Encystment Induces Down-Regulation of an Acetyltransferase-Like Gene in Acanthamoeba castellanii

Abstract

Acanthamoeba castellanii is a ubiquitous free-living amoeba. Pathogenic strains are causative agents of Acanthamoeba keratitis and granulomatous amoebic encephalitis. In response to adverse conditions, A. castellanii differentiate into cysts, which are metabolically inactive and resistant cells. This process, also named encystment, involves biochemical and genetic modifications that remain largely unknown. This study characterizes the role of the ACA1_384820 Acanthamoeba gene during encystment. This gene encodes a putative N-acetyltransferase, belonging to the Gcn5-related N-acetyltransferase (GNAT) family. We showed that expression of the ACA1_384820 gene was down-regulated as early as two hours after induction of encystment in A. castellanii. Interestingly, overexpression of the ACA1_384820 gene affects formation of cysts. Unexpectedly, the search of homologs of ACA1_384820 in the Eukaryota gene datasets failed, except for some species in the Acanthamoeba genus. Bioinformatics analysis suggested a possible lateral acquisition of this gene from prokaryotic cells. This study enabled us to describe a new Acanthamoeba gene that is down-regulated during encystment.

Overexpression of a G-protein coupled receptor-like gene affects encystment ofAcanthamoeba castellanii

“…Indeed, the mRNA encoding the TPBF was reported stable during the encystment (Orfeo and Bateman, 1998). As a control, we used the plasmid pTBPF-empty, which does not express of the coding sequence (Rolland et al, 2020). The growth of A. castellanii was not affected by the overexpression of the ACA1_384820 gene compared cells transfected with the control plasmid pTBPF-empty (Fig.…”

“…PCR primers are listed in the Table 1. The pTBPF-empty (Rolland et al, 2020) was used as a control vector.…”

“…Transfection of A. castellanii trophozoites were performed using the Viafect™ transfection reagent (Promega) as previously described (Rolland et al, 2020). Briefly, the cells were seeded into 24-well plates at a density of 1.25 x 10 5 cells per well in encystment medium.…”

“…The LightCycler FastStart DNA Master plus SYBR Green I (Roche Applied Science) was used to perform all quantitative RT-PCR (RT-qPCR) as previously described (Rolland et al, 2020). Reactions were prepared in a total volume of 10 µl containing 5 µl of 2X SYBR mix, 2 µl of H2O, 2 µl of diluted cDNA template, and 0.5 µl of 10 µM primers.…”

“…We incubated transfected amoebae within the encystment medium and evaluated the percentage of cysts using the calcofluor white stain at different time points (24 h, 48 h and 72 h). A. castellanii transfected with the pTBPF-empty was reported to encyst as efficiently as the untransfected amoebae (Rolland et al, 2020).…”