Poster abstracts

Poster number 37 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, The Ohio State University), Anice Sabag-Daigle (Department of Microbial Infection and Immunity, The Ohio State University), Andrew Schweiters (Department of Microbiology, The Ohio State University), Brian Ahmer (Department of Microbial Infection and Immunity, The Ohio State University), Venkat Gopalan (Department of Chemistry & Biochemistry, The Ohio State University)

Abstract:
The foodborne pathogen Salmonella enterica serovar Typhimurim (Salmonella) causes approximately 94 million enteric infections and 50,000 diarrheal deaths annually worldwide.1 There are no vaccines or antibiotics that specifically combat this bacterium. During infection, Salmonella exploits fructose-asparagine (F-Asn) as a carbon and nitrogen source.2 F-Asn is a product of an Amadori rearrangement that occurs during cooking and dehydration of raw foods.3 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.2,4 Importantly, the deglycase was identified as a promising drug target since its knock-out led to accumulation of its substrate and Salmonella self-poisoning.4,5 Our goal is to characterize regulation of the fra operon by FraR, the putative transcriptional factor 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 the fraB promoter in vivo, repressing the fra locus, and that binding of an inducer ligand to the FraR IBD triggers a conformational change to release the DNA from the DBD and permit transcription of the fra operon genes. Our recent biochemical and native mass spectrometry (nMS) studies with recombinant FraR provided insights into the inducer identity, the DNA and inducer binding affinity, and the binding stoichiometry.6 Building on these advances, we have identified key inducer binding residues in the IBD and the nucleotides in the DNA that are recognized by the DBD. To support our in vitro characterization, we are testing these mutants in vivo in vivo using a luciferase reporter system. Together, these data provide a model for how Amadori compound metabolism is regulated in a clinically significant bacterial pathogen and uncover thematic parallels in control of gene expression during utilization of unrelated nutrients.

References:
1) Majowicz et al. (2010) Clin. Infect. Dis. 50: 882-889
2) Ali et al. (2014) PLoS Pathog. 10: e1004209
3) Wu et al. (2017) J. Food. Ag. Chem. 66: 212-217
4) Sabag-Daigle et al. (2016) Sci. Reps. 6: 1-9
5) Sengupta et al. (2019) JMB 431: 4497-4513
6) Szkoda et al. (2022) JMB 434: 167480

Keywords: Salmonella, DNA, Amadori products