microorganisms

Review
Entamoeba histolytica—Gut Microbiota Interaction: More Than
Meets the Eye
Serge Ankri

Department of Molecular Microbiology, Ruth and Bruce Rappaport Faculty of Medicine, Haifa 31096, Israel;
[email protected]

Abstract: Amebiasis is a disease caused by the unicellular parasite Entamoeba histolytica. In most
cases, the infection is asymptomatic but when symptomatic, the infection can cause dysentery and
invasive extraintestinal complications. In the gut, E. histolytica feeds on bacteria. Increasing evidences
support the role of the gut microbiota in the development of the disease. In this review we will
discuss the consequences of E. histolytica infection on the gut microbiota. We will also discuss new
evidences about the role of gut microbiota in regulating the resistance of the parasite to oxidative
stress and its virulence.

Keywords: gut microbiota; entamoeba histolytica; resistance to oxidative stress; resistance to nitrosative
stress; virulence

1. Introduction



Amebiasis is caused by the protozoan parasite Entamoeba histolytica. This disease is


a significant hazard in underdeveloped countries with reduced socioeconomic and poor
Citation: Ankri, S. Entamoeba sanitation. It is assessed that amebiasis accounted for 55,500 deaths and 2.237 million
histolytica—Gut Microbiota disability-adjusted life years (the sum of years of life lost and years lived with disability)
Interaction: More Than Meets the Eye. in 2010 [1]. Amebiasis has also been diagnosed in tourists from developed countries who
Microorganisms 2021, 9, 581. return from vacation in endemic regions. Inflammation of the large intestine and liver
https://doi.org/10.3390/
abscess represent the main clinical manifestations of amebiasis. Amebiasis is caused by the
microorganisms9030581
ingestion of food contaminated with cysts, the infective form of the parasite. Following
excystation, the trophozoites migrates to the large intestine resulting in either asymptomatic
Academic Editor: Ryan J. Arsenault
colonization (90% of all infections) or causing bloody diarrhea. For unknown reasons, the
trophozoites can become virulent and invasive, cause amebic dysentery, and migrate to
Received: 23 February 2021
Accepted: 10 March 2021
the liver via the portal veins, where they cause hepatocellular damage. No vaccine against
Published: 12 March 2021
amebiasis currently exists; the drug of choice for treating amebiasis is metronidazole, which
may have severe side effects. Additionally, some clinical strains of E. histolytica are less
Publisher’s Note: MDPI stays neutral
sensitive to metronidazole, suggesting the emergence of metronidazole-resistant strains [2].
with regard to jurisdictional claims in
E. histolytica trophozoites proliferate in the intestinal lumen and phagocytose the resident
published maps and institutional affil- gut flora with a preference for some species like Lactobacillus ruminus [3]. At first glance,
iations. the interaction between E. histolytica and the gut microbiota can be perceived as a simple
interaction between a predator and its prey. Over the last few decades, this perception
has begun to change and some recent reviews in the field have emphasized the complex
interaction that occurs between the parasite and gut microbiota [4–6]. However, the role
of this interaction in the resistance of the parasite to environmental factors present in the
Copyright: © 2021 by the author.
Licensee MDPI, Basel, Switzerland.
gut which are tightly associated with the generation of reactive oxygen species (ROS) and
This article is an open access article
reactive nitrogen species (RNS) and on the pathogenesis of amebiasis has just emerged. In
distributed under the terms and this review, we will focus on this last aspect of the Entamoeba-gut microbiota interaction.
conditions of the Creative Commons
Attribution (CC BY) license (https://
2. Survey Methodology
creativecommons.org/licenses/by/ Literature searching aimed at collecting any published data about Entamoeba par-
4.0/). asites and the gut microbiota, resistance to oxidative stress (OS) and nitrosative stress

Microorganisms 2021, 9, 581. https://doi.org/10.3390/microorganisms9030581 https://www.mdpi.com/journal/microorganisms

Microorganisms 2021, 9, 581 2 of 12

(NS), virulence, crosstalk between E. histolytica, and gut microbiota. We searched literature
relevant to the topic of the articles using PubMed and Google Scholar. Key words such as
gut microbiota, amebiasis, resistance to oxidative stress, resistance to nitrosative stress, vir-
ulence, and probiotics were used to search. Then, screened articles were used as references
for this review.

3. The Human Large Intestine and Its Associated Microbiota

The human intestine is inhabited by more than 100 trillion bacteria representing 500
to 1000 species [7]. These bacteria lives in a mutualistic association with their host and
play a crucial role in producing vitamins and other metabolites that are essential to the
host [8]. The gut microbiota is closely related to human health and once the intestinal flora
is disturbed (gut microbiota dysbiosis), a series of intestinal diseases, such as inflammatory
bowel disease, and colorectal cancer can develop [9].
The bacterial microbiome of the adult human gut is colonized by Firmicutes and Bac-
teroidetes that represent 90% of gut microbiota [10]. The establishment of gut microbiota
is a dynamic process that begins even before birth to reach a stable level between 2 and
5 years of age [7]. Various factors influence the composition of gut microbiota following
birth including the mode of delivery, the diet (breastmilk vs. formula), hygiene, and antibi-
otic treatment [11]. Between childhood and adulthood, food culture, which significantly
differs between developed and developing countries, has a strong impact on the bacterial
microbiome [12]. In adulthood, the gut microbiota community is relatively stable but it
can change with age and diet. For example, older people present less Bacteroides, Bifi-
dobacterium, and Enterobacteriaceae in their gut and more Clostridium species compared
with younger adults [13]. Indeed, dietary fiber were found to increase faecal abundance of
Bifidobacterium and Lactobacillus species [14].

4. Change Occurring in the Large Intestine Microbiota Following Infection with

E. histolytica
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 [4]. This interaction
is very selective as only those bacteria with the appropriate recognition molecules are
ingested by the parasite [15]. It has been reported that association with specific intestinal
bacteria changes the E. histolytica cell surface architecture [16,17] and that phagocytosis
of pathogenic bacteria boosts E. histolytica cytopathogenicity, increases the expression
of Gal/GalNAc lectin on the cell surface, and boosts cysteine proteinase activity and
resistance to oxidative stress (OS) when E. histolytica trophozoites are co-cultured with
the enteropathogenic E.coli (EPEC) O55 [18] or Shigella dysenteriae [19]. Finally, bacteria-
induced augmentation of E. histolytica virulence seems to occur only when the trophozoites
phagocytose intact live cells [15]. The gut flora of patients suffering from amebiasis shows
a significant decrease in the population of Bacteroides, Clostridium coccoides, Clostridium
leptum, Lactobacillus, and Campylobacter and an increase in Bifidobacterium, while there
is no change in Ruminococcus compared to healthy patients [20]. Interestingly, the fecal
microbiota composition can be used as a predictive tool of Entamoeba colonization with an
accuracy of 79% [21]. Some of the taxa, like Clostridiales Ruminococcaceae or Prevotella
copri, which were central for the identification of patients infected with Entamoeba, have
been associated with inflammatory bowel disease [22,23]. It is still not clear how a specific
gut microbiota becomes associated with patients infected by Entamoeba. It is possible
that the colonization of the gut by Entamoeba is predisposed by the gut microbiota of the
host. Certain species of bacteria may also prevent the development of Entamoeba as it
has been suggested for the commensal Clostridia, segmented filamentous bacteria [24].
Alternatively, E. histolytica feeds preferentially on certain species of bacteria [3] which may
allow other species to proliferate.
Microorganisms 2021, 9, 581 3 of 12

5. Response of E. histolytica to OS
ROS play a key role in eliciting OS response in cells. They are capable of damaging
essential biomolecules in the cell such as DNA, proteins, lipids, and they primarily inhibit
cellular functions. Once formed, ROS leads to the oxidative damage of proteins thereby af-
fecting their structure and functional properties [25,26]. In the large intestine, the invading
E. histolytica trophozoites encounter OS. The sources of these stresses are fluctuations in
oxygen tension in the intestinal lumen and the generation of ROS by cells of the immune
system. Anti-amebic drugs like metronidazole and auranofin also induce oxidative damage
of proteins by inhibiting thioredoxin reductase, a central enzyme in the protection of the
parasite against OS [27]. Hydrogen peroxide (H2 O2 ) is capable of damaging proteins by
its interaction with thiol groups, which are present in the cysteine side chains as well as
with metal cofactors. E. histolytica lacks antioxidant enzymes, such as catalase, glutathione
reductase, and γ-glutamyl transpeptidase [28]. Thus, proteins such as the 29-kDa peroxire-
doxin [29] and iron-containing peroxide dismutase [30] aid in OS resistance. It has been
showed that E. histolytica strains sustain the exposure to OS better than avirulent strains,
due to the presence of peroxiredoxin [31,32]. OS resistance contributes to the pathogenic po-
tential of E. histolytica [33]. Additionally, OS leads to the oxidation of hundreds of proteins
in the parasite including proteins involved in redox homeostasis, lipid metabolism, small
molecule metabolism, carbohydrate derivative metabolism, and organonitrogen compound
biosynthesis [34,35]. Oxidation of these proteins often lead to their inhibition as reported for
glycolytic enzymes [36], virulence factors like the Galactose/N-acetylgalactosamine lectin
which is essential for the binding of the parasite to host cells [35], and arginase, an enzyme
that catalyzes the conversion of L-arginine to L-ornithine [35], a precursor of polyamine
synthesis [37]. Polyamines and their biosynthetic enzymes are considered essential for
growth and survival of unicellular parasites including Trypanosoma, Leishmania, and
Plasmodium [37]. One of these polyamines, putrescine, has been linked to OS resistance
and one of the proposed mechanism of OS resistance is based on its polycationic nature
that enables it to couple with nucleic acids and membrane phospholipids. Putrescine is
also free radical scavenger and an antioxidant [38]. The importance of putrescine and
other polyamines in the resistance of E. histolytica to OS has been proposed [35] but direct
experimental evidences to support this suggestion are still missing. OS induces a strong
inhibition of protein synthesis in the parasite [35]. Although the mechanism for this inhibi-
tion is still not understood, it probably involves a higher eukaryotes in the phosphorylation
of the initiation factor (eIF-2α) [39,40] and the oxidation of components of the parasite’s
translational machinery, such as ribosomal proteins and elongation factors which leads
to their inhibition [35,41]. At the transcriptomics level, OS triggers a complex response in
the parasite which involves the modulation of a large number of genes which encode pro-
teins with roles in translation, signaling/regulatory processes, metabolic/repair processes,
energy metabolism, stress response, and transport [18,42]. The regulation of expression
of genes which are responsive to OS mediated by H2 O2 is controlled by a transcription
factor EHI_108720 that binds to the AAACCTCAATGAAGA motif which is enriched in
promoters of H2 O2 -responsive genes [43].

6. Response of E. histolytica to OS in Presence of Bacteria

6.1. Effect of Bacteria on E. histolytica Transcriptome
It was proposed more than 30 years ago that bacteria can compensate the lack of
antioxidant enzyme in E. histolytica by complementing the parasite with such enzymes [15].
Excluding this work, the knowledge about the role of the gut microbiota on the resistance
of the parasite to OS was scant. Unexpected interactions between the parasite and the
bacteria that contribute to the resistance of the parasite to OS has been recently highlighted.
Interaction of E. histolytica with E. coli O55 (ratio 1:1000) confers resistance of the parasite to
OS [18]. At the transcriptomic level, E. coli O55 has almost no effect on gene expression in
the parasite. However, when the parasite is exposed to E. coli O55 and to OS, the combi-
nation of these two stimuli triggers a strong transcriptomic response that involves almost
Microorganisms 2021, 9, 581 4 of 12

50% of the parasite’s coding gene [18]. This transcriptomic response is very different to
the response of the parasite exposed to the OS alone. A general pattern of this combined
response is the “normalization” of the level of expression of many genes that have been
downregulated (including many ribosomal proteins) or upregulated (including oxidore-
ductases and several metabolic enzymes like glyceraldehyde-3-phosphate dehydrogenase
and malate dehydrogenase) by OS. Downregulation of ribosomal proteins expression is
a conserved mechanism to shut down unnecessary protein synthesis during stress [44].
In contrast, the upregulation of oxidoreductases and metabolic enzymes expression is a
mechanism that compensate the inhibition of activity of these essential enzymes for the
parasite following their oxidation [35]. The same “normalization” mechanism on gene
expression in the parasite has been observed with two other bacteria, Salmonella enterica
and Enterococcus faecalis but not with the probiotic Lactobacillus acidophilus. It is possible
that the production of H2 O2 by L. acidophilus [45] is detrimental to the parasite already
exposed to OS. The effect that bacteria have on gene expression in the parasite exposed
to OS goes beyond the “normalization” mechanism described above. Many leucine-rich
repeat (LRR) proteins that were downregulated in the presence of OS were upregulated
in the presence of bacteria and OS [18]. These LRR proteins which belong to the BspA
family of proteins present structural homologies with Toll-like receptors (TLRs). TLRs
are usually expressed on sentinel cells such as macrophages and dendritic cells and are
involved in the recognition of structurally conserved molecules derived from microbes [46].
The possibility that the ancient protozoan E. histolytica displays key characteristics of the
antibacterial response present in higher eukaryotes has been recently discussed [6,18].
However, the strong homology of sequence between these LRR proteins will make very
challenging the testing of their functionality as TLRs with the genetic tools that are actually
available to manipulate gene expression in E. histolytica [47]. The recent success to make
the CRISPR/Cas9 system work in E. histolytica at an episomal level provides hope for the
future study of these LRR proteins [48].

6.2. Effect of Chemical Molecules Originating from Bacteria

6.2.1. Short-Chain Fatty Acids (SCFAs)
Gut microbial dysbiosis causes changes in SCFAs production leading, for example, to
liver diseases [49] and neurodegenerative disorders [50]. The effect of chemical molecules
originating from bacteria on the physiology of Entamoeba parasites has been pioneered
by a study on SCFAs and their role in inhibiting encystation [51]. SCFAs are the main
metabolites produced in the colon by bacterial fermentation of dietary fibers and resistant
starch [52]. SCFAs inhibit OS in mammalian cells [53] and limit the genotoxic effect of
H2 O2 [54]. Based on this information, it will be very interesting to test in future the effect
of different SCFAs like butyrate or propionate on the resistance of the parasite to OS.

6.2.2. Oxaloacetate
Alpha-keto acids pyruvate, oxaloacetate, and alpha-ketoglutarate have a good H2 O2 -
scavenging activity [55]. The role of oxaloacetate produced by the enteropathogenic E. coli
O55 in protecting E. histolytica against OS has been recently demonstrated [34]. Malate
dehydrogenase (MDH), which catalyzes the formation of oxaloacetate from malate, is
essential for the protective effect to OS that E. coli O55 confers to E. histolytica. Two
mechanisms by which oxaloacetate is delivered to the parasite are possible: (i) Intrabacterial
oxaloacetate reach the parasite by phagocytosis of the bacteria and (ii) secreted E. coli MDH
are forming oxaloacetate in the environment and this oxaloacetate acts like a shield by
scavenging H2 O2 before it affects the parasite’s viability. Oxaloacetate also has a role in
promoting the virulence of the parasite, which confirmed previous observations about
the correlation between virulence of the parasite and its resistance to OS [33]. In future, it
will be interesting to test the protective effect of other alpha keto-acids produced by the
microbiota on the resistance of the parasite against OS. Other antioxidant metabolites are
produced by the gut microbiota like glutathione and folic acid [56]. Entamoeba histolytica
Microorganisms 2021, 9, 581 5 of 12

lacks glutathione reductase activity, the ability to synthesize glutathione de novo and the
ability to form trypanothione from taken up glutathione [57]. Therefore, the relevance
of glutathione produced by the gut microbiota to the resistance of the parasite to OS is
probably weak. In contrast, folic acid is one of the vitamins, which is currently added to
the culture media of E. histolytica [58]. In view of the ability of folic acid to scavenge free
radical [59], it will be interesting to test its ability to protect the parasite against OS.

6.2.3. Queuine
Queuine and 7-(((4.5-cis-dihydroxy-2-cyclopenten-1-yl)-amino)-methyl)-7-
deazaguanosine (queuosine—Q) are produced by bacteria. Q and its glycosylated deriva-
tives occur in position 34 of the anticodon of tRNAAsp , tRNAHis , tRNAAsn , and tRNATyr of
eubacteria and eukaryotes except for Saccharomyces cerevisiae [60,61]. Q is highly conserved
and found in plants, fishes, insects, and mammals. While many bacteria can synthesize
queuine (the nucleobase of Q) de novo, salvaging the prokaryotic Q precursors preQ0 and
preQ1 has recently been reported [62]. Eukaryotes are not capable of Q synthesis and they
rely on salvaging the queuine base as a Q precursor either by nutrition or by the intestinal
bacterial flora [63–65]. The effects of queuine on the physiology of E. histolytica have been
recently studied [66] and the main conclusions of this study are summarized in Figure
1. Queuine protects the parasite against OS and it antagonizes the negative effect that
OS has on translation by inducing the expression of genes involved in the OS response
like heat shock protein 70 (Hsp70), antioxidant enzymes such as alcohol dehydrogenases,
and proteins involved in the repair of oxidative DNA damage like RecQ helicase. On the
other hand, queuine impairs E. histolytica virulence by downregulating the expression of
cysteine proteases and other genes associated with virulence [66]. This is the first example
in Eukaryotes of an effect of queuine on the regulation of gene expression. In contrast
to oxaloacetate and other alpha-keto acids that rely on their ability to scavenge H2 O2
to protect E. histolytica against OS, queuine uses a much more complex mechanism that
depends on tRNA-guanine transglycosylase (TGT) activity. TGT is the main enzyme re-
sponsible for the formation of Q in the anticodon loop position 34 of tRNAAsp , tRNAHis ,
tRNAAsn , and tRNATyr . The enzyme exchanges G34 for the precursors. In contrast to
eubacterial TGT enzymes, all of which are homodimers, eukaryotic TGT enzymes, such
as human TGT, are heterodimers and consist of a Q tRNA-ribosyltransferase 1 (QTRT1)
and a Q tRNA-ribosyltransferase domain-containing 1 (QTRTD1) [67,68]. E. histolytica
TGT enzyme has been recently identified and forms a heterodimer composed of EhQTRT1
and EhQTRTD1. EhTGT is catalytically active and incorporates queuine into E. histolytica
tRNAs. Two mechanisms can possibly explain why queuine protects the parasite against
OS. The first mechanism relies on the reprograming of gene expression in the parasite
exposed to queuine. Genes involved in the resistance to OS like heat shock protein 70 (Hsp
70), antioxidant enzymes like alcohol dehydrogenases 2, and DNA repairing enzymes like
RecQ helicases have their expression upregulated in the presence of queuine [66]. Why
queuine leads to a reprograming of these genes is still an open question. It can be the result
of an increased transcription of these genes triggered by transcription factor(s) and/or by
an accumulation of these mRNAs in the parasite cultivated in presence of queuine. Work
is in progress to address this question. In the second mechanism that relies on studies
performed in S.pombe and mammals, Dnmt2 activity is stimulated by prior queuosine
incorporation at G34 of tRNAAsp GUC [69,70]. Q-modified tRNAAsp GUC is protected against
endonuclease cleavage and it is therefore preferentially used by the cells for the translation
of stress proteins. Data supporting the presence of this mechanism is E. histolytica which in-
cludes: (i) The exogenous supplementation of E. histolytica trophozoites with queuine leads
to hypermethylation of C38 in tRNAAsp GUC and (ii) hypermethylation of tRNAAsp GUC
catalyzed by the E. histolytica Dnmt2 homolog Ehmeth correlates with the resistance of
the parasite to OS [71]. The two mechanisms may be connected as U (U-GUN) ending
codons which are overrepresented in genes upregulated in the parasite exposed to queuine
including possible transcription factors and proteins involved in OS resistance [66].
Microorganisms 2021, 9, 581 6 of 12

Figure 1. Queuine produced by the gut microbiota regulates E. histolytica resistance to oxidative stress (OS) and virulence. 1.
Queuine which is produced in the human gut by the microbiota and released upon lysis of the bacteria can be salvaged by
E. histolytica. The mechanism used to uptake queuine is unknown. 2. Queuosine-modified tRNAs present inside bacteria can
enter inside the parasite following their phagocytosis. The parasite may rely on a dedicated enzymatic machinery to salvage
Q. One possible candidate for this function is DUF2419 (EHI_098190), an E. histolytica protein with structural similarity with
DNA glycosidases. Work is in progress to characterize the involvement of EhDUF2419 in the salvage of Q from bacteria.
3. Queuine regulates the transcriptome of the parasite by upregulating the expression of genes involved in the resistance
to OS and by downregulating the expression of genes involved in virulence [66]. 4. Protein synthesis is impaired in the
parasite exposed to OS [66]. In presence of queuine, OS does not block protein synthesis. This mechanism may help the
parasite to stand OS. 5. Queuosine-modified tRNAs are hypermethylated by Ehmeth, the amebic homolog of mammalian
Dnmt2 [66]. Hypermethylation of tRNAAsp GUC has been correlated with the resistance of the parasite to OS and nitrosative
stress (NS) [71,72]. It is possible that regulatory mechanisms described in 3 and 5 are linked [66]. This document has be
created with BioRender.com (accessed on 10 March 2021).

7. Response of E. histolytica to Nitrosative Stress (NS) and the Gut Microbiota

Following host invasion, the invading E. histolytica trophozoites are exposed to
nanomolar concentrations of nitric oxide (NO) that is produced in intestinal epithelial
cells by constitutive NO synthase [73] and as an intermediate in denitrification by the
intestinal microbiota [74]. Although exposure to low NO concentrations is insufficient to
kill the parasite [75], these low concentrations may strengthen its resistance to high NO
concentrations. Amebiasis is characterized by acute inflammation of the intestine with
the release of cytokines, such as tumor necrosis factor α, interleukin 8, interferon gamma,
and interleukin β, and the generation of micromolar concentrations of ROS (discussed
above) and reactive nitrogen species (RNS) from activated cells of the host’s immune
system. NO in micromolar concentrations is cytotoxic for E. histolytica, and this cytotoxic-
Microorganisms 2021, 9, 581 7 of 12

ity is implemented by S-nitrosylation of key metabolic enzymes and by fragmenting the

endoplasmic reticulum (ER) [76,77]. NO also inhibits cysteine proteases [77], which are
involved in differentiation, amino acid anabolism, inactivation of the host inflammatory
response, lysosomal transport, and invasion of the host’s tissues [78]. NO can also regu-
late the activity and function of proteins by S-nitrosylation of their cysteine residues [79].
A high-throughput proteomic analysis of S-nitrosylated (SNO) proteins in NO-exposed
E. histolytica using resin-assisted capture of SNO proteins [75], found that SNO proteins are
involved in glycolysis, translation, protein transport, and virulence. E. histolytica can adapt
to various stresses [80–82] including to progressive increases in the intestinal NO concen-
tration [83], which may occur in patients with inflammation of the large intestine [73] or
during the establishment of amebiasis [84].
Information about the role of the gut microbiota in protecting the host against NS is
scanty. The role of acetate and butyrate, two SCFAs produced by the gut microbiota, to
reduce NS in human islets and β cells after exposure to the apoptosis inducer and metabolic
stressor streptozotocin [85] is one of the few examples available in the literature. In contrast,
the ability of the gut microbiota to generate RNS is well discussed (for a recent review
see [86]). The gut bacteria can convert nitrites into nitrosamines which have carcinogenic
properties [87] and some food components present in meat and fish into trimethylamine.
In the liver, trimethylamine is converted to its oxidized form (trimethylamine N-oxide)
which have deleterious effects on cardiovascular and metabolic function [88].
Regarding E. histolytica, we did not found any protective effect of E. coli O55 on the
resistance of the parasite to NS [18]. The lack of protection may be explained by the fact
that E. coli O55 was not exposed to NS prior to its interaction with the parasite. E. coli
possesses three major enzymes to overcome NS: the soluble flavohaemoglobin Hmp, the
di-iron-center flavorubredoxin NorV with its NADH-dependent oxidoreductase NorW
(NorVW) and the cytochrome c nitrite reductase NrfA. The expression of these enzymes is
induced by the exposure of the bacteria to NS [89]. Consequently, it will be interesting to
measure the effect of E. coli O55 on the resistance of E. histolytica to NS by using bacteria
pre-exposed to NO. We have also addressed the role of queuine in protecting the parasite
against NS. Queuine did protect the parasite against NS to some extend but the variability
of the results among different experiments was very high (unpublished data).

8. E. histolytica Infection and Probiotics

Probiotics are live microorganisms that are intended to have health benefits when
consumed or applied to the body [90]. It has been proposed that the use of probiotics,
may present as complementary or as an alternative to the current treatment of amoebiasis.
The possible effect of probiotics in preventing amebiasis has been recently reviewed [91].
A number of studies have been conducted to test the effectiveness of the probiotic at
inhibiting adhesion of the protozoa to the intestinal mucosa surface [92,93]. More recently,
it has been proposed that Lactobacillus acidophilus [18], Lactobacillus casei and Enterococcus
faecium [94] are potent probiotics that can be used to fight amebiasis. How these probiotics
work against the parasite is still not well understood. For L. acidophilus, it has been
suggested that the ability of this bacteria to produce H2 O2 [45] contributes to its amebicidal
activity [18]. For Weissella paramesenteroides WpK, another lactic acid bacteria, amoebic
lesions caused by Entamoeba dispar are reduced in presence of this bacteria. The authors
proposed that W. paramesenteroides WpK4 works by strengthening the barrier function of
the caecal mucosa [95].

9. Concluding Remarks
Beyond the predator-prey relationship that exists between the parasite and the gut
microbiota evidences for a more complex interaction have emerged in the last decades. It
is still not clear if the microbiota is paving the way for the development of amebiasis or
if the disease is triggered by the dysbiosis caused by the parasite. It is probable that both
scenarios are taking place. Small molecules originating from the bacteria like oxaloacetate,
Microorganisms 2021, 9, 581 8 of 12

SCFA and queuine have proved to be important mediators between the bacteria and the
parasite. These bacterial molecules which can control the different aspects of the physiology
of the parasite may be exploited to manipulate the parasite and fight it. For example, the
fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for
preventing and/or treating amebiasis by providing queuine to the host as a postbiotic
(soluble factors secreted by live bacteria, or released after bacterial lysis that can be used to
improve host health [96]) or via probiotics. Such strategy has been proposed for example
with the gut bacteria Gordonibacter pamelaeae that produces the anticarcinogen urolithin [97].
SCFA, oxaloacetate and queuine represent probably the top of the iceberg of the molecules
used between the microbiota and the parasite to communicate. It is essential to perform
a systematic screen for such molecules in the future. Many challenges in studying the
microbiome in the context of human diseases exists including the choice of appropriate
experimental systems [98]. These challenges exist also in the study of the role of the
microbiota in amebiasis. It is essential in the future to develop a simple model to study
the interaction of the microbiota with the parasite in the gut. One such model that we are
currently investigating is a three-dimensional intestinal model that replicates the general
characteristics of the human colon. This model has been recently used to investigate the
early stage of invasion of the gut by E. histolytica trophozoites [99]. Finally, what can
be learned from the interaction taking place between E. histolytica and the microbiota
is certainly relevant to other parasitic protozoa and helminths which are also in a tight
relationship with the host’s intestinal microbiota. For example, the antioxidant properties
of oxaloacetate which is produced by the gut microbiota is also valid for the protection of
C. elegans by oxaloacetate against H2O2-induced oxidative stress [34].

Funding: The work was supported by the Israel Ministry of Health within the framework European
Research Area NETwork Infect-ERA (031L0004; AMOEBAC project), the Israel Science Foundation
(260/16), the ISF-NRF program (3208/19), the US–Israel Binational Science Foundation (2015211),
and the Niedersachsen program.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Turkeltaub, J.A.; McCarty, T.R., 3rd; Hotez, P.J. The intestinal protozoa: Emerging impact on global health and development. Curr.
Opin. Gastroenterol. 2015, 31, 38–44. [CrossRef] [PubMed]
2. Bansal, D.; Sehgal, R.; Chawla, Y.; Mahajan, R.C.; Malla, N. In vitro activity of antiamoebic drugs against clinical isolates of
Entamoeba histolytica and Entamoeba dispar. Ann. Clin. Microbiol. Antimicrob. 2004, 3, 27. [CrossRef] [PubMed]
3. Iyer, L.R.; Verma, A.K.; Paul, J.; Bhattacharya, A. Phagocytosis of Gut Bacteria by Entamoeba histolytica. Front. Cell. Infect.
Microbiol. 2019, 9, 34. [CrossRef]
4. Burgess, S.L.; Petri, W.A., Jr. The Intestinal Bacterial Microbiome and E. histolytica Infection. Curr. Trop. Med. Rep. 2016, 3,
71–74. [CrossRef]
5. Leon-Coria, A.; Kumar, M.; Chadee, K. The delicate balance between Entamoeba histolytica, mucus and microbiota. Gut Microbes
2020, 11, 118–125. [CrossRef] [PubMed]
6. Guillen, N. The interaction between Entamoeba histolytica and enterobacteria shed light on an ancient antibacterial response.
Cell. Microbiol. 2019, 21, e13039. [CrossRef]
7. Rodriguez, J.M.; Murphy, K.; Stanton, C.; Ross, R.P.; Kober, O.I.; Juge, N.; Avershina, E.; Rudi, K.; Narbad, A.; Jenmalm, M.C.; et al.
The composition of the gut microbiota throughout life, with an emphasis on early life. Microb. Ecol. Health Dis. 2015, 26, 26050.
[CrossRef] [PubMed]
8. Rowland, I.; Gibson, G.; Heinken, A.; Scott, K.; Swann, J.; Thiele, I.; Tuohy, K. Gut microbiota functions: Metabolism of nutrients
and other food components. Eur. J. Nutr. 2018, 57, 1–24. [CrossRef] [PubMed]
9. Carding, S.; Verbeke, K.; Vipond, D.T.; Corfe, B.M.; Owen, L.J. Dysbiosis of the gut microbiota in disease. Microb. Ecol. Health Dis.
2015, 26, 26191. [CrossRef] [PubMed]
10. Qin, J.; Li, R.; Raes, J.; Arumugam, M.; Burgdorf, K.S.; Manichanh, C.; Nielsen, T.; Pons, N.; Levenez, F.; Yamada, T.; et al.
A human gut microbial gene catalogue established by metagenomic sequencing. Nature 2010, 464, 59–65. [CrossRef] [PubMed]
Microorganisms 2021, 9, 581 9 of 12

11. Hasan, N.; Yang, H. Factors affecting the composition of the gut microbiota, and its modulation. PeerJ 2019, 7, e7502.
[CrossRef] [PubMed]
12. Milani, C.; Ferrario, C.; Turroni, F.; Duranti, S.; Mangifesta, M.; van Sinderen, D.; Ventura, M. The human gut microbiota and its
interactive connections to diet. J. Hum. Nutr. Diet. 2016, 29, 539–546. [CrossRef]
13. Xu, C.; Zhu, H.; Qiu, P. Aging progression of human gut microbiota. BMC Microbiol. 2019, 19, 236. [CrossRef]
14. Leeming, E.R.; Johnson, A.J.; Spector, T.D.; le Roy, C.I. Effect of Diet on the Gut Microbiota: Rethinking Intervention Duration.
Nutrients 2019, 11, 2862. [CrossRef] [PubMed]
15. Bracha, R.; Mirelman, D. Virulence of Entamoeba histolytica trophozoites. Effects of bacteria, microaerobic conditions, and
metronidazole. J. Exp. Med. 1984, 160, 353–368. [CrossRef] [PubMed]
16. Bhattacharya, A.; Ghildyal, R.; Prasad, J.; Bhattacharya, S.; Diamond, L.S. Modulation of a surface antigen of Entamoeba
histolytica in response to bacteria. Infect. Immun. 1992, 60, 1711–1713. [CrossRef] [PubMed]
17. Padilla-Vaca, F.; Ankri, S.; Bracha, R.; Koole, L.A.; Mirelman, D. Down regulation of Entamoeba histolytica virulence by monoxenic
cultivation with Escherichia coli O55 is related to a decrease in expression of the light (35-kilodalton) subunit of the Gal/GalNAc
lectin. Infect. Immun. 1999, 67, 2096–2102. [CrossRef]
18. Varet, H.; Shaulov, Y.; Sismeiro, O.; Trebicz-Geffen, M.; Legendre, R.; Coppee, J.Y.; Ankri, S.; Guillen, N. Enteric bacteria boost
defences against oxidative stress in Entamoeba histolytica. Sci. Rep. 2018, 8, 9042. [CrossRef] [PubMed]
19. Galvan-Moroyoqui, J.M.; Dominguez-Robles, M.D.; Franco, E.; Meza, I. The interplay between Entamoeba and enteropathogenic
bacteria modulates epithelial cell damage. PLoS Negl. Trop. Dis. 2008, 2, e266. [CrossRef] [PubMed]
20. Verma, A.K.; Verma, R.; Ahuja, V.; Paul, J. Real-time analysis of gut flora in Entamoeba histolytica infected patients of Northern
India. BMC Microbiol. 2012, 12, 183. [CrossRef] [PubMed]
21. Morton, E.R.; Lynch, J.; Froment, A.; Lafosse, S.; Heyer, E.; Przeworski, M.; Blekhman, R.; Segurel, L. Variation in Rural African
Gut Microbiota Is Strongly Correlated with Colonization by Entamoeba and Subsistence. PLoS Genet. 2015, 11, e1005658.
[CrossRef] [PubMed]
22. Morgan, X.C.; Tickle, T.L.; Sokol, H.; Gevers, D.; Devaney, K.L.; Ward, D.V.; Reyes, J.A.; Shah, S.A.; LeLeiko, N.; Snapper,
S.B.; et al. Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment. Genome Biol. 2012, 13, R79.
[CrossRef] [PubMed]
23. Iljazovic, A.; Roy, U.; Galvez, E.J.C.; Lesker, T.R.; Zhao, B.; Gronow, A.; Amend, L.; Will, S.E.; Hofmann, J.D.; Pils, M.C.; et al.
Perturbation of the gut microbiome by Prevotella spp. enhances host susceptibility to mucosal inflammation. Mucosal Immunol.
2021, 14, 113–124. [CrossRef] [PubMed]
24. Burgess, S.L.; Buonomo, E.; Carey, M.; Cowardin, C.; Naylor, C.; Noor, Z.; Wills-Karp, M.; Petri, W.A., Jr. Bone marrow dendritic
cells from mice with an altered microbiota provide interleukin 17A-dependent protection against Entamoeba histolytica colitis.
mBio 2014, 5, e01817. [CrossRef]
25. Aiken, C.T.; Kaake, R.M.; Wang, X.; Huang, L. Oxidative stress-mediated regulation of proteasome complexes. Mol. Cell Proteom.
2011, 10, R110.006924. [CrossRef]
26. Shacter, E. Quantification and significance of protein oxidation in biological samples. Drug Metab. Rev. 2000, 32, 307–326.
[CrossRef] [PubMed]
27. Shaulov, Y.; Nagaraja, S.; Sarid, L.; Trebicz-Geffen, M.; Ankri, S. Formation of oxidised (OX) proteins in Entamoeba histolytica
exposed to auranofin and consequences on the parasite virulence. Cell. Microbiol. 2020, 22, e13174. [CrossRef] [PubMed]
28. Tekwani, B.L.; Mehlotra, R.K. Molecular basis of defence against oxidative stress in Entamoeba histolytica and Giardia lamblia.
Microbes Infect. 1999, 1, 385–394. [CrossRef]
29. Sen, A.; Chatterjee, N.S.; Akbar, M.A.; Nandi, N.; Das, P. The 29-kilodalton thiol-dependent peroxidase of Entamoeba histolytica
is a factor involved in pathogenesis and survival of the parasite during oxidative stress. Eukaryot. Cell 2007, 6, 664–673.
[CrossRef] [PubMed]
30. Bruchhaus, I.; Tannich, E. Induction of the iron-containing superoxide dismutase in Entamoeba histolytica by a superoxide
anion-generating system or by iron chelation. Mol. Biochem. Parasitol. 1994, 67, 281–288. [CrossRef]
31. Iyer, L.R.; Singh, N.; Verma, A.K.; Paul, J. Differential expression and immunolocalization of antioxidant enzymes in Entamoeba
histolytica isolates during metronidazole stress. Biomed. Res. Int. 2014, 2014, 704937. [CrossRef] [PubMed]
32. Davis, P.H.; Zhang, X.; Guo, J.; Townsend, R.R.; Stanley, S.L., Jr. Comparative proteomic analysis of two Entamoeba histolytica
strains with different virulence phenotypes identifies peroxiredoxin as an important component of amoebic virulence. Mol.
Microbiol. 2006, 61, 1523–1532. [CrossRef]
33. Rastew, E.; Vicente, J.B.; Singh, U. Oxidative stress resistance genes contribute to the pathogenic potential of the anaerobic
protozoan parasite, Entamoeba histolytica. Int. J. Parasitol. 2012, 42, 1007–1015. [CrossRef]
34. Shaulov, Y.; Shimokawa, C.; Trebicz-Geffen, M.; Nagaraja, S.; Methling, K.; Lalk, M.; Weiss-Cerem, L.; Lamm, A.T.; Hisaeda, H.;
Ankri, S. Escherichia coli mediated resistance of Entamoeba histolytica to oxidative stress is triggered by oxaloacetate. PLoS
Pathog. 2018, 14, e1007295. [CrossRef] [PubMed]
35. Shahi, P.; Trebicz-Geffen, M.; Nagaraja, S.; Alterzon-Baumel, S.; Hertz, R.; Methling, K.; Lalk, M.; Ankri, S. Proteomic Identification
of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to
Oxidative Stress. PLoS Negl. Trop. Dis. 2016, 10, e0004340. [CrossRef] [PubMed]
Microorganisms 2021, 9, 581 10 of 12

36. Husain, A.; Sato, D.; Jeelani, G.; Soga, T.; Nozaki, T. Dramatic increase in glycerol biosynthesis upon oxidative stress in the
anaerobic protozoan parasite Entamoeba histolytica. PLoS Negl. Trop. Dis. 2012, 6, e1831. [CrossRef]
37. Phillips, M.A. Polyamines in protozoan pathogens. J. Biol. Chem. 2018, 293, 18746–18756. [CrossRef] [PubMed]
38. Groppa, M.D.; Benavides, M.P. Polyamines and abiotic stress: Recent advances. Amino Acids 2008, 34, 35–45. [CrossRef] [PubMed]
39. Walters, H.A.; Welter, B.H.; Sullivan, W.J., Jr.; Temesvari, L.A. Phosphorylation of eukaryotic initiation factor-2alpha in response
to endoplasmic reticulum and nitrosative stress in the human protozoan parasite, Entamoeba histolytica. Mol. Biochem. Parasitol.
2019, 234, 111223. [CrossRef]
40. Back, S.H.; Scheuner, D.; Han, J.; Song, B.; Ribick, M.; Wang, J.; Gildersleeve, R.D.; Pennathur, S.; Kaufman, R.J. Translation
attenuation through eIF2alpha phosphorylation prevents oxidative stress and maintains the differentiated state in beta cells.
Cell Metab. 2009, 10, 13–26. [CrossRef] [PubMed]
41. Brandes, N.; Reichmann, D.; Tienson, H.; Leichert, L.I.; Jakob, U. Using quantitative redox proteomics to dissect the yeast
redoxome. J. Biol. Chem. 2011, 286, 41893–41903. [CrossRef] [PubMed]
42. Vicente, J.B.; Ehrenkaufer, G.M.; Saraiva, L.M.; Teixeira, M.; Singh, U. Entamoeba histolytica modulates a complex repertoire
of novel genes in response to oxidative and nitrosative stresses: Implications for amebic pathogenesis. Cell. Microbiol. 2009, 11,
51–69. [CrossRef]
43. Pearson, R.J.; Morf, L.; Singh, U. Regulation of H2O2 stress-responsive genes through a novel transcription factor in the protozoan
pathogen Entamoeba histolytica. J. Biol. Chem. 2013, 288, 4462–4474. [CrossRef]
44. Zencir, S.; Dilg, D.; Rueda, M.P.; Shore, D.; Albert, B. Mechanisms coordinating ribosomal protein gene transcription in response
to stress. Nucleic Acids Res. 2020, 48, 11408–11420. [CrossRef] [PubMed]
45. Hertzberger, R.; Arents, J.; Dekker, H.L.; Pridmore, R.D.; Gysler, C.; Kleerebezem, M.; de Mattos, M.J. H(2)O(2) production in
species of the Lactobacillus acidophilus group: A central role for a novel NADH-dependent flavin reductase. Appl. Environ.
Microbiol. 2014, 80, 2229–2239. [CrossRef]
46. Kawasaki, T.; Kawai, T. Toll-like receptor signaling pathways. Front. Immunol. 2014, 5, 461. [CrossRef] [PubMed]
47. Zhang, H.; Pompey, J.M.; Singh, U. RNA interference in Entamoeba histolytica: Implications for parasite biology and gene
silencing. Future Microbiol. 2011, 6, 103–117. [CrossRef]
48. Kangussu-Marcolino, M.M.; Morgado, P.; Manna, D.; Yee, H.; Singh, U. Development of a CRISPR/Cas9 system in Entamoeba
histolytica: Proof of concept. Int. J. Parasitol. 2021, 51, 193–200. [CrossRef] [PubMed]
49. Giuffre, M.; Campigotto, M.; Campisciano, G.; Comar, M.; Croce, L.S. A story of liver and gut microbes: How does the intestinal
flora affect liver disease? A review of the literature. Am. J. Physiol. Gastr. Liver Physiol. 2020, 318, G889–G906. [CrossRef] [PubMed]
50. Giuffre, M.; Moretti, R.; Campisciano, G.; da Silveira, A.B.M.; Monda, V.M.; Comar, M.; di Bella, S.; Antonello, R.M.; Luzzati, R.;
Croce, L.S. You Talking to Me? Says the Enteric Nervous System (ENS) to the Microbe. How Intestinal Microbes Interact with the
ENS. J. Clin. Med. 2020, 9, 3705. [CrossRef] [PubMed]
51. Byers, J.; Faigle, W.; Eichinger, D. Colonic short-chain fatty acids inhibit encystation of Entamoeba invadens. Cell. Microbiol. 2005,
7, 269–279. [CrossRef]
52. Sun, Y.; O’Riordan, M.X. Regulation of bacterial pathogenesis by intestinal short-chain Fatty acids. Adv. Appl. Microbiol. 2013, 85,
93–118.
53. Huang, W.; Guo, H.L.; Deng, X.; Zhu, T.T.; Xiong, J.F.; Xu, Y.H.; Xu, Y. Short-Chain Fatty Acids Inhibit Oxidative Stress and
Inflammation in Mesangial Cells Induced by High Glucose and Lipopolysaccharide. Exp. Clin. Endocrinol. Diabetes 2017, 125,
98–105. [CrossRef]
54. Abrahamse, S.L.; Pool-Zobel, B.L.; Rechkemmer, G. Potential of short chain fatty acids to modulate the induction of DNA damage
and changes in the intracellular calcium concentration by oxidative stress in isolated rat distal colon cells. Carcinogenesis 1999, 20,
629–634. [CrossRef] [PubMed]
55. Bayliak, M.M.; Lylyk, M.P.; Vytvytska, O.M.; Lushchak, V.I. Assessment of antioxidant properties of alpha-keto acids in vitro and
in vivo. Eur. Food Res. Technol. 2016, 242, 179–188. [CrossRef]
56. Wang, Y.; Wu, Y.; Wang, Y.; Xu, H.; Mei, X.; Yu, D.; Wang, Y.; Li, W. Antioxidant Properties of Probiotic Bacteria. Nutrients 2017, 9,
521. [CrossRef] [PubMed]
57. Ariyanayagam, M.R.; Fairlamb, A.H. Entamoeba histolytica lacks trypanothione metabolism. Mol. Biochem. Parasitol. 1999, 103,
61–69. [CrossRef]
58. Diamond, L.S.; Harlow, D.R.; Cunnick, C.C. A new medium for the axenic cultivation of Entamoeba histolytica and other
Entamoeba. Trans. R. Soc. Trop. Med. Hyg. 1978, 72, 431–432. [CrossRef]
59. Joshi, R.; Adhikari, S.; Patro, B.S.; Chattopadhyay, S.; Mukherjee, T. Free radical scavenging behavior of folic acid: Evidence for
possible antioxidant activity. Free Radic. Bio Med. 2001, 30, 1390–1399. [CrossRef]
60. Fergus, C.; Barnes, D.; Alqasem, M.A.; Kelly, V.P. The queuine micronutrient: Charting a course from microbe to man. Nutrients
2015, 7, 2897–2929. [CrossRef]
61. Walden, T.; Reyniers, J.P.; Hiatt, V.; Farkas, W.R. Yeast cells cannot incorporate queuine into their tRNA. Proceedings of the Society
for Experimental Biology and Medicine. Soc. Exp. Biol. Med. 1982, 170, 328–332. [CrossRef] [PubMed]
62. Yuan, Y.; Zallot, R.; Grove, T.L.; Payan, D.J.; Martin-Verstraete, I.; Sepic, S.; Balamkundu, S.; Neelakandan, R.; Gadi, V.K.; Liu,
C.F.; et al. Discovery of novel bacterial queuine salvage enzymes and pathways in human pathogens. Proc. Natl. Acad. Sci. USA
2019. [CrossRef] [PubMed]
Microorganisms 2021, 9, 581 11 of 12

63. Katze, J.R.; Basile, B.; McCloskey, J.A. Queuine, a modified base incorporated posttranscriptionally into eukaryotic transfer RNA:
Wide distribution in nature. Science 1982, 216, 55–56. [CrossRef]
64. Ott, G.; Kersten, H.; Nishimura, S. Dictyostelium discoideum: A useful model system to evaluate the function of queuine and of
the Q-family of tRNAs. FEBS Lett. 1982, 146, 311–314. [CrossRef]
65. Farkas, W.R. Effect of diet on the queuosine family of tRNAs of germ-free mice. J. Biol. Chem. 1980, 255, 6832–6835. [CrossRef]
66. Nagaraja, S.; Cai, M.W.; Sun, J.; Varet, H.; Sarid, L.; Trebicz-Geffen, M.; Ankri, S.; Shaulov, Y.; Mazumdar, M.; Legendre, R.
Queuine is a nutritional regulator of Entamoeba histolytica response to oxidative stress and a virulence attenuator. mBio 2021,
12, 20. [CrossRef]
67. Chen, Y.C.; Kelly, V.P.; Stachura, S.V.; Garcia, G.A. Characterization of the human tRNA-guanine transglycosylase: Confirmation
of the heterodimeric subunit structure. RNA 2010, 16, 958–968. [CrossRef]
68. Stengl, B.; Meyer, E.A.; Heine, A.; Brenk, R.; Diederich, F.; Klebe, G. Crystal structures of tRNA-guanine transglycosylase (TGT)
in complex with novel and potent inhibitors unravel pronounced induced-fit adaptations and suggest dimer formation upon
substrate binding. J. Mol. Biol. 2007, 370, 492–511. [CrossRef]
69. Muller, M.; Hartmann, M.; Schuster, I.; Bender, S.; Thuring, K.L.; Helm, M.; Katze, J.R.; Nellen, W.; Lyko, F.; Ehrenhofer-Murray,
A.E. Dynamic modulation of Dnmt2-dependent tRNA methylation by the micronutrient queuine. Nucleic Acids Res. 2015, 43,
10952–10962. [CrossRef] [PubMed]
70. Ehrenhofer-Murray, A.E. Cross-Talk between Dnmt2-Dependent tRNA Methylation and Queuosine Modification. Biomolecules
2017, 7, 14. [CrossRef]
71. Fisher, O.; Siman-Tov, R.; Ankri, S. Pleiotropic phenotype in Entamoeba histolytica overexpressing DNA methyltransferase
(Ehmeth). Mol. Biochem. Parasitol. 2006, 147, 48–54. [CrossRef] [PubMed]
72. Hertz, R.; Tovy, A.; Kirschenbaum, M.; Geffen, M.; Nozaki, T.; Adir, N.; Ankri, S. The Entamoeba histolytica Dnmt2 homolog
(Ehmeth) confers resistance to nitrosative stress. Eukaryot. Cell 2014, 13, 494–503. [CrossRef]
73. Kolios, G.; Valatas, V.; Ward, S.G. Nitric oxide in inflammatory bowel disease: A universal messenger in an unsolved puzzle.
Immunology 2004, 113, 427–437. [CrossRef]
74. Vermeiren, J.; van de Wiele, T.; Verstraete, W.; Boeckx, P.; Boon, N. Nitric oxide production by the human intestinal microbiota by
dissimilatory nitrate reduction to ammonium. J. Biomed. Biotechnol. 2009, 2009, 284718. [CrossRef]
75. Hertz, R.; Lulu, S.B.; Shahi, P.; Trebicz-Geffen, M.; Benhar, M.; Ankri, S. Proteomic identification of S-nitrosylated proteins in the
parasite Entamoeba histolytica by resin-assisted capture: Insights into the regulation of the Gal/GalNAc lectin by nitric oxide.
PLoS ONE 2014, 9, e91518. [CrossRef]
76. Lin, J.Y.; Chadee, K. Macrophage cytotoxicity against Entamoeba histolytica trophozoites is mediated by nitric oxide from
L-arginine. J. Immunol. 1992, 148, 3999–4005.
77. Santi-Rocca, J.; Smith, S.; Weber, C.; Pineda, E.; Hon, C.C.; Saavedra, E.; Olivos-Garcia, A.; Rousseau, S.; Dillies, M.A.; Coppee,
J.Y.; et al. Endoplasmic reticulum stress-sensing mechanism is activated in Entamoeba histolytica upon treatment with nitric
oxide. PLoS ONE 2012, 7, e31777. [CrossRef]
78. Serrano-Luna, J.; Pina-Vazquez, C.; Reyes-Lopez, M.; Ortiz-Estrada, G.; de la Garza, M. Proteases from Entamoeba spp. and
Pathogenic Free-Living Amoebae as Virulence Factors. J. Trop. Med. 2013, 2013, 890603. [CrossRef] [PubMed]
79. Gould, N.; Doulias, P.T.; Tenopoulou, M.; Raju, K.; Ischiropoulos, H. Regulation of Protein Function and Signaling by Reversible
Cysteine S-Nitrosylation. J. Biol. Chem. 2013, 288, 26473–26479. [CrossRef]
80. Baumel-Alterzon, S.; Weber, C.; Guillen, N.; Ankri, S. Identification of dihydropyrimidine dehydrogenase as a virulence
factor essential for the survival of Entamoeba histolytica in glucose-poor environments. Cell. Microbiol. 2013, 15, 130–144.
[CrossRef] [PubMed]
81. Cabrera, H.A. Temperature adaptation of Entamoeba histolytica and its effect on virulence. Exp. Parasitol. 1958, 7,
276–284. [CrossRef]
82. Wassmann, C.; Hellberg, A.; Tannich, E.; Bruchhaus, I. Metronidazole resistance in the protozoan parasite Entamoeba histolytica
is associated with increased expression of iron-containing superoxide dismutase and peroxiredoxin and decreased expression of
ferredoxin 1 and flavin reductase. J. Biol. Chem. 1999, 274, 26051–26056. [CrossRef]
83. Trebicz-Geffen, M.; Shahi, P.; Nagaraja, S.; Vanunu, S.; Manor, S.; Avrahami, A.; Ankri, S. Identification of S-Nitrosylated (SNO)
Proteins in Entamoeba histolytica Adapted to Nitrosative Stress: Insights into the Role of SNO Actin and In vitro Virulence. Front.
Cell. Infect. Microbiol. 2017, 7, 192. [CrossRef] [PubMed]
84. Mortimer, L.; Chadee, K. The immunopathogenesis of Entamoeba histolytica. Exp. Parasitol. 2010, 126, 366–380. [CrossRef]
85. Hu, S.; Kuwabara, R.; de Haan, B.J.; Smink, A.M.; de Vos, P. Acetate and Butyrate Improve beta-cell Metabolism and Mitochondrial
Respiration under Oxidative Stress. Int. J. Mol. Sci. 2020, 21, 1542. [CrossRef] [PubMed]
86. Carlstrom, M.; Moretti, C.H.; Weitzberg, E.; Lundberg, J.O. Microbiota, diet and the generation of reactive nitrogen compounds.
Free Radic. Biol. Med. 2020, 161, 321–325. [CrossRef] [PubMed]
87. Rastogi, Y.R.; Saini, A.K.; Thakur, V.K.; Saini, R.V. New Insights into Molecular Links Between Microbiota and Gastrointestinal
Cancers: A Literature Review. Int. J. Mol. Sci. 2020, 21, 3212. [CrossRef]
88. Zeisel, S.H.; Warrier, M. Trimethylamine N-Oxide, the Microbiome, and Heart and Kidney Disease. Annu. Rev. Nutr. 2017, 37,
157–181. [CrossRef]
Microorganisms 2021, 9, 581 12 of 12

89. Arkenberg, A.; Runkel, S.; Richardson, D.J.; Rowley, G. The production and detoxification of a potent cytotoxin, nitric oxide, by
pathogenic enteric bacteria. Biochem. Soc. Trans. 2011, 39, 1876–1879. [CrossRef]
90. Ouwehand, A.C.; Salminen, S.; Isolauri, E. Probiotics: An overview of beneficial effects. Antonie Van Leeuwenhoek 2002, 82,
279–289. [CrossRef]
91. Nagaraja, S.; Ankri, S. Target identification and intervention strategies against amebiasis. Drug Resist. Updates 2019, 44, 1–14.
[CrossRef] [PubMed]
92. Mansour-Ghanaei, F.; Dehbashi, N.; Yazdanparast, K.; Shafaghi, A. Efficacy of saccharomyces boulardii with antibiotics in acute
amoebiasis. World J. Gastroenterol. 2003, 9, 1832–1833. [CrossRef]
93. Rigothier, M.C.; Maccario, J.; Gayral, P. Inhibitory activity of saccharomyces yeasts on the adhesion of Entamoeba histolytica
trophozoites to human erythrocytes in vitro. Parasitol. Res. 1994, 80, 10–15. [CrossRef]
94. Sarjapuram, N.; Mekala, N.; Singh, M.; Tatu, U. The Potential of Lactobacillus casei and Entercoccus faecium Combination as a
Preventive Probiotic Against Entamoeba. Probiotics Antimicrob. Proteins 2017, 9, 142–149. [CrossRef] [PubMed]
95. Prado, G.K.S.; Torrinha, K.C.; Cruz, R.E.; Goncalves, A.B.B.; Silva, C.A.V.; Oliveira, F.M.S.; Nunes, A.C.; Gomes, M.A.; Caliari,
M.V. Weissella paramesenteroides WpK4 ameliorate the experimental amoebic colitis by increasing the expression of MUC-2 and
the intestinal epithelial regeneration. J. Appl. Microbiol. 2020, 129, 1706–1719. [CrossRef] [PubMed]
96. Aguilar-Toala, J.E.; Garcia-Varela, R.; Garcia, H.S.; Mata-Haro, V.; Gonzalez-Cordova, A.F.; Vallejo-Cordoba, B.; Hernandez-
Mendoza, A. Postbiotics: An evolving term within the functional foods field. Trends Food Sci. Technol. 2018, 75, 105–114. [CrossRef]
97. Selma, M.V.; Beltran, D.; Garcia-Villalba, R.; Espin, J.C.; Tomas-Barberan, F.A. Description of urolithin production capacity from
ellagic acid of two human intestinal Gordonibacter species. Food Funct. 2014, 5, 1779–1784. [CrossRef] [PubMed]
98. Douglas, A.E. Which experimental systems should we use for human microbiome science? PLoS Biol. 2018, 16, e2005245.
[CrossRef] [PubMed]
99. Aguilar-Rojas, A.; Castellanos-Castro, S.; Matondo, M.; Gianetto, Q.G.; Varet, H.; Sismeiro, O.; Legendre, R.; Fernandes, J.;
Hardy, D.; Coppee, J.Y.; et al. Insights into amebiasis using a human 3D-intestinal model. Cell. Microbiol. 2020, 22, e13203.
[CrossRef] [PubMed]