Choice of Neuroendocrine Hormone and Appropriate Concentration

As would seem evident, the correct concentration of catecholamine neurohormone to use in an in vitro experiment should be the level that the bacteria are likely to encounter within their host. This has two provisos; firstly, the assumption that we can accurately determine the hormone concentrations at sites within the body, and secondly the assumption that bacterial pathogens will occupy just a single site during the course of an infection, and therefore will not be exposed to varying hormone concentrations. It is therefore important to realise that the concentrations of neuroendocrine hormones seen clinically are derived from fluid-based samples, such as plasma or urine, which are easy to obtain from a subject. However, the majority of the body's neurohormones are actually found in the tissue target where they act, and therefore it must be understood that the values obtained from circulatory specimens may give gross underestimates of (often several log orders lower than) the true levels in tissues or at mucosal surfaces where bacteria will interact with them (Leinhardt et al. 1993). Of course, this element of uncertainty will influence experimental design, most notably the choice of concentration(s) at which the specific neuroendocrine hormone under study should be tested (for further discussion, see Lyte 2004). Where the concentration of the neurohormone at the site of action in the body is unknown, or where it is predicted to be very variable, it would be prudent to perform dose responses analyses over a reasonably wide range of neurohormone concentrations. A typical dose response profile of catecholamine growth responsiveness for E. coli O157:H7 and Y. enterocolitica inoculated into serum-SAPI medium is shown in Fig. 16.3. As previously observed, epinephrine had little effect on growth of Y. enterocolitica, and on a concentration-dependent basis was seen to be less potent at stimulating growth of E. coli O157:H7 than either norepinephrine or dopamine (see also Fig. 16.3). Previous catecholamine dose response analyses for bacterial species such as E. coli, Salmonella, Yersinia, and the coagulase-negative staphylococci have suggested that initial test concentrations of 50 mM norepinephrine or dopamine, and 100 mM epinephrine (Fig. 16.3) would be suitable. Furthermore, we see that at very high concentrations of catecholamine (>200 mM) the effects upon growth can begin to become inhibitory (Fig. 16.3).

105 -1041030 1 5 10 20 50 100 200 500

Concentration mM

b Y.enterocolitica

Fig. 16.3 Dose-response effects of catecholamines on E. coli O157:H7 and Y enterocolitica. E. coli O157:H7 and Y enterocolitica were inoculated at approximately 102 CFU/ml into duplicate 1 ml aliquots of serum-SAPI containing the concentrations of the catecholamines shown and incubated for either 40 h (Y. enterocolitica) or 18 h (E. coli O157:H7), and enumerated for growth (CFU/ml) as described in Freestone et al. (2007a). The results shown for norepinephrine (NE), (grey bar), dopamine (Dop), (white bar) and epinephrine (Epi), (black bar), are representative data from two separate experiments; data points showed variation of less than 3%. This figure was taken with permission from Freestone et al. (2007a)

Catecholamines induce bacterial growth by providing access to host sequestered iron which in iron limited media, such as serum or blood, can then lead to clinically important effects (Coulanges et al. 1998; Freestone et al. 2000, 2002, 2003, 2007a, b, c; Neal et al. 2001, Lyte et al. 2003, Cogan et al. 2007). However, catecholamines

E. coli O157:H7

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10 20 50 Concentration mM

can also chelate iron and therefore given the role that iron, and in particular iron restriction, plays in the regulation of virulence factor expression, it is important to consider what the physiological effects of high concentrations of neurohormone would be in non-host-like media where iron is not limited. This is highlighted by the fact that some investigations of the effects of catecholamines on bacteria virulence have used catecholamine concentrations in the mM range. For example, Vlisidou et al. (2004) used 5 mM norepinephrine in Luria broth to investigate the effects of catecholamines on E. coli O157:H7 adherence to gut tissues. Though Luria broth is a relatively iron rich culture medium the addition of a 5 mM concentration of an iron chelator, such as norepinephrine, will undoubtedly result in the medium becoming more Fe limited. The possibility that iron limitation may have affected the observed results could be investigated by incubating cultures in iron-depleted Luria broth (made, for example, by Chelex pretreatment) or by the direct addition to test cultures of a ferric iron chelator such as dipyridyl or desferal. The chelation of iron by catecholamines in media containing free iron can also lead to the generation of oxygen-derived free radicals, a cell damaging process that has been implicated in the development of cancers or neurodegenerative diseases such as Parkinson's (Borisenko et al. 2000); therefore, particularly when using high catecholamine concentrations, it is important to consider the potential influence of oxidative stress on bacterial gene expression levels.

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