Insights into the Mechanisms of Lactobacillus acidophilus Activity against Entamoeba histolytica by Using Thiol Redox Proteomics

Amebiasis is an intestinal disease transmitted by the protist parasite, Entamoeba histolytica. Lactobacillus acidophilus is a common inhabitant of healthy human gut and a probiotic that has antimicrobial properties against a number of pathogenic bacteria, fungi, and parasites. The aim of this study was to investigate the amebicide activity of L. acidophilus and its mechanisms. For this purpose, E. histolytica and L. acidophilus were co-incubated and the parasite’s viability was determined by eosin dye exclusion. The level of ozidized proteins (OXs) in the parasite was determined by resin-assisted capture RAC (OX–RAC). Incubation with L. acidophilus for two hours reduced the viability of E. histolytica trophozoites by 50%. As a result of the interaction with catalase, an enzyme that degrades hydrogen peroxide (H2O2) to water and oxygen, this amebicide activity is lost, indicating that it is mediated by H2O2 produced by L. acidophilus. Redox proteomics shows that L. acidophilus triggers the oxidation of many essential amebic enzymes such as pyruvate: ferredoxin oxidoreductase, the lectin Gal/GalNAc, and cysteine proteases (CPs). Further, trophozoites of E. histolytica incubated with L. acidophilus show reduced binding to mammalian cells. These results support L. acidophilus as a prophylactic candidate against amebiasis.


Introduction
Amebiasis is an enormous global medical problem because of poor sanitary conditions and unsafe hygiene practices existing in many parts of the world. According to the World Health Organization, 50 million people in India, Southeast Asia, Africa, and Latin America suffer from amebic dysentery and amebiasis causes the death of at least 100,000 individuals each year. The main mode of transmission for amebiasis is the ingestion of food or water that is contaminated with feces containing E. histolytica cysts. After the cyst form has been swallowed by the host, excystation occurs in the intestinal lumen, followed by colonization of the large intestine by the trophozoites where they continue to divide and encyst. Eventually, both trophozoites and cysts are excreted in stools. Only 10% of the infected individuals will develop acute intestinal and extra-intestinal diseases. One possible explanation for this observation is the difference in the gut microbiota between individuals who may significantly influence the host's immune response in amebiasis and E. histolytica's virulence [1]. Over the last few decades, it has become evident that E. histolytica's pathogenicity is directly linked to the parasite's interaction with the gut microbiota [2], as the parasites are reported to feed on bacteria and cellular debris found in the large intestine [1]. However, such feeding is very selective, where only those bacteria with the appropriate recognition molecules are ingested by the parasite [3]. Amebiasis is characterized by acute inflammation of the intestine with release of pro-inflammatory

Detection of Oxidized Proteins (OXs) by Resin-Assisted Capture RAC (OX-RAC)
The detection of OXs by OX-RAC was performed on three biological replicates using a previously described protocol [6]. Captured proteins were eluted with 30-µL elution buffer containing 10 mM HEPES, 0.1 mM EDTA, 0.01 mM neocuproine, 0.1% sodium dodecyl sulfate (SDS), and 100 mM 2-mercaptoethanol for 20 min at room temperature. Proteins in a 10-µL aliquot of each eluent were resolved on a 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) gel. Each gel was then stained with silver (Pierce Silver Stain), and each gel slice was independently analyzed by MS. A protein was considered to be oxidized when its relative amount in the DTT-treated lysates was significantly more than that in the DTT-untreated lysates (p < 0.05 according to the results of an unpaired t-test).

In-Gel Proteolysis and MS Analysis
The proteins in the gel were reduced with 2.8mM DTT (60 • C for 30 min), modified with 8.8 mM iodoacetamide in 100 mM ammonium bicarbonate (in the dark and at room temperature for 30 min) and digested in 10% acetonitrile and 10 mM ammonium bicarbonate with modified trypsin (Promega, Beit Haemek, Israel) overnight at 37 • C. A second trypsin digestion was carried out for another 4 h at 37 • C.
The peptides were resolved by reverse-phase chromatography on 0.075 × 180-mm fused silica capillaries (JW) packed with Reprosil reversed phase material (Dr Maisch GmbH, Ammerbuch, Germany). The peptides were eluted with linear 60 min gradient of 5 to 28% 15 min gradient of 28 to 95% and 25 min at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0.15 µL/min. MS was performed by Q Exactive HF mass spectrometer (Thermo Fisher Scientific represented by BARGAL analytical instruments, Shoham, Israel) in a positive mode using a repetitively full MS scan followed by collision induces dissociation (HCD) of the 18 most dominant ions selected from the first MS scan. The mass spectrometry data were analyzed using the MaxQuant software 1.5.2.8, The Max Plank Institute of Biochemistry, Munich, Germany [24] vs. Entamoeba histolytica and Lactobacillus acidophilus proteomes from the Uniprot database with 1% FDR (false discovery rate). The data were quantified by label free analysis using the same software. Statistical analysis of the identification and quantization results was done using Perseus 1.6.7.0 software, The Max Plank Institute of Biochemistry, Munich, Germany [25]. A t-test between the groups with or without DTT was carried out, with the Benjamini-Hochberg correction for multiple testing. Proteins were considered as significantly changed if their p-value < 0.05, q-value < 0.05, and the fold change between the groups ≥ 1.

Classification of OXs According to Their Protein Class and Statistical Overrepresentation Test
The OXs were classified according to PANTHER Protein Class using the PANTHER Classification System software (http://www.pantherdb.org/, accessed on 28 July 2021) [26]. This classification of proteins derived from PANTHER/X molecular function ontology includes commonly used classes of protein families, many of which are not covered by GO molecular function.
Regarding the statistical overrepresentation test, the online system compares a list of genes of interest (in this work, genes encoding for OXs in E. histolytica trophozoites exposed to L. acidophilus) to a reference list (E. histolytica in database). The p-value calculation in the overrepresentation test is calculated automatically based on the number of genes expected in the test list for a particular PANTHER category, based on the reference list.

Adhesion Assay
The adhesion of E. histolytica trophozoites to HeLa cells (a kind gift from T. Kleinberger, Faculty of Medicine, Technion) was measured using a previously described protocol [28]. E. histolytica trophozoites (2 × 10 5 ) were incubated with live L. acidophilus (2 × 10 8 ), with heat-killed L. acidophilus (DN) (2 × 10 8 ), with paraformaldehyde-fixed L. acidophilus (PLA) (2 × 10 8 ) and with/without catalase (50 µg/mL) for 1 h at 37 • C and then transferred to paraformaldehyde-fixed HeLa cells monolayers for an additional hour of incubation at 37 • C. Trophozoites unattached to HeLa cell monolayers were washed once with phosphate buffer saline (PBS) buffer and the trophozoites attached to the HeLa cell monolayer were eluted with 500 µL of a solution of cold galactose (1%) in PBS and counted.

Results and Discussion
3.1. L. acidophilus Amebicide Activity Depends on the Formation of H 2 O 2 L. acidophilus is commonly found in the gastrointestinal tract of healthy humans. It is widely used as a food preservative and as a probiotic. L. acidophilus antimicrobial activity is caused by the production of antimicrobial peptides, including lactacins B, organic acid production such as lactic acids and H 2 O 2 (recently reviewed in [29]), and immune induction [30]. Whereas the antibacterial and antifungal activity [31,32] of L. acidophilus has been well illustrated, the antiparasitic properties of L. acidophilus have been less studied. Studies with mouse models of the diseases caused by Giardia lamblia [33], Toxocara canis [34], Trichinella spiralis [35], and Cryptosporidium parvum [36] reveal that a combination of probiotics and other probiotic strains is beneficial in the treatment and prevention of these parasites. In a recent study, we demonstrated that L. acidophilus is detrimental to E. histolytica but the amebicide mechanism was unknown [19]. In this study, we investigated whether H 2 O 2 generated by L. acidophilus is directly responsible for the amebicide activity. We first measured the ability of L. acidophilus to produce H 2 O 2 by the FOX assay. We found that overnight culture of L. acidophilus cultivated in MRS media with agitation produces 0.14 ± 0.3 mM H 2 O 2 . A viability assay was performed on E. histolytica trophozoites incubated either with L. acidophilus or with heat-killed L. acidophilus, which served as negative control. The viability of E. histolytica trophozoites was not affected when the parasite was incubated with L. acidophilus for 60 min ( Figure 1). However, the viability of E. histolytica trophozoites was significantly decreased by 50% when the parasite was incubated with L. acidophilus for 120 min. In contrast, the viability of E. histolytica trophozoites incubated with heat-killed L. acidophilus for 120 min was not impaired ( Figure 1). Next, we wanted to establish if the amebicidal activity of L. acidophilus was dependent on the formation of H 2 O 2 . We incubated E. histolytica and L. acidophilus in presence of catalase, an enzyme that catalyzes the decomposition of H 2 O 2 to H 2 O and O 2 [37]. We observed that the amebicidal activity of L. acidophilus was strongly reduced when catalase was added during the incubation of L. acidophilus with the parasite (Figure 1). Based on this finding, it strongly suggests that H 2 O 2 produced by L. acidophilus is the primary cause of parasite death.

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.

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 H 2 O 2 produced by L. acidophilus ( Figure 2B). These results indicate that the formation of OXs is triggered by H 2 O 2 produced by L. acidophilus. 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 H 2 O 2 released by the bacteria.
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][45][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].  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 H 2 O 2 [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.

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 H 2 O 2 produced by L. acidophilus inhibits the CPs. of CPs is dependent on the presence of free hemin, which can be released by quinoline drugs [62].

Adhesion of E. histolytica Trophozoites to HeLa Cells Is Impaired by L. acidophilus
E. histolytica trophozoites' ability to bind to mammalian cells is the initial step in the amebic infectious process [63]. In our experiment, trophozoites incubated with L. acidophilus exhibit reduced binding to HeLa cells compared to trophozoites incubated with heatkilled L. acidophilus or with paraformaldehyde-fixed L. acidophilus. However, the binding activity to HeLa cells of trophozoites incubated with L. acidophilus in the presence of catalase is comparable to the binding activity of heat-killed L. acidophilus or with paraformaldehyde-fixed L. acidophilus ( Figure 5). These data strongly suggest that the production of H2O2 by L. acidophilus inhibits E. histolytica's binding to HeLa cells rather than a competition between L. acidophilus and HeLa cells. The lectin Gal/GalNAc plays an essential role 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 H 2 O 2 results from the formation of sulfenic acid, which reacts with adjacent free thiol to form mixed disulfides. In addition, H 2 O 2 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 H 2 O 2 produced by L. acidophilus. As opposed to E. histolytica, where H 2 O 2 produced by L. acidophilus appears to inhibit CPs activity directly, in Plasmodium parasites, H 2 O 2 -mediated inhibition of CPs is dependent on the presence of free hemin, which can be released by quinoline drugs [62].
3.4. Adhesion of E. histolytica Trophozoites to HeLa Cells Is Impaired by L. acidophilus E. histolytica trophozoites' ability to bind to mammalian cells is the initial step in the amebic infectious process [63]. In our experiment, trophozoites incubated with L. acidophilus exhibit reduced binding to HeLa cells compared to trophozoites incubated with heat-killed L. acidophilus or with paraformaldehyde-fixed L. acidophilus. However, the binding activity to HeLa cells of trophozoites incubated with L. acidophilus in the presence of catalase is comparable to the binding activity of heat-killed L. acidophilus or with paraformaldehydefixed L. acidophilus ( Figure 5). These data strongly suggest that the production of H 2 O 2 by L. acidophilus inhibits E. histolytica's binding to HeLa cells rather than a competition between L. acidophilus and HeLa cells. The lectin Gal/GalNAc plays an essential role in parasite attachment to mammalian cells, including HeLa cells [64][65][66]. We previously demonstrated that oxidation of the carbohydrate-recognizing cysteine-rich domain (CRD) of Gal/GalNAc lectin renders it inactive [6]. We observed in this study that 170kDa Gal/GalNAc is one of the OXs produced in the parasite exposed to L. acidophilus. According to the MS analysis of OXs (Table S2), many cysteine residues are carbamidomethylated in the CRD of Gal/GalNAc lectin, which strongly suggests that they were oxidized leading to an impairment of the parasite's ability to bind mammalian cells (this work and [6]).

Conclusions
The results for this study show that the production of H2O2 by L. acidophilus causes oxidation of vital proteins in E. histolytica and ultimately results in parasite death. The present study emphasizes L. acidophilus' potential as a probiotic against amebiasis. However, in vivo trials are necessary to determine whether this probiotic has health benefits on humans when it is used alone or in combination with metronidazole.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Table S1: List of all OXs that were enriched by RAC in three independent experiments in E. histolytica trophozoites incubated with L. acidophilus. Table S2: List carbamidomethyl (C) sites in OXs. Table S3: Description of the parameters that are given in Table S1 and Table S2.   , and with/without catalase (50 µg/mL) for 1 h at 37 • C and then transferred to paraformaldehyde-fixed HeLa cell monolayers. Trophozoites attached to HeLa cells monolayers were counted. The number of trophozoites incubated with heat-killed L. acidophilus (WT + DN) that were bound to HeLa cells monolayer (around 75% of the original population) was obtained as 100%. The data represent two independent experiments performed in duplicate. **** p-value < 0.0001 by an unpaired Student's t-test.

Conclusions
The results for this study show that the production of H 2 O 2 by L. acidophilus causes oxidation of vital proteins in E. histolytica and ultimately results in parasite death. The present study emphasizes L. acidophilus' potential as a probiotic against amebiasis. However, in vivo trials are necessary to determine whether this probiotic has health benefits on humans when it is used alone or in combination with metronidazole.