*3.2. Resin-Assisted Capture (RAC) of Oxidized Proteins (OX) Coupled to Mass Spectrometry (OX–RAC) Analysis of E. histolytica Trophozoites Exposed to L. acidophilus*

In order to explore the amebicidal properties of *L. acidophilus*, we used OX–RAC to measure the levels of oxidized proteins (OXs) in *E. histolytica* trophozoites exposed to *L. acidophilus*. In absence of DTT treatment, OXs are not expected to bind to the thiopropyl resin [38]. We observed that the level of OXs in *E. histolytica* trophozoites exposed to heatkilled *L. acidophilus* culture is very low (Figure 2A). These results indicate that heat-killed culture of *L. acidophilus* do not trigger the formation of OXs in *E. histolytica* trophozoites. In contrast, a strong level of OXs was detected in *E. histolytica* trophozoites exposed to live *L. acidophilus* culture (Figure 2A). The addition of catalase during the interaction of *E. histolytica* trophozoites with *L. acidophilus* strongly inhibits the formation of OXs in the parasite, which confirms that the formation of OXs in the parasite is mediated by H2O2 produced by *L. acidophilus* (Figure 2B). These results indicate that the formation of OXs is triggered by H2O2 produced by *L. acidophilus*.

**Figure 2.** Detection of OXs by resin-assisted capture (OX–RAC) analysis of *E. histolytica.* Note: *E. histolytica* trophozoites were incubated with live *L. acidophilus* (L.a) or with heat-killed *L. acidophilus* (DN L.a) (**A**), with/without catalase (Cat.) (50 μg/mL) (**B**) for 2 h at 37 ◦C. Total protein lysate was prepared by lysing the trophozoites with 1% Igepal in PBS. The oxidized proteins in the cell lysates were subjected to RAC in the presence of 10 mM DTT (+DTT) or the absence of DTT (−DTT). The protein was resolved on a 12% SDS-PAGE and stained with silver stain.

The intensity of the protein bands were quantified by densitometry using Image J software [39]. The intensity of the OX-protein bands obtained in the presence of DTT in *E. histolytica* trophozoites incubated with live *L. acidophilus* was arbitrarily set to 1. It is important to note that the data presented in Figure 2A,B were obtained at two different times, and that the silver staining development time was different in each case.

Using MS, we identified 997 OXs in *E. histolytica* trophozoites incubated with *L. acidophilus* (Table S1), which were classified using PANTHER. The most abundant OX families belong to metabolite interconversion enzyme (PC00262), such as protein arginine N-methyltransferase (EHI\_158560), the galactose-specific adhesin 170kD subunit (EHI\_042370), and thioredoxin (EHI\_004490) (Figure 3A). *E. histolytica* lacks glutathione, so it relies mainly on thiol for its defense against OS [40]. Thioredoxin (TRX)/thioredoxin reductase (TRXR) also contributes to redox signaling in *E. histolytica* trophozoites as well as oxidative stress responses [41]. This ubiquitous mechanism of defense is present in many parasites, including *Schistosoma mansoni*, *Plasmodium falciparum*, *Giardia lamblia*, and *Trichomonas vaginalis* [41]. TRXs are small redox proteins of around 12 kD, which act as radical scavengers. In their active site, two cysteine residues are involved in the antioxidant system. The oxidation of these cysteine residues produces disulfide bonds, which will be reduced by TRXR. The presence of TRXs as OXs in *E. histolytica* exposed to *L. acidophilus* strongly suggests that the parasite is actively responding to H2O2 released by the bacteria.

The other abundant OX family belongs to the protein modifying enzyme (PC00260) such as cysteine proteinase CP5 (EHI\_168240), serine/threonine-protein phosphatase (EHI\_031240), or E3 ubiquitin-protein ligase (EHI\_050540) and the protein-binding activity modulator (PC00095) such as AIG1 family protein (EHI\_176700), inhibitors of serine proteinase domain-containing protein (SERPIN) (EHI\_119330), and the Rho family GTPase (EHI\_070730) (Figure 3A).

SERPINs control a broad range of biological processes, including pathogen evasion of the host defense system. Cathepsin G, a pro-inflammatory enzyme released by activated neutrophils, is inhibited by serpins [42]. *E. histolytica* expresses a SERPIN that interacts with human neutrophil cathepsin G [43]. In this work, we showed that EhSERPIN is one of the OXs present in *E. histolytica* exposed to *L. acidophilus*. Studies have suggested that SERPINs are redox-regulated by oxidation of cysteine residues in the reactive site loop of these enzymes or its vicinity [44–46]. The presence of carbamidomethylated cysteine residues in the vicinity of the reactive site loop of EhSERPIN (Table S2) [43] suggests that EhSERPIN is also redox-regulated. The effect of oxidation on EhSERPIN activity has yet to be determined.

A functional motility is critical to the survival of *E. histolytica* in order to both dislodge and phagocytose host cells as well as transport virulence factors intracellularly [47]. Rho GTPases play a critical role in the regulation of motility and phagocytic activity of *E. histolytica* [48]. There are several Rho GTPases present in the parasite, and we identified six of them (EHI\_126310, EHI\_013260, EHI\_197840, EHI\_029020, EHI\_129750, and EHI\_070730) as OXs. EhRho1 (EHI\_029020) regulates phagocytosis by regulating actin polymerization [49]. Numerous studies have shown that ROS regulate Rho GTPases activity [50]. Many Rho family GTPases contain a cysteine-containing motif (GXXXXGK[S/T]C) at their N-terminal, which is located directly adjacent to the phosphoryl-binding loop. Oxidation of the cysteine residue in this motif affects the nucleotide binding properties of these Rho GTPases [50]. According to the MS analysis of OXs (Table S2), this cysteine residue in the active site is not carbamidomethylated. Instead, we found that cysteine residues located at the C-terminal of these Rho GTPases are carbamidomethylated (Table S2). An ubiquitination region is present in the C-terminal region of many Rho GTPases that may regulate their stability [51]. In light of this information, it is tempting to speculate that the stability of these Rho GTPases is redox-dependent. An example of such regulation occurring in human endothelial cells is described here [52].

**Figure 3.** Protein analysis through evolutionary relationships (PANTHER) analysis of OXs in *E. histolytica* incubated with *L. acidophilus.* Note: (**A**) PANTHER sequence classification of the OXs identified in *E. histolytica* trophozoites co-incubated with *L. acidophilus*. (**B**) PANTHER statistical overrepresentation test of the OXs identified in *E. histolytica* trophozoites incubated with *L. acidophilus*.

Of the OXs in *E. histolytica* trophozoites incubated with *L. acidophilus* (Table S2), oxidoreductase (PC00176) and dehydrogenase (PC00092), such as glyceraldehyde-3-phosphate dehydrogenase (EHI\_008200), NAD(FAD)-dependent dehydrogenase (EHI\_099700), and pyruvate: ferredoxin oxidoreductase (EHI\_051060), are significantly enriched according to the PANTHER statistical overrepresentation test (Figure 3B). Pyruvate: ferredoxin oxidoreductase (EHI\_051060) is a Fe–S enzyme that catalyzes the oxidative decarboxylation of pyruvate [53]. This protein has also been identified as an OX in trophozoites exposed to

H2O2 [6], metronidazole, or auranofin [54]. In an oxidatively stressed parasite, pyruvate: ferredoxin oxidoreductase becomes strongly inhibited, resulting in an accumulation of pyruvate, which limits ATP production and causes parasite death [55]. Several cysteine residues present within the [4Fe–4S] clusters of close to them are carbamidomethylated suggesting that they are oxidized (Table S2). Destabilization of the Fe–S clusters integrity via oxidation of these cysteine residues in the parasite exposed to *L. acidophilus* will more certainly inactivate the enzyme and consequently contribute to the parasite death.

Other OXs, which are significantly enriched according to the PANTHER statistical overrepresentation test, include vesicle coat protein (PC00235), such as GOLD domaincontaining protein (EHI\_023070), beta2-COP (EHI\_088220) and coatomer subunit gamma (EHI\_040700) and protease (PC00190), such as EhCP-a1 (EHI\_074180) and EhCP-a4 (EHI\_ 050570) (Figure 3B).
