Combinatorial treatment with β-glucanase enzyme and chlorhexidine induces cysticidal effects in Acanthamoeba cyst.

Abstract

Acanthamoeba cysts have a cellulose cell wall made up of a solid layer of β-glucan, which confers resistance to the dormant phase of this microorganism. The ability of Acanthamoeba to change to this dormant phase causes difficulties in treating its infection at the cyst stage as compared to the trophozoite stage. Therefore, targeting cyst total mortality can help to prevent re-infection in patients. To ensure cysticidal treatment, a β-glucanase enzyme was introduced in vitro to the Acanthamoeba cyst, followed by a chlorhexidine solution treatment. β-glucanase enzyme and chlorhexidine dose-response analysis was performed based on cell wall integrity measurement. The treatment was also performed on human corneal epithelial cells to confirm the safety of the treatment in vitro. The surface morphology of the cysts was observed using scanning electron microscopy (SEM), while the protein alterations were determined using 1D protein analysis. The interaction of the β-glucanase enzyme with cellulose linkages was investigated based on molecular dosimetry. Incubation of the cyst for 24h at 8.75 units/ml of β-glucanase followed by 0.88μg/ml of chlorhexidine resulted in a substantial reduction in the total chlorhexidine used, which made it safer for human corneal epithelial cells. Ultrastructural changes revealed the reduction of the thickness in ectocyst and endocyst layers with the loss of the internal structure of the cyst. After combination treatment of chlorhexidine and β-glucanase, a decrease in the cyst protein from the size of 37 to 25kDa was observed. The enzyme-substrate interaction validated these results based on molecular docking between 1,4-β-D-glucan and 1,4- β-D-xylan with the β-glucanase enzyme. In silico analysis revealed that two catalytic glutamate residues (Glu160 and Glu267) are essential to catalysing the hydrolytic reaction. Molecular dynamic simulation analysis revealed that both ligands formed stable interactions throughout the simulation. This work concludes that the enzymatic approach combined with chlorhexidine is a novel and effective technique for ensuring the cysticidal effects against the Acanthamoeba cyst. The interaction of the chlorhexidine and β-glucanase enzyme on the surface of the cyst of amoeba resulted in the ecto-and endo cyst layer being damaged and confirmed the cysticidal effects.