**1. Introduction**

The protozoan parasite *Entamoeba histolytica* is the etiologic agent of amoebiasis, a significant hazard in countries with low socioeconomic status and poor sanitation. This disease accounted for 55,500 deaths and 2.237 million disability-adjusted life years in 2010 [1]. The main symptoms of amoebiasis are inflammation of the large intestine and liver abscesses. Infection occurs following the ingestion of food contaminated with cysts. Trophozoites that emerge from the cysts migrate to the large intestine. Asymptomatic colonization occurs in most cases (90% of all infections). Symptomatic infection is characterized by bloody diarrhea. Metronidazole (MNZ) is the drug currently used for invasive amoebiasis [2]. Inside the parasite, MNZ is reduced through the action of thioredoxin reductase (TrxR) to a nitro radical anion or to a nitroimidazole. This nitro group reduces O2, leading to the formation of cytotoxic reactive oxygen species (ROS) inside the parasite. The nitroimidazole can also modify cysteine containing proteins such as thioredoxin (Trx), leading to their inactivation [3]. There are numerous common side effects related to MNZ, including dizziness, heartburn, stomach cramps, trouble sleeping, and weight loss [4–6]. Treatment with MNZ is usually highly effective, but resistance to this drug has been reported in various bacteria [7,8] and protozoan parasites [9–11]. To address these drawbacks, new alternatives to MNZ have been initiated and AF has emerged as one of the most potent anti-protozoan parasites drugs. Initially, AF was a gold-containing compound developed in the 1970s for the treatment of rheumatoid arthritis [12]. Its mechanism of action as an antiarthritic gold drug remained controversial but it is assumed that it

**Citation:** Shaulov, Y.; Sarid, L.; Trebicz-Geffen, M.; Ankri, S. *Entamoeba histolytica* Adaption to Auranofin: A Phenotypic and Multi-Omics Characterization. *Antioxidants* **2021**, *10*, 1240. https:// doi.org/10.3390/antiox10081240

Academic Editor: Simone Carradori

Received: 21 June 2021 Accepted: 30 July 2021 Published: 2 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

works by inhibiting the activity of TrxR, a crucial enzyme involved in the maintenance of the redox homeostasis in the cell [13]. AF is also a potent anticancer agent [14] and has been found to be very efficient against a number of pathogens, including *Mycobacterium abscessus* [15], *Clostridium difficile* [16,17], vancomycin-resistant enterococci [18,19], and some additional multidrug resistant bacteria [20]. Auranofin is also very efficient against parasites, including the trematode *Schistosoma mansoni* [21,22], and protozoan parasites, including *Trichomonas vaginalis* [23], *Giardia lamblia* [24], and *E. histolytica* [25]. The mode of action of AF in protozoan parasites is not completely understood although it is assumed that TrxR is the main target of AF in *E. histolytica* [24,25]. In *G. lamblia*, this mechanism of action has been challenged by the significant TrxR activity that occurs in trophozoites exposed to high concentrations of auranofin [26]. Overexpression of TrxR in *G. lamblia* has no effect on the sensitivity of this parasite to AF [26]. AF can also target *E. histolytica* adenosine 5- -phosphosulfate kinase (EhAPSK), an essential enzyme in Entamoeba sulfolipid metabolism [27]. We recently showed that AF induced the formation of more than 500 oxidized proteins (OXs) in *E. histolytica*, including some crucial enzymes for redox homeostasis and cytoskeletal proteins, which are essential for *E. histolytica*'s cytoskeleton dependent virulence [28]. Knowledge about resistance to AF in bacteria and in protozoa is scarce. Recently, toxoplasma trophozoites resistant to AF (2 μM) were successfully generated through chemical mutagenesis. The authors identified point mutations in genes encoding redox-relevant proteins, such as superoxide dismutase and ribonucleotide reductase. However, recapitulation of these mutations in the parasite did not confer resistance to AF, suggesting that the mechanism of resistance is complex [29]. In this work, we used a multi-omics approach to characterize an *E. histolytica* strain that was made resistant to AF (AFAT) by progressively adapting the parasite to 2 μM of AF. At this concentration, the drug is lethal to non-adapted parasites [25,28].
