Molecular and Structural Basis Underlying Selective Targeting of Claudins by Clostridium perfringens Enterotoxin in Mammalian Gut

The bacterium Clostridium perfringens causes severe, sometimes lethal gastrointestinal disorders in humans, including enteritis and enterotoxemia. Type F strains produce an enterotoxin (CpE) that causes the 3 rd most common foodborne illness in the United States. CpE breaks down the gut barrier by recognizing then binding surface‐exposed receptors, triggering dissociation of tight junctions, multi‐protein complexes involved in cell/cell adhesion and paracellular ion transport in epithelium. These receptors, claudins, are a family of integral membrane proteins that fortify tight junctions through self‐assemblies within and between cells. Claudin assemblies maintain epithelial barriers normally but are disabled upon CpE binding, leading to tight junction disruption. Although it was known that claudin recognition was encoded in CpE's C‐terminal domain (cCpE) and that only some of the ~24 claudins present in mammalian genomes bind CpE, the molecular and structural basis for this subtype‐specific targeting by CpE was undetermined. Our objective, therefore, was to elucidate the origin of CpE selective targeting of claudins, as we hypothesized that subtle yet unknown molecular differences must exist between receptors and non‐receptors that were masked by sequence and structural conservation. To test this, we determined the crystal structure of a key receptor in humans, claudin‐4, in complex with cCpE; then measured the binding affinities, kinetics, and half‐lives of claudin/enterotoxin complexes and the cytotoxic effects of CpE on claudin‐expressing cells. Our findings reveal that CpE targets a cryptic motif conserved in receptive claudins and that this motif imparts high‐affinity CpE binding to these but not other subtypes—distinguishing receptors from non‐receptors. By correlating the binding residence times of claudin/CpE complexes that we determined to claudin expression patterns in the gut, we uncovered that the primary CpE receptors differ in mice and humans, due to sequential and thus structural changes to the target motif. These findings provide the molecular and structural elements CpE employs for selective targeting of claudins during pathogenicity of Clostridium perfringens in mammals and challenges the existing classification of CpE receptors in humans. Further, this work provides a framework for the design of new CpE‐based therapeutics that can be used for detection and directed destruction of cancer cells and tuning blood‐brain barrier permeability in drug delivery, and for the development of innovative strategies to treat CpE‐type gastrointestinal illnesses in domesticated mammals and humans.