An Acanthamoeba castellanii metacaspase associates with the contractile vacuole and functions in osmoregulation.

Acanthamoeba castellanii is a free-living protozoan. Some strains are opportunistic pathogens. A type-I metacaspase was identified in A. castellanii (Acmcp) and was shown to be expressed through the encystation process. The model organism, Dictyostelium discoideum, has been used here as a model for studying these caspase-like proteins. Separate cell lines expressing a GFP-tagged version of the full length Acmcp protein, as well as a deletion proline region mutant of Acmcp protein (GFP-Acmcp-dpr), have been introduced into D. discoideum. Both mutants affect the cellular metabolism, characterized by an increase in the growth rate. Microscopic imaging revealed an association between Acmcp and the contractile vacuole system in D. discoideum. The treatment of cells with selected inhibitors in different environments added additional support to these findings. This evidence shows that Acmcp plays an important role in contractile vacuole regulation and mediated membrane trafficking in D. discoideum. Additionally, the severe defect in contractile vacuole function in GFP-Acmcp-dpr mutant cells suggests that the proline-rich region in Acmcp has an essential role in binding this protein with other partners to maintain this process. Furthermore, Yeast two-hybrid system identified there are weak interactions of the Dictyostelium contractile vacuolar proteins, including Calmodulin, RabD, Rab11 and vacuolar proton ATPase, with Acmcp protein. Taken together, our findings suggest that A. castellanii metacaspase associate with the contractile vacuole and have an essential role in cell osmoregulation, which contributes to its attractiveness as a possible target for treatment therapies against A. castellanii infection.