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<p>The long-range goal of our work is to understand how the environment of the intestinal tract, produced by the microbiota, affects Salmonella virulence. The central hypothesis is that the resident microbiota of the large intestine create an environment that is repressive for Salmonella virulence gene expression through the production of fatty acids, and that these fatty acids signal Salmonella by means of existing metabolic and regulatory pathways in the pathogen. Our first objective is to characterize the repressive effects of fatty acids on Salmonella virulence. Specifically, we aim to identify the molecular mechanism by which these fatty acids have their effects. We will further determine how dietary manipulation affects Salmonella virulence in animals using models that are relevant to human disease and animal production. Specifically, we aim to:</p>
<p>1. Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion.</p>
<p>2. Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens.</p>
<p>3. Determine the importance of the intestinal environment to the prevention of infection by Salmonella in humans using an animal model of human disease.</p>
<p>NON-TECHNICAL SUMMARY:<br/> Salmonella is an important cause of foodborne illness in the United States. It is carried by many animal species and so can frequently contaminate meat, poultry and eggs. We hypothesize that the chemical environment of the animal intestine, generated by the bacteria that normally reside there, can affect the ability of Salmonella to cause disease. Specifically, we have found that fatty acids, by-products of intestinal bacteria, can inhibit the ability of Salmonella to penetrate animal tissues, a process called invasion. In this work, we will therefore identify the mechanism by which intestinal fatty acids prevent Salmonella invasion. We will then determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens. Finally, we will determine the importance of the intestinal environment to the prevention of
infection by Salmonella. We expect to determine the means by which these chemical compounds affect Salmonella and thus to gain insight into possible means to prevent disease caused by this pathogen. We anticipate that the long-term impact of this work will be the development of improved means to prevent the colonization of production animals with Salmonella and thus an increase in the safety of our food supply.
<p>APPROACH:<br/> Specific Aim 1: Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion.The approach employed here will be to use a combination of biochemical and genetic means to characterize the physical interaction of fatty acids with HilD, a central regulator of Salmonella virulence. The experiments are designed to determine the means by which fatty acids repress Salmonella invasion genes through HilD. We will begin with a genetic approach, identifying essential elements of hilD, and then progress using biochemical methods to characterize modifications of HilD. These experiments will focus on propionate, with extension to other fatty acids.Specific Aim 2: Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens.We will employ in this approach studies of in vivo Salmonella invasion gene expression
using gfp fusions followed by a feeding trial using a commercially available microencapsulated n-butyric acid feed additive. Here we will investigate the in vivo control of Salmonella virulence in the environment of the chicken intestinal tract by a prebiotic dietary supplement. We will use gfp reporter fusions in combination with biochemical analyses to determine the effects of n-butyric acid on Salmonella carriage.Specific Aim 3: Determine the importance of the intestinal environment to the prevention of infection by Salmonella.As our approach, we will use a well studied animal model of septicemic salmonellosis, the conventional mouse, in combination with the antibiotic-treated mouse model, in which the intestinal microbiota and fatty acid composition are altered to produce an aberrant large intestinal disease. Here we will investigate the in vivo control of Salmonella virulence in the
environment of the intestinal tract using the mouse as the experimental model. We will use gfp reporter fusions in combination with biochemical analyses to define the temporal and spatial variation in SPI-1 gene expression, determine what role fatty acids play in these expression changes, and find whether treatment with a dietary supplement known to exacerbate systemic salmonellosis does so by altering the expression of Salmonella invasion genes.