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

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.


Introduction
Acanthamoeba spp. are free-living amoebae, commonly found in diverse natural environments such as soil, water and air, and also in artificial facilities including tap water systems, cooling towers, sewage and air conditioning systems [1,2]. Pathogenic strains of Acanthamoeba spp. are causative agents of a rare fatal infection of the central nervous system, called granulomatous amoebic encephalitis, and of Acanthamoeba keratitis (AK), a progressive eye disease [3,4]. AK is generally associated with contact lens wearers and remains an elusive problem in spite of advances in antimicrobial chemotherapy and eye care [5].
Acanthamoeba's life cycle consists of two stages: a metabolically active trophozoite and an inactive dormant cyst [3]. The formation of cysts, also called encystment, is a reversible process that is induced by harsh conditions, such as lack of nutrients, or change in osmotic pressure or pH [6,7]. The cyst represents a resistant and resting form that protects the amoeba against adverse conditions such as heat, freezing and chemicals, and enables it to persist for many years [8,9]. hours after induction of the encystment. Results represent average values of three independent experiments, and error bars represent the standard error of the mean (± SEM). Statistical analysis was performed using the ordinary one-way ANOVA followed by Dunnett's post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) from ΔCt values. At each time point, means were compared to the '0 hour' condition.

The ACA1_384820 Gene Encodes a Putative N-acetyltransferase-like Protein That Presents Homologies with Prokaryotic Sequences
A bioinformatics analysis was performed to characterize the putative function of the ACA1_384820 gene. The ACA1_384820 gene encodes a putative protein of 345 amino acids [20]. The coding sequence of ACA1_384820 was amplified from the cDNA extracted from the A. castellanii str. Neff (ATCC 30010) and the nucleotide sequence obtained missed 21 nucleotides corresponding to 7 amino acids (Supplementary Data 1 and 2). This deletion did not change either the open reading frame or the putative active domain of the predicted protein.
The identification of specific domains with InterPro showed the presence of a Gcn5-related Nacetyltransferase (GNAT) domain in the C-terminal region. No additional domain was predicted. Accordingly, the ACA1_384820 protein could be a putative N-acetyltransferase, a family of proteins involved in the transfer of the acetyl group from acetyl-CoA to a substrate.
In order to find homologs of the ACA1_384820 protein, a Basic Local Alignment Search Tool (BLAST) analysis was performed against the National Center for Biotechnology Information (NCBI) Figure 1. The expression of the A. castellanii N-acetyltransferase-like genes is down-regulated during encystment. The relative expression of ACA1_127910, ACA1_164890, ACA1_215610, ACA1_350710, ACA1_164890, ACA1_384820 and cellulose synthase genes was assessed by RT-qPCR 0, 2, 4, 8 and 24 h after induction of the encystment. Results represent average values of three independent experiments, and error bars represent the standard error of the mean (± SEM). Statistical analysis was performed using the ordinary one-way ANOVA followed by Dunnett's post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) from ∆Ct values. At each time point, means were compared to the '0 h' condition.
The identification of specific domains with InterPro showed the presence of a Gcn5-related N-acetyltransferase (GNAT) domain in the C-terminal region. No additional domain was predicted. Accordingly, the ACA1_384820 protein could be a putative N-acetyltransferase, a family of proteins involved in the transfer of the acetyl group from acetyl-CoA to a substrate.

Overexpression of the ACA1_384820 Gene Affects the Formation of Cysts
To better characterize the function of the ACA1_384820 gene, we examined whether its overexpression could affect encystment. A plasmid with the ACA1_384820 coding sequence (pTBPF-ACA1_384820) was constructed. In this plasmid, the ACA1_384820 coding sequence was under the promoter of the TATA-binding protein promoter binding factor (TPBF) for constitutive expression [21]. Indeed, the mRNA of TPBF is stable during encystment [22]. Plasmids expressing eGFP (TBPF-eGFP) or nothing (TBPF-empty) were used as controls. Plasmids were transfected into A. castellanii, followed by selective treatment with Geneticin (G418). Cell death was assessed using Trypan blue and propidium iodide. Twenty percent of transfected cells were positive for both Trypan blue and propidium iodide, while all non-transfected cells were dead (Figure 2A-B). To determine whether ACA1_384820 was overexpressed within the transfected cells, we analyzed its expression by RT-qPCR. In cells transfected with pTBPF-ACA1_384820, an increased ACA1_384820 mRNA level was observed ( Figure 2C). In contrast, cells transfected with pTBPF-empty or pTBPF-eGFP displayed a similar level compared to untransfected cells ( Figure 2C).

Overexpression of the ACA1_384820 Gene Affects the Formation of Cysts.
To better characterize the function of the ACA1_384820 gene, we examined whether its overexpression could affect encystment. A plasmid with the ACA1_384820 coding sequence (pTBPF-ACA1_384820) was constructed. In this plasmid, the ACA1_384820 coding sequence was under the promoter of the TATA-binding protein promoter binding factor (TPBF) for constitutive expression [21]. Indeed, the mRNA of TPBF is stable during encystment [22]. Plasmids expressing eGFP (TBPF-eGFP) or nothing (TBPF-empty) were used as controls. Plasmids were transfected into A. castellanii, followed by selective treatment with Geneticin (G418). Cell death was assessed using Trypan blue and propidium iodide. Twenty percent of transfected cells were positive for both Trypan blue and propidium iodide, while all non-transfected cells were dead (Figure 2A-B). To determine whether ACA1_384820 was overexpressed within the transfected cells, we analyzed its expression by RT-qPCR. In cells transfected with pTBPF-ACA1_384820, an increased ACA1_384820 mRNA level was observed ( Figure 2C). In contrast, cells transfected with pTBPF-empty or pTBPF-eGFP displayed a similar level compared to untransfected cells ( Figure 2C).
In order to assess whether the overexpression of the ACA1_384820 gene altered the growth of amoebae, transfected and control cells were counted every 24 h for 72 h. A. castellanii growth was not affected by overexpression of the ACA1_384820 gene compared to untransfected cells, or those which were transfected with the two control plasmids pTBPF-eGFP and pTBPF-empty ( Figure 2D). Altogether, these results show that overexpression of the ACA1_384820 coding sequence did not disturb the growth of A. castellanii.  Results are the average of three independent experiments, and error bars represent the standard error of the mean (± SEM). Statistical analysis was performed using the ordinary one-way ANOVA followed by Dunnett's post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) with comparison to the condition 'untransfected Ac'.
In order to assess whether the overexpression of the ACA1_384820 gene altered the growth of amoebae, transfected and control cells were counted every 24 h for 72 h. A. castellanii growth was not affected by overexpression of the ACA1_384820 gene compared to untransfected cells, or those which were transfected with the two control plasmids pTBPF-eGFP and pTBPF-empty ( Figure 2D). Altogether, these results show that overexpression of the ACA1_384820 coding sequence did not disturb the growth of A. castellanii.
Finally, we tested the influence of ACA1_384820 overexpression on encystment of A. castellanii. We incubated transfected amoebae within the encystment medium and evaluated the percentage of cysts using the Calcofluor White stain at different time points (24, 48 and 72 h). Twenty-four hours after the induction of encystment, we observed a decrease in the percentage of cysts. This effect was more pronounced at 48 and 72 h (Figure 3). In conclusion, ACA1_384820 overexpression affects formation of cysts.
Finally, we tested the influence of ACA1_384820 overexpression on encystment of A. castellanii. We incubated transfected amoebae within the encystment medium and evaluated the percentage of cysts using the Calcofluor White stain at different time points (24, 48 and 72 h). Twenty-four hours after the induction of encystment, we observed a decrease in the percentage of cysts. This effect was more pronounced at 48 and 72 h (Figure 3). In conclusion, ACA1_384820 overexpression affects formation of cysts. Results are averages of three independent experiments, and error bars represent the standard error of the mean (± SEM). Statistical analysis was performed using ordinary one-way ANOVA followed by Dunnett's post hoc test (**p < 0.01) in comparison to the condition 'untransfected Ac'.

Discussion
The goal of this study was to find new proteins down-regulated during encystment of Acanthamoeba. The ACA1_384820 gene encodes for a putative acetyltransferase, according to the domain prediction showing the presence of a GNAT domain. GNATs are a very large family of enzymes, with more than 10,000 members, identified in all kingdoms of life [17]. GNATs catalyze the transfer of an acetyl group from acetyl-CoA to the primary amine of a large range of substrates, ranging from small molecules to macromolecules. More globally, among post-translational modifications (PTM), acetylation is able to modulate the function of target molecules implicated in numerous cellular processes, ranging from antibiotic resistance to gene regulation by histone acetylation for example [18]. Some N-acetyltransferases have been described as being involved in encystment of other eukaryotes. In Giardia intestinalis, the cyst-specific carbohydrate component (67%) of the cyst wall is a unique homopolymer composed of N-acetylgalactosamine (GalNAc). Its precursor, UDP-GalNAc is synthesized by a pathway of five inducible enzymes, including one glucosamine 6-phosphate N-acetyltransferase (EC 2.3.1.4, GNA) [23,24]. In Toxoplasma gondii, it has been shown that the knock-out of TgGCN5-A, a lysine acetyltransferase with a histone acetyltransferase activity, prevented up-regulation of 74% of stress response genes that are normally induced during alkaline stress-mediated encystment. Complementation of the TgGCN5-A knock-out restored this expression and the capacity to resist alkaline stress, underlining the role of a Nacetyltransferase in encystment [25]. These two examples demonstrate the involvement of GNAT members in encystment.
We found that expression of the putative Acanthamoeba GNATs was down-regulated during encystment. This decrease was transient or persisted depending on GNATs, suggesting an action at different times with different targets and outcomes. In the case of the ACA1_384820 gene, overexpression impaired formation of mature cysts. This protein might modulate molecules that

Discussion
The goal of this study was to find new proteins down-regulated during encystment of Acanthamoeba. The ACA1_384820 gene encodes for a putative acetyltransferase, according to the domain prediction showing the presence of a GNAT domain. GNATs are a very large family of enzymes, with more than 10,000 members, identified in all kingdoms of life [17]. GNATs catalyze the transfer of an acetyl group from acetyl-CoA to the primary amine of a large range of substrates, ranging from small molecules to macromolecules. More globally, among post-translational modifications (PTM), acetylation is able to modulate the function of target molecules implicated in numerous cellular processes, ranging from antibiotic resistance to gene regulation by histone acetylation for example [18]. Some N-acetyltransferases have been described as being involved in encystment of other eukaryotes. In Giardia intestinalis, the cyst-specific carbohydrate component (67%) of the cyst wall is a unique homopolymer composed of N-acetylgalactosamine (GalNAc). Its precursor, UDP-GalNAc is synthesized by a pathway of five inducible enzymes, including one glucosamine 6-phosphate N-acetyltransferase (EC 2.3.1.4, GNA) [23,24]. In Toxoplasma gondii, it has been shown that the knock-out of TgGCN5-A, a lysine acetyltransferase with a histone acetyltransferase activity, prevented up-regulation of 74% of stress response genes that are normally induced during alkaline stress-mediated encystment. Complementation of the TgGCN5-A knock-out restored this expression and the capacity to resist alkaline stress, underlining the role of a N-acetyltransferase in encystment [25]. These two examples demonstrate the involvement of GNAT members in encystment.
We found that expression of the putative Acanthamoeba GNATs was down-regulated during encystment. This decrease was transient or persisted depending on GNATs, suggesting an action at different times with different targets and outcomes. In the case of the ACA1_384820 gene, overexpression impaired formation of mature cysts. This protein might modulate molecules that negatively affect encystment in A. castellanii. Ongoing work consists of determining the molecular targets and activities Pathogens 2020, 9, 321 7 of 11 of the different GNATs. Further studies are needed to analyze the precise role of each acetyltransferases and how they are coordinated during encystment. Indeed, if several N-acetyltransferases are needed to prevent encystment, this could explain why overexpression of the ACA1_384820 gene does not completely block formation of cysts.
The analysis of amoeba genomes showed that the ACA1_384820 gene is conserved in some species of Acanthamoeba, but absent in Dictyostelium spp., Entamoeba spp. and Naegleria spp. The search for the protein sequence within other organisms using the BLAST tool have shown some identities with proteins mainly found in prokaryotes and belonging to the phyla of Chlorobacteria, Cyanobacteria and Firmicutes. In the environment, amoebae are in contact with numerous bacteria on which they graze [26]. Due to putative gene exchanges between amoebae and intracellular bacteria, it was not surprising to find an Acanthamoeba gene of prokaryotic origin. The study of the Acanthamoeba genome shows the presence of more than 450 genes, which corresponds to 2.9% of the genome, predicted to have arisen through lateral gene transfer [16]. Among these genes, we have ACA1_384820 and at least five genes that have been annotated as 'acetyltransferase, GNAT superfamily protein'. All these genes are down-regulated during encystment. These data suggest that amoebae could have acquired bacterial genes that are involved in encystment but further analysis is required to confirm the hypothesis.
In conclusion, we describe a new ACA1_384820 gene of which the expression is down-regulated during encystment of A. castellanii. Overexpression of ACA1_384820 affects formation of cysts. This protein encodes a putative N-acetyltransferase-like protein possibly acquired by lateral gene transfer from prokaryotes. Further studies are needed to determine the activity of this protein and its specific role in encystment of A. castellanii.

Encystment Assay
A. castellanii transfected and non-transfected trophozoites were seeded onto 24-well plates at a density of 5 × 10 4 cells per well in PAS buffer and incubated at 30 • C for 1 h for cell adhesion. , a dye that binds to cellulose, was incubated with live A. castellanii on a glass slide for 2 min at room temperature [27]. The cysts were observed by fluorescence microscopy (Olympus IX51). More than 800 cells were counted per condition and per experiment. This experiment was done in three independent replicates. supplemented with 20 µg/mL of G418 (Sigma) for two weeks. The concentration of G418 was then increased to 50 µg/mL to select the transfected population.
Five days after the increase of G418 concentration, cell viability was tested using Trypan blue and propidium iodide. For the Trypan blue experiments, cells were diluted in 2× Trypan blue (final concentration of 0.2%) and counted in triplicate for each condition using plastic counting slides FastRead 102 ® (Biosigma). For the propidium iodide experiments, cells were incubated with propidium iodide (10 µg/mL) and analyzed by flow cytometry (Cytoflex, Beckman Coulter). This experiment was done in three independent replicates.

Reverse Transcription-Quantitative PCR (RT-qPCR)
Total RNAs was extracted using the RNeasy Mini Kit (Qiagen). For samples incubated in encysting medium for at least 24 h, RNA extraction was preceded by physical lysis by bead-beating in tubes containing 500 mg of small diameter glass beads (100 µm) (Sigma) using Fastprep apparatus for 30 s (speed 5 m/s). The RNA samples were treated with RNase-free DNase I (TURBO DNA-free™ kit, Invitrogen) and reverse transcribed with the GoScript™ Reverse Transcriptase kit (Promega) according to the manufacturer's recommendations. The reverse transcription products were used to carry out real-time quantitative PCR. All primer sequences are shown in Table 2. Reverse Transcription-Quantitative PCR (RT-qPCR) was performed using the LightCycler ® FastStart DNA Master plus SYBR Green I (Roche Applied Science). Reactions were prepared in a total volume of 10 µL containing 5 µL of 2× SYBR mix, 2 µL of H 2 O, 2 µL of diluted cDNA template and 0.5 µL of 10 µM primers.
The reactions were performed under the following conditions: an initial denaturation step of 95 • C for 5 min, followed by a three-step thermal cycling profile comprising denaturation at 95 • C for 10 s, primer annealing at 60°C for 10 s and extension at 72°C for 10 s. This procedure was conducted for 45 cycles. To verify the specificity of the amplicon for each primer pair, a melting curve analysis was performed ranging from 65 to 95°C.

Bioinformatics Analysis of the ACA1_384820 Gene
Predictions of potential domains present on the protein were performed using online tool InterPro [30,31]. The search for putative homologous genes in other organisms were performed using the online bioinformatics tool BLASTp [32]. To generate alignment ( Supplementary Data 1 and 2), the sequences were analyzed by Multalin [33,34]. For amino acid sequences, the nucleotide sequences were translated using ExPasy tool [35].

Statistical Analysis
All results are averages of three independent experiments, and error bars represent the standard error of the mean (± SEM). Statistical analysis was performed using the ordinary one-way ANOVA followed by Dunnett's post hoc test (GraphPad Prism 6). For RT-qPCR experiments, statistical analyses were performed on ∆Ct values. Differences were considered statistically significant when p values were < 0.05 (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Funding: This work was supported by the Agence Nationale de la Recherche (ANR-17-CE13-00001-01 "Amocyst") and partly funded by the European Union and the region of Nouvelle Aquitaine through the "Habisan program" (CPER-FEDER).