Specificity of catecholamine-induced growth in Escherichia coli O157:H7, Salmonella enterica and Yersinia enterocolitica

Specificity of catecholamine-induced growth in Escherichia coli O157:H7,

Salmonella enterica and Yersinia enterocolitica

Primrose P.E. Freestone1, Richard D. Haigh1 and Mark Lyte2*

Department of Infection, Immunology and Inflammation, University of Leicester-Warwick

School of Medicine, Leicester, United Kingdom1 and Department of Pharmacy Practice,

School of Pharmacy, Texas Tech University Health Sciences Center, Lubbock, TX 79430,

USA2

* Corresponding author:

Dr. Mark Lyte

Department of Pharmacy Practice

Texas Tech University Health Sciences Center

3601 4th Street, STOP 8182

Lubbock, TX 79430-8162, USA

Telephone: (806) 743-4200, ext. 262

FAX: (806) 743-4209

Email: [email protected]

1

Abstract

The present study demonstrates that catecholamine responsiveness in Yersinia

enterocolitica, a bacterial pathogen whose infectious spectrum is principally limited to the gut,

is limited to norepinephrine and dopamine, and not epinephrine; this behavior contrasts with

observations for two pathogens with a wider extra-gastrointestinal spectrum, Escherichia coli

O157:H7 and Salmonella enterica, which respond to all three catecholamines. Epinephrine

showed lower potency than norepinephrine and dopamine in inducing growth of E. coli and S.

enterica, and was a potent antagonist of norepinephrine and dopamine growth

responsiveness in Y. enterocolitica. Given that only norepinephrine and dopamine and not

epinephrine containing neurons are found with the enteric nervous system, our results

suggest that certain of the more exclusive enteric pathogens may have developed response

systems preferentially for those neuroendocrine hormones that are produced by the enteric

nervous system as host-derived signals by which to sense the environment and initiate

pathogenic processes.

Keywords: norepinephrine, epinephrine, dopamine, iron, enteric pathogens

2

Introduction

The intersection of the fields of microbiology and neuroendocrinology represents an

interdisciplinary approach to the study of the infectious disease process that has been termed

microbial endocrinology (Lyte, 1993; Lyte, 2004). This field has as its central tenet that

microbes can use host hormones as environmental cues to initiate growth and pathogenic

processes. Microbial endocrinology research has focussed primarily on the role in the

infectious disease process of the catecholamines, a group of organic compounds responsible

for many different neuroendocrine signalling phenomena in multicellular organisms. The

catecholamine hormones epinephrine (Epi) and norepinephrine (NE) play an integral part of

the acute stress response in metazoa.

A role for catecholamine stress hormones in the infectious disease process was first

suggested in the early 1930’s (Lyte, 2004), but was not clearly elucidated until 1992 when the

first demonstration of the direct action of catecholamines on bacterial growth was shown by

Lyte and co-workers (Lyte & Ernst, 1992) and a theory proposed for a direct role of

catecholamine-bacterial interaction in the pathogenesis of infectious disease (Lyte, 1992,

Lyte, 1993). More recent interest in the role that catecholamines may play in bacterial

pathogenesis has been heightened by reports that NE, Epi and dopamine (Dop), can directly

stimulate bacterial growth and elaboration of virulence-associated factors, as well as induce

the production of autoinducer-like substances (Lyte & Ernst, 1992; Freestone, et al., 1999;

Kinney, et al., 1999; Kinney, et al., 2000; Neal, et al., 2001; Belay & Sonnenfeld, 2002;

Freestone, et al., 2002; Reissbrodt, et al., 2002; Lyte, et al., 2003).

The biochemical pathway for the synthesis of catecholamines is L-dopa (most

commonly from food-borne sources) → dopamine → norepinephrine → epinephrine. NE and

Dop containing sympathetic nerve terminals are distributed widely throughout the body,

3

including the intestinal tract where they make up part of the enteric nervous system (ENS)

(Costa, et al., 2000). Indeed, half of the NE present within the mammalian body is

synthesized and utilized within the ENS. Importantly, Epi is not synthesized within the ENS

and so far appreciable levels of Epi have not been detected within the gastrointestinal tract

(Costa, et al., 2000).

We, and others (Lyte & Ernst, 1992; Freestone, et al., 1999; Kinney, et al., 1999;

Kinney, et al., 2000; Neal, et al., 2001; Belay & Sonnenfeld, 2002; Freestone, et al., 2002;

Reissbrodt, et al., 2002; Lyte, et al., 2003) have demonstrated that the in vitro growth of a

number of bacterial species can be increased in the presence of one or more of the

catecholamines NE, Epi and Dop. However, there have been conflicting reports concerning

the ability of any one specific catecholamine to affect bacterial growth (Belay & Sonnenfeld,

2002; Belay, et al., 2003). Additionally, Sperandio and co-workers have reported that certain

aspects of the virulence of E. coli O157:H7 were regulated preferentially by Epi (Walters &

Sperandio, 2006), and suggested Epi may be a specific host hormonal cue for

enteropathogenic bacteria. In marked contrast, Lyte and Ernst (Lyte & Ernst, 1992) had

earlier found that another enteric pathogen, Y. enterocolitica, showed no apparent

responsiveness to Epi. However, the Lyte and Ernst study was performed with a single Y.

enterocolitica isolate, while the Walters and Sperandio report investigated Epi effects on the

virulence of high cell density cultures of E. coli O157:H7. To clarify whether there exists

preferences for catecholamine stress hormones amongst enteric bacteria that are reflective of

the host environment in which they are normally found, we undertook a comparative analysis

of the specificity of catecholamine responsiveness of three enteric pathogens characterized

by either their propensity to primarily inhabit the gut (Y. enterocolitica, or to colonize extra-

intestinal sites (E. coli O157:H7 and S. enterica).

4

Materials and methods

Bacterial strains and growth conditions

Recent clinical and reference (NCTC) isolates of Y. enterocolitica isolates were obtained from

Dr. Paddy Kimmit of the Leicester Public Health Laboratory, Leicester Royal Infirmary,

Leicester UK. S. enterica strain SL1344 was obtained from Dr. Jay Hinton, Institute of Food

Research, Norwich, UK. E. coli O157:H7 strain NCTC 12900 was used previously (Freestone,

et al., 2003). Serum-SAPI medium was prepared as described previously (Lyte & Ernst, 1992;

Freestone, et al., 1999), and had the following composition: 6.25 mM NH4NO3, 1.84 mM

KH2PO4, 3.35 mM KCl, 1.01 mM MgSO4 and 2.77 mM glucose, pH 7.5, supplemented with

30% (v/v) adult bovine serum) (Sigma, Poole, UK). DMEM medium, apo-forms of human

transferrin (Tf), epinephrine, dopamine and norepinephrine were all purchased from Sigma,

Poole, UK. 55FeCl3 (IES, specific activity 5 mCi/mg Fe), 3H-NE (TRK584,l-[7,8-3H]

norepinephrine) were obtained from Amersham Life Science, UK.

Catecholamine growth response and Epi antagonism assays

A serum-based medium was employed to more closely approximate the growth restrictive in

vivo conditions within a mammalian host (such as complement, antibodies, low nutrient

availability and Fe-restriction due to the presence of host iron sequestering proteins) (Lyte,

2004). Catecholamine (Dop, Epi and NE) dose response and antagonism assays were

performed in serum-SAPI supplemented with concentrations of the compounds as shown in

the text. Controls comprised equivalent volumes of the solvent used to dissolve the

catecholamine. To demonstrate that Epi was not directly inhibitory to Y. enterocolitica growth,

Epi antagonism of catecholamine-growth induction assays were also performed in the

presence of a concentration of Fe which overcomes the Fe-limitation of serum-SAPI (100 µM

5

Fe(NO3)3) (Burton, et al., 2002). Bacteria were inoculated into serum-SAPI at approximately

50-100 CFU per ml and incubated statically at 37°C in a 5% CO2 humidified incubator for 18

hrs in the case of E. coli O157:H7 and S. enterica, and 40 hours in the case of Y.

enterocolitica (which is slower growing in serum; Lyte & Ernst, 1992).

Dependence of catecholamine responsiveness on bacteria cell density

To examine correlations between bacterial cell density and specificity of catecholamine

responsiveness, one ml aliquots of overnight cultures of E. coli O157:H7, S. enterica and Y.

enterocolitica were pelleted by centrifugation at 5000 x g for 5 minutes, washed twice in warm

serum-SAPI, re-suspended in 1 ml of serum-SAPI and serially diluted to a nominal cell

density of less than 1 CFU per ml in serum-SAPI containing no additions (control), or 50 µM

NE, 50 µM Dop or 100 µM Epi. Cultures were incubated and enumerated for growth as

described above.

Analysis of effects of Epinephrine on catecholamine-mediated Y. enterocolitica

incorporation of iron from transferrin and uptake of NE

55Fe-Tf was prepared as described previously (Freestone, et al., 2000). Exponential bacteria

were inoculated at 108 CFU per ml into serum-SAPI supplemented with 2 x 105 counts per

minute (cpm) of 55Fe-Tf plus catecholamines added at the concentrations indicated in the

legend to Table 1. Cultures were incubated at 37°C in a 5% CO2 humidified incubator for 6

hrs, harvested by centrifugation at 5000 x g for 5 minutes, washed in PBS and assayed for

cell numbers and 55Fe incorporation using pour plate analysis and scintillation counting as

described previously (Freestone, et al., 2000). Assays were performed in triplicate on at least

two occasions; variation within individual assay sets was usually less than 5%, and between

experiments less than 10%.

6

To examine the effects of Epi on uptake of NE, exponential cultures of Y. enterocolitica

were harvested, washed twice in DMEM, and added at 2 x 108 CFU per ml to fresh DMEM

containing 50 μM NE plus 5 x 105 cpm per ml of 3H-NE and either no additions (control), or

100-300 μM Epi. Cultures were incubated statically for 6 hrs at 37°C in a 5% CO2 humidified

incubator (this time point was determined by conducting a prior time course of 3H-NE uptake

and represents the time required for maximal uptake), and then analyzed for growth and 3H-

NE uptake as described for the 55Fe incorporation assays above. 3H-NE uptake assays were

performed in duplicate on at least two occasions; variation within individual assay sets was

5% or less, and between experiments no more than 15%.

Statistical analysis

Where appropriate, statistical analysis was performed using an unpaired t-test in which a two-

tailed P value was calculated (Instat program, GraphPad Software, San Diego, CA, USA).

Statistical significance was defined as a P value of <0.05.

7

Results

Analysis of the specificity of catecholamine growth responsiveness in E. coli O157:H7

and Y. enterocolitica

During our initial investigations of the growth responsiveness of clinical bacterial isolates to

NE we occasionally found strains that had acquired mutations rendering them deficient in NE

recognition (Freestone, et al., 1999). In addition, we had also determined that bacterial

responses to catecholamine stress hormones are markedly concentration dependent (Lyte &

Ernst, 1992; Freestone, et al., 2002). We therefore investigated whether the lack of Epi

growth induction previously observed in a single Y. enterocolitica isolate (Lyte & Ernst, 1992)

was due either to a strain specific mutation, or simply because the concentrations of Epi

utilized were too low, by analyzing the responses of 14 Y. enterocolitica strains (including 5

NCTC strains) to a range of Epi concentrations. Figure 1A shows a typical growth response

profile of one of these 14 strains (NCTC 551) to increasing concentrations of Epi, compared

with similar concentrations of NE and Dop; an equivalent profile from E. coli O157:H7 is

shown in Figure 1B. It can clearly be seen that there is no growth responsiveness to

concentrations of Epi that are stimulatory to E. coli, indicating that the inability of Epi to affect

the growth of Y. enterocolitica does not appear to be a consequence of catecholamine

potency. The dose-response growth effect of the catecholamines for both Y. enterocolitica

and E. coli O157:H7 (Figure 1) also shows that on a concentration-dependent basis NE

exposure had the most potent ability among the catecholamines to stimulate bacterial growth.

8

Epinephrine acts as an antagonist of norepinephrine and dopamine growth induction

in Y. enterocolitica

It has previously been shown that catecholamines can act in a synergistic manner to promote

bacterial growth (Freestone, et al., 2002). The response of Y. enterocolitica to

catecholamines was further investigated by examining whether Epi could synergistically affect

NE and Dop-mediated growth enhancement. Figures 2A and 2B show that addition of Epi to

serum-SAPI assays of Y. enterocolitica supplemented with either NE or Dop resulted in a

concentration-dependent inhibition of NE and Dop growth induction (P<0.0001). This

unexpected inhibitory action of Epi appears to be due to a specific antagonism of both NE

and Dop-responsiveness and not Epi cytotoxicity, since addition of 100 µM Fe(NO3)3 to Epi-

supplemented Y. enterocolitica NE and Dop cultures relieved the Epi-induced growth

inhibition (Figures 2A and 2B). In contrast, inclusion of Epi into NE and Dop supplemented

cultures of E. coli O157:H7 (Figures 2C and 2D) or S. enterica (data not shown) acted only to

enhance growth as earlier reported (Freestone, et al., 2002).

Previously, we demonstrated that NE is internalized by bacteria during the NE-growth

induction process (Freestone, et al., 2000), prompting us to investigate whether Epi was

antagonizing Y. enterocolitica responses to NE by blocking entry of NE into the bacterial cell.

We found that uptake of 3H-NE in high cell density cultures of Y. enterocolitica treated with

growth inhibitory concentrations of Epi as used in Figure 2A) (100, 200 and 300 μM Epi) was

not significantly reduced (P>0.05). Internalisation of 3H-NE in control cultures was 1356

CPM/ml and in cells treated with 100-300 μM Epi 1284, 1222 and 1470 CPM/ml respectively

(standard deviations for each set of cultures were 64, 55, 70 and 90 CPM/ml) We also

analysed whether Epi might reduce uptake of 3H-NE in a active growth context using a low

cell density initial inoculum (around 102 CFU per ml) combined with 24 hrs incubation (to

9

enable the culture to grow to a cell density similar to that employed in the high cell density

uptake assay). Using this approach, we overall found slightly more 3H-NE internalisation

(around 2000 CPM/ml), but once again observed no significant effects of Epi on 3H-NE

cellular incorporation. We did find that Y. enterocolitica needs to be metabolically active to

assimilate NE, as a high cell density suspension of bacteria treated with 6 mM sodium azide

internalised very low levels of 3H-NE (approximately 50 CPM/ml of culture)). Similarly low cell-

associated levels of 3H-NE were also observed with heat-killed bacteria (around 40 CPM/ml).

Fractionation of 3H-NE-labelled bacteria into cytoplasm and membrane fractions also

revealed that over 95 % of the 3H-NE associated with the cell was cytoplasmic/periplasmic in

location (data not shown).

The data shown in Figures 1 and 2 illustrating Y. enterocolitica growth responses to

Epi were obtained using low cell density inocula, (around 102 CFU per ml). A previous report

(Walters & Sperandio, 2006) demonstrating apparent Epi specificity in E. coli O157:H7 used

very high cell density cultures (around 108 CFU per ml) that are not reflective of actual in vivo

infective doses (Tarr & Neill, 2001), causing us to question whether increasing the Y.

enterocolitica population density would affect its responsiveness to Epi. Because Y.

enterocolitica does not show growth responsiveness to Epi at low cell numbers, we chose a

high cell density iron-uptake assay to investigate this possibility since previous work from our

laboratories had demonstrated that Epi, NE and Dop stimulated bacterial growth via their

ability to facilitate iron removal from the host iron binding proteins Tf and lactoferrin

(Freestone, et al., 2000; Neal, et al., 2001; Freestone, et al., 2002; Lyte, et al., 2003). Table 1

shows that at higher cell densities (108 CFU per ml), Y. enterocolitica responds to Epi in the

same manner as E. coli and S. enterica, specifically utilizing it to access Fe bound to Tf. For

all 3 bacterial species increasing the concentration of Epi resulted in enhancement of 55Fe

10

incorporation, a trend also observed when cultures were supplemented with higher

concentrations of NE and Dop. This striking reversal in the response of Y. enterocolitica to

Epi suggests that the effect of Epi on Y. enterocolitica physiology may vary between cultures

of different population densities.

Catecholamine specificity in E. coli O157:H7, S. enterica and Y. enterocolitica is

dependent on population density

To determine if bacterial population density does influence the specificity of catecholamine

responsiveness, we examined growth of E. coli O157:H7, S. enterica and Y. enterocolitica

over an 8-log dilution curve using concentrations of Dop, NE and Epi optimal for growth

induction (Figure 1). Figure 3 shows the ability of the catecholamines to affect bacterial

growth in a serum-based medium is initially evident at low (<104 CFU per ml) cell densities

with the greatest differences observed at very low (<102 CFU per ml) cell densities. Although

Figure 3 shows that both E. coli O157:H7 and S. enterica are able to respond to Epi, at very

low population densities, which are reflective of the bacterial numbers that are likely to be

present at the initial stages of an infection, there exists an order of catecholamine preference

with growth responses to NE and Dop being at least a log-fold greater than those to Epi

(P<0.0001).

11

Discussion

The lack of Y. enterocolitica growth responsiveness to Epi is of great interest in that it

is impossible to reconcile this observation with a model of growth induction where

catecholamines are merely siderophore-like in their interaction with Tf/lactoferrin to provide

iron for bacterial growth. Intriguingly, the results shown in Figure 2 demonstrated that not

only did Epi fail to induce growth but that it could also antagonize the growth inducing effects

of both NE and Dop in Y. enterocolitica. To investigate how Epi was blocking NE and Dop

responsiveness in Y. enterocolitica, we used a high cell density iron uptake assay and found

that Y. enterocolitica could use Epi in a manner similar to that employed by E. coli O157:H7

and S. enterica, specifically, to mediate access to iron from Tf (Table 1). Indeed, Epi

concentrations markedly inhibitory to NE and Dop growth induction of Y. enterocolitica were

now able to significantly enhance acquisition of iron from Tf. Using 3H-NE we also found that

Epi does not block NE uptake thereby indicating that the inhibitory action of Epi is also not at

the level of uptake of catecholamines. Furthermore, the evidence from Epi antagonism of

both NE and Dop growth induction demonstrates that, in Y. enterocolitica at least, the

response pathways to NE and Dop must possess a common element that can be blocked by

Epi. This data also has experimental design implications; it suggests that, in the context of

analysis of microbe:host catecholamine interactions, caution should be exercised before

assuming that conclusions reached from in vitro observations of responses of high density

cultures can be directly extrapolated to the low population numbers typically present during

the initial stage of a foodborne infection (Tarr & Neill, 2001). Our data also indicates that the

bacterial growth response to catecholamines involves more than the simple provision of host-

derived iron, and suggests that a specific response pathway(s) may exist for each

catecholamine.

12

Importantly, the primary anatomical site of production and degradation of NE and Dop

in the body are the mesenteric organs which account for over 50% of the whole body total

(Eisenhofer, et al., 1996). While the level of catecholamines information is not available

within the enteric neuroepithelial synapses of the gastrointestinal tract, making it difficult to

estimate appropriate in vitro concentrations that are comparable to those that could exist in

vivo. Additionally, the use of plasma concentrations of catecholamines is not valid as

concentrations in the plasma reflect spillover from the tissues and therefore grossly

underestimate the local production that might be present within any specific target organ

(Leinhardt, et al., 1993). Nevertheless, it is emphasized that the level of catecholamines

tested in the bacterial growth studies reported in the present report are similar to those used

in other fields such as neurochemistry [e.g., to demonstrate the phosphorylation of synapsin I

by NE in the rat frontal cortex (Mobley & Greengard, 1985)] and in immunology [(e.g. the

identification of β-adrenergic receptor mediation of antibody production ((Sanders & Munson,

1984) and the inhibition of gamma interferon synthesis (Andrade-Mena, 1997)]. As the

gastrointestinal tract is also the principal anatomical site in which bacteria can routinely come

into contact with endogenously produced catecholamines (Eisenhofer, et al., 1996), the

convergence of these two systems, one neuroendocrine, one bacterial, suggests that an

interrelationship may exist that can contribute to the development of enteric bacterial disease.

What does the lack of Epi responsiveness in Y. enterocolitica, which is almost

exclusively an enteric pathogen with little invasion of extra-intestinal sites, tell us about the

potential role of neuroendocrine hormones in the infectious disease process in the gut?

While the presence of adrenergic and dopaminergic containing neurons has been well

demonstrated (Costa, et al., 2000) within the ENS, as well as local production of NE and Dop

13

(Meirieu, et al., 1986), there have been no reports of Epi or Epi-containing neurons. It has

been proposed (Walters & Sperandio, 2006) that Epi is the key host-derived hormonal signal

in the pathogenesis of enteric pathogens such as E. coli O157:H7; however if Epi were

present in concentrations equivalent to those of NE and Dop, our observation that Epi

antagonizes Y. enterocolitica growth responsiveness to these catecholamines would suggest

that this species should have significant problems in growing within the gut environment,

which it clearly does not. A recent report (Clarke, et al., 2006) which used in vitro constructs

to demonstrate that NE and Epi could bind to the E. coli O157:H7 two component regulator

sensor kinase QseC, proposed that this is the bacterial receptor for these catecholamines.

Interestingly, Y. enterocolitica (in common with most other pathogenic Yersinia species) does

not contain homologs for QseC (or the related receptor QseE). Indeed, BLAST searches for

matches to QseC/QseE in the microbial genome database revealed that of the Yersinia

species only Y. mollaretii possesses the qseC gene. If QseC does not exist in Y.

enterocolitica our data indicates that a different signal transduction system specific for NE

must exist in Y. enterocolitica.

Although it has been suggested (Walters & Sperandio, 2006) that sufficient quantities

of Epi could spill over from the circulatory system and somehow transverse the physical and

biological barriers separating the gut lumen from the host to affect bacteria, there are

currently no published clinical or animal studies of enteric neurophysiology to support this

assertion. Norepinephrine was the most potent catecholamine growth stimulator for all of the

three bacterial species examined, and we therefore further speculate that it is NE, rather than

Epi, that is likely to be the cross-communicating adrenergic signal molecule between host and

enteric pathogens and that the bacterial response to Epi observed in E. coli O157:H7

(Walters & Sperandio, 2006) is more likely related to its structural similarity to NE.

14

Acknowledgements

This work was supported by grant 064488/Z/01/Z from the Wellcome Trust (to P.P.E.F.) and

NIH grant MH-50431 (to M.L.).

15

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18

Figure legends

Figure 1. Dose-response effect of catecholamines on E. coli O157:H7 and Y.

enterocolitica.

E. coli O157:H7 and Y. enterocolitica NCTC strain 551 were inoculated at approximately 102

CFU per ml into duplicate 1-mL aliquots of serum-SAPI containing the concentrations of the

catecholamines shown and incubated for either 40 hours (Y. enterocolitica) or 18 hours (E.

coli O157:H7), and enumerated for growth (CFU per ml) as described in Materials and

methods. NE (grey bar), Dop (white bar), Epi (black bar). The results shown are

representative data from two separate experiments; data points showed variation of less than

3%. Results similar to those shown for E. coli O157:H7 were also obtained for S. enterica

(data not shown).

Figure 2. Ability of epinephrine to inhibit Y. enterocolitica growth induction by

norepinephrine and dopamine.

Y. enterocolitica NCTC strain 551 (panels A and B) and E. coli O157:H7 (panels C and D)

were inoculated at approximately 102 CFU per ml into duplicate 1 mL aliquots of serum-SAPI

containing the combination of catecholamines shown, and incubated for either 40 hours (Y.

enterocolitica) or 18 hours (E. coli O157:H7), and enumerated for growth (CFU per ml) as

described in Materials and Methods. The results shown are representative data from four

separate experiments; data points typically showed variation of no more than 3%; similar

result to those shown for Y. enterocolitica NCTC strain 551 were also seen with a Y.

enterocolitica clinical isolate and NCTC 1477 (data not shown).

Black bar, catecholamine/catecholamine combination only; light gray bar,

catecholamine/catecholamine combinations plus 100 μM Fe(NO3)3.

19

NE, 50 μM NE; NE+E100, 50 μM NE plus 100 μM Epi; NE+E200, 50 μM NE plus 200 μM Epi;

NE+E300, 50 μM NE plus 300 μM Epi; Dop, 50 μM Dop; Dop+E100, 50 μM Dop plus 100 μM

Epi; D+E200, 50 μM Dop plus 200 μM Epi; Dop+E300, 50 μM Dop plus 300 μM Epi. The

results shown are representative data from three separate experiments; data points typically

showed variation of no more than 3%.

Figure 3.

Bacterial population density influences catecholamine specificity.

Histograms A-C show the effects of varying inoculum size on the specificity of growth

response to catecholamines NE, Epi and Dop. Cultures were diluted in 10-fold steps into

serum-SAPI medium, incubated for either 18 hours (E. coli O157:H7 and S. enterica) or 40

hours (Y. enterocolitica NCTC 551) (and NCTC 1477, data not shown), and enumerated for

growth (CFU per ml) as described in Materials and Methods. The inoculum size of the E. coli

O157:H7 culture (panel A) was 5.29 x 108 CFU per ml; S. enterica (panel B) 2.84 x 108 CFU

per ml and Y. enterocolitica (panel C) 4.44 x 108 CFU per ml. The results shown are

representative data from four separate experiments; individual data points showed variation

of no more than 5%.

White bar, no additions (control); light grey bar, 50 µM NE; black bar, 100 µM Epi; diagonal

hatch, 50 µM Dop.

20

Table 1 Epinephrine does not inhibit the ability of norepinephrine and dopamine to mediate Y.

enterocolitica uptake of Tf-complexed iron.

Uptake of 55Fe from 55Fe-Tf (mean CPM/ml x 100)

Catecholamines µM Y. enterocolitica E. coli S. enterica

No additions (control) 3.63 14.77 15.95 NE (50) 27.81 213.21 198.10 NE (50) + Epi (100) 66.83 267.68 234.56 NE (50) + Epi (200) 97.18 297.33 256.90 NE (50) + Epi (200) 120.03 312.31 298.70 Dop (50) 14.03 247.68 208.77 Dop (50) + Epi (100) 21.45 263.12 267.27 Dop (50) + Epi (200) 44.09 296.89 299.01 Dop (50) + Epi (300) 41.76 301.78 300.98 NE (100) 31.33 263.43 229.67 NE (200) 80.24 288.81 234.89 NE (300) 12.47 323.12 310.08 Epi (100) 17.59 202.75 190.06 Epi (200) 37.50 240.00 238.51 Epi (300) 50.52 278.90 290.86 Dop (100) 24.03 245.12 213.89 Dop (200) 37.99 289.17 232.19 Dop (300) 72.25 292.19 265.90

Exponential cultures of Y. Enterocolitica strain 551, E. coli O157:H7 and S. enterica were

inoculated at approximately 2 x 108 CFU per ml into triplicate 1-ml aliquots of serum-SAPI

containing the combinations and concentrations of catecholamines shown in the Table plus

2.9 x 105 cpm of 55Fe-labelled transferrin. After incubation for 6 hours bacteria were

harvested, washed and measured for cellular uptake of 55Fe from 55Fe-transferrin (CPM) by

scintillation counting, as described in Materials and methods. The values shown represent the

21

means of bacterial 55Fe incorporation from triplicate 1 ml uptake assays; standard deviations

were between 1 and 6 % of the mean values shown in the table.. Similar results to those

shown for NCTC strain 551 were also observed with Y. enterocolitica NCTC strain 1477 (data

not shown). Analysis of cell numbers revealed no significant differences in growth levels

between control and catecholamine/antagonist supplemented cultures (data not shown).

22

23

E. coli O157:H7

1.E

1.E

1.E

1.E+06

1.E+07

1.E+08

.E+09

0 1 5 10 20 50 100 200 500

Concentration μM

+03

+04

+05

1 109

108

107

106

105

104

103

CFU

/ml

E. coli O157:H7E. coli O157:H7

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

.E+09

0 1 5 10 20 50 100 200 500

Concentration μM

1 109

108

107

106

105

104

103

CFU

/ml

E. coli O157:H7

Y. enterocolitica

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

0 1 5 10 20 50 100 200 500

Concentration μM

109

108

107

106

105

104

103

CFU

/ml

Y. enterocoliticaY. enterocolitica

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

0 1 5 10 20 50 100 200 500

Concentration μM

109

108

107

106

105

104

103

CFU

/ml

Y. enterocolitica

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

0 1 5 10 20 50 100 200 500

Concentration μM

109

108

107

106

105

104

103

CFU

/ml

109

108

107

106

105

104

103

109

108

107

106

105

104

103

CFU

/ml

Y. enterocolitica

Figure 1

A

B

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

24

1.0E+08

1.0E+09

Dop Dop+E100 Dop+E200 Dop+E300 Control

Fe

109

108

107

106

105

104

103

CFU

/ml

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

0E+08

1.0E+09

Dop Dop+E100 Dop+E200 Dop+E300 Control

1.Fe

109

108

107

106

105

104

1031.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

0E+08

1.0E+09

Dop Dop+E100 Dop+E200 Dop+E300 Control

1.Fe

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1. +08

1.0E+09

Dop Dop+E100 Dop+E200 Dop+E300 Control

109

108

107

106

105

104

103

CFU

/ml

1.

1.

1.

1.

1.

1.

1.

E+03

E+04

E+05

E+06

E+07

E+08

E+09

NE NE+E100 NE+E200 NE+E300 Control

109

108

107

106

105

104

103

CFU

/ml

1.

1.

1.

1.

1.

1.

1.

E+03

E+04

E+05

E+06

E+07

E+08

E+09

NE NE+E100 NE+E200 NE+E300 Control

109

108

107

106

105

104

103

CFU

/ml

109

108

107

106

105

104

103

109

108

107

106

105

104

103

CFU

/ml

0E

109

108

107

106

105

104

103

CFU

/ml

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1. +08

1.0E+09

Dop Dop+E100 Dop+E200 Dop+E300 Control

0E

109

108

107

106

105

104

103

109

108

107

106

105

104

103

CFU

/ml

B

D

Figure 2

1.0E

1.0E

1.0E

1.0E

1.0E

1.0E

1.0E

+03

+04

+05

+06

+07

+08

+09

NE NE+E100 NE+E200 NE+E300 Control

Fe

109

108

107

106

105

104

103

CFU

/ml

1.0E

1.0E

1.0E

1.0E

1.0E

1.0E

1.0E 09

NE NE+E100 NE+E200 NE+E300 Control+03

+04

+05

+06

+07

+08Fe

+109

108

107

106

105

104

1031.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E 09

NE NE+E100 NE+E200 NE+E300 Control

Fe

+109

108

107

106

105

104

103

109

108

107

106

105

104

103

CFU

/ml

A

C

25

S. enterica

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

-2 -3 -4 -5 -6 -7 -8 -9

Dilution factor

109

108

107

106

105

104

103

102

101

S. enterica109

108

107

106

105

104

103

102

101

CFU

/ml

S. enterica

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

E+09

-2 -3 -4 -5 -6 -7 -8 -9

Dilution factor

1. 109

108

107

106

105

104

103

102

101

109

108

107

106

105

104

103

102

101

S. enterica

E. coli O157:H7

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

E+09

-2 -3 -4 -5 -6 -7 -8 -9

Dilution Factor

109

108

107

106

105

104

103

102

101

CFU

/ml

1.E. coli O157:H7

109

108

107

106

105

104

103

102

101

CFU

/ml

E. coli O157:H7

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

E+09

-2 -3 -4 -5 -6 -7 -8 -9

Dilution Factor

1.E. coli O157:H7

109

108

107

106

105

104

103

102

101

Figure 3 109

108

107

106

105

104

103

102

101

CFU

/ml

A

B

Y. enterocolitica

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1 09

-2 -3 -4 -5 -6 -7 -8 -9

Dilution Factor

.E+

Y. enterocolitica109

108

107

106

105

104

103

102

101

CFU

/ml

Y. enterocolitica

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1 09

-2 -3 -4 -5 -6 -7 -8 -9

Dilution Factor

.E+

Y. enterocolitica

109

108

107

106

105

104

103

102

101

CFU

/ml

C