Lactococcus formosensis and its metabolite 4-acetamidobutanoic acid induced caspase-11 dependent myenteric neuronal pyroptosis in intractable functional constipation

Abstract

Intractable functional constipation (IFC), a severe form of chronic constipation characterized by slow transit and resistance to conventional treatments, posed a significant clinical challenge. Here, we identified Lactococcus formosensis (Lf), a Gram-positive bacterium prevalent in IFC patients, as a novel contributor to intestinal motility impairment. Clinically, IFC patients exhibited increased colonic mucosal colonization of Lf and significant myenteric neuronal loss and pyroptosis, particularly in excitatory choline acetyltransferase (ChAT)⁺ neurons, but not inhibitory neuronal nitric oxide synthase (nNOS)⁺ neurons. In mice, Lf and its supernatant impaired intestinal motility, reducing fecal water content, prolonging transit times, and inhibiting spontaneous contractions and maximum contractile force in isolated intestinal segments. RNA sequencing revealed that Lf disrupted neurochemical signaling, implicating neuronal damage in its pathogenic effects. Mechanistically, Lf and its supernatant selectively induced pyroptosis in ChAT⁺ neurons via caspase-1 activation. Metabolomic profiling identified 4-acetamidobutanoic acid (4-ABA) as a key metabolite shared between Lf supernatant and IFC patient feces. 4-ABA induced ChAT⁺ neuronal pyroptosis through a caspase-11-dependent pathway, further impairing intestinal motility, which was confirmed in Casp11⁻/⁻ mice. This study uncovered a previously unrecognized pathway of single microbiota-induced neuronal damage in IFC and provided a foundation for novel diagnostic and therapeutic strategies targeting gut microbiota-ENS interactions