Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of severe diarrheal disease in humans. Cattle are the natural reservoir of EHEC, and approximately 75% of EHEC infections in humans stem from bovine products. Many common bacterial pathogens, including EHEC, rely on chemical communication systems, such as quorum sensing (QS), to regulate virulence and facilitate host colonization. EHEC uses SdiA from E. coli (SdiAEC), an orphan LuxR-type receptor, to sense N-acyl l-homoserine lactone (AHL) QS signals produced by other members of the bovine enteric microbiome. SdiAEC regulates two phenotypes critical for colonizing cattle: acid resistance and the formation of attaching and effacing lesions. Despite the importance of SdiAEC, there is very little known about its selectivity for different AHL signals, and no chemical inhibitors that act specifically on SdiAEC have been reported. Such compounds would represent valuable tools to study the roles of QS in EHEC virulence. To identify chemical modulators of SdiAEC and delineate the structure–activity relationships (SARs) for AHL activity in this receptor, we report herein the screening of a focused library composed largely of AHLs and AHL analogues in an SdiAEC reporter assay. We describe the identity and SARs of potent modulators of SdiAEC activity, examine the promiscuity of SdiAEC, characterize the mechanism of a covalent inhibitor, and provide phenotypic assay data to support that these compounds can control SdiAEC-dependent acid resistance in E. coli. These SdiAEC modulators could be used to advance the study of LuxR-type receptor/ligand interactions, the biological roles of orphan LuxR-type receptors, and potential QS-based therapeutic approaches.

Protein‐carbohydrate interactions play critical recognition and signaling roles in a diverse array of biological processes—from stem cell differentiation to immune responses. However, the intermolecular forces that govern protein‐carbohydrate interactions have not been quantitatively elucidated. Given the dogma in the field and our data, the goal of this study is to understand the molecular basis for aromatic‐carbohydrate interactions. Previous bioinformatic analyses have shown that aromatic residues are markedly conserved and overrepresented in carbohydrate binding sites while aliphatic residues are dramatically underrepresented. Unlike aliphatic residues, aromatic amino acids present electronegative π‐electron systems that can interact with carbohydrate C–H bonds through CH–π interactions. We posited that if electrostatic contributions are important for CH–π interactions in protein–carbohydrate complexes, differences in the electronics of the aromatic systems and carbohydrate C–H bonds would determine participation in such interactions. We therefore sought to design a system to interrogate the molecular basis for aromatic‐carbohydrate interactions within proteins. Using the immunologically relevant lectin, galectin‐3, the stability and lactose binding affinity of galectin‐3 variants at CH‐π acceptor residue, W181, were assessed through differential scanning fluorimetry and isothermal titration calorimetry. We observed that the replacement of W181 with the smaller aromatic residues phenylalanine, tyrosine, and histidine diminished both protein stability and binding affinity, while replacement of W181 with an aliphatic methionine residue maintained protein stability but abrogated lactose binding. Supported by stability and binding data for the galectin‐3 variants, we conclude that CH‐π interactions are particularly important in promoting the stability of carbohydrate binding proteins as well as their affinity for binding β‐galactosides through the contribution of electrostatics versus hydrophobic interactions.Support or Funding InformationThis work was supported by a UW‐Madison L&S Honors Welton Undergraduate Research Fellowship.