Abstract:
Acanthamoeba is an opportunistic amphizoic protozoan which causes severe brain and eye infections. Virulence potential of Acanthamoeba is not constant and can change with growth conditions. DNA methylation, an epigenetic mechanism, is used by eukaryotes to control their gene expression. DNA-methyltransferase gene family (dnmt) was studied to determine the possibility of its involvement in programming Acanthamoeba pathogenicity. A virulence-attenuated Acanthamoeba isolate (designation: ATCC) was subjected to mouse passages to restore its pathogenicity; a virulence-reactivated isolate (designation: AC/5) was generated. Several established factors associated with Acanthamoeba virulence phenotype were examined to confirm the succession of reactivation process. Differential gene expression of dnmt between the ATCC and AC/5 isolates was performed by quantitative reverse transcription PCR (RT-qPCR). Silencing of dnmt in the AC/5 isolate was achieved by small interfering RNA (siRNA). Total RNAs extracted from the ATCC, AC/5 and siRNA-treated (designation: si-146) Acanthamoeba were subjected to RNA sequencing for transcriptomic studies. Pathogenicity assays demonstrated that the AC/5 isolate exhibited greater virulence potential after mouse passages. RT-qPCR revealed that the AC/5 isolate had an almost 15-fold increase in dnmt expression compared to the ATCC isolate (p ≤ 0.001). si-146 duplex significantly reduced the expression of dnmt in the AC/5 isolate by 30%. Comparative transcriptomic analysis identified differentially expressed genes (DEGs), with 1,717 genes in AC/5 versus ATCC; 778 genes in si-146 versus AC/5; and 1,513 genes in si-146 versus ATCC, respectively (log2 fold change of ≥ 2 or ≤ -2 and adjusted p-value of < 0.05). Functional analysis of DEGs in AC/5 versus ATCC and si-146 versus AC/5 using eukaryotic orthologous groups, gene ontology and Kyoto encyclopaedia of genes and genomes pathway revealed that a high percentage of DEGs was related to post-translational modifications, signal transduction, oxidation-reduction and metabolic processes. Of these, a wide range of genes associated with encystation, proteolysis, host-cell adhesion, oxidative stress defence, among others, was identified and could be transcriptionally regulated by dnmt. Decreased in the expression levels of these genes may account in part for Acanthamoeba reduced pathogenicity. These candidate genes could represent potential drug targets for therapeutic interventions. To the best of the author’s knowledge, this is the first genome-wide analysis of DNA methylation and its effects on gene expression in Acanthamoeba sp. The present data not only provided fundamental understanding of the mechanisms underlying Acanthamoeba virulence, but also identified potential targets for anti-acanthamoebic drugs.
