Inhibition of GABA metabolism by β-lactam antibiotics affects encystation in Acanthamoeba

Acanthamoeba keratitis (AK) is a painful corneal infection caused by pathogenic free-living amoebae of the genus Acanthamoeba, primarily affecting contact lens users with inadequate hygiene practices. Clinical manifestations include corneal infiltration, epithelial and stromal damage, and severe ocular pain, which may lead to vision loss or enucleation in advanced cases. Current treatments, such as polyhexamethylene biguanide (PHMB), often fail due to the parasite's ability to form drug-resistant cysts. In this study, metabolomic profiling revealed a significant accumulation of γ-aminobutyric acid (GABA) during encystation. Expression analysis of key genes associated with the GABA shunt pathway showed marked upregulation of glutamate dehydrogenase (GDH) and glutamate decarboxylase (GAD). Supplementation with exogenous GABA enhanced encystation rates and upregulated encystation-associated genes, including cellulose synthase I, autophagy-related protein 8, and encystation-mediating serine proteinase, suggesting that GABA may function as a signaling molecule regulating encystation. To explore potential anti-encystation strategies, β-lactam antibiotics, well-known GABAA receptor antagonists, were tested. Cefotaxime, carbenicillin, and penicillin G significantly inhibited cyst formation without inducing cytotoxicity, whereas non-β-lactam antibiotics showed no such effect. These findings suggest that the inhibitory effect is associated with the β-lactam ring structure and may involve interference with GABA-mediated signaling pathways. Collectively, our study reveals a critical role of GABA metabolism in Acanthamoeba encystation and highlights β-lactam antibiotics as potential adjunctive agents for overcoming cyst-associated drug resistance in AK treatment.