*3.2. Reponse of AFAT to OS, NS, and Cytopathic Activity*

The response of AFAT to OS was tested by exposing them to H2O2 (2.5 mM for 30 min) or to paraquat (2.5 mM for 12 h). We observed that AFAT are significantly more sensitive to H2O2 or to paraquat than WT trophozoites (Figure 1A). We also examined the resistance of AFAT to MNZ (5 μM for 24 h) and found that AFAT are significantly more sensitive to MNZ than WT trophozoites (Figure 1A). The sensitivity of AFAT to nitrosative stress (NS) was tested by exposing them to the NO donor S-nitrosoglutathion (GSNO) (350 μM for 2 h). We observed that AFAT are significantly more sensitive to NS than WT trophozoites (Figure 1A). The ability of AFAT to destroy a monolayer of mammalian cells (cytopathic activity) was also determined (Figure 1B). We observed that the cytopathic activity of AFAT

is impaired compared to that of WT trophozoites. Overall, these results indicate that, for *E. histolytica* trophozoites, adaptation to AF results in a loss of fitness.

**Figure 1.** (**A**) Viability of AFAT exposed to H2O2, paraquat, MNZ, or GSNO. WT and AFAT were exposed to 2.5 mM H2O2 for 30 min, 2.5 mM paraquat (PRQ) and 5 μM metronidazole (MNZ) for 24 h, or 350 μM GSNO for 2 h. All experiments were undertaken at 37 ◦C. Data are expressed as the mean ± standard deviation of three independent experiments that were performed in triplicate. The graph represents the ratio percentage of viable amoebas compared to WT. The viability of AFAT exposed to H2O2, PRQ, MNZ, or GSNO was significantly different (\* *p* < 0.05) from that of the WT according to the results of an unpaired Student's *t* test. (**B**) Cytopathic activity of AFAT. Data are displayed as the mean ± standard deviation of four independent experiments that were performed in triplicate. The cytopathic activity of AFAT was significantly different (\* *p* < 0.05) from that of the WT according to the results of an unpaired Student's *t* test.
