High-efficiency transfection ofAcanthamoeba castellaniiusing a cationic polymer

The free-living amoebaAcanthamoeba castellaniiis an ecologically, clinically, and evolutionarily important microorganism.A. castellaniiamoebae are directly pathogenic to humans and serve as reservoirs for bacterial pathogens (e.g.,Legionella pneumophila) but also regulate the proliferation of other microorganisms in the soil. Despite their importance, no reliable genetic system has been developed, hampering the use ofA. castellaniiand related species as model organisms. TransfectingA. castellaniiwith 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 ofA. castellaniiwith readily available and inexpensive polyethylenimines. We systematically explore the method’s parameters, obtaining up to 100-fold higher efficiency than currently used protocols. The method presented here provides a robust step towards a complete genetic toolbox forA. castellanii, hence expanding its use as a model organism.