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Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative, intracellular pathogen responsible for disease across a broad range of hosts and is a useful model for systemic infection. Salmonella is one of the major causes of food-borne infections. Poultry and farm products (meats, eggs, fresh produce, etc.) are the major source of Salmonella infections. Following ingestion of a contaminated food or water, the organism colonizes the host's intestinal epithelium and induces gastroenteritis (i.e., salmonellosis). Salmonellosis is an important public health problem in the United States and the World. According to the Centers for Disease Control and Prevention (CDC; Atlanta GA), the number of nontyphoidal Salmonella infections in USA is ~1,400,000 cases annually. Protection of the food and water supplies from Salmonella contamination is a major goal of any food safety program. There is an increasing interest in using vaccines against Salmonella serovars that are of public health concerns (i.e., S. Typhimurium and S. Enteritidis). However, most of the Salmonella strains currently in use, as live vaccines, are nutritional mutants that have a high probability of reversion. We believe that clear understanding of the virulence mechanisms and of the environmental conditions required for the expression of the virulence genes will provide better tools to combat Salmonellosis. <P>
Our long-term goal is to elucidate the regulatory networks in S. Typhimurium that are involved in the coordinated regulation of cellular metabolism, oxidative stress defenses, and pathogenesis in order to advance the development of novel strategies and therapeutics (e.g., vaccines) for the treatment and prevention of salmonellosis. <P>
The Expected outputs: 1) it will contribute to our basic understanding of the complex regulatory networks required for coordinating cellular metabolism and pathogenesis in response to changes in environmental stimuli (i.e., oxygen and iron in the host); 2) it will advance our understanding of the cooperative interactions among three major global regulatory elements (FNR, ArcA, and Fur) required for coordinating cellular metabolism, oxidative stress responses, and pathogenesis; 3) it will provide novel strategies and therapeutics for the prevention of salmonellosis and possibly other diseases; 4) it will provide a prototype/model system for basic understanding of protein-protein and protein-complexes/nucleic acid interactions and may provide new insight(s) on the general rules for other multi-layered, coordinately regulated circuits found in prokaryotes; and 5) it will provide the next generation of microbiologists and medical scientists with the training required to apply system biology approaches in solving biological and health related problems.
NON-TECHNICAL SUMMARY: Salmonella is a facultative, intracellular pathogen responsible for disease across a broad range of hosts. Salmonella is one of the major causes of food-borne infections. Poultry and farm products (meats, eggs, fresh produce, etc.) are the major source of Salmonella infections. Following ingestion of a contaminated food or water, the organism colonizes the host's intestinal epithelium and induces gastroenteritis (i.e., salmonellosis). Salmonellosis is an important public health problem in the United States and the World. According to the Centers for Disease Control and Prevention (CDC; Atlanta GA), the number of nontyphoidal Salmonella infections in USA is ~1,400,000 cases annually. Recently, there have been many reports on contaminated peanut butter, tomatos, and poultry / meat products. Protection of the food and water supplies from Salmonella contamination is a major goal of any food safety program. There is an increasing interest in using vaccines against Salmonella serovars that are of public health concerns (i.e., S. Typhimurium and S. Enteritidis). However, most of the Salmonella strains currently in use, as live vaccines, are nutritional mutants that have a high probability of reversion. We believe that clear understanding of the virulence mechanisms and of the environmental conditions required for the expression of the virulence genes will provide better tools to combat Salmonellosis. We plan to find out how this organism copes with its changing environment during infection. We need to know the factors that are involved in the coordinated regulation of cellular metabolism, oxidative stress defenses, and pathogenesis in order to advance the development of novel strategies and therapeutics (e.g., vaccines) for the treatment and prevention of salmonellosis. We believe that coordinated regulation of cellular redox (i.e., degree of oxygenation)and steady iron concentration in S. Typhimurium is important for its ability to cause illness. These planned studies are based on our recent findings (just published in Journal of Bacteriology - 2007) that FNR (the master regulator of conditions lacking oxygen- anaerobiosis) also regulates many of the S. Typhimurium virulence genes. Furthermore, an FNR mutant was attenuated (did not cause illness), in mice. Indeed, we have a Patent application (Patent Pending) for the use of this special mutant as a live vaccine in Poultry. We also know that iron is an essential nutrient for the microbe as well as for the host, and control of iron is essential for their survival. Based on these findings and on our previous experience in microbial biology and oxidative stress, we plan to study the combined global effects of the redox and iron regulators on cellular metabolism and pathogenesis in S. Typhimurium. The results will provide novel strategies and therapeutics for the prevention of salmonellosis and possibly other diseases.
<P>APPROACH: We have defined the genes that are regulated by the individual regulators of redox (FNR, ArcA) and of iron (Fur) alone. Now we are interested in identifying the genes that are only regulated by the combination of two or three of these global regulators (i.e., FNR ArcA, FNR Fur, ArcA Fur, or FNR ArcA Fur). In order to accomplish this goal, we will compare the transcriptome of each member of the combinatory mutants relative to the wild-type strain (seven mutants plus the wild-type). From this information, we will be able to deduce the global networks coordinately regulated by each and all of these three regulators, in S. Typhimurium. Selected genes of interest will include genes involved in both mtabolism and in virulence / pathogenesis. Knockout mutations will be generated in the selected genes of interest to determine/confirm their functions and roles in virulence. Standard microbiological and molecular biology/biochemistry approches will be used in these studies. DNA microarrays, DNA protein interactions, protein-protein interactions will be used throughout these studies. Data analysis of the DNA arrays will be conducted as done previously in our laboratory. Spots will be analyzed by adaptive quantification as previously done. Confidence intervals and P values (p ¡Ü 0.05) on the expression change will also be calculated using two systems: Pair wise comparisons, calculated using a two-tailed Student's t test. In this case, microarray data will be analyzed using the MEAN and TTEST procedures of SAS-STAT statistical software (SAS Institute, Cary, NC). We plan to use different experimental methods to identify and characterize protein-DNA, protein-protein, and protein complexes-DNA interactions. These methods rely on changes in the molecular weight (MW) of the complexes that affect their mobilities during gel electrophoresis and also depend on the use of specific antibodies to identify the members of the different complexes. To evaluate the cooperative binding of FNR, ArcA, and Fur to the promoters selected from the studies listed above, we will prepare DNA fragments (about 200-300 base pairs) containing the promoters of interest. We will mutagenize the predicted binding-sites using base substitutions (i.e., no deletions). We have previous experience with site-directed mutagenesis. The mutagenized DNA fragments will be sequenced to confirm the expected changes following site-directed mutagenesis. We will attempt to substitute the information rich bases with ones that are found with much less frequency. The effects of these binding-site knockouts on the binding of the corresponding transcription factor(s) will be evaluated by the fluorescence anisotropy and/or the band-shift assays. Also, the DNA fragments containing the binding-site knockouts will be tested for their ability (or inability) to compete with the un-mutagenized fragments in band-shift assays. We will also validate the potency of the FNR Vaccine strain (Patent Pending), and others (to be developed during the course of the proposed studies), to reduce infection by wild-type S. Typhimurium and related Salmonella serovars in Mice, Poultry and/or other animals.