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

Enterotoxigenic Escherichia coli (ETEC) causes significant diarrheal morbidity in humans and livestock, threatening both public health and agricultural productivity. 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). In vivo, PN (5 mg/kg) alleviated diarrhea in ETEC-infected mice, reduced intestinal cAMP and LT levels, restored villus structure, and normalized gut microbial composition. Co-administration with GM completely prevented diarrhea and enhanced therapeutic efficacy. Gene knockout and molecular dynamics analyses confirmed that PN interacts with LTb key residues (Arg13, Glu51, Gln61, Trp88, Asp91), disrupting toxin–host interactions. Collectively, these findings identify PN as a prototype natural LT inhibitor that mitigates ETEC infection through a non-bactericidal anti-virulence mechanism, offering a sustainable strategy to combat MDR enteric pathogens. KEYWORDS: Punicalin; ETEC; LT inhibitor; Bacterial resistance; Molecular dynamics simulation