: In drug discovery, target identification and elucidation of resistance mechanisms are essential. The pathogen strains resistant to a compound of interest are useful for these purposes. As the generation of drug-resistant strains is time-consuming and often burdensome, we generated the Entamoeba histolytica strain with a high accumulation rate of genetic mutations by introducing proofreading-deficient, error-prone DNA polymerase δ mutant gene under the regulation of a tetracycline-regulatable promoter. We validated this "mutator" strain by showing higher genetic mutations accumulated during in vitro passage and, as a proof of concept, by identifying genes and their mutations responsible for resistance against miltefosine. Whole-genome analyses of the mutator strain after 12, 33, and 66 weeks of cultivation in the presence of tetracycline revealed that mutations accumulated in a time-dependent fashion, and the mutation rate of the mutator strain was approximately 60-fold higher than the mock control strain. The highly miltefosine-resistant irreversible clones were isolated from mutator-66 weeks but not from mutator-12 weeks. Whole-genome sequencing analysis of the three miltefosine highly-resistant clones identified shared mutations in three candidate genes potentially responsible for miltefosine resistance. Among them, a mutation in P4-ATPase (EHI_096620N417K) was worth noting, as mutations in this gene have previously been implicated in miltefosine resistance in Leishmania and Saccharomyces. We further demonstrated that exogenous expression of EHI_096620N417K (P4-ATPase) and EHI_035500N182I (kinase) confers miltefosine resistance. The E. histolytica mutator is a powerful tool for elucidating resistance mechanisms and potentially the modes of action of existing and future drugs against amebiasis.

Objective: The protozoan parasite E. histolytica causes invasive amebiasis that is endemic in developing countries and is characterized by dysentery and liver abscesses. Metronidazole is the first-line therapeutic drug that has been used for a decade, although several adverse effects were well-documented and the risk of resistance was experimentally demonstrated. The development of alternative drugs with different modes of action is a prerequisite for future amebiasis control. To this end, elucidation of the mechanism of action and resistance of potential new antiamebic compounds is important but often challenging. To assist the process, we developed "mutator" with a high genetic mutation rate by exploitation of low-fidelity error-prone DNA polymerase δ. This genome-wide random mutagenesis system demonstrated in this study has many potentials, including rapid identification of mutations associated with resistance against new therapeutic candidates.