Acanthamoeba is an opportunistic pathogen that causes Acanthamoeba keratitis, granulomatous amoebic encephalitis, and other cutaneous diseases. The life cycle of Acanthamoeba consists of 2 stages of trophozoites and cysts. Under adverse environmental conditions, Acanthamoeba encysts, while the conditions become favorable for growth, it reverts to the trophozoite form. Acanthamoeba excystation is crucial for its proliferation and can lead to recurrent infections after incomplete treatment. To identify the factors involved in excystation, A. castellanii was subjected to either encystation- or excystation-inducing conditions, and gene expression profiles were compared using mRNA sequencing. A. castellanii samples were collected at 8 h intervals for analysis under both conditions. Differentially expressed gene analysis revealed that 1,214 and 1,163 genes were upregulated and downregulated, respectively, by more than 2-fold during early excystation. Five genes markedly upregulated in early excystation (ACA1_031140, ACA1_032330, ACA1_374400, ACA1_275740, and ACA1_112650) were selected, and their expression levels were confirmed via real-time PCR. Small interfering RNA (siRNA) targeting these 5 genes was transfected into Acanthamoeba and gene knockdown was validated through real-time PCR. The silencing of ACA1_031140, ACA1_032330, ACA1_374400, and ACA1_112650 inhibited excystation and suggested that these genes might be essential for excystation. Our findings provide valuable insights for suppressing Acanthamoeba proliferation and recurrence.

Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.