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

Abstract

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.

Extracellular vesicles and vesicle-free secretome of the protozoa Acanthamoeba castellanii under homeostasis and nutritional stress and their damaging potential to host cells

“…Huang and collaborators showed that proteins secreted by A. castellanii , induced the detachment of mammalian cells and assigned this effect to the M20/M25/M40 aminopeptidase superfamily proteins. These detached cells floated and were then ingested and killed by amoeba cells [ 84 ]. These proteins were not found in the EVs or EVs-free supernatant, but other molecules present in the secretome of A. castellanii could induce similar functions.…”

The Biochemical and Functional Characterization of M28 Aminopeptidase Protein Secreted by Acanthamoeba spp. on Host Cell Interaction

“…The matrix metalloproteases 2 and 7 of Bacillus cereus were cleaved with high efficiency at the Leu-Gly or Leu-Ala bond by the residue in the P1′ position [31]. A previous study showed that the M20/M25/M40 superfamily aminopeptidase protein, an aminopeptidase found in Asp can be hydrolyzed Leu-AMC more rapidly than Arg-AMC [24]. In this study, the results showed that Asp more rapidly hydrolyzed Leu-AMC than Arg-AMC, as well as the recombinant M28AP ( Figure 2B).…”

“…A previous study showed that the M20/M25/M40 superfamily aminopeptidase protein, an aminopeptidase that is found in Asp, causes cell line disruption after Acanthamoeba spp. and C6 cell co-culturing [24]. In general, the cell line causes organelle stress and induces apoptosis pathways due to parasite infection [35].…”

“…Acanthamoeba spp., at an approximate density of 1.0 × 10 6 parasites/mL, were washed three times and resuspended in PAS buffer for 4 h, after which the parasites were removed by centrifugation. The cell-free media containing secreted proteins was filtered through an Amicon ® Ultra-4 3K Centrifugal Filter Unit (Merck Millipore, Darmstadt, Germany) and then collected by centrifugation at 959× g for 75 min [24]. The protein concentration was measured with an ND-1000 (NanoDrop, Thermo, Waltham, MA, USA) and a protein assay (Bio-Rad, Hercules, CA, USA).…”

“…We previously showed that the M20/M25/M40 superfamily aminopeptidase protein, which is an aminopeptidase found in Acanthamoeba-secreted proteins (Asp), causes cell damage and disruption [24]. We also identified a novel protein that was secreted by Acanthamoeba spp., an M28 aminopeptidase (M28AP), as a target of the human innate immune defense [25,26].…”