High-efficiency transfection of Acanthamoeba castellanii using a cationic polymer

The free-living amoeba Acanthamoeba castellanii is an ecologically, clinically, and evolutionarily important microorganism. A. castellanii amoebae can be directly pathogenic to humans and serve as reservoirs for bacterial pathogens (e.g., Legionella pneumophila), but they also regulate the proliferation of other microorganisms in the soil. Despite their importance, no reliable genetic system has been developed, hampering the use of A. castellanii and related species as model organisms. Transfecting A. castellanii with plasmids is possible with commercial kits, but it is expensive, inefficient, and vulnerable to product discontinuation. In this contribution, we present a method for efficient transfection of A. castellanii with readily available and inexpensive polyethylenimines. We systematically explore the method's parameters, achieving up to 100-fold higher efficiency compared to currently used protocols. This method provides a robust step toward a complete genetic toolbox for A. castellanii, thereby expanding its utility as a model organism.