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. Author manuscript; available in PMC: 2014 Dec 26.
Published in final edited form as: Eur J Pharmacol. 2006 Apr 6;539(0):116–124. doi: 10.1016/j.ejphar.2006.03.081

Mucosally-directed adrenergic nerves and sympathomimetic drugs enhance non-intimate adherence of Escherichia coli O157:H7 to porcine cecum and colon

Chunsheng Chen a, Mark Lyte b, Mark P Stevens c, Lucy Vulchanova a, David R Brown a
PMCID: PMC4277206  NIHMSID: NIHMS69989  PMID: 16687138

Abstract

The sympathetic neurotransmitter norepinephrine has been found to increase mucosal adherence of enterohemorrhagic Escherichia coli O157:H7 in explants of murine cecum and porcine distal colon. In the present study, we tested the hypothesis that norepinephrine augments the initial, loose adherence of this important pathogen to the intestinal mucosa. In mucosal sheets of porcine cecum or proximal, spiral and distal colon mounted in Ussing chambers, norepinephrine (10 µM, contraluminal addition) increased mucosal adherence of wild-type E. coli O157:H7 strain 85–170; in the cecal mucosa, this effect occurred within 15 – 90 min after bacterial inoculation. In addition, norepinephrine transiently increased short-circuit current in cecal and colonic mucosal sheets, a measure of active anion transport. Norepinephrine was effective in promoting cecal adherence of a non-O157 E. coli strain as well as E. coli O157:H7 eae or espA mutant strains that are incapable of intimate mucosal attachment. Nerve fibers immunoreactive for the norepinephrine synthetic enzyme dopamine β-hydroxylase appeared in close proximity to the cecal epithelium, and the norepinephrine reuptake blocker cocaine, like norepinephrine and the selective α2-adrenoceptor agonist UK-14,304, increased E. coli O157:H7 adherence. These results suggest that norepinephrine, acting upon the large bowel mucosa, modulates early, non-intimate adherence of E. coli O157:H7 and probably other mucosa-associated bacteria. Sympathetic nerves innervating the cecocolonic mucosa may link acute stress exposure or psychostimulant abuse with an increased microbial colonization of the intestinal surface. This in turn may alter host susceptibility to enteric infections.

Keywords: Intimin, Type III secretion system, Mucosa-adherent bacteria, Norepinephrine, Cocaine, Enteric nervous system

1. Introduction

Enterohemorrhagic Escherichia coli O157:H7 is an important human pathogen that has been isolated from contaminated water as well as meat products. It produces hemorrhagic colitis after oral ingestion, and Shiga toxin-producing strains may additionally cause acute renal failure or neurological disturbances especially in young, elderly or immunocompromised individuals (Nataro and Kaper, 1998). E. coli O157:H7 possesses a pathogenicity island, termed the locus of enterocyte effacement. This locus encodes a type III secretion system which introduces virulence-associated proteins into host epithelial cells via a hollow filamentous extension of the needle complex encoded by the espA gene (Roe et al., 2003). One important protein is the translocated intimin receptor, which is delivered into epithelial cells and interacts with its cognate ligand, intimin, encoded by the eae gene in the locus of enterocyte effacement and expressed on the bacterial outer membrane (Campellone and Leong, 2003). Intimin interactions with the translocated intimin receptor and E. coli O157:H7 -induced changes in the epithelial cytoskeleton contribute to intimate mucosal adherence and the production of characteristic attaching and effacing lesions involving the cecum and large intestine (Moxley, 2004). Intimin also appears to determine the tropism of E. coli O157:H7 towards the mucosa of the large intestine (Stevens and Wallis, 2005). In addition to their role in intimin receptor translocation, espA filaments have been shown to act as an initial adhesin of E. coli O26:H- (Ebel et al., 1998).

Norepinephrine, at micro- to milli-molar concentrations and long (> 4 hr) exposure periods, has been reported to stimulate E. coli O157:H7 growth (Lyte and Nguyen, 1997), epithelial adherence (Vlisidou et al., 2004), and virulence (Lyte et al., 1996). Although the mechanisms underlying this direct interaction of norepinephrine or other biogenic amines with E. coli O157:H7 are incompletely defined, they appear to include increased bacterial iron transport (Freestone et al., 2003) and modulation of quorum sensing with potential upregulation of virulence factors (Clarke and Sperandio, 2005). In addition to their actions on enteric bacteria, norepinephrine or other substances released in response to acute stress in the host may act upon the intestinal mucosa to alter interactions between luminal microorganisms and epithelial cells. The results of recent investigations tend to support this hypothesis. Both norepinephrine and adrenocorticotrophic hormone have been reported to enhance E. coli O157:H7 adherence to the porcine distal colon through interactions with mucosal α-adrenergic and melanocortin receptors respectively (Green et al., 2004; Schreiber and Brown, 2005).

The main objective of the present study was to test the hypothesis that norepinephrine and other sympathomimetic drugs modulate the initial, loose adherence of E. coli O157:H7 and other mucosa-adherent, non-O157 E. coli, rather than the E. coli O157:H7 -mediated processes of epithelial cytoskeletal reorganization and formation of attaching/effacing lesions. To this end, pharmacological experiments were performed using E. coli O157:H7 strains manifesting genetically-engineered mutations of the espA and eae genes. In addition, our initial observations with norepinephrine were extended through the determination of the large intestinal sites and time course of norepinephrine action and a confirmation of the adrenergic receptors mediating bacterial adherence.

2. Materials And Methods

2.1. Bacteria

E. coli O157:H7 strains used in this study were from the laboratory of Dr. Mark Stevens. E. coli O157:H7 strain 87–170 nalR (code 95) is a spontaneous Shiga toxin-negative, nalidixic acid-resistant derivative E. coli O157:H7 strain 84–289, which was originally isolated from a food handler in a Canadian nursing home (Tzipori et al., 1987). Strain 85–170 has previously been reported to adhere to porcine ileal explants in an eae-dependent manner and to induce the formation of attaching and effacing lesions (Girard et al., 2005). E. coli O157:H7 85–170 nalR harboring a non-polar deletion of eae (strain ICC170; code 93) has been described previously (Fitzhenry et al., 2002). Strain 85–170 nalR espA::kanR (code 99) contains an insertion of the kanamycin resistance gene from plasmid pUC4K in espA and was constructed by allelic exchange using the positive-selection suicide vector pCVD442. Mutants lacking eae and espA were verified by Southern blotting and confirmed to respectively lack intimin or the filamentous type III translocon by Western blotting and immunofluorescence microscopy using specific antisera.

Commensal strains of porcine non-O157 E. coli were obtained by plating homogenized colonic mucosa from normal pigs onto Fluorocult agar (EM Science, Gibbstown, NJ) supplemented with 100 µg/ml streptomycin sulfate to isolate E. coli strains that were resistant to this antibiotic drug. 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 (Heizmann et al., 1998). Presumptive colonies of E. coli that did not have the appearance of E. coli O157:H7 were randomly chosen from Fluorocult plates following overnight incubation and were streaked onto Luria-Bertani (LB) agar plates supplemented with 100 µg/ml streptomycin. Following 24 h incubation at 37° C, individual colonies were picked from these plates and their identities confirmed as Escherichia 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). One strain of non-O157 E. coli (#4) was used in the present study.

Bacteria were stored as glycerol stocks at −80° C. For each experiment, bacteria were grown to stationary phase following overnight incubation in LB broth at 37° C in a humidified 5% CO2 atmosphere.

2.2. Animals and tissue preparation

Tissues were obtained from weaned Yorkshire-Landrace pigs of each sex; animals were 6–10 weeks old (10–18 kg body weight) and received food and water ad libitum. Each pig was anesthetized 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), and subsequently euthanized with intravenous Beuthanasia-D® (0.5 ml/kg; Schering-Plough Animal Health, Union, NJ) in accordance with approved University of Minnesota Animal Use and Care Committee protocols. A midline laparotomy was performed to expose the intestine. Explants were obtained from 10 cm of the terminal cecum; the proximal colon starting 15 cm distal to the cecal-colonic junction; the spiral colon starting approximately 120 cm distal to the cecal-colonic junction; and the distal colon above the terminal 10–15 cm of rectum and anus. These tissues were selected for investigation because the large intestine is an important site for E. coli O157:H7 colonization and attachment/effacement in swine, particularly the cecum, distal colon and rectum (Wales et al., 2005; Best et al., 2006). Tissues were placed in ice-cold oxygenated (95% O2, 5% CO2) physiological tissue preservation solution (ionic composition in mM: Na+, 130; K+, 6.0; Mg2+, 0.7; Ca2+, 3.0; HCO3, 19.6; HPO4−, 0.29, H2PO4−, 1.3; D-glucose, 11.0) which was maintained at pH 7.4.

2.3. Measurement of transepithelial electrical parameters

Each tissue was stripped of its underlying circular and longitudinal smooth muscle, and the resulting mucosal explant containing the inner submucosal plexus was mounted between two Ussing half-chambers (World Precision Instruments, Sarasota, FL) having a flux area of 1.0 cm2. D-Glucose and mannitol were added to the contraluminal and luminal bathing medium respectively at a final bath concentration of 10 mM. Both luminal and contraluminal reservoirs contained 10 ml of buffered, standard porcine physiological saline solution similar in composition to porcine extracellular fluid (composition in mM: NaCl, 130.0; KCl, 6.0; CaCl2, 3.0; MgCl2, 0.7; NaHCO3, 20.0; NaH2PO4, 0.29; Na2HPO4, 1.3) and gassed with 95% O2 and 5% CO2 at 39° C (porcine core temperature). Tissues were voltage-clamped (World Precision Instruments, Sarasota, FL) for measurement of short-circuit current (Isc, in µA/cm2), a measure of active, electrogenic transepithelial ion transport. Tissue electrical conductance (Gt, in mSiemens/cm2), a measure of mucosal ionic permeability, was calculated from potential difference and Isc by Ohm’s law. Experiments commenced after the Isc had stabilized.

2.4. Drugs

l-(−)-Norepinephrine bitartrate, propranolol hydrochloride, phentolamine mesylate, yohimbine HCl, prazosin HCl, l-phenylephrine HCl, and d,l-isoproterenol HCl were obtained from Sigma Chemical Co, (St. Louis, MO). 5-Bromo-N-(4,5-dihydro-1H-imidazol-2-yl)quinoxalin-6-amine (UK-14,304) was obtained from Tocris Bioscience (Ellisville, MO) and cocaine HCl (100 mg/ml) was obtained from Roxane Laboratories (Columbus, OH). Stock solutions of drugs were made in distilled water, with the exception of UK-14, 304 which was solubilized in dimethylsulfoxide, and yohimbine, prazosin and cocaine which were dissolved in ethanol; stock solutions were stored frozen and aliquots were serially diluted with water at each experimental session. Norepinephrine and propranolol were freshly prepared in distilled water prior to each experiment.

2.5. E. coli O157:H7 exposure and measurement of bacterial adherence in tissue mucosal sheets

Fifteen min after the addition of drugs to the contraluminal chamber, wild-type or mutated E. coli O157:H7 or commensal, enteroadherent E. coli in the stationary phase of growth were diluted 1:10 in PBS, and 100 µl of this diluted stock added to the luminal bath volume of 10 ml (1:100). The exact number of bacteria added was determined by serial spread plating of luminal bathing fluid samples onto Fluorocult E. coli O157 agar. The final density of bacteria achieved in the luminal bathing medium varied between 106 – 107 colony forming units (CFU)/ml.

Following mucosal exposure to the bacteria for 90 min, tissues were removed from Ussing chambers and mucosal adherence of E. coli O157:H7 was determined after the method of Knutton et al. (1989). In a previous study of porcine distal colonic explants, intracellular internalization of E. coli O157:H7 was negligible (Green et al., 2004). Tissues were removed from Ussing chambers, weighed, and washed three times in PBS (pH 7.4) to remove non-adherent bacteria. They were then homogenized using a Brinkman Polytron (Model PT 10–35; Kinematica AG, Littau, Switzerland) and dilutions of 1:1 and 1:10 were prepared and spread-plated on Fluorocult E. coli O157:H7 agar supplemented with 25 µg/ml nalidixic acid to select for E. coli O157:H7 nalR strain 87–170 and derivatives, or with 100 µg/ml streptomycin sulfate to select for streptomycin-resistant non-O157 E. coli. Following incubation of plates at 37° C for 24 h, the number of bacterial colonies (green coloration for E. coli O157:H7 and yellow coloration for non-O157 E. coli) were enumerated in CFUper gram of tissue and transformed to log10 values.

2.6. Immunohistochemistry

Segments of cecum were pinned mucosa side up on a Silgard-coated surface and fixed in modified Zamboni’s 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 double-labeled with a rabbit polyclonal antibody against bovine dopamine β-hydroxylase (DBH; 1:1,000; Immunostar, Hudson, WI) and a mouse monoclonal antibody against the neuronal marker protein gene product 9.5 (Biogenesis, Poole, England). The staining was visualized with indocarbocyanine (Cy3)-conjugated donkey anti-rabbit and cyanine (Cy2)-conjugated donkey anti-mouse secondary antisera (Jackson Immunoresearch Laboratories, West Grove, PA). Images were collected using a confocal laser-scanning microscope (Bio-Rad MRC 1000) and processed using Adobe Photoshop (version 6.0.1, Adobe Systems, San Jose, CA).

2.7. Statistical analysis

All data are expressed as mean ± standard error of the mean. Statistical analyses of data were performed using the PRISM computer software program (Version 4.0a; GraphPad Software, Inc., San Diego, CA). Single comparisons between control and treatment means were made with a paired or unpaired Student’s two-tailed t test when appropriate. Comparisons of multiple means were made by an analysis of variance (ANOVA) with Tukey’s test. The minimum level for statistical significance was set at P < 0.05.

3. Results

3.1. Localization of norepinephrine action on E. coli O157:H7 adherence in the porcine large intestine

Wild-type E. coli O157:H7 adhered to mucosal explants from cecum and colon after its exposure to the luminal aspect of these tissues for 90 min (Fig. 1). Bacterial adherence did not differ significantly among large intestine segments (F = 1.9; 3,71 df, one-way ANOVA). Baseline Isc and Gt were similar among mucosal explants from different large intestine segments as well (Table 1). Added to the contraluminal bath at a concentration of 10 µM, norepinephrine significantly increased E. coli O157:H7 adherence over 90 min in mucosal explants from cecum and colon relative to adherence measured in control tissues untreated with norepinephrine (Fig. 1). It also produced a transient increase in Isc which was significantly greater in distal colon explants than in the other segments of the porcine large intestine (Table 2).

Fig. 1.

Fig. 1

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Effect of norepinephrine (NE) on mucosal adherence of E. coli O157:H7 (Shiga toxin-negative Nalr strain 85–170) to porcine cecum (CM), proximal colon (PC), spiral colon (SC) and distal colon (DC). The aligned dot plot represents E. coli O157:H7 adherence to individual control tissues untreated with norepinephrine (unfilled circles) and to tissues contraluminally exposed to 10 µM norepinephrine (filled circles) from each intestinal site. Wild-type E. coli O157:H7 85–170 nalR were added to the luminal medium bathing mucosal explants at an inoculum of 2.0 ± 0.3 × 106 CFU/ml and remained in contact with each tissue for 90 min. Wide horizontal bars indicate mean values and shorter horizontal bars indicate standard error of the mean for each condition. *P < 0.05 and ** P ≤ 0.001 vs. control mean, t test; n = 11 – 24 tissues from 4 – 6 pigs.

Table 1.

Baseline short-circuit current (Isc) and electrical conductance (Gt) in porcine mucosal explants in the presence of E. coli O157:H7 85–170 nalR

Tissue Isc (µA/cm2), mean ± SE Gt (mS/cm2), mean ± SE n/Na
Cecum 5.3 ± 1.2 19.9 ± 1.9 14/6
Proximal colon 4.3 ± 0.5 19.6 ± 1.6 10/5
Spiral colon 4.5 ± 0.6 19.6 ± 2.7 17/8
Distal colon 5.2 ± 1.0 14.9 ± 2.9 12/6
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a

Total number (n) of tissues tested from N pigs.

Table 2.

Effect of 10 µM norepinephrine on short-circuit current in porcine mucosal explants in the presence of E. coli O157:H7 85-170 nalR

Tissue Peak ΔIsc (μA/cm2) n/Na
Cecum 37 ± 4 22/8
Proximal colon 14 ± 3 24/8
Spiral colon 17 ± 3 25/8
Distal colon 80 ± 16* 18/6
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a

Total number (n) of tissues tested from N pigs.

*

Significantly greater than proximal three segments (P < 0.001, Tukey test). Mean ΔGt in response to norepinephrine ranged from 1.9 ± 0.8 (distal colon) to 2.8 ± 0.7 mS/cm2 (cecum), but did not differ statistically among intestinal segments.

3.2. Time course of norepinephrine action on E. coli O157:H7 adherence in porcine cecal mucosa

Bacterial adherence to the cecal mucosa increased as the luminal exposure period to wild-type E. coli O157:H7 lengthened; E. coli O157:H7 adherence after 90 min of bacterial exposure was significantly greater than after 15, 30 or 60 min of exposure (P < 0.05, Tukey test). Contraluminal norepinephrine (10 µM) significantly increased cecal adherence of E. coli O157:H7 relative to norepinephrine-untreated control tissues at 15 – 90 min of E. coli O157:H7 exposure (Fig. 2).

Fig. 2.

Fig. 2

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Time course of the adherence-enhancing effect of norepinephrine (NE) in porcine cecal mucosa. Wild-type E. coli O157:H7 85–170 nalR were added to the luminal medium bathing mucosal explants at an inoculum of 9.9 ± 4.2 × 106 CFU/ml and remained in contact with each tissue represented in the aligned dot plot for the time periods indicated on the abscissa. In the absence of norepinephrine (unfilled circles), there was a significant time-related increase in bacterial adherence (F = 7.45, P = 0.0008, 31 total df). In some tissues (filled circles), norepinephrine was added to the contraluminal bathing medium to achieve a bath concentration of 10 µM. Wide horizontal bars indicate mean values and shorter horizontal bars indicate standard error of the mean for each condition. *P < 0.05 and **P < 0.01 vs. control mean (unfilled circles), t test; n = 8 tissues from 8 pigs.

3.3. Effect of norepinephrine action on cecal adherence of E. coli O157:H7 eae and espA mutants

Over a period of 90 min, both E. coli O157:H7 85–170 nalR mutants adhered to the mucosal surface of cecal explants, although the number of the adherent E. coli O157:H7 eae mutant was significantly less than that of wild-type E. coli O157:H7 (Fig. 3). Nevertheless, norepinephrine at a contraluminal concentration of 10 µM increased adherence of both E. coli O157:H7 mutants to the cecal mucosa relative to norepinephrine-untreated control tissues (Fig. 3). Norepinephrine significantly enhanced adherence of the E. coli O157:H7 espA mutant to the distal colonic mucosa as well (Table 3). Moreover, it increased cecal adherence of a non-O157 strain of E. coli (Fig. 3).

Fig. 3.

Fig. 3

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Norepinephrine-enhanced adherence of E. coli O157:H7 does not require intimin or protein translocation into host cells. Bacteria were added to the luminal medium bathing mucosal explants at inocula of 22.4 ± 6.0 × 106 CFU/ml (E. coli O157:H7 nalR, wild type; n= 5), 12.6 ± 7.6 × 106 CFU/ml (O157:H7 nalR eae mutant; n= 5), 46.8 ± 7.6 × 106 CFU/ml (85–170 nalR espA::kanR; n= 8) or 43.1 ± 19.3 × 106 CFU/ml of a streptomycin-resistant, non-O157:H7 E. coli strain isolated from porcine colon (n = 4). They remained in contact with each tissue represented in the aligned dot plot for 90 min. In some tissues (filled shapes), norepinephrine (NE) was added to the contraluminal bathing medium to achieve a final concentration of 10 µM. Wide horizontal bars indicate mean values and shorter horizontal bars indicate standard error of the mean for each condition. ‡P < 0.05 vs. wild-type E. coli O157:H7 85–170 Nalr strain, Tukey’s test. **P < 0.01 and ***P < 0.001 vs. norepinephrine-untreated control tissues (unfilled shapes), t test; n = 9 – 36 tissues from 5–12 pigs.

Table 3.

Effect of 10 μM norepinephrine on adherence of an E. coli O157:H7 85–170 nalR espA mutant (in log10 CFU/g, mean ± S.E.M.) to distal colonic mucosa

Time interval of mucosal E. coli O157:H7 exposure (min) Control Norepinephrinea
30 3.70 ± 0.10 3.82 ± 0.10*
60 3.64 ± 0.08 4.04 ± 0.08*
90 3.89 ± 0.05 4.30 ± 0.05**
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n = one pair of tissues from each of 6 pigs/each time point

a

Added to contraluminal bathing medium

*

P < 0.05 and

**

P < 0.01 vs. norepinephrine-untreated control tissues (paired t test)

3.4. Characterization of adrenergic receptors mediating E. coli O157:H7 adherence to cecal mucosa

The effect of norepinephrine on mucosal adherence of E. coli O157:H7 in the porcine distal colon was previously found to be mediated by α2-adrenoceptors (Green et al., 2004). To confirm and extend this finding to the porcine cecum, the effects of selective adrenergic receptor antagonists and agonists were examined. In cecal mucosa explants, the effects of norepinephrine (10 µM, contraluminal administration) on wild-type E. coli O157:H7 adherence were inhibited significantly by the α-adrenoceptor antagonist phentolamine, but not by the β-adrenoceptor antagonist propranolol (Fig. 4, top). Furthermore, they were inhibited by the α2-adrenoceptor antagonist yohimbine, but not by the α1-adrenoceptor antagonist prazosin (Fig. 4, middle).

Fig. 4.

Fig. 4

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Characterization of adrenergic receptors mediating mucosal adherence of E. coli O157:H7 in porcine cecum. Top. Effect of 10 µM norepinephrine in the absence (NE) and presence of 0.1 µM phentolamine (PTL/NE) or propranolol (PPL/NE). Bacteria were added to the luminal medium bathing mucosal explants at an inoculum of 16.5 ± 4.8 × 106 CFU/ml and remained in contact with each tissue represented in the aligned dot plot for 90 min. All drugs were added to the contraluminal bathing medium. **P < 0.01 vs. control mean (unfilled circles) and ‡‡P < 0.01 vs. norepinephrine mean (black filled circles), one-way ANOVA with Tukey’s test; n = 7– 12 tissues from 7 pigs. Middle. Effect of 10 µM norepinephrine in the absence (NE) and presence of 30 µM prazosin (PRZ/NE) or yohimbine (YHM/NE). Bacteria were added to the luminal medium bathing mucosal explants at an inoculum of 9.0 ± 2.5 × 106 CFU/ml and remained in contact with each tissue for 90 min. All drugs were added to the contraluminal bathing medium. *P < 0.05 and **P < 0.01 vs. control mean (unfilled circles) and ‡‡P < 0.01 vs. norepinephrine mean (black filled circles), one-way ANOVA with Tukey’s test; n = 8 tissues from 6 pigs. Bottom. Adherence-promoting effect of selective adrenergic agonists. Bacteria were added to the luminal medium bathing mucosal explants at an inoculum of 8.4 ± 4.4 × 106 CFU/ml and remained in contact with each tissue for 90 min. Each agonist was added to the contraluminal bathing medium to achieve a final concentration of 10 µM. ***P < 0.001 vs. control mean (unfilled circles), one-way ANOVA with Tukey’s test; n = 6 – 8 tissues from 5 pigs. In each panel, wide horizontal bars indicate mean values and shorter horizontal bars indicate standard error of the mean for each condition.

The highly-selective α2-adrenoceptor agonist UK14,304 significantly increased wild-type E. coli O157:H7 adherence to the porcine cecal mucosa after its addition to the contraluminal bathing medium at a concentration of 10 µM; in contrast, neither the α1-adrenoceptor agonist phenylephrine nor the β-adrenoceptor agonist isoproterenol altered E. coli O157:H7 adherence (Fig. 4, bottom).

3.5. Localization of adrenergic nerve fibers in cecal mucosa and effects of cocaine on E. coli O157:H7 adherence

The localization of adrenergic nerve fibers in the cecal mucosa was demonstrated using immunohistochemistry. Antisera against dopamine β-hydroxylase (DBH), the last enzyme in the norepinephrine synthetic pathway, labeled nerve fibers in submucosal ganglia and throughout the mucosa. The neuronal localization of DBH staining was confirmed by double-labeling with the neuronal marker protein gene product 9.5 (PGP), which showed overlap of DBH and PGP immunoreactivities (Fig. 5, top).

Fig. 5.

Fig. 5

Fig. 5

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Endogenous norepinephrine modulates cecal adherence of E. coli O157:H7. Top. Localization of noradrenergic nerve fibers in the mucosa of porcine cecal explant by immunohistochemistry. Double labeling for dopamine β-hydroxylase (DBH; left) and protein gene product 9.5 (PGP; right) demonstrated the presence of DBH-immunoreactive (-ir) nerve fibers within PGP-ir nerve bundles in submucosal ganglia (arrowheads), in the vicinity of crypts (small arrows) as well as within the villous lamina propria. Bottom. Concentration-effect relationship for the norepinephrine re-uptake blocker cocaine in enhancing adherence. Bacteria were added to the luminal medium bathing mucosal explants at an inoculum of 6.1 ± 1.1 × 106 CFU/ml and remained in contact with each tissue represented in the aligned dot plot for 90 min. Cocaine was added to the contraluminal bathing medium to achieve the bath concentrations indicated on the abscissa (gray filled squares). Wide horizontal bars indicate mean values and shorter horizontal bars indicate standard error of the mean for each condition. ***P < 0.001 vs. mean log CFU/g of E. coli O157:H7 adhering to cecal mucosa explants not treated with cocaine (concentration = 0, unfilled squares), one-way ANOVA with Tukey’s multiple comparisons test; n = 8 – 10 tissues from 5 pigs.

The psychostimulant drug cocaine is a potent blocker of norepinephrine reuptake into adrenergic nerve terminals (Fleckenstein et al., 2000). At contraluminal concentrations ≥ 10 µM, cocaine significantly increased E. coli O157:H7 85–170 nalR adherence to the cecal mucosa (Fig. 5, bottom). The estimated 50% effective concentration of cocaine was 14.5 µM based on best fit analysis of the cocaine concentration-effect curve. Cocaine similarly increased E. coli O157:H7 adherence in explants of distal colonic mucosa as well (mean log10 CFU/gm E. coli O157:H7 85–170 nalR recovered from 6 pairs of distal colonic mucosa untreated and pretreated with 10 µM cocaine was respectively 2.59 ± 0.18 and 3.04 ± 0.10, P = 0.008, paired t test).

4. Discussion

Young pigs are an important host for attaching-effacing E. coli and have been used in several previous investigations as an animal model for the study of E. coli O157:H7 pathogenesis (Wales et al., 2005). The present findings indicate that a strain of E. coli O157:H7 is capable of adhering to the mucosae of the porcine cecum and three subregions of the colon after a relatively short (90 min) period of luminal exposure. This is likely to occur at the surface epithelium of these tissues (Green et al., 2004). Adherence of E. coli O157:H7 85–170 nalR was reduced in the absence of the outer membrane adhesin intimin, a finding consistent with those of recent studies using porcine ileal explants cultured in vitro and the same bacterial strains (Girard et al., 2005). In experimental animals inoculated with E. coli O157:H7, the microorganism produces attaching-effacing lesions in, and can be cultured from, both the cecum and colon (Francis et al., 1986; Grauke et al., 2002). In explants from all large intestinal segments examined, norepinephrine increased both Isc and mucosal adherence of E. coli O157:H7. These effects are similar to those reported previously in the mouse cecum (Chen et al., 2003), a finding suggesting that they are not restricted to a particular host species. Peak Isc responses to norepinephrine, which were highest in porcine distal colonic mucosa, have been attributed to active chloride secretion and are mediated by α1-adrenoceptors that are likely to be localized on colonic crypt cells (Traynor et al., 1991). As the tissues were voltage-clamped, the potential difference produced by anion secretion was automatically nullified and an ionic gradient to support water flux could not develop. Moreover, the muscarinic cholinergic antagonist carbachol rapidly increases Isc but has no effect on E. coli O157:H7 adherence in the porcine distal colon (D.R. Brown, unpublished observations). Therefore, the observed norepinephrine-induced increase in Isc per se would not be expected to alter bacterial adherence.

A previous report (Green et al., 2004) indicated that norepinephrine promotes colonic adherence of both Shiga toxin-producing E. coli O157:H7 (strain EDL933) and, as observed in the present study, toxin-negative E. coli O157:H7 strains (e.g. strain 700728). Thus, it is unlikely that Shiga toxins play a role in this effect of norepinephrine. E. coli O157:H7 infection is generally acknowledged to occur in three stages: initial, loose attachment to host epithelial cells; EspA-mediated protein translocation to host cells; and intimate adherence and pedestal formation (Wales et al., 2005). Three findings strongly suggest that norepinephrine acts at the earliest stage of E. coli O157:H7 adherence to the intestinal epithelium of the host. First, norepinephrine increased the number of E. coli O157:H7 adhering to the cecal mucosa as early as 15 min after bacterial inoculation of the luminal bathing fluid. Second, norepinephrine increased the cecal adherence of an E. coli O157:H7 mutant strain that was incapable of expressing intimin. Third, norepinephrine increased cecal and colonic adherence of an E. coli O157:H7 espA mutant, which lacks the ability to deliver adherence-promoting type III secreted proteins to host cells. We hypothesize that norepinephrine enhances the early, non-intimate attachment of E. coli O157:H7 to the epithelium of the cecum and colon. Although the process of intimate attachment has been well characterized in enterohemorrhagic and enteropathogenic E. coli, considerably less is known about the factors mediating early, non-intimate adherence. These include flagellae (Best et al., 2005, 2006), long polar fimbriae (Jordan et al., 2004), and OmpA protein (Torres and Kaper, 2003). It is notable that norepinephrine increased adherence of a non-O157 E. coli to the cecal mucosa. In a previous investigation (Green et al., 2004), colonic adherence of a rodent-adapted E. coli strain as well as a different non-O157 E. coli isolated from pigs was not increased by norepinephrine. It is possible that norepinephrine alters the expression or clustering of host epithelial adhesion molecules that interact with one or more bacterial adherence determinants expressed not only by E. coli O157:H7 but also by some non-O157 strains of mucosa-adhering E. coli or other species of bacteria.

In the porcine cecum, the adherence-promoting effect of norepinephrine appears to be mediated by α-adrenoceptors. The α-adrenergic antagonist phentolamine, but not the β-adrenoceptor antagonist propranolol, prevented norepinephrine action. In addition, the norepinephrine effect was prevented by the selective α2-adrenoceptor antagonist yohimbine, and mimicked by the selective α2-adrenergic agonist UK-14,304. Alpha2-adrenoceptors, which are coupled to inhibition of adenylate cyclase, appear to mediate norepinephrine-induced E. coli O157:H7 adherence to the porcine distal colonic mucosa as well (Green et al., 2004). In the latter tissue, norepinephrine action remains unaltered in the presence of the axonal conduction blocker saxitoxin, a finding which suggests that the relevant adrenergic receptors are located on non-neuronal cells in the intestinal mucosa, most probably epithelial cells. Alpha2-adrenoceptors have been identified previously on both primary and transformed colonic epithelial cells (Senard et al., 1990; Valet et al., 1993).

The present immunohistochemical studies indicate that sympathetic nerve fibers immunoreactive for the norepinephrine-synthesizing enzyme dopamine β-hydroxylase are localized in close proximity to the cecal epithelium. These fibers likely arise from paravertebral ganglia and therefore originate from outside the intestinal wall (Janig and McLachlan, 1987). The psychostimulant drug cocaine, which increases endogenous norepinephrine concentration at neuro-epithelial synapses, increased adherence of E. coli O157:H7 to the cecal mucosa. The presence of presumptive noradrenergic nerve fibers in the cecal mucosa and ability of cocaine to mimic the effect of norepinephrine on E. coli O157:H7 adherence suggest that sympathetic nerves innervating the mucosa may play a role in the short-term regulation of epithelium-microbe interactions. Due to their potent vasoconstrictive activity, cocaine and other psychostimulant drugs such as methamphetamine can produce ischemic colitis (Cappell, 2004), but there appear to be no reports documenting an increased incidence in intestinal infections following the acute administration of these drugs. On the other hand, physical or mental stress appears to alter bacterial adherence to the intestinal mucosa (Alverdy et al., 2005). As acute exposure to stressful stimuli is accompanied by increases in intestinal sympathetic outflow (Bhatia and Tandon, 2005), norepinephrine and α2-adrenoceptors in the large intestine may serve to link host responses to stress with an increased susceptibility to enteric infections. Finally, some bacterial strains might alter enteric neurotransmission as a means of competing with other microorganisms in colonizing the intestinal mucosa. For example, Clostridium difficile is a non-invasive, exterotoxigenic bacterium that can colonize the large intestine after disruption of the normal microfloral environment by antibiotic treatment. Toxin A from C. difficile appears to inhibit transmission in enteric sympathetic nerves (Xia et al., 2000). The cellular mechanisms by which sympathetic nerves modulate bacterial interactions with cecal and colonic epithelia and the potential role that norepinephrine-induced bacterial adherence might play in psychostimulant abuse or disease states affecting the large bowel clearly warrant additional investigations.

Acknowledgments

We thank Melissa A. Casey and H. Noel Opitz for excellent technical assistance. This investigation was supported by National Institutes of Health grants AI-44918 and MH-50431 (M.L.) and DA-10200 (D.R.B.). M.P.S. gratefully acknowledges the support of the Biotechnology & Biological Sciences Research Council, UK (C518022). Salary support for C.C. was provided by NIH training grant T32 DA-007239.

Footnotes

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