Salmonella enterica serovar Typhimurium (Salmonella) is one of the most significant food-borne pathogens affecting humans and agriculture. It has long been thought that nutrient utilization systems of Salmonella would not make effective drug targets because there are simply too many nutrients available to Salmonella in the intestine. However, we have discovered that during growth in the inflamed intestine Salmonella relies heavily on a single nutrient - fructose-asparagine (F-Asn), which is present at high concentrations in human foods. Mutants that cannot acquire F-Asn are severely attenuated suggesting that F-Asn is the primary nutrient utilized by Salmonella during inflammation. No other organism has been reported to synthesize or utilize this compound, although we suspect that a few other pathogens and members of the normal gut microbiota can utilize it. The apparent lack of F-Asn utilization pathways in mammals and most other bacteria suggests a specific and potent therapeutic target for Salmonella. The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1- SPI2- or ttrA mutants, respectively). We hypothesize that if Salmonella can initiate inflammation (or enters a gut that is already slightly inflamed), it can begin tetrathionae respiration during F-Asn catabolism and thereby outcompete the normal microbiota, which are doubly compromised by the inflammation and their ability to only ferment (but not respire) F-Asn. We will test this central postulate and build the foundation for two types of therapeutics to block Salmonella acquisition of F-Asn. In our first specific aim, we will investigate the role of a asparaginase (FraE), kinase (FraD) and deglycase (FraB) in F-Asn utilization. Through biochemical characterization of the individual reactions catalyzed by these Fra enzymes and development of high-throughput assays, we expect to facilitate future screens that will identify small molecule inhibitors of these enzymes. We hypothesize that the FraR transcription factor is a repressor. Therefore, preventing its release from the fra operon promoter would also be of therapeutic interest. We propose to determine the natural inducer of FraR and determine the DNA binding sites of FraR in the fra operon. In the second aim, we plan to employ a combination of metagenomics, selective growth in the presence of F-Asn, and bioinformatics to test the idea that in healthy gut communities there are select members of the microbiota that utilize F-Asn and prevent Salmonella from acquiring this nutrient. Finally, we expect our findings on the enzymology and regulation of F-Asn utilization in Salmonella, and possible competing intestinal microbes, to inform our efforts to design new probiotic bacteria that can reduce the severity and duration of Salmonella infection in mice. Overall, our efforts will lead to a better understanding of Salmonella growth in the inflamed intestine and to novel therapeutics.
The metabolism of fructose-asparagine is required for Salmonella to thrive in the intestine, making the acquisition system a novel drug target. We will learn which intestinal organisms compete with Salmonella for fructose-asparagine and why they fail, which will facilitate the development of novel probiotics that can prevent or treat Salmonell infections. We will also determine the enzymatic mechanisms used by Salmonella to grow on fructose-asparagine, which will facilitate high-throughput screens for inhibitors of these enzymes.
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