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. 2011 Sep 1;2(5):267–273. doi: 10.4161/gmic.2.5.18256

Polyethylene glycol diminishes pathological effects of Citrobacter rodentium infection by blocking bacterial attachment to the colonic epithelia

Wentao Qi 1, Suhasini Joshi 1, Christopher R Weber 2, Ramesh K Wali 1, Hemant K Roy 1, Suzana D Savkovic 1,✉
PMCID: PMC3242793  PMID: 22067938

Abstract

Infections from enteric bacteria such as enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are a public health threat worldwide. EPEC and EHEC are extracellular pathogens, and their interaction with host surface receptors is critical to the infection process. We previously demonstrated that polyethylene glycol (PEG) downregulates surface receptors in intestinal cells. Here we show that PEG decreases β1-integrin, the surface receptor in intestinal cells that is critical for EPEC and EHEC attachment. We hypothesized that PEG would inhibit the attachment of these enteric pathogens to host cells and improve clinical signs of infection. We found that attachment of the mouse enteric pathogen Citrobacter rodentium, which belongs to the same group of pathogens as EPEC and EHEC, was attenuated by the concurrent presence of PEG. Pretreatment with PEG, without concurrent presence during infection, also reduced bacterial attachment. This finding was further supported in vivo such as that PEG administered by gavage daily during infection as well as prior to infection significantly decreased C. rodentium in the colon and improved the appearance of the infected colon in mice. In addition, PEG decreased the β1-integrin in colonic mucosa and reduced the C. rodentium-induced activation of epidermal growth factor receptors. PEG also significantly reduced infection-induced colonic inflammation. Finally, PEG efficiently reduced C. rodentium shedding from the colon during infection. In conclusion, PEG can be an efficient and safe preventive agent against EPEC and EHEC infections.

Key words: PEG, prophylaxis, enteropathogenic bacteria, C. rodentium, infection

Introduction

Enteropathogenic bacterial infections are one of the most common health threats worldwide. Such infections are common in developing countries, and their impact in industrialized countries has increased dramatically in the past few decades as a result of food processing and travel.1,2 Virulent strains of Escherichia coli such as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are responsible for a significant proportion of bacterial enteric infections.3 Whereas infections with EPEC cause infantile diarrhea,4,5 EHEC, an emerging zoonotic pathogen, causes diarrhea that can lead to hemorrhagic colitis and hemolytic uremic syndrome.6,7 Although the mechanisms of their infection have come to be better understood over the past few decades,7,8 EPEC and EHEC remain serious threats to human health, demanding a new preventive approach.

Both EPEC and EHEC are extracellular pathogens that induce attaching and effacing (A/E) lesions in host cells.9 Another pathogen from the A/E group with significant genetic similarity to EPEC and EHEC is the mouse pathogen Citrobacter rodentium, which is frequently used as a model for studying mechanisms of infection and host response.10,11 These pathogens infect host cells using a type III secretion system (T3SS) encoded by the locus of enterocyte effacement pathogenicity island.12,13 T3SS is needle-like extension that attaches to the host cell membrane, allowing bacteria to directly translocate effector molecules to the cell.14,15 During infection, these bacterial effector molecules interact with host cell molecules. For example, the bacterial effector intimin interacts with β1-integrin,16–18 whereas the effector OspE interacts with integrin-linked kinase.18 These interactions with integrin generate signals in host cells18 that disrupt the intestinal barrier.18,19 Inhibiting interaction with integrin may be an efficient strategy for preventing enteric infections.

Polyethylene glycol (PEG), a polymer of ethylene oxide, is a nontoxic agent available commercially in various molecular weights. Moderate intake of low-molecular weight PEG (17–25 g per day) provides symptomatic relief of chronic constipation.20,21 Because of its outstanding safety record, PEG is often used as a carrier for systemic pharmaceutical drugs.22,23 We previously demonstrated that low-molecular weight PEG has chemopreventive properties against colorectal cancer through the downregulation of surface receptors critical to tumor growth.24 Because surface integrins are essential for EPEC and EHEC attachment, we hypothesized that PEG would decrease the amount of integrins in the host intestinal membrane and as a consequence attenuate bacterial attachment and prevent infection.

This study demonstrates that PEG donwnregulates β1-integrin and effectively inhibits C. rodentium attachment to colonic epithelia whether introduced prior to infection or during infection. PEG gavages (0.4 g/kg of body weight) attenuate C. rodentium-induced signaling pathways in colonic epithelia, attenuate inflammation and improve the clinical signs of infection. Moreover, PEG inhibits C. rodentium shedding in the stool and thus has the potential to prevent the spread of infection to other hosts. Based on these findings, we speculate that low-molecular weight PEG can serve as an efficient agent against EPEC and EHEC infections.

Results

PEG downregulates β1-integrin and reduces C. rodentium attachment to mouse colonic cells.

Infection with A/E pathogens is mediated by the interaction of their T3SS apparatus members with host cell surface β1-integrin.16–18 Because our previous findings demonstrated that PEG downregulates surface receptors on intestinal cells,24 we hypothesized that PEG would decrease the amount of surface β1-integrin and thus attenuate attachment of enteric pathogens from the A/E group. This study showed that PEG reduces β1-integrin (Fig. 1A) and decreases C. rodentium attachment to mouse CMT-93 monolayers (Fig. 1B). The presence of PEG did not decrease C. rodentium exponential growth (Fig. 1C) or its ability to attach (Fig. 1D,f), suggesting that bacteria were unaffected by this compound. However, exposure of intestinal monolayers to PEG prior to infection without the concurrent presence of PEG during infection was sufficient to reduce C. rodentium attachment (Fig. 1D,e). Moreover, PEG added after infection (2 h) decreases attachment of C. rodentium to the host cells (Fig. 1D,g). These data show that C. rodentium attachment to mouse intestinal monolayers was protected by adding PEG prior, during or after initial infection. Staining of tight junction protein occludin showed that PEG at this condition did not overtly disrupt the epithelium. Next we assessed the role of PEG in protecting against C. rodentium infection in vivo.

Figure 1.

Figure 1

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Polyethylene glycol (PEG) decreases the amount of β1-integrin and inhibits C. rodentium attachment to host cells. (A) Protein from intestinal HT-29 cells treated with 5% PEG for various lengths of time was immunoblotted with antibodies against β1-integrin and actin (loading control). During PEG treatment, the amount of β1-integrin in HT-29 cells decreased. (B) CMT-93 monolayers were incubated with various concentrations of PEG for 3 h and infected with C. rodentium for 5 h in the presence of PEG. Diluted aliquots of cell lysates were plated on LB agar plates. The graph shows that 5% and 10% PEG significantly attenuated bacterial attachment to CMT-93 monolayers. This experiment was repeated three times; one representative experiment is shown (n = 4, *p < 0.05). (C) C. rodentium culture diluted in fresh tissue medium (serum and antibiotic free) was grown in the presence of 1%, 5% and 10% PEG. Bacterial growth, determined by OD660, was not affected by the presence of PEG (n = 6). (D) Concurrent presence of PEG during infection, pretreatment with PEG before infection or treatment with PEG after occurring infection decreased C. rodentium attachment to CMT-93 monolayers. Immunofluorescent staining of bacterial LPS and host cell occludin in monolayers with or without C. rodentium infection and with or without PEG (60x objective): (a) control, (b) PEG (5%) for 5 h, (c) C. rodentium infected 5 h, (d) monolayers treated with 5% PEG for 3 h and infected with C. rodentium for 5 h (e) monolayers treated with 5% PEG for 3 h, washed and infected with C. rodentium without the presence of PEG, (f) C. rodentium grown in the presence of 5% PEG for 3 h, washed and used to infect monolayers for 5 h, (g) PEG was added (final concentration of 5%) into monolayers infected with C. rodentium for 2 h.

PEG attenuates C. rodentium attachment to colonic mucosa of mice.

C. rodentium is a mouse enteric pathogen that attaches to colonic mucosa, causing diarrhea, inflammation and epithelial hyperproliferation.25–27 We assessed whether the administration of PEG in a concentration insufficient to induce diarrhea (0.4 g/kg of body weight) 20,21 improves clinical signs of C. rodentium infection in the mouse colon. Colonic stool pellets from infected mice that received concurrent PEG treatment (PEG-CR) or received PEG before infection [PEG(pre)-CR] appeared normal compared with those mice that were infected with C. rodentium alone (Fig. 2A). The colon contents of mice that received only PEG were similar to those of the control, with no signs of soft stool or diarrhea. Colonic weight increased in C. rodentium-infected mice (CR) (by 46 ± 3%), was significantly reduced when mice were treated with PEG (PEG-CR) and was attenuated when they received PEG only before infection [PEG(pre)-CR] (Fig. 2B). Furthermore, the number of C. rodentium colonies attached to colonic mucosa was assessed on MacConkey plates and confirmed by polymerase chain reaction as previously described in reference 28. Colonic mucosa of mice infected with C. rodentium with concurrent PEG treatment (PEG-CR) or only pretreatment with PEG [PEG(pre)-CR] showed a significant decrease in the number of colonies (CR: 58 ± 18 CFU × 103/g mucosal protein, PEG-CR: 4 ± 1 CFU × 103/g mucosal protein, PEG(pre)-CR: 15 ± 3 CFU × 103/g mucosal protein; Fig. 2C), confirming the in vivo data shown in Figure 1D. Together, these data support the fact that PEG efficiently reduces the attachment of C. rodentium to colonic mucosa and improves infected colons macroscopically.

Figure 2.

Figure 2

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PEG improves the clinical appearance of the infected colon and reduces the number of C. rodentium colonies attached to colonic mucosa. Mice were divided into the following groups: control, PEG treated (PEG), C. rodentium infected (CR), C. rodentium infected with concurrent PEG treatment (PEG-CR), and PEG pretreated and then infected with C. rodentium without further presence of PEG [PEG(pre)-CR]. (A) The appearance of the colon was assessed for the various experimental groups 14 d post-infection (p.i.). In contrast to C. rodentium infection, treatment or pretreatment with PEG resulted in stool formation that was similar to that of the control. (B) The lengths and weights (g/cm) of colons cleaned of stool and extensively washed were expressed as a percentage of the change relative to the control. PEG reduced the amount of increased colonic weight due to C. rodentium infection. (C) Colons from the various experimental groups were cleaned, extensively washed and scraped mucosa was dissolved in lysis buffer. The aliquots of scraped mucosa were plated on MacConkey plates. The number of colonies was significantly lower for PEG or pretreatment with PEG. Data are represented by Log5 graph (n = 9, *p < 0.05).

PEG decreases C. rodentium-induced signals in colonic epithelia.

Findings that PEG reduces β1-integrin in colonic cells in vitro (Fig. 1A) led us to further assess the effect of PEG on reducing β1-integrin in colonic mucosa. When mice received PEG daily, expression of β1-integrin in colonic mucosa decreased significantly (control = 100 ± 6%, PEG = 38 ± 8%; Fig. 3A), further supporting the in vitro data in Figure 1A. We previously demonstrated that C. rodentium infection induces expression and activation of EGFR in mouse colonic mucosa.29 Thus, we further assessed whether PEG inhibits C. rodentium-induced EGFR activation in colonic epithelia. Increased expression of 208 ± 23% (and phosphorylation) of EGFR in colonic mucosa induced by C. rodentium infection (CR) was reduced to 81 ± 14% in the presence of PEG (PEG-CR) (Fig. 3B), and the EGFR level was similar to that in uninfected colonic mucosa. PEG alone decreases EGFR expression in vitro, as we have previously shown in reference 24, whereas pretreatment with PEG [PEG(pre)-CR] modestly reduces C. rodentium-induced EGFR expression. Taken together, these data support the fact that PEG is effective at inhibiting signals generated from the cell membrane induced by C. rodentium infection.

Figure 3.

Figure 3

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PEG inhibits signaling pathways induced by C. rodentium infection. (A) PEG decreased the amount of β1-integrin in colonic mucosa. Mice received PEG by gavage for 3 weeks and immunoblot from scraped mucosa showed decreased expression of β1-integrin relative to the control (n = 4, *p < 0.05). (B) Increased EGFR expression and phosphorylation in C. rodentium-infected (14 d.p.i.) colon was attenuated by PEG gavage. Protein from scraped colonic mucosa of various experimental groups control, PEG treated (PEG), C. rodentium infected (CR), C. rodentium infected with concurrent PEG treatment (PEG-CR) and PEG pretreated and then infected with C. rodentium without further presence of PEG [PEG(pre)-CR] was immunoblotted for EGFR and phosphorylated EGFR. The graph shows a densitometric analysis (n = 4, *p < 0.05).

PEG inhibits C. rodentium-induced inflammation in the mouse colon.

C. rodentium infection induces infiltration of inflammatory cells in the colon, causing an increase in colonic weight.26,30 Above data showed that presence of PEG attenuates this effect of C. rodentium (Fig. 2B), suggesting that PEG may inhibit inflammation induced by infection. Hematoxylin and eosin staining and inflammatory scoring revealed that C. rodentium infection (CR) induces infiltration of inflammatory cells into the lamina propria and epithelia and induces formation of crypt abscesses in the mouse colon (Fig. 4). Colons of infected mice treated concurrently with PEG (PEG-CR) showed some lymphocytes in the lamina propria, but no active intraepithelial inflammation was detected. When mice received PEG only prior to the infection [PEG(pre)-CR], inflammation was also reduced (Fig. 4). These data demonstrate histologically that PEG normalizes inflammation induced by C. rodentium in the mouse colon.

Figure 4.

Figure 4

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PEG attenuates C. rodentium-induced colonic inflammation. Colonic inflammation in mice was assessed in control, PEG treated (PEG), C. rodentium infected (CR), C. rodentium infected with concurrent PEG treatment (PEG-CR), and PEG pretreated and then infected withC. rodentium without further presence of PEG [PEG(pre)-CR]. Intensity of inflammation in colonic tissue stained with hematoxylin and eosin was blindly scored by a pathologist. Treatment with PEG significantly lowered colonic inflammation induced by C. rodentium infection (14 d.p.i.), and pretreatment with PEG was sufficient to reduce colonic inflammation (the number of mice in control, PEG treated and CR infected was six in each group, while in groups where CR infected were treated or pretreated with PEG the number of mice was nine, *p < 0.05).

C. rodentium shedding from the mouse colon is decreased by PEG.

As an extracellular pathogen, C. rodentium multiplies on the apical side of host cells and spreads to infect neighboring cells or is shed though the stool to infect other hosts.31 Because PEG reduced the number of C. rodentium colonies attached to colonic mucosa as shown in Figure 2C, we hypothesized that it would also decrease bacterial shedding into the stool. The concurrent presence of PEG (PEG-CR) significantly reduced the number of bacterial colonies shed via the stool at days 7 and 14 post-infection (Fig. 5A and B). Also, pre-treatment with PEG [PEG(pre)-CR] resulted in a decrease in the number of C. rodentium colonies in the stool (CR: 28.5 ± 7.8 × 106 CFU, PEG-CR: 0.6 ± 0.1 × 106 CFU, PEG(pre)-CR: 1.2 ± 0.4 × 106 CFU per g of stool at day 7). These data support that PEG effectively decreases bacterial shedding from mouse colon.

Figure 5.

Figure 5

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PEG decreases C. rodentium shedding in the stool. Stool was collected from the C. rodentium infected (CR), C. rodentium infected with concurrent PEG treatment (PEG-CR), and PEG pretreated and then infected with C. rodentium without further presence of PEG [PEG(pre)-CR] at days (A) 7 and (B) 14 d.p.i. The number of colonies grown on MacConkey plates was expressed on a Log5 scale graph (n = 9, *p < 0.05). During infection, the presence (PEG-CR) or pretreatment [PEG(pre)-CR] of PEG significantly reduced the number of bacterial colonies found in the stool compared with C. rodentium infection alone.

Discussion

Infections by A/E group bacteria such as EPEC and EHEC are a major health threat worldwide and contribute to mortality in both children and adults. In the past decade a number of studies have helped explain the mechanisms of such infections and host responses. However, these enteric pathogens remain a serious danger and have inspired the development of novel approaches for preventing infections. Here we used a C. rodentium mouse model to assess the ability of PEG to prevent infections by A/E pathogens. We showed that low-molecular weight PEG downregulates β1-integrin, a host cell surface receptor critical for A/E pathogen attachment.16–19 Pretreatment of colonic epithelia with PEG, concurrent presence of PEG during infection, or PEG treatment after the start of infection decreases the attachment of C. rodentium to colonic epithelia. We also presented evidence that PEG attenuates EGFR signaling and improves clinical signs of disease in mouse colon infected with C. rodentium. Moreover, PEG efficiently reduces bacterial shedding into the stool, which is critical for preventing the spread of infection to other hosts. All together, these findings demonstrate that PEG efficiently attenuates infection and the shedding of C. rodentium, pathogen from A/E group along with EPEC and EHEC.

It has been demonstrated that the binding of effector molecules from A/E pathogens to host cell proteins such as β1-integrin is a critical part of the infection process.17,18 During infection, β1-integrin relocalizes from the basolateral to the apical membrane to interact with bacterial intimin, which is essential for disruption of intestinal barrier function.19 This study demonstrates that PEG decreases the amount of β1-integrin in intestinal epithelia and that pretreatment with PEG is sufficient to reduce attachment of C. rodentium. It has been demonstrated that high-molecular weight PEG inhibits the attachment of Pseudomonas to host cells by forming a physical barrier between the bacteria and host cells;32 however, this type of PEG is not for systemic oral use. Moreover, Henry-Stanley and Wells demonstrated that low-molecular weight PEG inhibits the interactions of some strains of E. coli and Candida with the intestinal epithelium, but the mechanisms behind this were unclear.33 It is important to note that longer expose of colonic cells to low-molecular weight PEG inhibits proliferation;24 however, this in vitro study used a shorter incubation time and thus did not show any effect of PEG on cell viability which is supported by unaltered localization of tight junction occludin. Here we show that PEG-mediated disruption of bacterial attachment to host cells is associated with decreased β1-integrin expression, providing evidence of a possible mechanism.

Infection with C. rodentium induces signaling pathways in colonic epithelia, leading to proliferation and inflammation of colonic epithelia.26,30 EPEC and C. rodentium induce the activation of membrane EGFR in colonic epithelia;29,34 which is also activated by inflammatory stimuli.35 This study shows that PEG inhibits C. rodentium-induced EGFR expression and activation. Because PEG pretreatment inefficiently inhibits C. rodentium-induced EGFR activation, we speculate that even a small number of bacteria attached to colonic mucosa are sufficient to activate EGFR. Another characteristic of EPEC, EHEC and C. rodentium infections is the infiltration of inflammatory cells into intestinal epithelia.26,30,36,37 This study showed that the presence of PEG efficiently inhibits C. rodentium-induced inflammation in the mouse colon, further supporting the inhibitory role of PEG. Also, these extracellular pathogens multiply on the surface of infected cells, infecting neighboring cells, or are shed via the stool to infect other hosts.31 We found that PEG has the ability to protect the host from bacterial infection (as illustrated by the dramatic reduction in C. rodentium-induced colonic inflammation) and reduces the presence of bacteria in the stool, thus likely can reduce spread of infections to other host. Prevention and treatment of EHEC and EPEC infections has been the subject of many studies. Recent attempts at using antibodies from hyperimmunized cows did not show significant therapeutic benefit in the treatment of EPEC and ETEC infections.38 One approach to treating EHEC infection involves Gb3 receptor analogs that bind shiga toxins produced by EHEC.39 However, this treatment is expensive and is not applicable for prophylaxis during outbreaks. Furthermore, it has been suggested that glycomacropeptide inhibits EHEC infection by binding to bacteria, thus preventing attachment to the host cell,40 but the effect of this compound on intestinal bacterial flora is not clear. This study shows that PEG has no effect on bacteria but that the modified surface membrane of host cells instead leads to resistance to bacterial attachment.

In conclusion, PEG efficiently prevents C. rodentium attachment to host cells, improves clinical signs of infection, and inhibits bacterial shedding in the stool. This study supports a prophylactic role for PEG against infection with enteropathogenic bacteria that needs to be further assessed for the specific pathogens EPEC and EHEC. PEG may be effective at curtailing EPEC and EHEC outbreaks.

Material and Methods

Tissue culture.

Mouse colonic CMT-93 cells (American Type Culture Collection) were propagated in DMEM medium (Gibco) with 10% fetal bovine serum (Gibco) at 37°C with 5% CO2. Human colon HT-29 cells (American Type Culture Collection) were propagated in McCoy's 5A medium (Sigma) supplemented with 10% fetal bovine serum (Gibco). Monolayers were deprived of serum overnight prior to experiments.

PEG treatment.

For in vitro studies, 5% PEG (molecular weight = 3,350; Sigma) dissolved in serum-free, antibiotic-free DMEM was added to monolayers 3 h before infection. For in vivo studies, 5% PEG (200 µl) per 0.4 g/kg of body weight, which is equivalent to the 17 g per day used to relieve chronic constipation in humans,20,21 was introduced to mice daily by oral gavage for 7 d prior to infection. One experimental group continually received PEG daily during infection, whereas the other group PEG(pre) did not.

Citrobacter rodentium strain.

C. rodentium (formerly Citrobacter freundii biotype 4,280 and Citrobacter genomospecies 9) was obtained from David Schauer (Massachusetts Institute of Technology). Bacteria were stored at −80°C in Luria-Bertani broth (LB) containing 50% (vol/vol) glycerol. C. rodentium cultures were grown overnight in LB broth at 37°C. Bacteria were diluted (1:33) in DMEM (serum free, antibiotic free) and allowed to grow to mid-log growth phase (OD660 nm ∼ 0.5) overnight.

C. rodentium infection in vitro.

Bacterial pellets, grown as described above, were resuspended in serum- and antibiotic-free DMEM media. Approximately 4 × 107 bacteria were applied to mouse intestinal epithelial monolayers grown in 24-well plates. Infected monolayers were incubated at 37°C in 5% CO2.

C. rodentium infection in vivo.

C57BL/6J mice (4–6 weeks old) were used for in vivo studies. Mice were infected with C. rodentium as previously described in reference 41, in accordance with approved animal care protocols of NorthShore University HealthSystem Research Institute.

Bacterial colony counts.

Monolayers infected with bacteria were washed five times with phosphate-buffered saline and lysed with 1% Triton X-100, and diluted aliquots were plated on Luria Bertani (LB) agar plates. For in vivo experiments, scraped colonic mucosa was lysed, the fresh stool particles homogenized in phosphate-buffered saline and aliquots plated on MacConkey agar plates. C. rodentium colonies were recognized as pink with a white rim after incubation overnight at 37°C. C. rodentium colonies grown on LB or MacConkey plates were randomly selected and confirmed by polymerase chain reaction as previously described in reference 28.

Immunofluorescent staining.

Infected monolayers were extensively washed and fixed with 3.7% paraformaldehyde. Monolayers were incubated with anti-lipopolysaccharide (LPS) antibody (Santa Cruz Biotechnology) and anti-occludin antibody (Santa Cruz Biotechnology) following incubation with Alexa 488 conjugated secondary antibody (Invitrogen). Coverslips were mounted using Prolong Gold antifade reagent (Invitrogen), and images were captured using a Nikon Confocal Microscope C1 and analyzed with EZ-C1 software (Nikon).

Protein extraction.

Total protein was extracted from scraped mucosa using cell lysis buffer (Cell Signaling Technology) supplemented with a protease inhibitor cocktail (Sigma). Protein concentration was determined using the Bradford assay (Bio-Rad), and aliquots were stored at −20°C.

Immunoblot.

Total protein (40 µg) was separated by sodium dodecyl sulfate PAGE and transferred to nitrocellulose membranes (Bio-Rad) as previously described in reference 29. Primary antibodies against EGFR, pEGFR, β-actin (Santa Cruz Biotechnology), and β1-integrin (Cell Signaling Technology) were used. Secondary antibodies linked to horseradish peroxidase (Cell Signaling Technology) were visualized using electrochemiluminescence plus protein gel blotting detection reagents (GE Healthcare).

Histological analysis.

Distal segments of colons were fixed in formalin and embedded in paraffin, and tissue sections (5 µm thick) were stained with hematoxylin and eosin. The degree of inflammation was evaluated according to the following criteria: 0 = no architectural distortion or infiltrates; 1 = architectural distortion, increased lamina propria lymphs, no activity; 2 = increased lamina propria granulocytes without definite intraepithelial granulocytes (i.e., no activity); 3 = intraepithelial granulocytes (i.e., activity) without crypt abscesses; 4 = crypt abscesses in less than 50% of crypts; 5 = crypt abscesses in more than 50% of crypts or erosion/ulceration.

Statistical analysis.

Data were analyzed using Student's t-test and expressed as means ± SEM. Differences were considered significant at p < 0.05.

Acknowledgements

This work was supported in part by a Senior Investigator Award from the Crohn's and Colitis Foundation of America (CCFA #1953), a NorthShore University HealthSystem and University of Chicago collaborative grant, and National Institutes of Health Grant (C.R.W.) K08DK088953. Drs. Savkovic, Wali and Roy are shareholders in Pegasus BioSolutions LLC.

Abbreviations

EPEC

enteropathogenic E. coli

EHEC

enterohemorrhagic E. coli

A/E

attaching and effacing

T3SS

type III secretion system

PEG

polyethylene glycol

EGFR

epithermal growth factor receptors

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest has been disclosed.

References

  • 1.DuPont HL. The growing threat of foodborne bacterial enteropathogens of animal origin. Clin Infect Dis. 2007;45:1353–1361. doi: 10.1086/522662. [DOI] [PubMed] [Google Scholar]
  • 2.Chak A, Banwell JG. Traveler's diarrhea. Gastroenterol Clin North Am. 1993;22:549–561. [PubMed] [Google Scholar]
  • 3.Cravioto A, Trujillo F, Leon LA, Hernandez JM, Eslava C. Infections caused by enteropathogenic Escherichia coli. Gac Med Mex. 1996;132:611–615. [PubMed] [Google Scholar]
  • 4.Mäki M, Vesikari T, Gronroos P. Enterotoxigenic and invasive Escherichia coli as causes of childhood diarrhoea in Finland. Acta Paediatr Scand. 1980;69:219–224. doi: 10.1111/j.1651-2227.1980. tb07064.x. [DOI] [PubMed] [Google Scholar]
  • 5.Robins-Browne RM, Still CS, Miliotis MD, Richardson NJ, Koornhof HJ, Freiman I, et al. Summer diarrhoea in African infants and children. Arch Dis Child. 1980;55:923–928. doi: 10.1136/ adc.55.12.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Cohen MB, Giannella RA. Hemorrhagic colitis associated with Escherichia coli O157:H7. Adv Intern Med. 1992;37:173–195. [PubMed] [Google Scholar]
  • 7.Garmendia J, Frankel G, Crepin VF. Enteropathogenic and enterohemorrhagic Escherichia coli infections: translocation, translocation, translocation. Infect Immun. 2005;73:2573–2585. doi: 10.1128/IAI.73.5.2573-85.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Schmidt MA. LEEways: tales of EPEC, ATEC and EHEC. Cell Microbiol. 2010;12:1544–1552. doi: 10.1111/j.1462-5822.2010.01518.x. [DOI] [PubMed] [Google Scholar]
  • 9.Knutton S, Baldwin T, Williams PH, McNeish AS. Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun. 1989;57:1290–1298. doi: 10.1128/iai.57.4.1290-1298.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Borenshtein D, McBee ME, Schauer DB. Utility of the Citrobacter rodentium infection model in laboratory mice. Curr Opin Gastroenterol. 2008;24:32–37. doi: 10.1097/MOG.0b013e3282f2b0fb. [DOI] [PubMed] [Google Scholar]
  • 11.Wiles S, Dougan G, Frankel G. Emergence of a ‘hyper-infectious’ bacterial state after passage of Citrobacter rodentium through the host gastrointestinal tract. Cell Microbiol. 2005;7:1163–1172. doi: 10.1111/j.1462-5822.2005.00544.x. [DOI] [PubMed] [Google Scholar]
  • 12.Elliott SJ, Wainwright LA, McDaniel TK, Jarvis KG, Deng YK, Lai LC, et al. The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol Microbiol. 1998;28:1–4. doi: 10.1046/j.1365-2958.1998.00783.x. [DOI] [PubMed] [Google Scholar]
  • 13.Hueck CJ. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev. 1998;62:379–433. doi: 10.1128/mmbr.62.2.379-433.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Jarvis KG, Giron JA, Jerse AE, McDaniel TK, Donnenberg MS, Kaper JB. Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation. Proc Natl Acad Sci USA. 1995;92:7996–8000. doi: 10.1073/pnas.92.17.7996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Jarvis KG, Kaper JB. Secretion of extracellular proteins by enterohemorrhagic Escherichia coli via a putative type III secretion system. Infect Immun. 1996;64:4826–4829. doi: 10.1128/iai.64.11.4826-4829.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kenny B. Phosphorylation of tyrosine 474 of the enteropathogenic Escherichia coli (EPEC) Tir receptor molecule is essential for actin nucleating activity and is preceded by additional host modifications. Mol Microbiol. 1999;31:1229–1241. doi: 10.1046/j.1365-2958.1999.01265.x. [DOI] [PubMed] [Google Scholar]
  • 17.Frankel G, Lider O, Hershkoviz R, Mould AP, Kachalsky SG, Candy DC, et al. The cell-binding domain of intimin from enteropathogenic Escherichia coli binds to beta1 integrins. J Biol Chem. 1996;271:20359–20364. doi: 10.1074/jbc.271.34.20359. [DOI] [PubMed] [Google Scholar]
  • 18.Kim M, Ogawa M, Fujita Y, Yoshikawa Y, Nagai T, Koyama T, et al. Bacteria hijack integrin-linked kinase to stabilize focal adhesions and block cell detachment. Nature. 2009;459:578–582. doi: 10.1038/nature07952. [DOI] [PubMed] [Google Scholar]
  • 19.Muza-Moons MM, Koutsouris A, Hecht G. Disruption of cell polarity by enteropathogenic Escherichia coli enables basolateral membrane proteins to migrate apically and to potentiate physiological consequences. Infect Immun. 2003;71:7069–7078. doi: 10.1128/IAI.71.12.7069-78.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Chaussade S, Minic M. Comparison of efficacy and safety of two doses of two different polyethylene glycolbased laxatives in the treatment of constipation. Aliment Pharmacol Ther. 2003;17:165–172. doi: 10.1046/j.1365-2036.2003.01390.x. [DOI] [PubMed] [Google Scholar]
  • 21.DiPalma JA, DeRidder PH, Orlando RC, Kolts BE, Cleveland MB. A randomized, placebo-controlled, multicenter study of the safety and efficacy of a new polyethylene glycol laxative. Am J Gastroenterol. 2000;95:446–450. doi: 10.1111/j.1572-0241.2000.01765.x. [DOI] [PubMed] [Google Scholar]
  • 22.Kommareddy S, Amiji M. Biodistribution and pharmacokinetic analysis of long-circulating thiolated gelatin nanoparticles following systemic administration in breast cancer-bearing mice. J Pharm Sci. 2007;96:397–407. doi: 10.1002/jps.20813. [DOI] [PubMed] [Google Scholar]
  • 23.Vinogradov SV, Zeman AD, Batrakova EV, Kabanov AV. Polyplex Nanogel formulations for drug delivery of cytotoxic nucleoside analogs. J Control Release. 2005;107:143–157. doi: 10.1016/j.jconrel.2005.06.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Wali RK, Kunte DP, Koetsier JL, Bissonnette M, Roy HK. Polyethylene glycol-mediated colorectal cancer chemoprevention: roles of epidermal growth factor receptor and Snail. Mol Cancer Ther. 2008;7:3103–3111. doi: 10.1158/1535-7163.MCT-08-0434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Barthold SW. Autoradiographic cytokinetics of colonic mucosal hyperplasia in mice. Cancer Res. 1979;39:24–29. [PubMed] [Google Scholar]
  • 26.Luperchio SA, Schauer DB. Molecular pathogenesis of Citrobacter rodentium and transmissible murine colonic hyperplasia. Microbes Infect. 2001;3:333–340. doi: 10.1016/S1286-4579(01)01387-9. [DOI] [PubMed] [Google Scholar]
  • 27.Mundy R, MacDonald TT, Dougan G, Frankel G, Wiles S. Citrobacter rodentium of mice and man. Cell Microbiol. 2005;7:1697–1706. doi: 10.1111/j.1462-5822.2005.00625.x. [DOI] [PubMed] [Google Scholar]
  • 28.Savkovic SD, Villanueva J, Turner JR, Matkowskyj KA, Hecht G. Mouse model of enteropathogenic Escherichia coli infection. Infect Immun. 2005;73:1161–1170. doi: 10.1128/IAI.73.2.1161-70.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Qi W, Weber CR, Wasland K, Roy H, Wali R, Joshi S, et al. Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells. Am J Physiol Gastrointest Liver Physiol. 2011;300:264–272. doi: 10.1152/ ajpgi.00416.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.MacDonald TT, Frankel G, Dougan G, Goncalves NS, Simmons C. Host defences to Citrobacter rodentium. Int J Med Microbiol. 2003;293:87–93. doi: 10.1078/1438-4221-00247. [DOI] [PubMed] [Google Scholar]
  • 31.Higgins LM, Frankel G, Douce G, Dougan G, MacDonald TT. Citrobacter rodentium infection in mice elicits a mucosal Th1 cytokine response and lesions similar to those in murine inflammatory bowel disease. Infect Immun. 1999;67:3031–3039. doi: 10.1128/iai.67.6.3031-3039.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Wu L, Zaborina O, Zaborin A, Chang EB, Musch M, Holbrook C, et al. High-molecular-weight polyethylene glycol prevents lethal sepsis due to intestinal Pseudomonas aeruginosa. Gastroenterology. 2004;126:488–498. doi: 10.1053/j.gastro.2003.11.011. [DOI] [PubMed] [Google Scholar]
  • 33.Henry-Stanley MJ, Wells CL. Polyethylene glycol influences microbial interactions with intestinal epithelium. Shock. 2009;31:390–396. doi: 10.1097/SHK.0b013e31818348a5. [DOI] [PubMed] [Google Scholar]
  • 34.Roxas JL, Koutsouris A, Viswanathan VK. Enteropathogenic Escherichia coli-induced epidermal growth factor receptor activation contributes to physiological alterations in intestinal epithelial cells. Infect Immun. 2007;75:2316–2324. doi: 10.1128/IAI.01690-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Yamaoka T, Yan F, Cao H, Hobbs SS, Dise RS, Tong W, et al. Transactivation of EGF receptor and ErbB2 protects intestinal epithelial cells from TNF-induced apoptosis. Proc Natl Acad Sci USA. 2008;105:11772–11777. doi: 10.1073/pnas.0801463105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Westerholt S, Hartung T, Tollens M, Gustrau A, Oberhoffer M, Karch H, et al. Inflammatory and immunological parameters in children with haemolytic uremic syndrome (HUS) and gastroenteritis-pathophysiological and diagnostic clues. Cytokine. 2000;12:8227. doi: 10.1006/cyto.1999.0624. [DOI] [PubMed] [Google Scholar]
  • 37.Savkovic SD, Koutsouris A, Hecht G. Attachment of a non-invasive enteric pathogen, enteropathogenic Escherichia coli, to cultured human intestinal epithelial monolayers induces transmigration of neutrophils. Infect Immun. 1996;64:4480–4487. doi: 10.1128/iai.64.11.4480-4487.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Casswall TH, Sarker SA, Faruque SM, Weintraub A, Albert MJ, Fuchs GJ, et al. Treatment of enterotoxigenic and enteropathogenic Escherichia coli-induced diarrhoea in children with bovine immunoglobulin milk concentrate from hyperimmunized cows: a double-blind, placebo-controlled, clinical trial. Scand J Gastroenterol. 2000;35:711–718. doi: 10.1080/003655200750023372. [DOI] [PubMed] [Google Scholar]
  • 39.Orth D, Grif K, Zimmerhackl LB, Wurzner R. Prevention and treatment of enterohemorrhagic Escherichia coli infections in humans. Expert Rev Anti Infect Ther. 2008;6:101–108. doi: 10.1586/14787210.6.1.101. [DOI] [PubMed] [Google Scholar]
  • 40.Nakajima K, Tamura N, Kobayashi-Hattori K, Yoshida T, Hara-Kudo Y, Ikedo M, et al. Prevention of intestinal infection by glycomacropeptide. Biosci Biotechnol Biochem. 2005;69:2294–2301. doi: 10.1271/bbb.69.2294. [DOI] [PubMed] [Google Scholar]
  • 41.Snoeks L, Weber CR, Turner JR, Bhattacharyya M, Wasland K, Savkovic SD. Tumor suppressor Foxo3a is involved in the regulation of lipopolysaccharideinduced interleukin-8 in intestinal HT-29 cells. Infect Immun. 2008;76:4677–4685. doi: 10.1128/IAI.00227-08. [DOI] [PMC free article] [PubMed] [Google Scholar]

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