Identification of a Pyruvate Ferredoxin Oxidoreductase in <i>Acanthamoeba castellanii</i> Cysts: A Key Enzyme in Cyst Energy Homeostasis

Pyruvate ferredoxin oxidoreductase (PFOR) is the main enzyme responsible for pyruvate decarboxylation under anaerobic conditions. This enzyme is very well characterized in a wide range of microorganisms, such as anaerobic bacteria and microaerophilic parasites; however, the presence of this enzyme in free-living amoebas (FLAs) has not been demonstrated. Acanthamoeba castellanii (A. castellanii) is an FLA that exhibits trophozoite and cyst forms during its life cycle. The trophozoite form possesses functional mitochondria that are responsible for ATP synthesis. The cyst form possesses a rudimental mitochondrial structure that seems to be not functional and anaerobically synthesizes ATP. In this study, we described the presence of a PFOR in A. castellanii (known as AcPFOR). The structure of this enzyme is very similar to that of PFOR, which has been characterized in other microorganisms, and the main domains responsible for the enzymatic activity of PFOR are present in AcPFOR. The cyst forms exhibited increased expression and enzymatic activity of PFOR. This enzyme is inhibited by nitazoxanide (a PFOR inhibitor), and drug administration was able to inhibit the encystment process by overstimulating autophagy. The inhibition of the enzyme also affects cyst viability, thus resulting in the inhibition of the excystation process. In conclusion, we demonstrated the importance of PFOR in A. castellanii cysts energy homeostasis, thereby indicating that this enzyme may be an interesting therapeutic target.