Pathogenic Acanthamoeba castellanii Secretes the Extracellular Aminopeptidase M20/M25/M40 Family Protein to Target Cells for Phagocytosis by Disruption

Abstract

<i>Acanthamoeba</i> is free-living protist pathogen capable of causing a blinding keratitis and granulomatous encephalitis. However, the mechanisms of <i>Acanthamoeba</i> pathogenesis are still not clear. Here, our results show that cells co-cultured with pathogenic <i>Acanthamoeba</i> would be spherical and floated, even without contacting the protists. Then, the <i>Acanthamoeba</i> protists would contact and engulf these cells. In order to clarify the contact-independent pathogenesis mechanism in <i>Acanthamoeba</i>, we collected the <i>Acanthamoeba</i>-secreted proteins (Asp) to incubate with cells for identifying the extracellular virulent factors and investigating the cytotoxicity process. The Asps of pathogenic <i>Acanthamoeba</i> 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 <i>Acanthamoeba</i> and C6 cell co-culturing for 6 h. Pre-treating the Asp with leucine aminopeptidase inhibitor and the specific antibodies of <i>Acanthamoeba</i> 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 <i>Acanthamoeba</i> secreted extracellular aminopeptidases in the <i>Acanthamoeba</i> pathogenesis process. This study provides information regarding clinically pathogenic isolates to target specific molecules and design combined drugs.