Zinc protects against shiga-toxigenic Escherichia coli by acting on host tissues as well as on bacteria
© Crane et al.; licensee BioMed Central Ltd. 2014
Received: 28 February 2014
Accepted: 21 May 2014
Published: 5 June 2014
Zinc supplements can treat or prevent enteric infections and diarrheal disease. Many articles on zinc in bacteria, however, highlight the essential nature of this metal for bacterial growth and virulence, suggesting that zinc should make infections worse, not better. To address this paradox, we tested whether zinc might have protective effects on intestinal epithelium as well as on the pathogen.
Using polarized monolayers of T84 cells we found that zinc protected against damage induced by hydrogen peroxide, as measured by trans-epithelial electrical resistance. Zinc also reduced peroxide-induced translocation of Shiga toxin (Stx) across T84 monolayers from the apical to basolateral side. Zinc was superior to other divalent metals to (iron, manganese, and nickel) in protecting against peroxide-induced epithelial damage, while copper also showed a protective effect.
The SOS bacterial stress response pathway is a powerful regulator of Stx production in STEC. We examined whether zinc’s known inhibitory effects on Stx might be mediated by blocking the SOS response. Zinc reduced expression of recA, a reliable marker of the SOS. Zinc was more potent and more efficacious than other metals tested in inhibiting recA expression induced by hydrogen peroxide, xanthine oxidase, or the antibiotic ciprofloxacin. The close correlation between zinc’s effects on recA/SOS and on Stx suggested that inhibition of the SOS response is one mechanism by which zinc protects against STEC infection.
Zinc’s ability to protect against enteric bacterial pathogens may be the result of its combined effects on host tissues as well as inhibition of virulence in some pathogens. Research focused solely on the effects of zinc on pathogenic microbes may give an incomplete picture by failing to account for protective effects of zinc on host epithelia.
Zinc has been tested for its ability to treat and prevent diarrheal diseases in many large field trials over a period of over 4 decades[1–3] and has generally been found effective. Nevertheless, the protective mechanism of zinc has remained elusive. For example, most of the articles on zinc and enteric pathogens emphasize the essential nature of this metal and imply that zinc would enhance enhance the virulence of the pathogen[4, 5] rather than help the host. It is often suggested that zinc acts via the immune system, but actual studies on zinc and immune responses are more nuanced and show that zinc can impair as well as enhance immune functions[7–10]. Instead of invoking zinc effects on immunity, we and others have shown that zinc can have pathogen-specific protective effects by acting directly on enteric bacteria including enteropathogenic E. coli (EPEC), Shiga-toxigenic E. coli (STEC), and enteroaggregative E. coli (EAEC)[11–13]. Recently, Mukhopadhyay and Linstedt reported that manganese could block the intracellular trafficking of Shiga toxin 1 (Stx1) and thus inhibit its ability to kill susceptible host cells. This prompted us to reexamine the effects of zinc on host cells and to compare the effects of zinc with that of other divalent metals, including manganese.
STEC includes older names and subsets including enterohemorrhagic E. coli, EHEC, and Verotoxigenic E. coli, VTEC. STEC is the main cause of episodic “E. coli outbreaks” which are usually food-borne and often attract a great deal of attention in the news media[15–17]. As the name implies, these strains produce potent cytotoxins such as Stx1 or Stx2, or both. Absorption of Stx from the gastrointestinal tract can lead to severe extra-intestinal effects, including kidney failure, brain damage, and death. Antibiotics often make STEC infections worse by virtue of their ability to induce Stx production[18, 19] and so are considered contraindicated in STEC infection. The severe sequelae of STEC infection has prompted many to seek additional treatments, sometimes by heroic measures that might rescue patients from the throes of full-blown disease, such as hemolytic-uremic syndrome (HUS)[20, 21]. In contrast, we thought it would make more sense to intervene earlier in the course of STEC infection and prevent STEC infections from progressing to severe disease. Safe and inexpensive measures such as supplementation with oral zinc or other metals therefore seemed attractive as options. In contrast to our previous studies emphasizing the effects of zinc and other metals on the pathogenic bacteria, in this study we began by comparing zinc and other metals for protective effects on host epithelial cells, using T84 colonic cells grown as polarized monolayers. We found that zinc increased the trans-epithelial electrical resistance (TER) of the T84 cell monolayers; TER serves as a measure of epithelial integrity and of the barrier function provided by tight junctions. Zinc also protected monolayers from damage induced by hydrogen peroxide, an oxidant host defense that is released in 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 SOS response. While other metals occasionally mimicked zinc’s effects in one particular attribute or another, zinc was unique in its ability to simultaneously 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.
Bacterial strains used
Bacterial strains used
stx2; stx2c United States 2006 spinach-associated outbreak strain.
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
LEE4-lacZ reporter strain
LEE4 encodes the EPEC and EHEC secreted proteins (Esps)
LEE5 encodes Tir and intimin
Used as susceptible host strain for bacteriophage plaque assays.
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.) We adjusted the concentration of hydrogen peroxide used to damage the monolayers based on the TER at the start of the experiment: 2 mM H2O2 was used for monolayers with resistances of 1000–1500 Ω, and 3 mM H2O2 for monolayers with resistances above 1500 Ω. TER values are reported in ohms (Ω). To obtain values in Ω · cm2, one would multiply by the area (1.12 cm2). For monolayer experiments, we removed serum-containing medium and performed the experiments in serum-free medium. Delta TER (ΔTER) is defined as the TERfinal – TERinitial; TER and Stx translocation measurements were done in quadruplicate wells and are shown as means ± SD.
Stx toxin translocation assay
We measured translocation of Stx2 from the upper chamber to lower chamber in T84 cells grown in Transwell inserts (apical-to-basolateral) as described by Acheson et al.. T84 cells are insensitive to the toxic effects of Stx, at least in part due to low or absent expression of the Gb3 glycolipid receptors for Stx1 and Stx2; intestinal epithelia in humans and other mammals also show nil expression of Gb3. As a source of Stx2 we used crude supernatants of STEC strain Popeye-1, subjected to sterile filtration, and containing 1 to 1.5 μg/mL of Stx2. Crude supernatant was used because other soluble factors present in STEC supernatants, including EHEC secreted protein P (EspP) increase the ability of Stx to translocate across monolayers by the trans-cellular route[29, 30]. This crude supernatant would be expected to contain Stx2c as well as Stx2. Stx supernatants were diluted to a final concentration of Stx2 in the upper chamber of between 50,000 to 100,000 pg/mL in various experiments done over several months. Stx2 addition was delayed until 2 h after the oxidant in order to avoid denaturing the Stx by oxidation. Medium from the lower chambers was collected at various times and Stx2 measured by enzyme immunoassay (EIA) as described using the Premier EHEC toxin EIA kit (Meridian Biosciences, Cincinnati, OH). Purified Shiga toxin 2 toxoid was a kind gift of Dr. Alison Weiss, Univ. of Cincinnati, and was used to create standard curves to allow better quantitation. To provide context, in monolayers damaged with 3 mM H2O2, the amount of Stx2 translocated across the monolayer at 24 h averaged 7.0 ± 4.8% of the amount originally added. Hypoxanthine + XO triggered a similar amount of Stx2 translocation: 8.5 ± 3.0% at 24 h (mean ± SD of 5 experiments).
Miller assay for expression of β-galactosidase in bacterial reporter strains
Strain JLM281, the reporter strain containing the recA-lacZ construct was used to measure recA expression in response to inducing antibiotics, zinc and other metals. We used a version of the Miller assay adapted to 96 well plates for higher throughput. However, we used 0.1% hexadecyltrimethylammonium bromide (HTA-Br) detergent alone, without chloroform or sodium dodecyl sulfate (SDS), to permeabilize the bacteria. The buffers used are described in a Open WetWare website at http://openwetware.org/wiki/Beta-Galactosidase_Assay_%28A_better_Miller%29.
Agar overlay assay for bacteriophage plaques by modified spot assay
We used wild-type STEC strains as the source of bacteriophage for these experiments. STEC bacteria were subcultured at a dilution of 1:100 into antibiotic-free DMEM medium from an overnight culture. After 1 h of growth at 37°C with 300 rpm shaking, additions such as ciprofloxacin or zinc were made and the tubes returned to the shaker incubator for 5 h total. The STEC suspension was clarified by centrifugation, then subjected to sterile filtration using syringe-tip filters. The STEC filtrate was diluted 1:10 in DMEM medium, then serial 2-fold dilutions were made to yield dilutions of 1:20, 1: 40, 1: 80 and so on. The recipient strain, E. coli MG1655, was subcultured at 1: 50 from overnight and grown in LB broth for 3 hours. Soft LB agar was prepared using LB broth supplemented with 0.5% agar and 0.5 mM MgSO4. The soft agar was melted by microwave heating, and kept warm at 45°C on a heater block. The MG1655 culture was diluted 1: 10 into the soft agar and 5 ml of the bacteria-containing agar was overlaid on top of the agar of regular LB agar plate and allowed to solidify. Then 3 μl aliquots of the diluted STEC filtrates were spotted on top of the agar overlay. Plaques were visualized after 16 h of additional incubation at 37°C. Any faint zone of clearing was counted as a plaque. The highest dilution of STEC filtrate that produced a plaque was recorded as the plaque titer.
Rabbit infection experiments
No new rabbit infection experiments were performed for this study. We used photographs from the archives of our previous animal experiments to create the illustration in final figure. Nevertheless, all of our past and ongoing animal work has been scrutinized and approved by the animal care committee (IACUC) of the University at Buffalo.
Data analysis and statistics
Error bars shown on graphs and in Tables are standard deviations. Statistical signficance was tested by ANOVA using the Tukey-Kramer post-test for multiple comparisons.
We recently reported that the xanthine oxidase (XO) enzyme pathway is activated in response to EPEC and STEC infection. Infection with these pathogens triggers a release of nucleotides and nucleosides into the gut lumen, and XO itself is also released into the lumen of the intestine as a result of damage inflicted by these pathogens. XO catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid, with both steps creating one molecule of hydrogen peroxide. As previously reported by Wagner for oxidant molecules generated from neutrophils, XO-generated H2O2 increases the production of Stx from STEC strains. Since H2O2 is known to be able to damage intestinal epithelia[32, 33], we thought this would be a relevant model to test whether zinc or other metals could protect against oxidant damage, since zinc has been reported to reported to help restore intestinal barrier function following other insults. We used T84 cells grown to confluency in polarized monolayers in Transwell inserts as previously reported. We measured trans-epithelial electrical resistance (TER), an index of intestinal barrier function, as well as H2O2-induced translocation of Stx2 from apical to basolateral chambers.
To summarize Figures 1,2 and3, zinc increased the TER in undamaged cells, and protected intestinal monolayers against the drop in TER induced by DMSO, by hydrogen peroxide, and that induced by XO plus hypoxanthine. Zinc also protected against oxidant-induced translocation of Stx2 across the monolayers at 0.1 to 0.3 mM concentration. These protective effects of zinc are attributable to actions of zinc on the host tissues, not on bacteria. None of the four other metals tested (iron, manganese, copper, or nickel) protected against oxidant-induced decrease in TER, but copper was still able to reduce Stx2 translocation across monolayers (Figure 3D). Our results did not support the idea, advanced by Mukhopadhyay and Linstedt, that manganese was the metal with the greatest promise for protection against STEC infection in the clinical setting. Zinc still seemed to be a candidate for such studies, but to address this more fully we compared zinc and other metals for their ability to block bacterial signaling and stress-response pathways associated with virulence.
Stx production and release in STEC bacteria is strongly regulated by the SOS stress response system in E. coli[18, 38]. In contrast, Stx production is quite insensitive to commonly mentioned signaling pathways such as quorum sensing, and to transcription factors such as the LEE-encoded regulator (Ler) and Plasmid-encoded regulator (Per)[25, 39–41]. This is not surprising since stx1 and stx2 are encoded on phages similar to phage lambda, and these phage genes are strongly activated by the DNA damage triggered by certain antibiotics, hydrogen peroxide[22, 42], or ultraviolet light. An early, reliable, and quantifiable marker of the SOS response is the expression of recA[43, 44]. We hypothesized that zinc’s ability to inhibit Stx production arises from its ability to inhibit the SOS response and recA. To test this, we measured recA expression using a recA-lacZ reporter gene construct using the Miller assay method and compared those results with metals ability to inhibit Stx production.
Effect of zinc on the bacteriophage yield from STEC bacteria by phage plaque assay on E. coli MG1655 as host strain
Donor/source strain for bacteriophage
Growth condition (in DMEM Medium)
Fold reduction by zinc
TSA14; O26:H11, Stx1+; harbors phage H19B
control, no additives
+ 0.4 mM Zn
no plaques, < 1:10
> 2-fold decrease
+ 4 ng/ml cipro
+ 4 cipro + 0.4 mM Zn
control, no additives
+ 0.6 mM Zn
> 2-fold decrease
+ 8 ng/ml cipro
+ 8 cipro + 0.4 mM Zn
+ 8 cipro + 0.6 mM Zn
EDL933; O157:H7; Stx1+, Stx2+;
+ 0.6 mM Zn
Harbors phages H19B and 933 W
+ 10 ng/ml cipro
+ 10 cipro + 0.6 mM Zn
≥ 16-fold decrease
+ 0.6 mM Zn
+ 10 ng/ml cipro
+ 10 cipro + 0.6 mM Zn
Discussion and conclusions
Our understanding of the roles of divalent metals as regulators of bacterial pathogenesis has lagged behind that of other molecules such as quorum sensing auto-inducers and transcriptional regulators such as H-NS and Ler. Most of the work on transporters and metabolism of zinc and other metals has been done with non-pathogenic laboratory strains of E. coli[50–52], which makes the results difficult to extrapolate to strains which are professional intestinal or extra-intestinal pathogens. For example, STEC expresses several different metal uptake and zinc export genes not present in laboratory E. coli strains[4, 5, 53, 54] so STEC’s response to bioactive metals often differs from non-pathogenic E. coli. In addition, the specialized Type III secretion system (and Type VI secretion system in EAEC) used to deliver effectors into host cells may serve as an “Achilles’ heel” in these pathotypes because the membrane secretion machinery causes them to become hypersusceptible to some stressful stimuli such as the envelope stress response[27, 56]. Furthermore, many of the reports on zinc in enteric bacteria only focus on the essential nature of this metal for the pathogen[4, 57], without consideration of how zinc might also benefit the host. In addition, many reports do not distinguish between the growth-and-fitness promoting effects of zinc on pathogens at the low concentrations usually present (1 to 50 μM) versus the higher, stress-inducing concentrations of zinc that can occur during zinc supplementation (0.1 to 0.4 mM). In general, it appears that host cells are better able to survive--- and thrive--- in the presence of these higher zinc concentrations that are deleterious to E.coli and other enteric bacteria ([58, 59], and Figures 1,2 and3 of this study). Moreover, studies that have actually tested zinc for infection outcomes using cultured cell models or animal models have generally shown that zinc benefits the host more than the pathogen, resulting in a reduction in severity of disease[11, 13, 48, 60]. Indeed, Botella et al. recently showed that zinc is mobilized in macrophages and concentrated in phagosomes as part of the host defense against Mycobacterium tuberculosis. This is relevant to the gut because zinc is also concentrated in the secretory granules of Paneth cells[62, 63], specialized cells in the intestinal crypts involved in antimicrobial defenses.
The discovery that zinc specifically inhibits virulence factor expression by some pathogens and not others has led us to emphasize that zinc’s effects may be pathogen-specific. We may have to temper that emphasis, however, because Figures 1 and2 of this study show zinc may strengthen the intestinal epithelial barrier against oxidant damage and this might extend zinc’s protection to organisms that are not specifically affected by zinc. Zinc may have mild protective effects against multiple diarrheal pathogens via its effects on enterocytes, and then also have additional protective activity against specific pathogens such as EPEC, STEC, EAEC, and Campylobacter.
Mukhopadhyay and Linstedt reported that manganese was able to block the intracellular trafficking of Stx1 through the Golgi apparatus of Stx-susceptible HeLa cells engineered to overexpress the glycolipid Gb3; by doing so MnCl2 appeared to block the toxic effects of Stx1. Hope that manganese could be used as a treatment for STEC infection diminished, however, when Gaston et al. and additional work by Mukhopadhyay et al. showed that the protective effects of manganese did not extend to Stx2[65, 66]. Gaston and colleagues also showed that manganese was more toxic, both in cultured cells and in mice, than was reported by Mukhopadhyay and Linstedt. Our results show that manganese, unlike zinc, shows no protective effects on epithelial barrier function (measured as TER) or on Stx2 translocation across intestinal monolayers (Figure 3). Manganese did not inhibit ciprofloxacin-stimulated Stx2 production from STEC bacteria, unlike zinc (Figure 3A and B) and copper, and did not have any effect on recA expression (Figure 4F) or the SOS- induced bacterial elongation response (Additional file1: Figure S1). Manganese has been shown to up-regulate expression of the Esps in STEC and to increase basal Stx toxin production, so manganese has real potential to cause more harm than good in STEC infection. In addition, the neurotoxicity of manganese, which is worse in children and young animals, could exacerbate the Stx-induced encephalopathy that can accompany severe cases of STEC infection. Based on the literature mentioned and our results here, it appears that zinc is more likely to have therapeutic effects against STEC than manganese.
Copper also appears to have the ability to inhibit Stx production in an recA-independent fashion (Figure 4G and Ref.), which is plausible given that recA-independent pathways are known to regulate Stx. Copper, like zinc, also was able to block Stx2 translocation across intestinal monolayers (Figure 3F). Although copper is more toxic to humans than is zinc (based on the inverse ratios of the tolerable Upper Limits of these metals from the Food and Nutrition Board of the Institute of Medicine, available at https://fnic.nal.usda.gov/dietary-guidance/dietary-reference-intakes/dri-tables it is possible that copper might be combined with zinc to obtain additive effects via recA- dependent and recA-independent effects on STEC bacteria.
Additional file2: Table S1 summarizes the effects of zinc and four other metals in STEC and EPEC infection, based on results reported in this study as well as previous work by other investigators and our own laboratory. As can be seen from Additional file2: Table S1, no other metal quite matches zinc in the wide number of different beneficial effects it exerts on host cells and inhibitory effects it exerts on the pathogen, although copper also shows some beneficial effects. In contrast, manganese, iron, and nickel all have the potential to worsen one or more aspects of STEC’s interactions with host cell (Additional file2: Table S1).
EPEC adherence to host intestinal cells is heaviest in the ileum and cecum, and STEC adheres most strongly in the cecum and large intestine. Therefore, drugs or metals with limited absorption in the upper gastrointestinal tract would be ideal candidates for intervening at Phases 1 or 2 of Figure 7, because they would have to attain sufficient concentrations in the lumen of the distal gut; zinc salts fall into this category.
In the 3rd phase of Figure 7, Stx which has crossed the epithelial barrier binds to and begins to kill susceptible host cells, especially endothelial cells. Figure 7, lower portion, shows a higher power view of an intestinal blood vessel which has been affected by Stx2, showing adherence of polymorphonuclear leukocytes on the lumen of the endothelium (green arrows), as well as leukocytes which have been recruited into the wall of the vessel itself (blue arrow, showing a true vasculitis). When a similar process occurs in blood vessels elsewhere severe extra-intestinal complications can ensue. It appears that more research will be needed before we can declare we have drugs capable of blocking the 3rd Phase of Stx action[14, 65], and Additional file2: Table S1.
Figure 7 illustrates possible points at which metals might act after STEC enters the intestinal tract of the host. Metals which prove too toxic to use in vivo in humans might still find use, however, in the “pre-ingestion” phase of STEC, i.e., in agricultural practices, during germination of sprouts, or during food processing to limit STEC adherence to fresh foods or block virulence. Indeed, copper has already attracted attention for its antimicrobial properties in this regard[78, 79]. Divalent metals deserve additional research attention as inhibitors of bacterial virulence and enhancers of host defenses.
We thank Dr. Jay Mellies, Reed College, Portland, OR, for the gift of reporter strains JLM281, JLM165, and KMTIR3. Thomas A. Veeder and Anushila Chatterjee also contributed to this research during their laboratory rotations. We thank the National Institutes of Health (NIH) for financial support via grants RO1 AI 81528 and AI R21 102212.
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