Influence of the neuroendocrine stress hormones on the carriage and virulence of zoonotic bacterial pathogens in farm animals. THIS GRANT IS A SUPPLEM

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For over a century correlations between stress, nutrition and susceptibility to microbial infection have been recognised. Whilst this may be partially explained by the suppression of immune function following trauma or malnutrition, it is now clear that bacterial pathogens can directly sense neuroendocrine hormones released by the host under stress and respond by activating growth and the expression of virulence factors. Our proposal seeks to address the impact of neuroendocrine hormones on the carriage and virulence of enterohaemorrhagic Escherichia coli and Salmonella enterica in target food-producing animals and to dissect the molecular mechanisms by which the bacteria sense and respond to such molecules. It builds on recent observations by our group that norepinephrine (NE), which is released by the enteric nervous system under stress, augments E. coli O157:H7-induced enteritis and adherence in the intestines of calves. Invasion of porcine intestinal explants by Salmonella enterica serovar choleraesuis is also stimulated by NE, indicating that Gram- negative pathogens have evolved conserved strategies to sense and respond to neuroendocrine stress hormones. We believe that this may partially explain the sporadic incidence of excreting and diseased animals within herds and contribute to the increased shedding of zoonotic pathogens following nutritional stress and transportation. Neurochemicals can also be consumed in the diet in quantities sufficient to alter host physiology. Recent studies have established that tyramine and dopamine, which are abundant in certain foods and can be produced by certain enteric bacteria, also augment the adherence of E. coli O157:H7 to intestinal explants. Tyramine appears to stimulate EHEC adherence via a different mechanism to norepinphrine, since expression of the adhesin intimin is not induced. Remarkably, it was recently shown that adrenergic receptor antagonists can inhibit NE-induced invasion of intestinal explants by E. coli O157:H7 and S.choleraesuis and the expression of bacterial virulence factors in vitro. These are exciting findings, however the mechanism of action and relative influence of hormones and antagonists in the context of persistent or acute infections by these organisms remains to be determined. Here we seek to integrate in vitro and in vivo approaches to dissect the role of neurochemicals produced by the host under stress or consumed in the diet in the carriage and virulence of two major zoonotic bacterial pathogens. The influence of NE and tyramine and of alpha- and beta-adrenergic receptor antagonists on E coli O157:H7- and S. typhimurium-induced enteritis, adherence and invasion will be studied using well established ligated intestinal loop models in calves or pigs. We will then assess the influence of neurotoxin-induced stress and feeding a neurochemical or antagonist supplemented diet on the pathogenicity of S. typhimurium and EHEC following oral infection. In parallel we will perform experiments with intestinal explants is using changers to dissect the relative importance of neurochemicals or antagonists acting on bacteria as opposed to intestinal cells in these events. Established whole-genome microarrays for E coli O157:H7 and S. typhimurium will be used to dissect the global transcriptional response to NE and tyramine and the results integrated with our existing knowledge of the numerous bacterial genes required for intestinal colonisation. In addition we aim to identify bacterial factors involved in sensing neuroendocrine hormones and signal transduction by screening existing libraries of E coli O157;H7 and S.typhimrium mutants with defined intestinal colonisation phenotypes for their ability to respond to NE in vitro.