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Am J Physiol Gastrointest Liver Physiol 287: G1238-G1246, 2004; doi:10.1152/ajpgi.00471.2003
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MUCOSAL BIOLOGY

Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa

Benedict T. Green,1,* Mark Lyte,2,3,* Chunsheng Chen,2 Yonghong Xie,2 Melissa A. Casey,1 Anjali Kulkarni-Narla,1 Lucy Vulchanova,1 and David R. Brown1

1Pharmacology Section, Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul 55108-6010; 2Department of Surgery, Minneapolis Medical Research Foundation/Hennepin County Medical Center, Minneapolis 55404; and 3Department of Biological Sciences, Minnesota State University–Mankato, Mankato, Minnesota 56001

Submitted 6 November 2003 ; accepted in final form 13 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Enteric neurotransmitters can modulate the biodefensive functions of the intestinal mucosa, but their role in mucosal interactions with enteropathogens is not well defined. Here we tested the hypothesis that norepinephrine (NE) modulates interactions between enterohemorrhagic Escherichia coli O157:H7 (EHEC) and the colonic epithelium. Mucosal sheets from porcine distal colon were mounted in Ussing chambers. Drugs and an inoculum of either Shiga toxin-negative or -positive EHEC were added to the contraluminal and luminal bathing medium, respectively. After 90 min, adherent bacteria were quantified by an adherence assay and by immunohistochemical methods; short-circuit current (Isc) was measured continuously to assess changes in active ion transport. NE-treated tissues exhibited concentration-dependent increases in Isc and EHEC adherence. NE did not alter adherence of a rodent-adapted, noninfectious E. coli strain or two porcine-adapted non-O157 E. coli strains. The actions of NE on EHEC adherence but not Isc were prevented by the {alpha}-adrenergic antagonist yohimbine and the PKA activator Sp-8-bromoadenosine-3',5'-cyclic monophosphorothioate. Like NE, the PKA inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate or indirectly acting sympathomimetic agents increased EHEC adherence. Nerve fibers immunoreactive for the NE-synthesizing enzymes tyrosine hydroxylase and dopamine {beta}-hydroxylase appeared to innervate the colonic epithelium. EHEC-like immunoreactivity on the colonic surface had the appearance of bacterial microcolonies and increased after NE treatment by a phentolamine-sensitive mechanism. Through interactions with {alpha}2-adrenergic receptors, NE appears to increase EHEC adherence to the colonic mucosa. Changes in sympathetic neural outflow may alter intestinal susceptibility to infection.

sympathetic nervous system; colonocyte; {alpha}-adrenergic receptor; protein kinase A; enteritis

THE HUMAN INTESTINAL TRACT encompasses an extensive surface area ~300–400 m2, which constitutes the largest interface between the host and microorganisms. It is colonized by at least 400 species of commensal bacteria at densities reaching 1011 organisms/ml of luminal fluid (29). The intestine has developed a diverse array of innate protective mechanisms that allow it to coexist benignly with resident flora, yet effectively remove pathogenic microorganisms (12). Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a highly virulent enteric pathogen that is acquired by ingestion of contaminated food or water (2). The microorganism can produce acute gastroenteritis and severe hemorrhagic colitis, and its expression of bacteriophage-associated Shiga toxins is associated with the hemolytic uremic syndrome, which can lead to acute renal failure (35). One target for EHEC infection is the colonic mucosa. After its initial "loose" adherence to colonic surface cells, EHEC alters the epithelial actin cytoskeleton by delivering virulence proteins into host cells through a type III secretion system and produces characteristic attaching and effacing lesions (16). EHEC also targets the follicle-associated epithelium of Peyer's patches in the small intestine (11, 27).

Neurons containing norepinephrine (NE) are located in prevertebral sympathetic ganglia located in the lumbar portion of the spinal cord. They send fiber projections to intramural neurons and nonneuronal target cells within the wall of the distal colon (14). Through interactions with {alpha}- and {beta}-adrenergic receptors, NE modulates intestinal smooth muscle contractility, submucosal blood flow, and active transepithelial ion transport (24). Extrinsic sympathetic input to the gut mucosa may function to regulate adaptive immune responses to luminal antigens as well (10). NE concentrations in the intestinal lumen increase in response to surgery and other stressors and play an important role in the early stages of sepsis (1, 18). At high concentrations, NE has been shown to directly enhance the growth rate of commensal E. coli as well as the growth and virulence characteristics of EHEC (20–22, 30). Although enteric neurotransmitters and hormones can affect active ion transport and other putative biodefensive functions of the intestinal mucosa, their role in modulating mucosal interactions with enteropathogens has not been well-defined (31).

We tested the hypothesis that NE plays a role in modulating interactions between EHEC and the colonic epithelium. The porcine distal colon was chosen as a biomedical model because it is functionally homologous to the human colon (33) and is susceptible to infection by a number of human enteropathogens, including EHEC (26). Moreover, NE transiently increases active chloride secretion through interactions with colonocyte {alpha}1-adrenergic receptors in this tissue (37). In the present study, we examined the ability of NE to alter EHEC adherence to the colonic epithelium through a mechanism of action independent of mucosal ion transport.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Bacterial strains and growth conditions. Bacteria were stored in 20% (vol/vol) glycerol/PBS until time of culture. An inoculum of Escherichia coli O157:H7 [EHEC; Shiga toxin-negative strain 700728 American Type Culture Collection (Manassas, VA) or the Shiga toxin-producing strain EDL933] or a nonpathogenic, rodent-adapted E. coli (streptomycin-resistant strain M-21; Ref. 40) were grown overnight in Luria-Bertani (LB) medium at 37°C in a humidified 5% CO2 atmosphere. Shiga toxins produced by EHEC have been reported to induce apoptosis in epithelial cells; therefore, most experiments were carried out by using a Shiga toxin-negative strain of E. coli O157:H7 (strain 700728) to eliminate this potential variable (15). Inocula were diluted 1:9 in PBS and added to the luminal bathing medium. Strain 700728 EHEC and E. coli M-21 were used at luminal inocula of 5.96 x 105 ± 2.81 x 105 colony forming units (CFU)/ml and 1.52 x 106 ± 1.23 x 106 CFU/ml, respectively, as determined by spread plating.

Commensal porcine non-O157 E. coli was obtained by plating homogenized colonic mucosa from normal pigs onto Fluorocult agar supplemented with 100 µg/ml streptomycin sulfate. The selective isolation and differentiation capabilities of Fluorocult medium for Enterobacteriaceae, especially E. coli O157:H7, which are achieved by a combination of fluorogenic and chromogenic substrates, have been well described to identify relevant bacteria from a variety of sources (13, 23). Presumptive colonies of E. coli that did not have the appearance of E. coli O157:H7 were randomly chosen from Fluorocult plates after overnight incubation and were streaked onto LB agar plates supplemented with 100 µg/ml streptomycin to isolate E. coli strains resistant to this antibiotic drug. After 24-h incubation at 37°C, individual colonies were picked from streptomycin sulfate-supplemented LB plates, and their identities were confirmed as E. coli using the API-20E Enteric Identification System (BioMerieux, Hazelwood, MO). Colonies were further determined to represent non-O157 E. coli with the use of an E. coli O157 latex agglutination-based diagnostic test kit (Oxoid, Ogdensburg, NY).

Drugs. Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate (Rp-8-Br-cAMPS) and Sp-8-bromoadenosine-3',5'-cyclic monophosphorothioate (Sp-8-Br-cAMPS) were obtained from Alexis Biochemicals (San Diego, CA). All other drugs were obtained from Sigma (St. Louis, MO). Yohimbine and prazosin were dissolved in methanol. The other drugs were solubilized in physiological saline solution (composition indicated in Tissue preparation and measurement of transepithelial ion transport) as concentrated stock solutions and stored at –80°C; NE and propranolol solutions were prepared immediately before use.

Agonists were added to the luminal or contraluminal media bathing colonic mucosa sheets 5 min before bacterial exposure; adrenergic receptor antagonists were added to the contraluminal bathing medium 10 min before agonist addition. When given alone, Rp-8-Br-cAMPS and Sp-8-Br-cAMPS were added to the contraluminal bathing medium 5 min before EHEC exposure; in NE experiments, Sp-8-Br-cAMPS was added 10 min before addition of NE.

Animals. Tissues were obtained from outbred Yorkshire/Landrace-crossed pigs of each sex that were 5–9 wk old and weighed between 10 and 18 kg. Pigs had continuous access to water and nonmedicated feed and were not fasted before death. They were sedated with an intramuscular injection of tiletamine hydrochloride-zolazepam (Telazol; 8 mg/kg, Fort Dodge Laboratories, Fort Dodge, IA), in combination with xylazine (3 mg/kg). They were subsequently euthanized by barbiturate overdose in accordance with approved University of Minnesota Institutional Animal Care and Use Committee protocols. A midline laparotomy was performed to expose the intestine, and a 7-cm segment of the distal colon extending orad from the internal anal sphincter was isolated.

Tissue preparation and measurement of transepithelial ion transport. The colonic epithelium was stripped of underlying smooth muscle coats, and the mucosa with attached submucosa was mounted in Ussing chambers (1- or 2-cm2 flux area). The serosa and smooth muscle layers of an excised colonic segment were removed by blunt dissection, and the remaining submucosa-mucosa was mounted between two Lucite Ussing-type half chambers. Mucosal sheets were bathed on both luminal and contraluminal aspects in 10 ml of a buffered, physiological saline solution (composition in mM: 130 NaCl, 6 KCl, 3 CaCl2, 0.7 MgCl2, 20 NaHCO3, 0.29 NaH2PO4, and 1.3 Na2HPO4,) that was continuously oxygenated with 95% O2-5% CO2 delivered by gas lift and maintained at pH 7.4 and 39°C (porcine core temperature). D-Glucose and mannitol (10 mM) were added to the contraluminal and luminal bathing media, respectively.

Short-circuit current (Isc, in µA/cm2) was monitored continuously across each mucosa-submucosal sheet with an automatic voltage clamp apparatus (model TR100; JWT Engineering, Overland Park, KS or model EVC-4000; World Precision Instruments, Sarasota, FL). Experiments were initiated after the basal Isc had stabilized (25–35 min). Throughout each experiment, the transepithelial voltage was periodically adjusted to 5 mV, and the resulting current change was used to calculate the tissue conductance (Gt) by Ohm's law. Isc was measured immediately before drug administration and at the peak of drug action.

Bacterial adherence and gentamicin resistance assays. Bacterial adherence to the colonic mucosa was determined by the method of Knutton et al. (17). Mucosal sheets were removed from Ussing chambers after 90 min of luminal exposure to bacteria. Each tissue was weighed and washed three times in PBS (pH 7.4). In some cases, tissues were divided in half, weighed, and assayed for both bacterial adherence and bacterial internalization. All tissues were subsequently homogenized by using a Brinkmann Polytron and spread-plated on differential and selective media for O157:H7 (Fluorocult agar, EM Science, Gibbstown, NJ) and M-21 (MacConkey agar; Difco, Detroit, MI, containing 1 mg/ml streptomycin sulfate). For experiments that examined the ability of commensal non-O157 E. coli obtained from pigs as described above in Bacterial strains and growth conditions to adhere to the colonic mucosa, homogenates were plated on Fluorocult agar supplemented with 100 µg/ml streptomycin sulfate

To assess the intracellular invasion of EHEC, colonic sheets removed from Ussing chambers after 90 min of EHEC exposure were incubated at 37°C in a humidified 5% CO2 atmosphere in a gentamicin solution (100 µg/ml in PBS) for 80 min to eliminate extracellular bacteria after the method of Elsinghorst (6). The tissues were subsequently homogenized and spread-plated in a manner identical to that performed in bacterial adherence assays.

Visualization and quantitation of bacterial adherence. Bacterial aggregates immunoreactive for the O-antigen of E. coli O157:H7 that adhered to whole mounts of the colonic mucosa obtained from seven pigs were examined and quantified by confocal laser-scanning microscopy after luminal exposure to E. coli O157:H7 in Ussing chambers. Some tissues were unexposed to bacteria or pretreated contraluminally with 10 µM NE in the absence or presence of 1 µM phentolamine. Mucosal sheets were removed from the chambers after 90 min of EHEC exposure and gently washed three times in PBS (pH 7.4). EHEC on the colonic mucosa was visualized and quantified by immunofluorescence. Briefly, whole mounts of colonic sheets were incubated in PBS containing 2.5% bovine serum albumin (Sigma) for 30 min at room temperature on an orbit shaker. Whole mounts were then incubated for 2 h at room temperature with a FITC-conjugated goat anti-E. coli 0157:H7 affinity-purified antibody directed toward the O-antigen of E. coli O157:H7 (1:20 dilution; BacTrace antibody; Kirkegaard and Perry Laboratories, Gaithersburg, MD). After brief washes in PBS, whole mounts were incubated for 30 min at room temperature with Texas Red-conjugated antibody to DNAse (Molecular Probes, Eugene, OR) to visualize the colonic surface. After additional washes in PBS, whole mounts were fixed in 2% paraformaldehyde (Sigma) for 1 h and refrigerated in PBS until visualized.

Images from five randomly selected, nonoverlapping fields, each representing a total visual field of 1.5 mm2, were acquired by using Comos software (version 6.05.8; Comos Bio-Rad, Hercules, CA) and further processed employing NIH Image software (version 1.6.0) and Adobe Photoshop (version 6.0.1, Adobe Systems, San Jose, CA). An average of 14 sections (using an average z-step of 6 µm) were made through each field. The area occupied by EHEC immunoreactivity in each field was quantified by using NIH Image software (version 1.6.0) and expressed as the area of adherent EHEC immunoreactivity per tissue in square pixels.

Immunohistochemical localization of adrenergic neural elements. Standard immunofluorescence methods were used to detect immunoreactivities for tyrosine hydroxylase (TH) and dopamine {beta}-hydroxylase (DBH), key catalytic enzymes in NE biosynthesis (19). After being rinsed, segments of distal colon were opened along the mesenteric border, pinned mucosal side up on a Silgard-coated surface, and fixed in fixative (4% paraformaldehyde and 0.2% picric acid) for 2 h at room temperature. The tissue was rinsed extensively in PBS and incubated in 10% sucrose for a minimum of 24 h before cryostat sectioning. Cryostat sections (20 µm) were triple-labeled with a mouse monoclonal antibody raised against rat TH (1:1,000, Immunostar, Hudson, WI), a rabbit polyclonal antibody raised to bovine DBH (1:1,000, Immunostar), and a rat monoclonal antibody against the tight junction protein zonula occludens-1 (Chemicon, Temecula, CA), that allowed visualization of the epithelial cell layer. To confirm neuronal staining, some sections were double-labeled with anti-DBH and a mouse monoclonal antibody against the neuronal marker protein gene product 9.5 (Biogenesis, Poole, England). Triple-labeled preparations were visualized with indocarbocyanine (Cy3)-conjugated donkey anti-mouse, cyanine (Cy2)-conjugated donkey anti-rat and indodicarbocyanine (Cy5)-conjugated donkey anti-rabbit secondary antisera (Jackson Immunoresearch Laboratories, West Grove, PA). Double-labeled preparations were visualized with Cy3- and Cy2-conjugated donkey anti-rabbit and donkey anti-mouse antiserum, respectively. Control experiments consisted of omission of primary antibodies from the staining protocol or substitution of an unrelated antibody for the primary antibody; these experiments resulted in the absence of a specific pattern of immunoreactivity. A minimum of five sections from four pigs were used to determine patterns of TH- and DBH-like immunoreactivity in the porcine distal colon. Sections were scanned by using Bio-Rad confocal laser-scanning microscope (CLSM 1024, Bio-Rad) attached to a Nikon microscope. The different fluorophores were imaged sequentially. Images were acquired and processed as described above.

Data analysis. Data are expressed as means ± SE of colony forming units per gram of tissue, Isc (in µA/cm2), Gt (in milliSiemens/cm2) or area encompassed by adherent immunoreactive EHEC per tissue in square pixels. Statistical analyses of data were performed by using the PRISM computer software program (Version 3.0; GraphPad Software, San Diego, CA). Comparisons between a control mean and a single treatment mean were made by unpaired t-tests with Welch's correction used for unequal variances. Comparisons of a control mean with multiple treatment means were made by one-way or two-way ANOVA followed by Dunnett's test where appropriate. In all cases, the limit for statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Baseline electrical parameters. Under baseline conditions, Isc and Gt in uninfected, isolated sheets of colonic mucosa-submucosa averaged 2 ± 2 µA/cm2 and 13 ± 1 mS/cm2, respectively (n = 405 tissues from 37 pigs). At 90 min, Isc remained unchanged in EHEC-infected tissues, but Gt increased relative to earlier time points. However, this increase in Gt was similar in untreated tissues serving as controls and tissues treated with NE (Table 1). The contraluminal addition of NE at 10 µM produced a rapid elevation in Isc with a mean duration of 46 ± 8 min before return to baseline values (n = 8 tissues from 4 pigs); at 90 min, all NE-treated tissues had returned to baseline Isc (Table 1). The luminal addition of E. coli O157:H7 strain 700728 did not produce a significant, acute change in either Isc or Gt.


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  		Table 1. Time-related changes in the electrical properties of colonic tissues exposed to luminal EHEC in the absence and presence of 10 µM norepinephrine in contraluminal bathing medium

 
Concentration-dependent actions of NE on interactions of the colonic mucosa with EHEC. EHEC strain 700728 adhered to, but did not substantially invade the colonic mucosa. After a 90-min period of EHEC exposure, the number of bacteria internalized in and adhering to the mucosa under control conditions was 45 ± 19 and 5,779 ± 1,369 CFU/g tissue, respectively (n = 11 tissues from 7 pigs). The contraluminal addition of NE over a concentration range of 0.1–10 µM increased luminal adherence of the toxin-negative EHEC strain to sheets of colonic mucosa in a concentration-dependent fashion (Fig. 1). The contraluminal concentrations of NE producing EC50 on EHEC adherence and Isc were significantly different (EC50s for increasing adherence and Isc were 1.32 and 0.82 µM with 95% confidence limits of 1.27–1.37 and 0.74–0.91 µM, respectively). However, NE at a contraluminal concentration of 10 µM had no effect on EHEC internalization (31 ± 21 CFU/g recovered from 5 gentamicin-treated tissues from 3 pigs). When added to the luminal bathing medium, 10 µM NE had no significant effect on EHEC adherence (8,678 ± 1,875 and 11,541 ± 1,791 CFU/g, respectively, in 15 control and 14 NE-treated tissues from 9 pigs; P > 0.05, t-test).



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  		Fig. 1. Concentration-effect relationship for the actions of norepinephrine (NE) in the porcine colonic mucosa. A: effects of NE on enterohemorrhagic Escherichia coli O157:H7 (EHEC) adherence. B: effects of NE on short-circuit current (Isc). Data represent means ± SE of counts of adherent EHEC [in colony forming units (CFU)/g; 6–19 tissues from 6–18 pigs] or peak changes in Isc (in µA/cm2; 6–42 tissues from 6–39 pigs) measured after the contraluminal addition of NE at the log10 molar concentrations indicated.

 
NE also significantly increased mucosal adherence of Shiga toxin-positive E. coli O157:H7 strain EDL933 added in an inoculum of 4,820 ± 3,191 CFU/ml of luminal bathing medium. Bacterial adherence was 10,973 ± 2,737 and 89,034 ± 10,113 CFU/g in tissues untreated or contraluminally pretreated with 10 µM NE, respectively (P < 0.05, t-test, n = 3 and 4 tissues respectively, from 3 pigs). Two streptomycin-resistant strains of non-0157:H7 E. coli were isolated from porcine colon and grown in a manner similar to E. coli 0157:H7 also adhered to the colonic mucosa, but NE in the contraluminal bathing medium did not significantly change mucosal adherence of these organisms (number of colonic non-EHEC E. coli strain no. 1 in the absence and presence of 10 µM NE = 2,333 ± 928 and 5,464 ± 1,099 CFU/g tissue, respectively; P > 0.05, unpaired t-test, 4 control and NE-treated tissues from 4 pigs; number of colonic non-EHEC E. coli strain no. 2 in the absence and presence of 10 µM NE = 6,769 ± 3,309 and 8,837 ± 2,886 CFU/g tissue, respectively; P > 0.05, t-test, 3 control and NE-treated tissues from 3 pigs). Moreover, NE had no effect on mucosal adherence of rodent-adapted, nonpathogenic E. coli M-21 (Fig. 2A). In a separate series of gentamicin-resistance assay experiments, colonic epithelial tissues were exposed to an inoculum of 17,366 ± 11,780 CFU/ml of E. coli M-21 for 90 min. The number of internalized bacteria were 4,870 ± 3,410 and 1,476 ± 513 CFU/g in tissues untreated or pretreated contraluminally with NE, respectively (n = 20 untreated and 15 NE-treated tissues from 8 and 6 pigs, respectively).



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  		Fig. 2. Effects of phentolamine (PTL) or saxitoxin (STX) on NE-induced EHEC adherence. A: effects of NE on the adherence of E. coli O157 (solid bars) or E. coli M-21 (patterned bars) to porcine colonic mucosa. Tissues were untreated (control), treated with 10 µM NE, or treated with 1 µM PTL (PTL/NE) before administration of NE. Bars represent the means ± SE counts of adherent EHEC (in CFU/g) in 14 control tissues (from 8 pigs), 12 tissues (from 8 pigs) treated with NE, and 16 tissues (from 8 pigs) treated with PTL and NE. In 1 animal, a tissue serving as a control had 259,176 EHEC CFU/g compared with a mean of 10,874 ± 3,037 CFU/g for 14 other tissues serving as controls. Data obtained in all tissues from this animal were excluded from further analysis. The mean counts of adherent M-21 (in CFU/g) were obtained in 6 control tissues (from 3 pigs) and 6 tissues (from 3 pigs) treated with NE. B: effects of STX on the action of NE. Tissues were untreated (control), treated with 0.1 µM STX before administration of 10 µM NE, or treated with 0.1 µM STX alone. Bars represent the means ± SE counts of adherent EHEC (in CFU/g) in 14 control tissues (from 9 pigs), 7 tissues (from 7 pigs) treated with both STX and NE, and 6 tissues (from 4 pigs) treated with STX alone. *P < 0.05 vs. EHEC adherence in control tissues, #P < 0.05 vs. control tissues exposed to EHEC, as determined by unpaired t-tests with Welch's correction.

 
Saxitoxin, a neuronal Na+ channel blocker, did not alter EHEC adherence or Isc after its contraluminal addition at 0.1 µM. Moreover, it did not prevent the actions of NE on EHEC adherence (Fig. 2B) or Isc (data not shown).

Adrenergic receptors mediating NE action on EHEC adherence. The adrenergic receptor-type mediating the effects of NE on EHEC adherence was characterized with receptor-selective antagonists. At a contraluminal concentration of 1 µM, the {alpha}-adrenergic antagonist phentolamine prevented the action of 10 µM NE on EHEC adherence (Fig. 2A). At 1 µM, the {beta}-adrenergic antagonist propranolol had no effect (data not shown). Furthermore, the effect of NE was inhibited in tissues pretreated with the {alpha}2-adrenergic antagonist yohimbine at a contraluminal concentration of 0.3 µM, but not the {alpha}1-adrenergic antagonist prazosin (Fig. 3). However, yohimbine did not alter the ability of NE to increase Isc ({Delta}Isc = 82 ± 10 µA/cm2 in 46 NE-treated tissues from 38 pigs and 104 ± 27 µA/cm2 in 8 tissues treated both with yohimbine and NE from 8 pigs).



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  		Fig. 3. Effect of selective {alpha}-adrenergic antagonists on NE-induced EHEC adherence. Tissues were either untreated (control), treated with 10 µM NE, or treated with 0.3 µM prazosin (PRZ/NE) or yohimbine (YHB/NE) before NE administration. The antagonists and NE were added to the contraluminal bathing medium 15 and 5 min, respectively, before luminal exposure to EHEC in the stationary growth phase. Bars represent the means ± SE counts of adherent EHEC (in CFU/g) in 12 control tissues (from 7 pigs), 8 tissues (from 8 pigs) treated with NE, 8 tissues (from 8 pigs) treated with PRZ and NE, and 8 tissues (from 8 pigs) pretreated with YHB/NE. *P < 0.05 vs. control mean, Dunnett's test.

 
Yohimbine-sensitive {alpha}2-adrenergic receptors are coupled through the GTP-binding protein G{alpha}i to decreases in intracellular cAMP production and secondary decreases in protein kinase A (PKA) activity. To test the hypothesis that direct inhibition of PKA activity would mimic the action of NE on EHEC adherence, mucosal sheets were treated contraluminally with either 3 µM Rp-8-Br-cAMPS or Sp-8-Br-cAMPS before luminal EHEC exposure. These membrane-permeant cAMP analogs inhibit and stimulate PKA activity, respectively (9). As shown in Fig. 4, pretreatment with Rp-8-Br-cAMPS, but not Sp-8-Br-cAMPS increased EHEC adherence. However, Sp-8-Br-cAMPS pretreatment appeared to prevent the action of NE on bacterial adherence (Fig. 4). Neither PKA modulator had significant effects on baseline Isc or Gt (data not shown). Moreover, the mucosal Isc response to NE in tissues pretreated with Sp-8-Br-cAMPS was not significantly different from tissues treated with 10 µM NE alone ({Delta}Isc = 82 ± 10 µA/cm2 in 46 NE-treated tissues from 38 pigs, and 66 ± 25 µA/cm2 in 6 Sp-8-Br-cAMPS- and NE-treated tissues from 3 pigs, P > 0.05, t-test).



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  		Fig. 4. The effect of PKA modulators on EHEC adherence to the porcine colonic mucosa. Tissues were untreated (control) or pretreated contraluminally with either the PKA inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate (Rp-8-Br-cAMPS) (3 µM) or the PKA activator Sp-8-bromoadenosine-3',5'-cyclic monophosphorothioate (Sp-8-Br-cAMPS; 3 µM). Some tissues were treated contraluminally with 3 µM Sp-8-Br-cAMPS in combination with 10 µM NE. Bars represent the means ± SE counts of adherent EHEC (in CFU/g) in 16 control tissues (from 10 pigs), 13 tissues (from 8 pigs) treated with Rp-8-Br-cAMPS alone, 10 tissues (from 8 pigs) treated with Sp-8-Br-cAMPS alone, and 6 tissues (from 3 pigs) treated with Sp-8-Br-cAMPS and NE. *P < 0.05 vs. control mean, Dunnett's test.

 
Visualization of adherent EHEC in NE-pretreated and untreated colonic sheets. Whole mounts of mucosal sheets serving as controls that were not exposed to EHEC did not exhibit immunoreactivity toward the O-antigen of E. coli O157:H7 (Fig. 5A). In contrast, tissues exposed to luminal EHEC displayed numerous immunoreactive aggregates that appear to represent E. coli O157:H7 microcolonies (Fig. 5, B–D). Sheets of colonic mucosa treated contraluminally with 10 µM NE before luminal EHEC exposure manifested a greater number of immunoreactive aggregates (Fig. 5, C and E) than those either not treated with NE or pretreated with 1 µM phentolamine before NE administration (Fig. 5, B, D, and E).



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  		Fig. 5. Visualization of EHEC-like immunoreactivity on the luminal surface of porcine colonic mucosa. A: whole mount of colonic mucosal surface that was not exposed to EHEC. B: whole mount of mucosal surface exposed to EHEC for 90 min but untreated with drugs. Immunoreactive microcolonies (green) can be seen. C: mucosal surface of a tissue pretreated contraluminally with 10 µM NE 5 min before luminal EHEC exposure for 90 min. A relative increase in the number of adherent microcolonies compared with B can be observed. D: mucosal surface of a tissue pretreated contraluminally with 1 µM PTL and 10 µM NE and subsequently exposed to EHEC for 90 min. Note a similar number of microcolonies as seen in B and a lesser number than that seen in C. Bar = 50 µm. E: quantitation of EHEC immunoreactivity adhering to the mucosal surfaces of colonic sheets from 6 pigs. Values represent the mean area occupied by adherent, immunoreactive EHEC per tissue in square pixels as determined in each of 5 nonoverlapping visual fields of 1.5 mm2 area per treatment condition in tissues from 6 pigs (*P < 0.05 vs. control mean, Dunnett's test).

 
Immunohistochemical localization of noradrenergic nerve fibers in porcine colonic mucosa and modulation of EHEC adherence by endogenous NE. Nerve fibers immunoreactive for the NE-synthesizing enzymes TH and DBH were observed in the submucosal plexuses and mucosa of the porcine distal colon (Fig. 6A) and throughout the myenteric plexus and circular muscle layer (data not shown). There was frequent colocalization of TH- and DBH-immunoreactivities in colonic nerve fibers (Figs. 6, B–D). Fine, varicose nerve fibers exhibiting both TH and DBH immunoreactivities were observed throughout the colonic villi. They were often seen terminating near colonic epithelial cells (Fig. 6A).



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  		Fig. 6. Immunohistochemical localization of noradrenergic innervation in an oblique longitudinal section from the porcine distal colon. A: colonic mucosa was triple-labeled for dopamine {beta}-hydroxylase (DBH; red), tyrosine hydroxylase (TH; green) and the tight-junction protein zonula occludens-1 (ZO1; blue), which permitted visualization of epithelial cells. DBH- and TH-immunoreactive (-ir) fibers were present in the colonic villi and often appeared to terminate in close proximity to epithelial cells (small arrowhead). Only very fine fibers could be distinguished in the most apical portion of the villi (small arrow). Colocalization of DBH and TH labeling (yellow) was evident in submucosal ganglia (large arrowhead) and in nerve bundles (large arrow) near crypts (C) in the base of the villi. B–D: imaging at high magnification demonstrated that DBH-ir (B) and TH-ir (C) were colocalized in varicose nerve fibers (D). Asterisks in A and D indicate the region of A presented in B–D. The image is a stack of 6 optical sections collected at 1-µm intervals. Scale bars: A, 100 µm; B–D, 10 µm.

 
To test the hypothesis that endogenous NE is capable of increasing the epithelial adherence of EHEC, tissues were pretreated with the NE reuptake blocker desipramine and the monoamine oxidase inhibitor pargyline. These drugs in combination mimicked the effect of NE on EHEC adherence (Fig. 7). The contraluminal addition of the NE-releasing agent tyramine did not further increase EHEC adherence (Fig. 7). The effect of these drugs on EHEC adherence was abolished in tissues pretreated with phentolamine (Fig. 7). The three sympathomimetic agents in combination did not produce significant changes in Isc or Gt (data not shown).



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  		Fig. 7. The effect of indirectly acting sympathomimetic drugs on EHEC adherence to the porcine colonic mucosa. Tissues were pretreated contraluminally with the monoamine oxidase inhibitor pargyline (PRG; 300 µM) and the NE reuptake blocker desipramine (DSP; 10 µM). Some tissues were additionally treated with the NE-releasing agent tyramine (TYR, 10 µM) or the {alpha}-adrenergic antagonist PTL (1 µM). Bars represent the means ± SE counts of adherent EHEC (in CFU/g) in 6 control tissues (from 3 pigs), 3 tissues (from 3 pigs) treated with PRG and DSP, 3 tissues (from 3 pigs) pretreated with PRG, DSP, and TYR, and 3 tissues (from 3 pigs) pretreated with PTL in combination with PRG, DSP, and TYR. *P < 0.05 vs. control mean, Dunnett's test.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 REFERENCES
 
Pigs have been used previously as an animal model for studies of E. coli O157:H7 infections (4, 26, 38). The ability of enteric neurotransmitters, such as NE, to modulate mucosal interactions with EHEC and other pathogens has not been examined previously in the pig. The results presented demonstrate that the catecholamine NE significantly increased EHEC adherence to muscle-stripped sheets of porcine distal colonic mucosa. NE has previously been shown to transiently increase Isc in this tissue. This effect has been attributed to an increase in net chloride secretion and appears to be mediated by prazosin-sensitive, {alpha}1-adrenergic receptors (37). In the present investigation, NE increased both Isc and the adherence of Shiga toxin-positive and -negative strains of E. coli O157:H7 to the colonic mucosa. The potency of NE in elevating Isc was 1.6-fold greater than its potency in promoting the adherence of EHEC, and both effects were resistant to the axonal conduction blocker saxitoxin, indicating that they may result from direct interactions of NE with colonocytes rather than through indirect actions through enteric neurons. The {alpha}2-adrenergic antagonist yohimbine inhibited the effects of NE on EHEC adherence. Moreover, by acting downstream of {alpha}2-adrenergic receptors, the PKA activator Sp-8-Br-cAMPS appeared to antagonize the action of NE on bacterial adherence. In contrast, mucosal Isc responses to NE were resistant to yohimbine, and PKA modulators had no effect on colonic ion transport. Therefore, it appears that the effect of NE on EHEC adherence is mediated by {alpha}2-adrenergic receptors that are negatively coupled to PKA activity in colonocytes. On the basis of these results, we hypothesize that NE increases active ion transport and bacterial adherence in colonic epithelial cells through different mechanisms and cellular sites of action.

NE increased EHEC adherence after its addition to the contraluminal, but not luminal bathing medium. This finding is consistent with a site of action for the catecholamine at the basolateral aspect of the colonic epithelium and indicates that NE does not interact directly with EHEC during the relatively short 90-min duration of the assay. Indeed, a previous report (21) indicated that a minimum time period of 4 to 6 h is required for NE to directly influence EHEC growth and virulence. The relatively short time course of NE action suggests that the catecholamine may be promoting the initial loose adherence of EHEC to the mucosa, rather than EHEC attachment and effacement. Much less is known about the process of initial EHEC adherence compared with attachment and effacement. Recently, a fimbrial operon of E. coli O157:H7 has been discovered, which appears to play a role in microcolony formation and bacterial interactions with epithelial cells (36). Moreover, flagella of the closely related enteropathogenic E. coli appear to mediate adherence of this organism to epithelial cells and flagellum production may be induced by host signals (8). We hypothesize that NE-mediated decreases in PKA activity may modulate the expression of host protein(s) mediating the process of initial EHEC adherence to the mucosa. Although a phenomenon such as this has not hitherto been reported, NE has been shown to modulate the expression of intercellular adhesion molecule-1 in leukocytes through a {beta}2-adrenergic receptor-mediated mechanism (34).

The ability of NE to promote the adherence of both Shiga toxin-positive and -negative strains of E. coli O157:H7 indicates that the expression of these exotoxins is not an important factor in EHEC adherence. Moreover, NE appeared to selectively modulate the adherence of pathogenic E. coli, because it had no effect on either the adherence of nonpathogenic rodent- or porcine-adapted non-0157 strains of E. coli. The rodent-adapted commensal E. coli M-21 has been shown previously to be internalized by Caco-2 cells at counts similar to those determined by gentamicin resistance assay in the present study (39, 40). Internalization of this microorganism may represent a normal sampling phenomenon undertaken by the colonic mucosa. In isolated porcine Peyer's patches, NE increases the intracellular internalization of EHEC and Salmonella enterica serovar choleraesuis by a phentolamine-sensitive mechanism (11). In the porcine colonic mucosa, however, NE had no effect on the intracellular internalization of EHEC.

Previous investigations of E. coli O157:H7 adherence in pigs have employed the microorganism at inocula several orders of magnitude greater than that used in the present study. In these reports, EHEC was observed to adhere to both crypt and villous subregions of the small intestinal epithelium as well as to surface cells and glandular crypts of the colon (7, 38). This pattern of adherence, particularly in the less-accessible crypts, may have been due to a relatively high number of bacteria and long periods of bacterial exposure to the intestinal mucosa. In comparison, enteropathogenic strains of E. coli have been shown to adhere predominately to the upper half of the villous epithelium in the porcine small intestine and to surface cells of the colon (5, 25, 32). It will be of interest to determine in future studies whether the adherence-promoting action of NE extends to other enteropathogens, including strains of enteropathogenic E. coli, as well as to different species of colonic microflora.

Nerve fibers immunoreactive for TH and DBH, enzymes that catalyze NE synthesis in neurons, were observed to innervate the colonic mucosa. These fibers probably emanate from neurons in the caudal mesenteric ganglion as almost 85% of TH-immunoreactive cells in this ganglion project to the porcine colon and rectum (28). A combination of the indirectly acting sympathomimetic agents desipramine, pargyline, and tyramine mimicked the action of NE on EHEC adherence, but did not affect Isc. Moreover, the {alpha}-adrenergic antagonist phentolamine prevented the effects of exogenous NE and the sympathomimetic agents. These data suggest that NE present in adrenergic nerve fibers innervating the colonic mucosa may be released in quantities sufficient to increase EHEC adherence through an action on colonocyte {alpha}-adrenergic receptors, particularly in tissues treated with NE reuptake and degradation inhibitors. The inability of the indirectly acting sympathomimetic drugs to alter Isc might be attributed to the predominant localization of TH/DBH-immunoreactive nerve fibers near colonic surface cells rather than in the colonic crypts, a site for active chloride secretion.

The enteric nervous system appears to modulate epithelial defense and repair processes associated with intestinal infection (31). However, little is known concerning the effects of enteric neurotransmitters and hormones on the interactions between microbes and the intestinal mucosa. Because NE also increases adherence of EHEC to the cecal mucosa of mice (3), the phenomenon investigated here appears not to be restricted to the colon or to a particular animal species. We hypothesize that changes in sympathetic neural outflow to the colon may increase susceptibility of the host to infection by enteropathogens such as EHEC.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported, in part, by National Institutes of Health Grants R01-DA-10200, R01-AI-44918, and T32-DA-007239.


   ACKNOWLEDGMENTS
 
Present address of B. T. Green: United States Department of Agriculture, Agricultural Research Service, Clay Center, Nebraska 68933-0166 (E-mail address: green{at}email.marc.usda.gov).

The authors thank Dr. Carol L. Wells (Dept. of Laboratory Medicine and Pathology, University of Minnesota) for generously providing the M-21 rodent gut commensal strain of E. coli for this investigation, Dr. Sanford Weisberg (University of Minnesota Statistical Consulting Service) for expert advice on data analysis, and Eric Stewart for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. R. Brown, Univ. of Minnesota, Dept. of Veterinary and Biomedical Sciences, 1988 Fitch Ave., St. Paul, Minnesota 55108-6010

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* B. T. Green and M. Lyte contributed equally to this work. Back


   REFERENCES
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 DISCUSSION
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 REFERENCES
 

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