Poster abstracts

Poster number 46 submitted by Blake Szkoda

Elucidating the regulatory mechanism of FraR, a transcription factor in Salmonella enterica critical for pathogenesis

Blake E. Szkoda (The Ohio State Biochemistry Program), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
The foodborne pathogen Salmonella enterica serovar Typhimurim (Salmonella) causes 94 million enteric infections and 50,000 diarrheal deaths annually worldwide. There are no vaccines or drugs against this pathogen. During infection, Salmonella exploits fructose-asparagine (F-Asn) as a carbon and nitrogen source. F-Asn is a product of an Amadori rearrangement that occurs during cooking and dehydration of raw foods. F-Asn is metabolized by Salmonella using three enzymes and a transporter encoded by the fra operon. The roles of a periplasmic asparaginase, cytoplasmic kinase, and a cytoplasmic deglycase in F-Asn catabolism are now established. Importantly, the deglycase was identified as a promising drug target since a knock-out strain accumulates the substrate and leads to Salmonella self-poisoning. This work was undertaken to characterize control of the fra operon gene expression by FraR, the putative transcriptional regulatory protein in this locus. FraR is predicted to contain an N-terminal DNA-binding domain (DBD) and a C-terminal inducer-binding domain (IBD). We hypothesize that FraR binds to a sequence within the fra operon in vivo and acts as a transcriptional repressor, and that binding of a yet unidentified inducer to the FraR IBD triggers a conformational change to release the DNA from the DBD and permit transcription of the fra structural genes. This hypothesis was tested by purifying FraR recombinantly (from Escherichia coli), using maltose-binding protein as a chaperone, and assessing FraR-DNA binding affinity (+/- putative inducers) with gel-shift assays. Our biochemical studies, which provide insights into the DNA-binding properties of FraR and the inducer’s identity, provide a first glimpse into the regulation of Amadori metabolism in a clinically significant bacterial pathogen and uncovers thematic parallels in control of gene expression during utilization of unrelated nutrients.

References:
Majowicz et al. (2010) Clin. Infect. Dis. 50: 882-889
Wu et al. (2017) J. Food. Ag. Chem. 66: 212-217
Ali et al. (2014) PLoS Pathog. 10: e1004209
Sabag-Daigle et al. (2016) Sci. Reps. 6: 1-9

Keywords: Salmonella, DNA, Amadori Products