Structural and Functional Basis of GusR‐Mediated Regulation of ß‐Glucuronidase Expression in Enterobacteriaceae Gastrointestinal Pathobionts

Abstract

Gastrointestinal (GI) microbial dysbioses have been linked to the on‐set or exacerbation of mammalian metabolic disorders, inflammatory bowel diseases, and colorectal cancer, particularly with increases in the levels of Proteobacteria. Furthermore, elevated activities of microbial ß‐Glucuronidase (GUS) enzymes correlates to colon carcinogenesis and poor outcomes in cancer treatment. Here, we determine the structural and functional basis for the regulation of GUS expression in Proteobacterial pathobionts of the Enterobacteriaceae family, species Escherichia coli and Salmonella enterica. In the GI microbiota, only the Enterobacteriaceae maintain an operon of GUS and glucuronide transporters under the control of a glucuronide repressor GusR, and presumably this operon can provide a growth advantage in the competitive intestinal milieu. The crystal structures of E. coli GusR and S. enterica GusR in complexes with glucuronidated ligands were determined to 2.0 Å resolution. Isothermal titration calorimetry (ITC) was used to determine E. coli GusR's DNA binding affinity to the GusABC operon operator site and identify an endogenous glucuronide ligand with low micro‐molar affinity. Structural‐guided GusR ligand binding pocket mutants coupled with ITC pinpointed residues essential for glucuronide binding. Finally, we confirm using in vivo cell‐based assay that GusR robustly induces GUS activity in the presence of GusR‐targeted ligands. Taken together, these data provide the first molecular insights into the control of GUS expression by a family of pathobionts that proliferate in the GI during disease.Support or Funding InformationNational Science Foundation Graduate Research Fellowship & National Institute of Health