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.
KeywordsEnterohemorrhagic E. coli O157:H7 Hemolytic-uremic syndrome SOS response Diarrheal diseases Xanthine oxidase Manganese Copper
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.
- Bhutta ZA, Bird SM, Black RE, Brown KH, Gardner JM, Hidayat A, Khatun F, Martorell R, Ninh NX, Penny ME, Rosado JL, Roy SK, Ruel M, Sazawal S, Shankar A: Therapeutic effects of oral zinc in acute and persistent diarrhea in children in developing countries: pooled analysis of randomized controlled trials. Am J Clin Nutr. 2000, 72: 1516-1522.PubMedGoogle Scholar
- Sazawal S, Black R, Bhan M, Bhandari N, Sinha A, Jalla S: Zinc supplementation in young children with acute diarrhea in India. N Engl J Med. 1995, 333: 839-844.View ArticlePubMedGoogle Scholar
- Patel A, Mamtani M, Dibley MJ, Badhoniya N, Kulkarni H: Therapeutic value of zinc supplementation in acute and persistent diarrhea: a systematic review. PLoS One. 2010, 5: e10386-PubMed CentralView ArticlePubMedGoogle Scholar
- Gabbianelli R, Scotti R, Ammendola S, Petrarca P, Nicolini L, Battistoni A: Role of ZnuABC and ZinT in Escherichia coli O157:H7 zinc acquisition and interaction with epithelial cells. BMC Microbiol. 2011, 11: 36-PubMed CentralView ArticlePubMedGoogle Scholar
- Porcheron G, Garenaux A, Proulx J, Sabri M, Dozois CM: Iron, copper, zinc, and manganese transport and regulation in pathogenic Enterobacteria: correlations between strains, site of infection and the relative importance of the different metal transport systems for virulence. Front Cell Infect Microbiol. 2013, 3: 90-PubMed CentralView ArticlePubMedGoogle Scholar
- Prasad AS: Zinc: mechanisms of host defense. J Nutr. 2007, 137: 1345-1349.PubMedGoogle Scholar
- Karlsen TH, Sommerfelt H, Klomstad S, Andersen PK, Strand TA, Ulvik RJ, Åhrén C, Grewal HMS: Intestinal and systemic immune responses to an oral cholera toxoid B subunit whole-cell vaccine administered during zinc supplementation. Infect Immun. 2003, 71: 3909-3913.PubMed CentralView ArticlePubMedGoogle Scholar
- Wellinghausen N, Martin M, Rink L: Zinc inhibits interleukin-1-dependent T cell stimulation. Eur J Immunol. 1997, 27: 2529-2535.View ArticlePubMedGoogle Scholar
- Schlesinger L, Arevalo M, Arredondo S, Lonnerdal B, Stekel A: Zinc supplementation impairs monocyte function. Acta Paediatr. 1993, 82: 734-738.View ArticlePubMedGoogle Scholar
- Provinciali M, Montenovo A, Stefano G, Colombo M, Daghetta L, Cairati M, Veroni C, Cassino R, Torre FD, Fabris N: Effect of zinc or zinc plus arginine supplementation on antibody titre and lymphocyte subsets after influenza vaccination in elderly subjects: a randomized controlled trial. Age Ageing. 1998, 27: 715-722.View ArticlePubMedGoogle Scholar
- Crane J, Naeher T, Shulgina I, Zhu C, Boedeker E: Effect of zinc in enteropathogenic Escherichia coli infection. Infect Immun. 2007, 75: 5974-5984.PubMed CentralView ArticlePubMedGoogle Scholar
- Crane JK, Byrd IW, Boedeker EC: Virulence inhibition by zinc in shiga-toxigenic escherichia coli. Infect Immun. 2011, 79: 1696-PubMed CentralView ArticlePubMedGoogle Scholar
- Medeiros P, Bolick D, Roche J, Noronha F, Pinheiro C, Kolling G, Guerrant R: The micronutrient zinc inhibits EAEC strain 042 adherence, biofilm formation, virulence gene expression and epithelial cytokine responses benefiting the infected host. Virulence. 2013, 4: 624-633.PubMed CentralView ArticlePubMedGoogle Scholar
- Mukhopadhyay S, Linstedt AD: Manganese blocks intracellular trafficking of shiga toxin and protects against shiga toxicosis. Science. 2012, 335: 332-335.View ArticlePubMedGoogle Scholar
- Frank C, Werber D, Cramer JP, Askar M, Faber M, Heiden M, Bernard H, Fruth A, Prager R, Spode A, Wadl M, Zoufaly A, Jordan S, Kemper MJ, Follin P, Mueller L, King LA, Rosner B, Buchholz U, Stark K, Krause G: Epidemic profile of shiga-toxin-producing escherichia coli O104:H4 outbreak in Germany. N Eng J Med. 2011, 365: 1771-1780.View ArticleGoogle Scholar
- Buchholz U, Bernard H, Werber D, Bohmer MM, Remschmidt C, Wilking H, Delere Y, an der Heiden M, Adlhoch C, Dreesman J, Ehlers J, Ethelberg S, Faber M, Frank C, Fricke G, Greiner M, Hohle M, Ivarsson S, Jark U, Kirchner M, Koch J, Krause G, Luber P, Rosner B, Stark K, Kuhne M: German outbreak of Escherichia coli O104:H4 associated with sprouts. N Engl J Med. 2011, 365: 1763-1770.View ArticlePubMedGoogle Scholar
- Gould LH, Mody RK, Ong KL, Clogher P, Cronquist AB, Garman KN, Lathrop S, Medus C, Spina NL, Webb TH, White PL, Wymore K, Gierke RE, Mahon BE, Griffin PM: Increased recognition of non-O157 Shiga toxin-producing Escherichia coli infections in the United States during 2000–2010: epidemiologic features and comparison with E. coli O157 infections. Foodborne Pathog Dis. 2013, 10: 453-460.View ArticlePubMedGoogle Scholar
- Kimmitt P, Harwood C, Barer M: Toxin gene expression by Shiga toxin-producing Escherichia coli: the role of antibiotics and the bacterial SOS response. Emerg Infect Dis. 2000, 6: 458-466.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang X, McDaniel A, Wolf L, Keusch G, Waldor M, Acheson D: Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice. J Infect Dis. 2000, 181: 664-670.View ArticlePubMedGoogle Scholar
- Colic E, Dieperink H, Titlestad K, Tepel M: Management of an acute outbreak of diarrhoea-associated haemolytic uraemic syndrome with early plasma exchange in adults from southern Denmark: an observational study. Lancet. 2011, 378: 1089-1093.View ArticlePubMedGoogle Scholar
- Andreoli SP, Trachtman H, Acheson DWK, Siegler RL, Obrig TG: Hemolytic uremic syndrome: epidemiology, pathophysiology, and therapy. Pediatr Nephrol. 2002, 17: 293-298.View ArticlePubMedGoogle Scholar
- Wagner PL, Acheson DWK, Waldor MK: Human neutrophils and their products induce shiga toxin production by enterohemorrhagic escherichia coli. Infect Immun. 2001, 69: 1934-1937.PubMed CentralView ArticlePubMedGoogle Scholar
- Crane JK, Naeher TM, Broome JE, Boedeker EC: Role of host xanthine oxidase in infection Due to enteropathogenic and shiga-toxigenic escherichia coli. Infect Immun. 2013, 81: 1129-1139.PubMed CentralView ArticlePubMedGoogle Scholar
- Mellies JL, Haack KR, Galligan DC: SOS regulation of the type III secretion system of enteropathogenic Escherichia coli. J Bacteriol. 2007, 189: 2863-PubMed CentralView ArticlePubMedGoogle Scholar
- Mellies JL, Elliott SJ, Sperandio V, Donnenberg MS, Kaper JB: The Per regulon of enteropathogenic Escherichia coli : identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler). Mol Microbiol. 1999, 33: 296-306.View ArticlePubMedGoogle Scholar
- Haack KR, Robinson CL, Miller KJ, Fowlkes JW, Mellies JL: Interaction of Ler at the LEE5 (tir) operon of enteropathogenic Escherichia coli. Infect Immun. 2003, 71: 384-392.PubMed CentralView ArticlePubMedGoogle Scholar
- Mellies J, Thomas K, Turvey M, Evans N, Crane J, Boedeker EC, Benison G: Zinc-induced envelope stress diminishes type III secretion in enteropathogenic Escherichia coli. BMC Microbiol. 2012, 12: 123-PubMed CentralView ArticlePubMedGoogle Scholar
- Acheson DWK, Moore R, De Breucker S, Lincicome L, Jacewicz M, Skutelsky E, Keusch GT: Translocation of Shiga toxin across polarized intestinal cells in tissue culture. Infect Immun. 1996, 64: 3294-3300.PubMed CentralPubMedGoogle Scholar
- In J, Lukyanenko V, Foulke-Abel J, Hubbard AL, Delannoy M, Hansen A-M, Kaper JB, Boisen N, Nataro JP, Zhu C: Serine protease EspP from enterohemorrhagic escherichia coli is sufficient to induce shiga toxin macropinocytosis in intestinal epithelium. PLoS One. 2013, 8: e69196-PubMed CentralView ArticlePubMedGoogle Scholar
- Malyukova I, Murray KF, Zhu C, Boedeker E, Kane A, Patterson K, Peterson JR, Donowitz M, Kovbasnjuk O: Macropinocytosis in Shiga toxin 1 uptake by human intestinal epithelial cells and transcellular transcytosis. Am J Physiol. 2008, 296: G78-G92.Google Scholar
- Griffith KL, Jr Wolf RE: Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays. Biochem Biophys Res Commun. 2002, 290: 397-402.View ArticlePubMedGoogle Scholar
- Wang N, Wang G, Hao J, Ma J, Wang Y, Jiang X, Jiang H: Curcumin ameliorates hydrogen peroxide-induced epithelial barrier disruption by upregulating heme oxygenase-1 expression in human intestinal epithelial cells. Dig Dis Sci. 2012, 57: 1792-1801.View ArticlePubMedGoogle Scholar
- Yu W, Beaudry S, Negoro H, Boucher I, Tran M, Kong T, Denker BM: H2O2 activates G protein, α 12 to disrupt the junctional complex and enhance ischemia reperfusion injury. Proc Natl Acad Sci USA. 2012, 109: 6680-6685.PubMed CentralView ArticlePubMedGoogle Scholar
- Rodriguez P, Darmon N, Chappuis P, Candalh C, Blaton MA, Bouchaud C, Heyman M: Intestinal paracellular permeability during malnutrition in guinea pigs: effect of high dietary zinc. Gut. 1996, 39: 416-422.PubMed CentralView ArticlePubMedGoogle Scholar
- Jepson MA: Disruption of epithelial barrier function by H2O2: distinct responses of Caco-2 and Madin-Darby canine kidney (MDCK) strains. Cell Mol Biol (Noisy-le-Grand). 2003, 49: 101-112.Google Scholar
- Peng L, He Z, Chen W, Holzman I, Lin J: Effects of butyrate on intestinal barrier function in a Caco-2 cell monolayer model of intestinal barrier. Pediatr Res. 2007, 61: 37-41.View ArticlePubMedGoogle Scholar
- Velazquez OC, Lederer HM, Rombeau JL: Butyrate and the colonocyte. Production, absorption, metabolism, and therapeutic implications. Adv Exp Med Biol. 1997, 427: 123-134.View ArticlePubMedGoogle Scholar
- Bielaszewska M, Idelevich EA, Zhang W, Bauwens A, Schaumburg F, Mellmann A, Peters G, Karch H: Effects of antibiotics on shiga toxin 2 production and bacteriophage induction by epidemic escherichia coli O104:H4 strain. Antimicrob Agents Chemother. 2012, 56: 3277-3282.PubMed CentralView ArticlePubMedGoogle Scholar
- Spears K, Roe A, Gally D: A comparison of enteropathogenic and enterohaemorraghic Escherichia coli pathogenesis. FEMS Microbiol Lett. 2006, 255: 187-202.View ArticlePubMedGoogle Scholar
- Elliott S, Sperandio V, Giron J, Shin S, Mellies J, Wainwright L, Jutcheson S, McDaniel T, Kaper J: The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun. 2000, 68: 6115-6126.PubMed CentralView ArticlePubMedGoogle Scholar
- Sperandio V, Mellies JL, Nguyen W, Shin S, Kaper JB: Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli. Proc Natl Acad Sci USA. 1999, 96: 15196-15201.PubMed CentralView ArticlePubMedGoogle Scholar
- Łoś JM, Łoś M, Węgrzyn A, Węgrzyn G: Hydrogen peroxide-mediated induction of the Shiga toxin-converting lambdoid prophage ST2-8624 in Escherichia coli O157:H7. FEMS Immunol Med Microbiol. 2010, 58: 322-329.PubMedGoogle Scholar
- Vareille M, de Sablet T, Hindré T, Martin C, Gobert A: Nitric oxide inhibits Shiga-toxin synthesis by enterohemorrhagic Escherichia coli. Proc Natl Acad Sci USA. 2007, 104: 10199-10204.PubMed CentralView ArticlePubMedGoogle Scholar
- Fuchs S, Muhldorfer I, Donohue-Rolfe A, Kerenyi M, Emody L, Alexiev R, Nenkov P, Hacker J: Influence of RecA on in vivo virulence and Shiga toxin 2 production in Escherichia coli pathogens. Microb Pathog. 1999, 27: 13-23.View ArticlePubMedGoogle Scholar
- Kaneko Y, Thoendel M, Olakanmi O, Britigan B, Singh P: The transition metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial and antibiofilm activity. J Clin Invest. 2007, 117: 877-888.PubMed CentralView ArticlePubMedGoogle Scholar
- DuPont H, Sullivan P, Pickering L, Haynes G, Ackerman P: Symptomatic treatment of diarrhea with bismuth subsalicylate among students attending a Mexican university. Gastroenterology. 1977, 73: 715-718.PubMedGoogle Scholar
- Johnson P, Ericsson C, DuPont H, Morgan D, Bitsura J, Wood L: Comparison of loperamide with bismuth subsalicylate for the treatment of acute travelers’ diarrhea. JAMA. 1986, 255: 757-760.View ArticlePubMedGoogle Scholar
- Xie Y, He Y, Irwin PL, Jin T, Shi X: Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni. Appl Environ Microbiol. 2011, 77: 2325-2331.PubMed CentralView ArticlePubMedGoogle Scholar
- Mellies JL, Barron AMS, Carmona AM: Enteropathogenic and Enterohemorrhagic Escherichia coli Virulence Gene Regulation. Infect Immun. 2007, 75: 4199-4210.PubMed CentralView ArticlePubMedGoogle Scholar
- Outten C, O’Halloran T: Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis. Science. 2001, 292: 2488-2491.View ArticlePubMedGoogle Scholar
- Outten CE, Outten FW, O’Halloran TV: DNA distortion mechanism for transcriptional activation by ZntR, a Zn(II)-responsive MerR homologue in escherichia coli. J Biol Chem. 1999, 274: 37517-37524.View ArticlePubMedGoogle Scholar
- Yamamoto K, Ishihama A: Transcriptional response of escherichia coli to external zinc. J Bacteriol. 2005, 187: 6333-6340.PubMed CentralView ArticlePubMedGoogle Scholar
- Torres AG, Payne SM: Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol. 1997, 23: 825-833.View ArticlePubMedGoogle Scholar
- Lim J, Lee KM, Kim SH, Kim Y, Kim SH, Park W, Park S: YkgM and ZinT proteins are required for maintaining intracellular zinc concentration and producing curli in enterohemorrhagic Escherichia coli (EHEC) O157:H7 under zinc deficient conditions. Int J Food Microbiol. 2011, 149: 159-170.View ArticlePubMedGoogle Scholar
- Bower S, Rosenthal KS: The bacterial cell wall: the armor, artillery, and achilles heel. Infect Dis Clin Pract. 2006, 14: 309-317. 310.1097/1001.idc.0000240862.0000274564.0000240857View ArticleGoogle Scholar
- Vogt SL, Raivio TL: Just scratching the surface: an expanding view of the Cpx envelope stress response. FEMS Microbiol Lett. 2012, 326: 2-11.View ArticlePubMedGoogle Scholar
- Gielda LM, DiRita VJ: Zinc competition among the intestinal microbiota. MBio. 2012, 3: 1-7.View ArticleGoogle Scholar
- Bratz K, Golz G, Riedel C, Janczyk P, Nockler K, Alter T: Inhibitory effect of high-dosage zinc oxide dietary supplementation on Campylobacter coli excretion in weaned piglets. J Appl Microbiol. 2013, 115: 1194-1202.View ArticlePubMedGoogle Scholar
- Zhang P, Carlsson M, Schneider N, Duhamel G: Minimal prophylactic concentration of dietarry zinc compounds in a mouse model off swine dysentery. Anim Health Res Rev. 2001, 2: 67-74.PubMedGoogle Scholar
- Roselli M, Finamore A, Garaguso I, Britti MS, Mengheri E: Zinc oxide protects cultured enterocytes from the damage induced by Escherichia coli. J Nutr. 2003, 133: 4077-4082.PubMedGoogle Scholar
- Botella H, Peyron P, Levillain F, Poincloux R, Poquet Y, Brandli I, Wang C, Tailleux L, Tilleul S, Charrière GM, Waddell Simon J, Foti M, Lugo-Villarino G, Gao Q, Maridonneau-Parini I, Butcher Philip D, Castagnoli Paola R, Gicquel B, de Chastellier C, Neyrolles O: Mycobacterial P1-type ATPases mediate resistance to zinc poisoning in human macrophages. Cell Host Microbe. 2011, 10: 248-259.PubMed CentralView ArticlePubMedGoogle Scholar
- Giblin LJ, Chang CJ, Bentley AF, Frederickson C, Lippard SJ, Frederickson CJ: Zinc-secreting paneth cells studied by ZP fluorescence. J Histochem Cytochem. 2006, 54: 311-316.View ArticlePubMedGoogle Scholar
- Dinsdale D: Ultrastructural localization of zinc and calcium within the granules of rat Paneth cells. J Histochem Cytochem. 1984, 32: 139-145.View ArticlePubMedGoogle Scholar
- Patel A, Dibley M, Mamtani M, Badhoniya N, Kulkarni H: Influence of zinc supplementation in acute diarrhea differs by the isolated organism. Int J Pediatr. 2010, 2010: 671587-PubMed CentralView ArticlePubMedGoogle Scholar
- Gaston MA, Pellino CA, Weiss AA: Failure of manganese to protect from shiga toxin. PLoS One. 2013, 8: e69823-PubMed CentralView ArticlePubMedGoogle Scholar
- Mukhopadhyay S, Redler B, Linstedt AD: Shiga toxin–binding site for host cell receptor GPP130 reveals unexpected divergence in toxin-trafficking mechanisms. Mol Biol Cell. 2013, 24: 2311-2318.PubMed CentralView ArticlePubMedGoogle Scholar
- Beltrametti F, Kresse AU, Guzmán CA: Transcriptional regulation of the esp genes of enterohemorrhagic escherichia coli. J Bacteriol. 1999, 181: 3409-3418.PubMed CentralPubMedGoogle Scholar
- Moreno JA, Yeomans EC, Streifel KM, Brattin BL, Taylor RJ, Tjalkens RB: Age-dependent susceptibility to manganese-induced neurological dysfunction. Toxicol Sci. 2009, 112: 394-PubMed CentralView ArticlePubMedGoogle Scholar
- Imamovic L, Muniesa M: Characterizing RecA-independent induction of shiga toxin2-encoding phages by EDTA treatment. PLoS One. 2012, 7: e32393-PubMed CentralView ArticlePubMedGoogle Scholar
- Rao RK, Baker RD, Baker SS, Gupta A, Holycross M: Oxidant-induced disruption of intestinal epithelial barrier function: role of protein tyrosine phosphorylation. Am J Physiol. 1997, 273: G812-G823.PubMedGoogle Scholar
- Perez LM, Milkiewicz P, Ahmed-Choudhury J, Elias E, Ochoa JE, Sanchez Pozzi EJ, Coleman R, Roma MG: Oxidative stress induces actin-cytoskeletal and tight-junctional alterations in hepatocytes by a Ca2+ -dependent, PKC-mediated mechanism: protective effect of PKA. Free Radic Biol Med. 2005, 40: 2005-2017.View ArticleGoogle Scholar
- Demehri F, Barrett M, Ralls M, Miyasaka E, Feng Y, Teitelbaum D: Intestinal epithelial cell apoptosis and loss of barrier function in the setting of altered microbiota with enteral nutrient deprivation. Front Cell Microbiol. 2013, 3: 1-15.Google Scholar
- Bleich M, Shan Q, Himmerkus N: Calcium regulation of tight junction permeability. Ann N Y Acad Sci. 2012, 1258: 93-99.View ArticlePubMedGoogle Scholar
- Ma TY, Tran D, Hoa N, Nguyen D, Merryfield M, Tarnawski A: Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: role of cytoskeletal involvement. Microsc Res Tech. 2000, 51: 156-168.View ArticlePubMedGoogle Scholar
- Finamore A, Massimi M, Conti Devirgiliis L, Mengheri E: Zinc deficiency induces membrane barrier damage and increases neutrophil transmigration in Caco-2 cells. J Nutr. 2008, 138: 1664-1670.PubMedGoogle Scholar
- Wang X, Valenzano MC, Mercado JM, Zurbach EP, Mullin JM: Zinc supplementation modifies tight junctions and alters barrier function of CACO-2 human intestinal epithelial layers. Dig Dis Sci. 2013, 58: 77-87.View ArticlePubMedGoogle Scholar
- Moran JR, Lewis JC: The effects of severe zinc deficiency on intestinal permeability: an ultrastructural study. Pediatr Res. 1985, 19: 968-973.View ArticlePubMedGoogle Scholar
- Warnes SL, Caves V, Keevil CW: Mechanism of copper surface toxicity in Escherichia coli O157:H7 and Salmonella involves immediate membrane depolarization followed by slower rate of DNA destruction which differs from that observed for Gram-positive bacteria. Environ Microbiol. 2012, 14: 1730-1743.View ArticlePubMedGoogle Scholar
- Wilks SA, Michels H, Keevil CW: The survival of Escherichia coli O157 on a range of metal surfaces. Int J Food Microbiol. 2005, 105: 445-454.View ArticlePubMedGoogle Scholar
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