*3.3. E. histolytica CP Activity Is Impaired by L. acidophilus*

In order to gain information on the consequence of *L. acidophilus*-mediated-oxidation on the activity of proteins that were identified in the OX–RAC analysis, we decided to focus here on the CPs. When trophozoites are incubated with live *L. acidophilus*, CPs activity is strongly inhibited (Figure 4). However, this activity is not inhibited when trophozoites are incubated with *L. acidophilus* in the presence of catalase (Figure 4). The addition of DTT in lysates of trophozoites incubated with live *L. acidophilus* partially restored CP activity. Based on these results, it could be assumed that the *L. acidophilus*-mediated-oxidation of CPs' catalytic cysteine residues inhibits CPs, while their reduction by DTT restores the activity. Indeed, the fact that adding catalase to trophozoites incubated with *L. acidophilus* prevents the inhibition of CPs confirms that H2O2 produced by *L. acidophilus* inhibits the CPs.

**Figure 4.** CPs activity of *E. histolytica* trophozoites. Note: *E. histolytica* trophozoites were incubated with heat-killed *L. acidophilus* (DN) or with live *L. acidophilus* (LA), and with/without catalase (50 μg/mL) for 2 h at 37 ◦C. Total protein was prepared and CPs activity was measured. One unit of CP activity was defined as the number of micromoles of substrate digested per minute per milligram of protein. CP activity performed without DTT of *E. histolytica* trophozoites incubated with heat-killed *L. acidophilus* (WT + DN) was obtained as 100% and it corresponds to 0.31 units. The data represent two independent experiments performed in triplicate. \* *p*-value < 0.05 by an unpaired Student's *t*-test. \*\*\* *p*-value < 0.001 by an unpaired Student's *t*-test.

CPs are essential for the growth of *E. histolytica* trophozoites and their inhibition by inhibitors of the CPS, such as E64d, causes their death [56]. In this study, we found that many CPs, including EhCP-a1 (EHI\_074180), EhCP-a4 (EHI\_050570), EhCP-a5 (EHI\_168240), and EhCP8 (EHI\_010850), are oxidized, and that *E. histolytica* CPs activity are inhibited when the parasite is incubated with *L. acidophilus*. Some of these OXs CPs, such as EhCP-A1 and EhCP-A5, are highly expressed in *E. histolytica* [57] and are involved in rosette formation, hemolysis, and erythrocyte digestion [58]. The expression of EHI\_010850 (EhCP-8) is upregulated when the parasite is incubated in the presence of hemoglobin, which suggests CP-8 is involved in iron uptake by the parasite [59]. The mechanisms that lead to oxidants inhibiting CPs have recently been examined [60]. For example, inhibition of papain by H2O2 results from the formation of sulfenic acid, which reacts with adjacent free thiol to form mixed disulfides. In addition, H2O2 inhibits cathepsin B by targeting the active site residue (Cys25) to form either sulfenic acid or sulfonic acid around 70% of the time. *E. histolytica* CPs contain four active-site residues, namely Gln, Cys, His, and Asn, the cysteine residue at the active site being present in all *E. histolytica* CPs [61]. According to the MS analysis of OXs (Table S2), this cysteine residue in the active site is carbamidomethylated, which strongly suggests that it was oxidized. By itself, this observation would explain why *E. histolytica*'s CP activity is inhibited by H2O2 produced by *L. acidophilus*. As opposed to *E. histolytica*, where H2O2 produced by *L. acidophilus* appears to inhibit CPs activity directly, in *Plasmodium* parasites, H2O2-mediated inhibition of CPs is dependent on the presence of free hemin, which can be released by quinoline drugs [62].
