compare changes in envelope stress marker (A) and LEE operon (B) expression between zinc acetate treatment and vehicle treatment, and statistical significance is indicated by asterisks (two-tailed t test; *, P < 0.05). All values are compared to those of the internal control, rrsB. At least three biological samples were independently prepared and assayed. Dashed lines indicate a 1.0-fold change or no change in expression. Any bars above the line indicate increased expression, while bars below the line indicate decreased expression.

FIG 4

Inhibition of RpoE-sensitized EPEC to 0.3 mM zinc acetate. EPEC bacteria containing pRseA, in order to inhibit stimulation of the RpoE envelope stress response, were grown in DMEM with 100 μg/ml ampicillin selection for 90 min in the absence (−) and presence (+) of 0.2% arabinose (Ara). Either a dilution of 1% glacial acetic acid or zinc acetate (final concentration, 0.3 mM) was then added directly to the culture. The rate of bacterial growth for the first 120 min in the absence (A) and presence (C) of zinc is presented. Cultures grown in the absence (B) and presence (D) of zinc were serially diluted and plated on an LB agar-ampicillin selection plate in triplicate. After overnight incubation at 37°C, CFU counts per milliliter were determined for each plate, and statistically significant differences are indicated by asterisks (two-tailed t test; *, P < 0.05).

FIG 5

Induction of RpoE or truncated RpoE alone was sufficient to suppress LEE virulence. EPEC bacteria carrying pTrc99a (empty vector), pLC245 (wt RpoE), and pTrunc RpoE were grown in DMEM supplemented with 1 mM IPTG at 37°C with aeration for 2 h. RNA was extracted and reverse transcribed into cDNA as described in the text. REST 2009 (Roche) software was used to compare changes in envelope stress marker and LEE operon expression due to RpoE (A) or truncated RpoE (B) induction to the levels of the empty vector control. Statistical significance is indicated by asterisks (two-tailed t test; *, P < 0.05). Dashed lines indicate a 1.0-fold change or no change in expression. Any bars above the line indicate increased expression, while bars below the line indicate decreased expression. All values are compared to those of the internal control, rrsB. At least three biological samples were independently prepared and assayed. (C) The immunoblot assays were conducted as described in Materials and Methods. Cell lysates of EPEC carrying the pTrc99a plasmid control (lane 1), pLC245 carrying the wt RpoE (lane 2), or pTrunc RpoE (lane 3) are indicated. Mouse primary antibodies were used to probe against EspC, Tir, and DnaK (loading control). Data presented are representative of least two independent experiments for each assay.

FIG 6

EPEC virulence was suppressed by RpoE and truncated RpoE induction. EPEC strain E2348/69 carrying pTrc99a (empty vector), pLC245 (wt RpoE), or pTrunc RpoE or the CVD452 (ΔescN) strain, defective for type III secretion and A/E lesion formation, was grown in DMEM with 1 mM IPTG for 2 h. Bacterial culture or DMEM alone was then added to 80% confluent HEp-2 cells seeded on coverslips and coincubated for 4 h. Cells were fixed, permeabilized, and stained with FITC-phalloidin and DAPI counterstain. Samples were recorded by confocal microscopy and quantified as described in Materials and Methods. Representative images from each treatment are presented: HEp-2 cells only (A), HEp-2 cells with E2348/69 pTrc99a (B), HEp-2 cells with E2348/69 pLC245 (C), HEp-2 cells with E2348/69 pTrunc RpoE (D), and HEp-2 cells with strain CVD452 (E). White arrows point at A/E lesion formations identified by colocalization of DAPI-stained bacterial DNA and accumulated phalloidin-stained actin filaments. The percentages of cells with A/E lesions were quantified (F), and statistically significant differences are indicated by asterisks (two-tailed t test; *, P < 0.05).

FIG 7

Model of envelope stress-mediated regulation of EPEC virulence. The RpoE-dependent envelope stress response mediates regulation of EPEC virulence by zinc. Zinc activates both the RpoE and Cpx envelope stress pathways. It was previously demonstrated that Cpx downregulates the LEE (37), and we show that RpoE downregulates both LEE genes and the non-LEE EspC, a serine protease autotransporter protein (38–40). Our data suggest that LEE regulation by RpoE is indirect, and thus additional regulators remain to be identified.