Acanthamoeba is free-living protist pathogen capable of causing a blinding keratitis and granulomatous encephalitis. However, the mechanisms of Acanthamoeba pathogenesis are still not clear. Here, our results show that cells co-cultured with pathogenic Acanthamoeba would be spherical and floated, even without contacting the protists. Then, the Acanthamoeba protists would contact and engulf these cells. In order to clarify the contact-independent pathogenesis mechanism in Acanthamoeba, we collected the Acanthamoeba-secreted proteins (Asp) to incubate with cells for identifying the extracellular virulent factors and investigating the cytotoxicity process. The Asps of pathogenic Acanthamoeba express protease activity to reactive Leu amino acid in ECM and induce cell-losing adhesion ability. The M20/M25/M40 superfamily aminopeptidase protein (ACA1-264610), an aminopeptidase be found in Asp, is upregulated after Acanthamoeba and C6 cell co-culturing for 6 h. Pre-Treating the Asp with leucine aminopeptidase inhibitor and the specific antibodies of Acanthamoeba M20/M25/M40 superfamily aminopeptidase could reduce the cell damage during Asp and cell co-incubation. These results suggest an important functional role of the Acanthamoeba secreted extracellular aminopeptidases in the Acanthamoeba pathogenesis process. This study provides information regarding clinically pathogenic isolates to target specific molecules and design combined drugs.
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