Genetically engineered probiotics, able to in situ deliver therapeutically active compounds while restoring gut eubiosis, could represent an attractive therapeutic alternative in
Clostridium difficile infection (CDI).
Palmitoylethanolamide is an endogenous
lipid able to exert immunomodulatory activities and restore epithelial barrier integrity in human models of
colitis, by binding the
peroxisome proliferator-activated receptor-α (PPARα). The aim of this study was to explore the efficacy of a newly designed PEA-producing probiotic (pNAPE-LP) in a mice model of C. difficile toxin A (TcdA)-induced
colitis. The human N-acyl-
phosphatidylethanolamine-specific
phospholipase D (NAPE-
PLD), a key
enzyme involved in the synthesis of PEA, was cloned and expressed in a Lactobacillus paracasei that was intragastrically administered to mice 7 days prior the induction of the
colitis. Bacteria carrying the empty vector served as negative controls (pLP).In the presence of
palmitate, pNAPE-LP was able to significantly increase PEA production by 27,900%, in a time- and concentration-dependent fashion. Mice treated with pNAPE-LP showed a significant improvement of
colitis in terms of histological damage score, macrophage count, and
myeloperoxidase levels (-53, -82, and -70.4%, respectively). This was paralleled by a significant decrease both in the expression of toll-like receptor-4 (-71%), phospho-p38
mitogen-activated protein kinase (-72%),
hypoxia-inducible factor-1-alpha (-53%), p50 (-74%), and p65 (-60%) and in the plasmatic levels of
interleukin-6 (-86%),
nitric oxide (-59%), and
vascular endothelial growth factor (-71%). Finally,
tight junction protein expression was significantly improved by pNAPE-LP treatment as witnessed by the rescue of zonula occludens-1 (+304%),
Ras homolog family member A-
GTP (+649%), and
occludin expression (+160%). These protective effects were mediated by the specific release of PEA by the engineered probiotic as they were abolished in PPARα knockout mice and in wild-type mice treated with pLP. Herein, we demonstrated that pNAPE-LP has therapeutic potential in CDI by inhibiting colonic
inflammation and restoring
tight junction protein expression in mice, paving the way to next generation probiotics as a promising strategy in CDI prevention.