**4. Discussion**

In our previous work, we demonstrated that AF triggers OS inside *E. histolytica* trophozoites, resulting in the oxidation of more than 500 proteins, including many redox enzymes that are essential for controlling the intracellular levels of ROS in the parasite [28,42,43]. Here, we characterized *E. histolytica* trophozoites that were adapted to 2 μM AF. Adaptation of *E. histolytica* to AF leads to the upregulation and downregulation of hundreds of genes, which

suggests that the mechanism of adaptation is complex. Drug resistance is often mediated by a drug's molecular target gene overexpression [44,45]. Consequently, we expected that *E. histolytica* TrxR (EhTrxR), the assumed main target of AF [25], would be one of the upregulated genes in AFAT. However, transcriptomics of AFAT indicates that this was not the case. Indeed, the overexpression of EhTrxR did not confer to *E. histolytica* resistance to AF. This information raises a question about why EhTrxR expression is not upregulated as a simple mechanism to resist AF. One possible answer is that, as for *Giardia lamblia*, TrxR is not the primary target of AF in *E. histolytica* [26]. This is supported by the absence of the detection of EhTrxR among OXs in AFAT (this work) and acute AF trophozoites [28].

It is also possible that the fitness cost for *E. histolytica* to overexpress TrxR during adaptation to AF resistance is too high. EhTrxR can generate H2O2 from molecular oxygen, leading to the formation of reactive species [46]. Therefore, it is possible that the production of H2O2 resulting from EhTrxR overexpression combined with OS triggered by AF [28] during the adaptation process cannot be tolerated by the parasite.

In this work, we found that only two genes upregulated in AFAT have their products oxidized in AFAT. In contrast, 77 genes upregulated in AFAT have their product oxidized in acute AF trophozoites [28]. The upregulation of these 77 genes in AFAT may be essential for the adaption of the parasite to AF by replacing their oxidized-inactivated products by reduced-activated proteins. The relevance of this mechanism for some of these 77 genes is discussed in the following.

Pyruvate:ferredoxin oxidoreductase (EHI\_051060), NADP-dependent alcohol dehydrogenase (EHI\_107210), and Fe-ADH domain-containing protein (EHI\_198760), which encode for proteins involved in redox regulation: These redox enzymes depend on cysteine residues for their activity [47–49]. The oxidation of these cysteine residues impairs their activity [47,50].

Genes that encode the protein-binding activity modulator, such as Ras guanine nucleotide exchange factor (EHI\_035800), Rho guanine nucleotide exchange factor (EHI\_005910), or Ras GTPase-activating protein (EHI\_105250): These proteins have their product oxidized in acute AF trophozoites [28]. G proteins are involved in vesicular trafficking and cytoskeleton regulation [51]. Redox regulation of G-proteins have been well documented [52] and their oxidation impairs *E. histolytica*'s motility [28].

Genes that encode protein-modifying enzymes such as protein kinase domain-containing proteins (EHI\_186820) (EHI\_101280) and Protein kinase (EHI\_188110), which are also oxidized in acute AF trophozoites [28]: Protein kinases have been associated with the virulence and phagocytic activity of *E. histolytica* [53]. The redox regulation of protein kinases is well established [54], and it has been demonstrated that AF can directly inhibit protein kinase C by interacting with thiol groups present in the catalytic site [55].

Genes that encode actin or actin-binding cytoskeletal proteins are upregulated in AFAT and oxidized in acute AF trophozoites [28]: In our previous work, we showed that AF induces the oxidation of *E. histolytica* cytoskeletal proteins and consequently inhibits the formation of F-actin [28]. Consequently, it appears that the parasite upregulated the expression of actin-binding cytoskeletal proteins as a mechanism to adapt to AF by replacing oxidized cytoskeletal proteins that were formed during the process of adaptation to AF. The low level of F-actin in acute AF trophozoites and the normal level of F-actin in AFAT (this work) support this hypothesis.

The fact that *E. histolytica* can adapt to AF illustrates the remarkable ability of *E. histolytica* to adapt to drugs [56,57] and environmental stresses [32,58]. The fitness cost paid by the parasite to adapt to AF resembles collateral sensitivity, which occurs when the acquisition of resistance to one antibiotic produces increased susceptibility to a second antibiotic [59]. AFAT are more sensitive to OS, paraquat, MNZ, and GSNO than WT trophozoites. Resistance to OS in *E. histolytica* involves the upregulation of 29 kDa peroxiredoxin [60] and iron-containing peroxide dismutase expression, which is also involved in the resistance to MNZ [10,61]. The level of expression of 29 kDa peroxiredoxin and iron-containing peroxide dismutase is globally the same in WT and in AFAT, which sug-

gests that the sensitivity of AFAT to OS and MNZ is not caused by a reduced level of these redox enzymes' expressions. As discussed above, many oxidized proteins in AFAT have their level of expression upregulated. The fitness cost observed in AFAT may be due to numerous factors, including the rerouting of protein synthesis toward oxidized proteins, or substrate wasting that results from target overexpression [62]. In hydroxamic acid analog pan-histone deacetylase inhibitor-resistant leukemia cells, overexpression of the target protein heat shock protein 90 (HSP90) revealed collateral sensitivity to the HSP90 inhibitor 17-*N*-allylamino-17-demethoxygeldanamycin [63].
