*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 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 in parasite attachment to mammalian cells, including HeLa cells [64–66]. 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]).

**Figure 5.** Binding activity assay of *E. histolytica* trophozoites. Note: *E. histolytica* trophozoites were incubated with live *L. acidophilus* (LA), with heat-killed *L. acidophilus* (DN), with paraformaldehydefixed *L. acidophilus* (PLA), 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.

#### **4. 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 https://www.mdpi.com/article/10 .3390/antiox11050814/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 Tables S1 and S2.

**Author Contributions:** Conceptualization: L.S. and S.A.; methodology, L.S., E.Z., J.Y., M.T.-G. and S.A.; software, L.S. and S.A.; validation, L.S., E.Z., J.Y., M.T.-G. and S.A.; formal analysis, L.S., E.Z., M.T.-G. and S.A.; investigation, L.S., E.Z., J.Y., M.T.-G. and S.A.; resources, S.A.; data curation, L.S. and S.A.; writing—original draft preparation, L.S. and S.A.; writing—review and editing, L.S., E.Z., J.Y., M.T.-G. and S.A.; visualization, S.A.; supervision, S.A.; project administration, S.A.; funding acquisition, S.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Israel Science Foundation (3208/19) and the Ministry of Science and Technology, Israel (1020546).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and Supplementary Material.

**Acknowledgments:** We thank the staff of the Smoler Proteomics Center at the Technion for their technical help.

**Conflicts of Interest:** The authors declare no conflict of interest.
