Are any of the Effects of Catechols on Bacteria Catecholamine Specific

Although a wide range of catechols are able to stimulate the growth of certain bacteria in minimal, iron-complexed media, there is at least one report of heightened specificity. The growth of Yersinia enterocolitica is stimulated by norepinephrine

E.coll 0157:H7

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Fig. 4.2 Growth modulation by dietary catechols in laboratory culture media. E. coli O157:H7 (a) and S. enterica (b) were inoculated at approximately 102 CFU ml-1 into duplicate 1 ml volumes of Luria broth containing the concentrations of the catechols shown, incubated for 10 h and enumerated for growth by measurement of the absorbance of the cultures at 600 nm. Catechin, caffeic and chlorogenic acids are measured in mM units, while tannic acid is measured in units of |g ml-1. The results shown are representative data from two separate experiments; data points showed

Fig. 4.3 Growth modulation by dietary catechols in serum-based media. E. coli O157:H7 and S. enterica were inoculated at approximately 102 CFU ml-1 into duplicate 1 ml aliquots of serumSAPI containing the concentrations of the catechols shown, incubated for 18 h, and enumerated for growth (CFU ml-1). Catechin, caffeic and chlorogenic acids are measured in mM units, while tannic acid is measured in units of |ig ml-1. The results shown are representative data from two separate experiments; data points showed variation of less than 5%. Catechin (black bar); Caffeic acid (grey bar); Chlorogenic acid (white bar); Tannic acid (diagonal hatch). Reproduced from Freestone et al. (2007c), with permission

Fig. 4.3 Growth modulation by dietary catechols in serum-based media. E. coli O157:H7 and S. enterica were inoculated at approximately 102 CFU ml-1 into duplicate 1 ml aliquots of serumSAPI containing the concentrations of the catechols shown, incubated for 18 h, and enumerated for growth (CFU ml-1). Catechin, caffeic and chlorogenic acids are measured in mM units, while tannic acid is measured in units of |ig ml-1. The results shown are representative data from two separate experiments; data points showed variation of less than 5%. Catechin (black bar); Caffeic acid (grey bar); Chlorogenic acid (white bar); Tannic acid (diagonal hatch). Reproduced from Freestone et al. (2007c), with permission

Fig. 4.2 (continued) variation of less than 5%. Catechin (black bar); Caffeic acid (grey bar); Chlorogenic acid (white bar); Tannic acid (diagonal hatch). (c) examines the mechanistic basis of the growth inhibition by tannic acid of E. coli O157:H7. A similar methodology to that used in (a) and (b) was employed, except that the culture media were supplemented with either no additions (black bar), 50 mM Tris-HCl, pH 7.5 (grey bar), 100 mM ferric nitrate (white bar) and 50 mM Tris-HCl, pH 7.5 and 100 mM ferric nitrate (diagonal hatch). Cultures were corrected for absorbance due to media components. The results shown are representative data from two separate experiments; data points showed variation of less than 5%. Reproduced from Freestone et al. (2007c), with permission

Fig. 4.4 Structures of flavanols: (+)-catechin (I), (-)-epicatechin (II, R =H), (-)-epigallocatechin (II, R=OH), (-)-epicatechin-3-gallate (III, R=H) and (-)-epigallocatechin-3-gallate (III R=OH). Reproduced from Hollman and Arts (2000), © Society of Chemical Industry, with permission

Fig. 4.4 Structures of flavanols: (+)-catechin (I), (-)-epicatechin (II, R =H), (-)-epigallocatechin (II, R=OH), (-)-epicatechin-3-gallate (III, R=H) and (-)-epigallocatechin-3-gallate (III R=OH). Reproduced from Hollman and Arts (2000), © Society of Chemical Industry, with permission and dopamine but not by epinephrine (Freestone et al. 2007a). It has been postulated that this specificity reflects the fact that Y. enterocolitica infection is principally limited to the gut where epinephrine-containing neurones are not found. This study also showed that epinephrine was a less potent growth inducer for E. coli 0157:H7, with 50 mM epinephrine being required to elicit the stimulation of growth produced by 20 mM norepinephrine or dopamine. Thus, although a general growth response to catecholic substances appears to be common, more specificity may exist than is presently apparent.

In addition to the growth stimulation by neuroendocrine catecholamines, there are also several reports of bacteria utilising these catecholamines as signalling molecules in the activation of expression of colonisation and/or virulence factors. The question therefore arises as to whether these effects are specific to catecholamines or whether similar responses might also be elicited by dietary catechols. Expression of the outer-membrane enterobactin transporter BfeA of Bordetella bronchiseptica is activated in response to its substrate by the AraC family transcriptional regulator, BfeR. Anderson and Armstrong (2006) demonstrated that BfeA expression could

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Fig. 4.5 Dietary catechols are able to remove Tf-complexed iron. Urea gels illustrate the effect on the iron-binding status of Tf incubated for 18 h at 37°C in the presence of increasing concentrations of the catechol compounds shown. (a) Caffeic acid (lower legend mM); (b) catechin (lower legend |tM); (c) chlorogenic acid (lower legend mM); (d) tannic acid (lower legend mg ml-1). Lane M contains iron-free (Apo-Tf), monoferric with iron in the N-terminal or C-terminal domains (Fe-Tf and Tf-Fe, respectively), and saturated (Fe2-Tf) isoforms as markers. +Fe (1 mM ferric nitrate). Reproduced from Freestone et al. (2007c), with permission also be activated by norepinephrine as well as by several other catecholamines and by the non-amine catechols, pyrocatechol and 3,4-dihydroxymandelic acid. It remains to be established whether catechols derived from the diet might also activate BfeA expression.

Although the activation of B. bronchiseptica BfeA does not appear to be cate-cholamine-specific, there is some evidence of catecholamine specificity in Borrelia burgdorferi, the causative agent of Lyme disease, in which expression of outer-surface protein A (OspA) is induced by epinephrine and norepinephrine (Scheckelhoff et al. 2007). This induction is blocked by a competitive inhibitor of human b-adrenergic receptors, suggesting that the mechanism is catecholamine-specific. However, since no non-amine catechols were tested, there remains in principle the possibility that other catechols could be bound by, and activate, the as-yet-uncharacterised adrenergic receptor. Importantly, no effect on B. burgdorferi growth was seen with either epinephrine or norepinephrine, suggesting that this signalling pathway is independent of iron acquisition.

Additional evidence for the presence of bacterial adrenergic receptors comes from work with E. coli 0157:H7. In this bacterium, two response regulators, QseA and QseB, activate virulence gene expression in response to epinephrine, norepinephrine and the bacterial autoinducer AI-3. Binding of epinephrine directly activates QseC, the cognate histidine kinase for QseB, and this activation is blocked by the a-adren-ergic antagonist, phentolamine, but not by the b-adrenergic antagonist, propanolol (Clarke et al. 2006). However, both a- and b-adrenergic receptor antagonists block the activation of LEE (Locus of Enterocyte Effacement) gene expression by QseA (Sperandio et al. 2003). This difference in antagonist specificity implies that E. coli 0157:H7 possesses an additional, as yet undiscovered, adrenergic-receptor, which is responsible for activating QseA.

Clarke et al. (2006) established that the periplasmic signal-sensing domain of QseC is strongly conserved across a range of bacteria, including Shigella sp., Salmonella sp., Erwinia carotovora, Haemophilus influenzae, Pasteurella multo-cida, Actinobacillus pleuropneumoniae and Psychrobacter sp., and that it shares no primary sequence homology with classical G-protein-coupled adrenergic receptors. It will now be of particular interest to establish the structural features and binding properties of this domain and to discover the extent to which it might also bind, if at all, other catecholic molecules of biological interest. However, the observation that phentolamine antagonizes the effects of norepinephrine upon QseC (Clarke et al. 2006), yet does not block the growth stimulation of E. coli 0157-H7 that is seen in response to several dietary catechols when they are provided under iron-restricted conditions (Freestone, unpublished), suggests that growth stimulation by catechols may be mechanistically separate from effects that are mediated via QseC.

The evidence discussed in the last two chapters suggests that catechols in general can stimulate growth of bacteria through their ability to increase the bioavail-ability of iron. However, studies with inhibitors of adrenergic receptors indicate that there are additional bacterial signalling pathways that are likely to be specific for neuroendocrine catecholamines. There is also evidence that these catecholamines may stimulate growth in a manner independent of iron release from transferrin. It was recently shown in E. coli 0157:H7 that adrenergic and dopaminergic receptor antagonists that block growth stimulation by norepinephrine, epinephrine and dop-amine do not block 55Fe uptake from 55Fe transferrin (Freestone et al. 2007b). The presence of the a-adrenergic receptor antagonists did, however, block norepineph-rine uptake. However, these effects may still be linked to iron provision since the addition of ferric nitrate overcame the antagonist blockade of growth induction.

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