In Vitro Inhibition of Bacterial Interactions With Acanthamoeba castellanii by a Monoclonal Antibody Against...

Introduction Acanthamoeba castellanii causes amoebic keratitis (AK), primarily in contact lens wearers, and severe lesions may require corneal transplantation. Direct contact between the amoeba and the cornea during AK induction is important for the pathogenic process of the amoeba. From a clinical perspective, identifying molecular factors that regulate this contact-dependent interaction is essential for understanding disease progression and for developing preventive or therapeutic strategies for AK. Methods This study analyzed the effect of treatments with galactose and monoclonal antibodies against the galactose-binding protein (GBP) on the interactions between bacteria and A. castellanii. A. castellanii trophozoites were pre-incubated with monosaccharides (galactose, mannose, or glucose; 100 mM) or monoclonal antibodies against GBP for one hour, followed by co-incubation with pathogenic Escherichia coli O157:H7 (one hour) or non-pathogenic E. coli DH5α (four hours). Bacterial association and invasion were quantified using colony-forming unit (CFU)-based assays following cell lysis, and proteolytic activity was evaluated using gelatin zymography. Statistical analysis was performed using Student's t-test, with P<0.05 considered significant. Results The amoeba-bacteria association after the incubation treatment with mannose was 3.4 times lower than with no treatment. However, the association after the galactose treatment was about 0.7 times lower than with no treatment. In particular, incubation with monoclonal antibodies to GBP showed results very similar to those of mannose. E. coli O157:H7's invasion of the amoebae was reduced by about three times as compared to the association reaction. This reduction in invasion suggests that disruption of lectin-mediated binding may limit the ability of pathogenic bacteria to persist within amoebae. The effects of the incubation treatments with monoclonal antibodies against galactose, mannose, and GBP were so similar that they were almost incomparable. E. coli DH5α's association reaction with galactose was slightly lower than that in the monosaccharide-untreated (no treatment) group. However, it was confirmed that monoclonal antibodies against GBP could reduce the amoeba-bacteria association about 3.65 times more than no treatment. The incubation treatment with antibodies to GBP showed an increase in proteolytic enzyme expression to a degree very similar to that of the mannose treatment group. Conclusion Taken together, these findings indicate that GBP plays a key role in contact-dependent interactions relevant to amoebic pathogenicity. Targeting GBP-mediated pathways may represent a clinically meaningful approach to attenuating host tissue damage and microbial interactions associated with AK.