Autophagy protein 12 plays an essential role in Acanthamoeba encystation

Autophagy is a well conserved, catabolic process in eukaryotic cells. Previously, we identified two novel ubiquitin like conjugation systems (Atg12 and Atg8) in the autophagy process of Acanthamoeba castellanii. To obtain more specific information on the Atg12 ubiquitin like conjugation system during encystation of Acanthamoeba, we characterized the function of Atg12. Knockdown of AcAg12 in trophozoites resulted in inhibition of cyst formation. Analysis of subcellular localization showed that AcAtg12 was evenly distributed in the trophozoites during early encystation, started to accumulate partially as dots or fragments, and then co-localized with the vesicle of the autophagic structure. However, the mRNA expression of AcAtg12 was maintained at a constant level during encystation as well as in trophozoites. Ultrastructural analysis with TEM showed that AcAtg12-knockdown cells showed vacuolization, lack of cyst wall formation, and numerical decline of autophagic structures, compared with the control cells. Interestingly, these knockdown cells began to round-up and swell, and then burst at 144 h post encystation. Taken together, our results might provide a better understanding of the Atg12 UBL conjugation system in Acanthamoeba and other cyst forming protozoan parasites.