Metabolic Pathways in Salmonella enterica

We are investigating the individual roles of an asparaginase, a kinase, a deglycase, and a putative transcriptional regulator in the utilization of fructose-asparagine (F-Asn), an Amadori product, by Salmonella. In particular, we are exploring whether this distinctive metabolic capability may be exploited to design narrow-spectrum anti-Salmonella therapeutics.

I. NIH R01AI116119 (December 2014 – November 2019)

Title
Salmonella, colonization resistance, and fructose-asparagine

Narrative
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 Salmonella 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.

Senior personnel
•   Brian M.M. Ahmer (OSU, lead PI)
•   Edward J. Behrman, Venkat Gopalan, Kelly C. Wrighton, Vicki H. Wysocki (OSU, PIs)

Lab members involved in this project
•   Sravya Kovvali (Graduate Student, OSU Microbiology Graduate Program)
•   Blake E. Szkoda (Graduate Student, Ohio State Biochemistry Program)

Methods
•   In our laboratory: protein overexpression and purification; enzyme kinetic studies; DNA—protein footprinting; fluorescence polarization (FP) binding assays
•   In collaboration with other PIs: in vivo studies (Ahmer); synthesis of Amadori products (Behrman); protein crystallography (Charles Bell, OSU); protein modeling (Steffen Lindert, OSU); mass spectrometry (Wysocki)

Recent Publications
•   Sengupta A, Wu J, Seffernick JT, Sabag-Daigle A, Thomsen N, Chen T-H, Di Capua A, Bell CE, Ahmer BMM, Lindert S, Wysocki VH, and Gopalan V*. (2019) Integrated use of biochemical, native mass spectrometry, computational and genome-editing methods to elucidate the mechanism of a Salmonella deglycase. J. Mol. Biol., in press.
•   Wu J°, Sabag-Daigle A°, Borton MA^, Kop LFM^, Szkoda BE^, Deatherage Kaiser BL, Lindemann SR, Renslow RS, Wei S, Nicora CD, Weitz KK, Kim Y-M, Adkins JN, Metz TO, Boyaka P, Gopalan V, Wrighton KC, Wysocki VH*, and Ahmer BMM*. (2018) Salmonella-mediated inflammation eliminates competitors for fructose-asparagine in the gut. Infect. Immun., 86: e00945-17. °joint first authors; ^joint second authors
•   Wu J, Sabag-Daigle A, Metz TO, Deatherage Kaiser BL, Gopalan V, Behrman EJ, Wysocki VH*, and Ahmer BMM*. (2018) Measurement of fructose–asparagine concentrations in human and animal foods. J. Agric. Food Chem., 66: 212-217.
•   Sabag-Daigle A, Wu J, Borton MA, Sengupta A, Gopalan V, Wrighton KC, Wysocki VH, and Ahmer BMM*. (2018) Identification of bacterial species that can utilize fructose-asparagine. Appl. Environ. Microbiol., 84: e01957-17.
•   Sabag-Daigle A, Sengupta A, Blunk HM, Biswas PK, Cron MC, Bogard AJ, Behrman EJ, Gopalan V, and Ahmer BMM*. (2017) Salmonella FraE, an asparaginase homolog, contributes to fructose-asparagine but not asparagine utilization. J. Bacteriol., 199: e00330-17.
•   Biswas PK, Behrman EJ*, and Gopalan V*. (2017) Characterization of a Salmonella sugar kinase essential for utilization of fructose-asparagine. Biochem. Cell Biol., 95: 304-309.
•   Sabag-Daigle A, Blunk HM, Sengupta A, Wu J, Bogard AJ, Ali MM, Stahl C, Wysocki VH, Gopalan V, Behrman EJ, and Ahmer BMM*. (2016) A metabolic intermediate of the fructose-asparagine utilization pathway inhibits growth of a Salmonella fraB mutant. Sci. Rep., 6: 28117. [SI]

II. NIH R01AI140541 (August 2018 – July 2022)

Title
Salmonella-specific therapeutics

Narrative
Non-typhoidal salmonellosis is a serious threat in the United States and globally. Because Salmonella cells lacking the FraB deglycase of the fructose-asparagine (F-Asn) utilization pathway accumulate a toxic intermediate during F-Asn metabolism, FraB is a valid and exploitable drug target. Our objectives in this proposal are to identify and characterize potent inhibitors of FraB, and provide a foundation for our long-term goal of developing a novel therapeutic for non-typhoidal salmonellosis.

Senior personnel
•   Brian M.M. Ahmer (OSU, lead PI)
•   Venkat Gopalan (OSU, PI)
•   Edward J. Behrman, Charles E. Bell, Steffen Lindert (OSU, co-Is)

Lab members involved in this project
•   Sravya Kovvali (Graduate Student, OSU Microbiology Graduate Program)
•   Jamison Law (Graduate Student, Ohio State Biochemistry Program)

Methods
•   In our laboratory: high-throughput biochemical screening, validation, and characterization of small molecule inhibitors
•   In collaboration with other PIs: in vivo screening (Ahmer); synthesis of fructose-asparagine derivatives and lead optimization (Behrman); protein crystallography (Bell); in silico structure-based virtual screening (Lindert); mass spectrometry (Vicki Wysocki, OSU)