A Novel Anti-Virulence Strategy: Targeting Heat-Labile Enterotoxin with Punicalin to Combat Multidrug-Resistant ETEC

The extensive use of antibiotics, such as gentamicin (GM), has accelerated the emergence of multidrug-resistant (MDR) ETEC strains, underscoring the urgent need for resistance-independent therapeutic strategies. The heat-labile enterotoxin (LT), a key virulence determinant mediating bacterial adhesion and cAMP elevation, represents an attractive target for anti-virulence intervention.

Using computer-aided drug design (CADD) with LTb (PDB: 1EFI) as the target, 3,875 natural compounds were screened, yielding 17 high-affinity candidates. In vitro assays in IPEC-J2 cells identified Punicalin (PN) as the most potent inhibitor, significantly reducing ETEC-induced cytotoxicity, adhesion, and cAMP accumulation without affecting bacterial viability (MIC = 1024 μg/mL). PN, alone or in combination with GM, markedly decreased inflammatory cytokines (IL-6, TNF-α, IL-1β).