**5. Conclusions**

We showed that *E. histolytica* trophozoites can be easily selected to resist toxic concentrations of AF in vitro. Adaptation to AF reduces the fitness of *E. histolytica*, as seen in a decreased growth rate and virulence, and a sensitivity to OS, NS, and MNZ. Overexpression of genes whose products are sensitive to AF-mediated oxidation may represent an important step in the adaptation process to AF, and EhTrxR does not appear to be central to this process.

AF is FDA approved for the treatment of rheumatoid arthritis but has not been yet used as an antimicrobial drug in the field. The ability of *E. histolytica* to adapt to amebicidal concentrations of AF raises concerns about the future use of this drug as an antiamebic compound. Our omics data provide the basis for the development of strategies to limit the emergence of resistance against AF. One possible strategy suggested by our data is to promote dual antibiotic therapy (AF + MNZ) vs. single AF therapy, because adaptation to AF leads to more MNZ sensitivity in *E. histolytica*.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/antiox10081240/s1. Table S1: transcriptomics of WT trophozoites vs. AFAT. Table S2: legend of Table S1. Table S3: comparative analysis of OXs in AFAT vs. OXs in acute AF; OXs in AFAT vs. gene products upregulated in AFAT and OXs in acute AF vs. gene products upregulated or downregulated in AFAT.

**Author Contributions:** Conceptualization: Y.S., S.A.; methodology, Y.S., S.A.; software, Y.S., S.A.; validation, Y.S., L.S., M.T.-G., S.A.; formal analysis, Y.S., L.S., M.T.-G., S.A.; investigation, Y.S., L.S., M.T.-G., S.A.; resources, S.A.; data curation, Y.S., S.A.; writing—original draft preparation, Y.S., S.A.; writing—review and editing, Y.S., L.S., M.T.-G., S.A.; visualization, S.A.; supervision, S.A.; project administration, S.A.; funding acquisition, S.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Israel Science Foundation (260/16), the ISF-NRF program (3208/19), the US–Israel Binational Science Foundation (2015211), and the Niedersachsen program.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** RNA-Seq data have been deposited at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo accessed on 28 July 2021) under the accession number GSE178520.

**Acknowledgments:** We thank the staff of the Microscopy Imaging facility, the genomics Core Facility Laboratory, and the Smoler Proteomics Center at the Technion for their technical help.

**Conflicts of Interest:** The authors declare no conflict of interest.
