Oxygen and Redox Reactions Contribute to the Protection of Free-Living and Parasite Helminths against Pathogens and/or Host Response

Abstract

Millions of years ago, the reductive atmosphere environment of Earth was replaced by an oxidative one because of redox reactions. These conditions allowed aerobic organisms to populate the planet and control the toxicity of oxygen. Aerobic organisms began to produce reactive oxygen species (ROS) via oxygen redox reactions and used them for their physiology process. Free-living helminths appeared in the early Paleozoic era and parasite helminths in the late same era. Free-living helminths, such as Caenorhabditis elegans and earthworms, have been used as host models to understand their micro pathogen defenses, particularly those associated with ROS. We speculate that the micro pathogens of C. elegans are equivalent to the parasite helminth hosts in terms of generating a worm’s defense response. Therefore, parasite helminths may share similar defense mechanisms to humans, as in C. elegans. This last observation suggests the existence of a conservative pathogen protection process for centuries. This review discusses the evolution of oxygen molecules and redox reactions, as well as of the Earth’s atmosphere, and changes over time in the protection of helminths mechanisms. These mechanisms have been so successful that have improved our understanding and have had a positive impact on humans.

1. Introduction

There are two types of helminths: free-living and parasitic helminths. For decades, free-living helminths have been used as models in studies on mechanisms used to survive against the pathogenic effects of micro pathogens. Because of the evolutionary link between free-living helminth defenses and human innate immunity, this research is highly relevant to humans [1].

On the other hand, little is known about the micro pathogens that affect animal helminth parasites, particularly in the adult form, despite coexisting with large numbers of microorganisms in the intestine of their host. This gap in our understanding is problematic because of the damage that helminth parasites can inflict on the health of their hosts, including humans and livestock.

Identifying the defense mechanism that helminth parasites use against their micro pathogens, as is known for free-living helminths, would be extremely useful. However, this is technically impossible, despite indirect information suggesting that helminth parasites develop defense mechanisms against micro pathogens as a result of the long periods of time they spend inside the intestine of their hosts [2].

One observation relevant to helminth parasites, in relation to the defense mechanisms used by free-living helminths, is that of aerobic organism conditions. Under these conditions, free-living helminths survive against their micro pathogens using in some situations the toxic capacity of the oxygen molecule to induce oxidative stress [3].

The defense mechanisms of helminths against micro pathogens are important in the study of the evolution of helminths from their ancient origins to the modern day. Understanding these mechanisms will provide insights into oxidative mechanisms and reduction-oxidation reactions (redox) more generally, both of which are chemical events present in the defense mechanisms of any pathogen.

2. The origin of helminths

Helminths are free-living parasitic invertebrate metazoan organisms. They include nematodes (round worms), trematodes (flukes), cestodes (tapeworms), and acanthocephalans (thorny-headed worms). The fossil record provides evidence that ectoparasitic helminths (e.g., worm-like pentastomid arthropods) have existed since the early Paleozoic era (542–444 million years (My)), while endoparasitic helminths (cestodes) arose during, or possibly even before, the late Paleozoic era (416–251 My) [4]. Therefore, the origins of helminths, all from free-living and parasitic organisms, were derived from a world in which the atmospheric conditions were initially reductive before transforming to oxidative [5].

3. The “rusting” of the Earth

The amount of oxygen (O₂) in the atmosphere before the Paleozoic era was at levels <0.001% of those present in the atmosphere today. However, during the Paleozoic and after this era, free oxygen was spawned by cyanobacteria producing land releasing it as a by-product of photosynthesis [6], causing the Great Oxidation Event (GOE), which dramatically changed the composition of the Earth’s life forms and led to the near extinction of anaerobic organisms. The GOE is believed to have input sufficient oxygen into the atmosphere to allow for the evolution of animal respiration Figure 1.

Figure 1.

Earth atmosphere modification and consequences on living organisms. Cyanobacteria are associated with the Great Oxidation Event (GOE) on Earth. Then redox reactions contribute to the development of reactive oxygen, nitrogen, and sulfur species (ROS, RNS, RSS). High concentrations of these are avoided through glutathione (GSH)/thioredoxin (TrxR) systems, but low species concentrations are necessary for signal transduction pathway cells to control gene expressions.

On the other hand, if cyanobacteria were fundamental for the “rusting” of the Earth, redox reactions (electron transfer mechanism or redox) would still be relevant, in particular for the physiology of aerobic organisms.

In the Hadean eon (4.6 billion years ago), redox reactions were a response to the large amounts of energy in the primitive Earth resulting from cosmic and geophysical reactions occurring at the time [7].

4. Energy flow theory

The energy flow theory proposed by Harold Morowitz is useful for explaining the origin of life [8]. In the primitive Earth, millions of reduction-oxidation reactions took place, one of which occurred between molecular hydrogen (reductor) and carbon dioxide (oxidant). This redox reaction was not spontaneous. Therefore, primitive organisms, such as helminths, acquired the skills needed to manage this reaction via enzyme catalysis. The citric acid or Krebs cycle is one such example. In addition to the citric acid cycle, aerobic organisms, such as helminths, developed a group of metabolic cycles to obtain their capacity to manage oxygen because of their dual contrasting molecular characteristics Figure 2.

Figure 2.

Redox throughout life’s evolution. Two different groups of molecules that originate redox reactions. Principally, metals, in the early Earth contributed to its oxidation. Redox enzymes in organism, including helminths, contributed to their homeostasis.

5. The contrasting oxygen molecule

As described previously, although molecular oxygen is vital for aerobic organisms, it is also a toxic mutagenic gas due to the production of intermediary oxygen molecules and reactive oxygen species (ROS) [9]. The toxicity of oxygen arises from its chemical electron acceptability by redox mechanisms, producing superoxide radicals (O•∙), hydrogen peroxide (H₂O₂), hydroxyl radicals (•OH), and singlet oxygen (O₂), also known as reactive oxygen species (ROS). When concentration of ROS exceeds the capacity of the cells’ defense systems, this results in the phenomenon of oxidative stress, which is characterized by an increase in the reduction potential or a large decrease in the reducing capacity of the cellular redox couples.

Oxidative stress is associated with damage to biological molecules. ROS can oxidize amino acid chains and cross-link proteins, as well as oxidize protein backbones. The highly reactive hydroxyl radical (•OH) reacts with DNA via the addition of double bonds of DNA bases and by the abstraction of a hydrogen atom from the methyl group of thymine and each of the C∙H bonds of 2-deoxyribose. Furthermore, ROS also induces the process of lipid peroxidation in lipoprotein particles or membranes, giving rise to a variety of products, including short chain aldehydes, such as malondialdehyde or 4-hydroxynonenal, alkanes, alkenes, conjugated dienes, and a variety of hydroxides and hydroperoxides.

One way to understand how oxidative stress works in free-living helminths is to appreciate the process by which these organisms can be affected by bacterial virulence. This observation is clear from the studies developed in C. elegans, which produced hundreds of mutant worms with enough different genes [10] and mutants for the study of all aspects of this organism. Therefore, this worm is an excellent host model for the study of the evolutionarily conserved mechanisms of microbial pathogens.

Based on this, microbes that cause diseases in mammalian hosts have also been shown to be important for diseases in C. elegans, and as a terrestrial microbiome, C. elegans can be fed not only with the auxotrophic Escherichia coli strain OP50, which is harmless to the worm, but also with a variety of pathogenic bacteria. This means that a way to understand how a worm can get a disease is just to see them without movement in culture in the presence of the pathogen bacteria. Specifically, it was observed that C. elegans was killed when the Pseudomonas aeruginosa PA14 strain or another pathogenic microbe was provided as a food source [11].

A historical summary of the major results obtained in the study of the C. elegans model is as follows: (a) Sifri et al. [12] identified C. elegans as a new, simple, and cheap model organism for the study the pathogenesis of the Gram-negative bacteria P. aeruginosa and Salmonella enterica serovar Typhimurium. (b) Garsin et al. [13] showed that the Gram-positive bacteria Enterococcus faecalis and Streptococcus pneumoniae kill C. elegans. (c) Hodgkin et al. [14] demonstrated that the genetically amenable nematode C. elegans is ideally suited to identify host factors. Therefore, the biological complementarity between C. elegans and pathogenic microorganisms is well suited for the study of bacterial virulence, not least because of the vast bacterial strains available for this aim.

6. How helminths, particularly C. elegans, are killed by pathogens

To answer this question, several research groups have developed nematode bacteria experimental systems. Their results can be grouped into five different mechanisms: (1) Colonization: The worm is killed slowly through an infection-like process, which correlates with the accumulation of bacteria within the worm’s intestine [15]. (2) Infection persistence: In this mechanism, contact between the worm and live bacterial cells is necessary as they accumulate in the intestinal tract of the animal host. Additionally, the proliferation of bacterial cells inside the worm intestine is also needed to establish a persistent infection. This mechanism suggests that some bacterial species may adhere to the intestinal receptors in worms [16]. (3) Invasive: Bacterial cells, such as S. enterica, use a type III secretion system to invade C. elegans [17]. In this worm, there are two major surfaces that act as nutritious bacterial interfaces: the first is at the chemosensory and olfactory neurons of the amphids, and the second is at the apical surface of intestinal cells. (4) Biofilms: These occur when bacteria form an obstructive matrix over the animal pharyngeal opening, which accumulates over time and prevents normal feeding and nutrition [18]. (5) Toxin-mediated killing: Bacterial pathogens kill worms via the production of diffusible toxins. Under these conditions, no bacterial but rather a soluble toxin is necessary to kill the worm [19]. For the first four mechanisms, direct contact with the pathogen microbes is necessary. The fifth mechanism requires that a soluble toxin reaches the worm. These mechanisms indicate that toxin-mediated killing associated with ROS products is feasible, as all redox reactions are developed under soluble conditions. For example, if the C. elegans mutants mev-1(kn1) and rad-8(mn163) are fed with the human opportunistic pathogen P. aeruginosa strain PA14, they will be killed. This is because P. aeruginosa secretes phenazines, and this organic compound exerts its toxic effect on C. elegans mutants undergoing redox cycling for ROS production.

7. How helminths defend themselves against pathogens

Although the mechanisms by which different bacteria affect the resistance of worm to pathogens are poorly understood, helminths have developed a number of different procedures to survive: (1) Behavioral defense: In this case, the worm detects olfactory stimuli, recognizes odors, and modifies its behavior by olfactory learning and imprinting [20]. (2) Barrier mechanism: The muscular pharynx grinder provides a physical barrier against pathogens, which protects them by disrupting the engulfed microbes [21]. (3) Production of soluble molecules: Examples of antimicrobial proteins and peptides in response to microbial infection [22]. (4) Direct inhibition of pathogens: Exerts a commensal-mediated protective effect on C. elegans [23]. (5) RNA interference: Orsay virus (OV) is a natural pathogen of C. elegans. The worm develops specific protection against this virus via the antiviral RNAi response. This mechanism not only inhibits vertical OV transmission, but also promotes transgenerational inheritance of antiviral immunity [24]. (6) Innate immunity involving signaling pathways: Specific responses that protect and repair against the collateral damage caused by ROS are critical for a successful attack against pathogens. Thus, there is a connection between the generation of ROS by Ce-Duox1/BLI-3 and the upregulation of a protective transcriptional response by SKN-1 [25]. (7) Oxidative damage: The secretion of ROS by gut epithelia [25]. ROS are known to be involved in tissue damage. This is because of the imbalance between the antioxidizing defenses of the organisms and the oxygen intermediaries produced in cells during aerobic metabolism and/or host inflammatory defenses against some pathogens.

NADPH oxidases, whose biological function lies in electron transport, are also a major source of ROS. These enzymes are multi-pass transmembrane proteins that catalyze the reduction of extracellular or luminal oxygen by intracellular NADPH to generate superoxide anions (O₂•) [26]. NADPH oxidases have been discovered in macrophages as a defense mechanism against pathogens, but today it is known that they are widely distributed in different kingdoms with multiple biological functions. The importance of these enzymes in aerobic organisms has led to the discovery of the NOX/DUOX family of NADPH oxidases, which includes three NOX subfamilies: ancestral type, NOX5-like, and DUOX [27]. DUOX isoforms that presumably developed from the NOX5-like subfamily are known as dual oxidases because they have both a peroxidase homology domain and a gp91phox domain. This last domain is the heme-binding subunit of the superoxide-generating NADPH oxidase, the catalytic moiety; thus, DUOXs produce anion superoxide (O₂•) and hydrogen peroxide (H₂O₂) by transferring one and two electrons, respectively, from intracellular NADPH to extracellular oxygen. DUOX is the only type of NOX present in C. elegans. The worm encodes two Duox genes (bli-3 and duox-2) that share an amino acid sequence with 94% identity to each other and approximately 30% to human Duox 1 and 2. It is known that C. elegans intestinal cells, like mammalian phagocytes, produce ROS, such as O₂• and H₂O₂, via DUOXs as an antimicrobial response [3].

Therefore, C. elegans may be able to exert lipid peroxidation in the lipid membrane of micro pathogens in an effort to kill them, as has been described in prokaryotes and other eukaryote parasite-host relationships in the past.

8. The importance of lipid peroxidation

Lipid peroxidation comprises a chain of reactions involving the oxidative degradation of lipids. It is the process in which free radicals, such as O₂•, “steal” electrons from the lipids in cell membranes, resulting in cell damage. This process evolved from a free radical chain reaction mechanism, which comprised three steps: initiation, propagation, and termination. In the first step, O₂• interacts with polysaturated fatty acids. This O₂• is dismuted by superoxide dismutase, and in addition to hydrogen atoms, it breaks down into ordinary molecular oxygen and H₂O₂. Then, H₂O₂ in the presence of Fe²⁺ produces hydroxyl anions (OH•) via the Fenton reaction. The OH• takes away allylic hydrogens from the polyunsaturated fatty acid chains to obtain a radical carbon (L•). Then, the easy reaction with oxygen molecules by L• gives rise to the peroxyl radical (LOO•). When hydrogens are removed from polyunsaturated fatty acid neighbors, this LOO• results in the formation of lipid hydroperoxide (LOOH). The propagation step occurs when LOO• interacts with other polyunsaturated fatty acids, resulting in the formation of further lipid radicals and H₂O₂. Additionally, the catalysis of H₂O₂ by Fe²⁺ makes results in the formation of alkoxy and peroxy radicals during propagation step, with this secondary free radical production beginning another lipid hydrogen peroxide chain. Termination occurs when two radicals are conjugated, the result of which is a non-radical product.

The C. elegans model provides an opportunity to gain insights into how free-living helminths and parasite helminths exert this strategy to protect themselves against oxidative stress, even if the ROS are self-produced, as in C. elegans, or by the host response, as in human parasite helminths. In addition, the concept of lipid peroxidation can be explained practically since lipid peroxidation starts with the same oxidative molecules in any organism.

Due the short life of C. elegans compared with humans, the exertion of lipid peroxidation against micro pathogens could be considered an acute response, if a short one, as it is sufficient to damage the worm’s own tissues. In other words, collateral damage can result from the processes by which worms are trying to kill micro pathogens. In human helminthiasis is a rule to see the development of chronic infections besides lipid peroxidation.

9. DUOX/NADPH oxidases in toxicity and signal transduction

C. elegans is known to express antioxidant genes for protection against its auto-oxidative response, as described previously [28]. Human helminth parasites may also exert similar procedures for protection against oxidative stress. Therefore, understanding how C. elegans resist their own protective oxidative response could provide insights into how helminthiasis chronicity evolved in humans.

In this sense, Hoeven et al. [25] found that aerobic organism evolution works in a balanced dualism. For example, when the Earth’s atmosphere became oxidant, living forms, including older forms of free-living helminths, developed an extremely complex cellular signal mechanism to manage oxygen toxicity. This permitted them to kill their adversary while surviving the collateral damage at the same time; this strategy is very clever and clearly observed in C. elegans. Upon exposure to P. aeruginosa and E. faecalis, C. elegans uses DUOX/1BLI-3 to kill pathogens by producing ROS. In addition, as DUOX/1BLI-3 is activated to kill pathogens found in the intestine of helminths, SKN-1 (Skinhead family member 1) transcription factor, a member of the Cap’n’collar (CNC) protein family, is simultaneously activated to avoid tissue damage [29].

Transcription factors belonging to this group of proteins play a crucial role in protecting cells against oxidative stress. Under physiological conditions, they remain in the cytoplasm in the inactive form or are degraded. However, under oxidative stress conditions, they are translocated to the nucleus and bind to DNA in the antioxidant response element (ARE) motif. Consequently, genes encoding cytoprotective proteins, such as low-molecular-weight antioxidant proteins (i.e., thioredoxin, ferritin, and metallothionein), responsible for protecting cells against the action of ROS, are transcribed. C. elegans SKN-1 has been extensively studied, with studies finding that this transcription factor is orthologous with the nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2). They are both members of the CNC subfamily of the basic leucine zipper (bZip) transcription factors [30].

Both transcription factors are highly conserved proteins with functions similar to those of the promoters of oxidative-stress-related genes. In fact, Nrf2 and SKN-1 regulate phase II detoxification genes needed to defend against oxidative stress and electrophilic xenobiotics. With this detoxification system, worms can solubilize lipophilic xenobiotics or endobiotics via cytochrome P450s (CYPs) and short-chain dehydrogenases (SDHs), two classic enzymes of the phase I detoxification step. Reactive products, including ROS originating from the original toxic molecules, are detoxified, either via metabolization or conjugation, by the phase II system using UDP-glucuronosyl/glucosyl transferases (UDP) or glutathione transferases (GSTs), among others. Afterward, conjugated toxins are eliminated from cells by phase III proteins, including ATP-binding cassette (ABC) and other transporters.

Thus, similar to Nrf2, SKN-1 controls many critical detoxification processes directly as glutathione transferase enzymes (GSTs).

10. Glutathione transferases (GST) and their importance in detoxification

From an evolutionary point of view, these enzymes emerged over two billion years ago. Based on structural and functional criteria, they can be grouped into four different families: cytoplasmic, microsomal, mitochondrial, and bacterial.

Glutathione transferases are ubiquitous in prokaryotes and eukaryotes, indicating their protective and functional importance. These transferases are a large superfamily of supergene isoenzymes that play important roles in cell detoxification. These enzymes use electrophiles to catalyze the nucleophilic addition of the thiol of reduced glutathione (l-g-glutamyl-l-cysteinyl-glycine) (GSH) to electrophilic centers in organic compounds. The resulting glutathione conjugates are rendered more water-soluble to facilitate their eventual elimination. A wide variety of endogenous (e.g., by-products of reactive oxygen species activity) and exogenous (e.g., polycyclic aromatic hydrocarbons) electrophilic substrates have been identified. In addition, the detoxification functions of these enzymes have been observed not only in one but two mechanisms: passive detoxification and active detoxification. The former, as mentioned by Kostaropoulos et al. [31], refers to a detoxification mechanism characterized by an absence of catalytic function, such as the binding of potentially toxic non-substrate ligands, including porphyrins and lipid peroxides. In fact, GSTs were originally named “ligandins” due to their passive role in detoxification.

Ligandin activity exhibited by GST isoforms was first suggested as a result of the observed affinity for bilirubin, an azo dye carcinogen, and a metabolite of cortisone. The second mechanism was developed by catalytic activity, as described previously Table 1.

Ligand	I50 (mM)	Ki (mM)	%F (mM)
Mesoporphyrin	00.0012–0.1	15, 30, 45	0.0003–0.014
Prothoporphyrin	0.002–0.064	12, 24, 36	0.0012–0.027
Coproporphyrin	0.0005–0.005	0.0015, 0.0045	0.0002–0.014
Hematin	0.0007–0.012	0.002, 0.004	0.00012–0.003
trans,trans-2,4-hexadienal	0.0001–10	1.5, 3	0.003–1.9
trans,trans-2,4-nonadienal	0.0001–10	0.45, 0.9	0.0016–0.35
trans,trans-2,4-decadienal	0.0001–10	0.01, 0.1	0.0016–2.6
Arachidic acid	0.001–0.25	ND	0.0016–0.2
Palmitic acid	0.0001–1	ND	0.0016–0.27
Cholic acid	0.0001–1	ND	0.003–0.045
Chenodeoxycholic acid	0.0001–0.2	ND	0.001–0.011
Lithocholic acid	0.0001–1	ND	0.025–0 .2

Table 1.

Conditions for inhibition Ts26GST catalytic activity and spectrofluorometric assays.

I₅₀, is a parameter giving the inhibitory concentration causing 50% inhibition; Ki is the inhibition catalytic constant value; %F, is the % of Ts26GST intrinsic fluorescence quenching; ND, not determined. (Exp. Par. 2014;138:63-70).

Glutathione transferases in cestodes were identified several years ago. Initially, these cestode transferase isoforms were associated with the detoxified procedures in several organisms, including C. elegans [32, 33]. However, because almost all GSTs have GSH as a nucleophilic substrate, and this is the central redox agent of most aerobic organisms, GST functions encompass other purposes, as reported by Ferguson and Bridge in the C. elegans model [34].

11. The redox couples in enzyme functions

As mentioned before, the reduced form of glutathione (GSH) serves as a ubiquitous nucleophile for the conversion of a variety of electrophilic substances under physiological conditions. This is possible when GSH is oxidized to glutathione disulfide (GSSG) by a reaction that involves the transfer of electrons between two species; in other words, when it is affected by the redox reaction.

GSH/GSSG is an example of millions of redox couples that are chemically similar or different, present in cells, organs, tissues, biological fluids, and cell organelles. A considerable number of these redox couples could be linked to each other to form a set of related redox couples, or redox couples that work independently. These reactions are achieved by capturing the energy released via oxidation to build cellular and organismic structures, maintain these structures (some avoid pathogenic action), and provide energy for the processes they support.

The production of a large number of redox couples in aerobic organisms occurs by enzymes and proteins of the glutaredoxin and thioredoxin systems, the former using GSH and the latter thioredoxin (Trx) [35].

12. The glutaredoxin and thioredoxin systems

The glutaredoxin system is composed of glutathione reductase (GR or GSR), glutathione (GSH), and glutaredoxin (Grx), while the thioredoxin system comprises thioredoxin reductase (TrxR) and thioredoxin (Trx). The glutaredoxin and thioredoxin systems are likely to have evolved very early in aerobic organisms. Owing to the cysteine moiety of GSH, the entire system is based on common sulfur biochemistry. Therefore, it requires an electron relay, linking the universal reducing agent NADPH to thiol/disulfide metabolism, and a thiol-containing adapter molecule (GSH, which is considered as a universal adaptor) to transfer electrons to a set of different acceptors, such as flavoproteins, which are widely used as electron relays.

Hence, it is not surprising that the reducing equivalents from NADPH enter the glutathione system either with the help of the FAD-dependent enzyme glutathione reductase (GR) or the thioredoxin reductase/thioredoxin couple (TrxR/Trx).

Glutaredoxin protein (Grx) was first described in crude enzyme preparations from beef liver by Racker [36] in 1955. Grxs are small (12–18 kDa) GSH-disulfide oxidoreductase members of the thioredoxin family, which includes the cytosolic (Grx1) and mitochondrial (Grx2) isoforms. Oxidized Grxs are reduced by GSH. According to its active site domain, Grxs are classified as dithiols (CPY/FC motif) and monothiols (CGFS motif), wherein monothiols can contain single or multiple monothiol Grx domains. Dithiol Grxs regulate the redox state of various proteins by catalyzing the reversible reduction of oxidized disulfides. For this purpose, Grxs use both cysteine residues from their active sites. In contrast, the monothiol Grxs reduce mixtures of disulfides (glutathionylation) formed between GSH and the thiols of proteins or other small compounds, using the cysteine residues from the active sites in their amino terminals.

With regard to the glutaredoxin genes of C. elegans, five have been annotated: glrx-3, glrx-5, glrx-10, glrx-21, and glrx-22 [37]. Except for glrx-5, which is predicted to be a mitochondrial glutaredoxin, the other annotated glutaredoxins are expected to be found in the cytosol. Based on phylogenetic analysis, the C. elegans GLRX-3 isoform has been postulated to be an ortholog of the mammalian GLRX3 protein kinase C-interacting cousin of thioredoxin (PICOT), suggesting that it exerts a protective mechanism against DNA-damage-inducing agents, such as some micro pathogens, by acting as an upstream positive regulator of ATR-dependent signaling pathways. On the other hand, a recent report demonstrated that C. elegans exhibits changes in the protein S-thiolation patterns (i.e., S-glutathionylation and S-cysteinylation) of targeted cysteine residues. This evidence suggests that glutaredoxins may provide an evolutionarily conserved mechanism for the catalysis of the reversal of S-glutathionylation, preventing the irreversible oxidation of protein thiols in C. elegans derived from micro pathogens.

Recently, the human and pig helminth parasite, Taenia solium, was cloned, expressed, and characterized for the first time as glutaredoxin (r-TsGrx1) [38]. The full-length DNA of the TsGrx1 gene comprised one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. Conventional enzymes, such as tioredoxin reductase (TrxR) and glutathione reductases (GR or GSR), do not exist in Theridion solium. However, the presence of a protein hybrid tioredoxin glutathione reductase (TGR), with tioredoxin and glutathione reductase activity, as described below, makes it possible for the components of Trx and Grx systems in platyhelminth (flatworms) to work as observed in organisms that have independent enzymes for those functions.

Protein S-glutathionylation by glutaredoxins is a widely distributed posttranslational modification of thiol groups with glutathione, which can function as a redox-sensitive switch to mediate redox regulation and signal transduction. Therefore, the presence of Grxs in C. elegans and T. solium contributes to our understanding of the micro pathogen activation of redox-regulatory processes in these helminths.

GR (also termed GSR, as mentioned before) is a flavoenzyme of the pyridine nucleotide-disulfide oxidoreductase family (EC 1.6.4.2, now 1.8.1.7). This enzyme recycles reduced GSH from its oxidized form GSSG. However, this function was also developed for the thioredoxin system acting as a backup, a trait that is conserved from bacteria to mammals, highlighting its physiological relevance, including protection against toxicity, in both systems.

Glutathione reductase is a GR-isoform from prokaryote and eukaryotes that form stable homodimers of ~110 kDa. From a structural point of view, each subunit is organized into four domains (FAD binding, NADPH binding, central, and interface) and possesses an N-terminal flexible segment of 18 amino acids with a cysteine residue at position 2.

In C. elegans, the gsr-1 gene encodes the GSR enzyme, which produces two protein isoforms (GSR-1a and GSR-1b) [39], and its expression of GSR-1 is modulated by the SKN-1 transcription factor.

The GSR-1 gene is vital in C. elegans because it supports its embryonic development, and there is no alternative molecule for this purpose. In many organisms, the thioredoxin system exerts GSSG reduction in the absence of GSR. As described previously, this appears not to be the case for C. elegans, even though both systems have been shown to cooperate in other processes, such as worm molting.

Therefore, the C. elegans thioredoxin and glutaredoxin systems share common functions but also have specific non-overlapping roles in worm physiology. The thioredoxin system was first recognized in the early 1960s as a reductant of methionine sulfoxide and PAPS (3 H-phos- phoadenosine-5 H-phosphosulfate) in yeast and ribonucleotides in E. coli.

Thioredoxin reductase (EC 1.6.4.5) (TrxR) was originally identified in E. coli as part of the ribonucleoside diphosphate reductase system. TrxR catalyzes the reversible electron transfer from NADPH to oxidized thioredoxin (thioredoxin-S2), a 12 kDa protein containing a single oxidation-reduction active disulfide bond. The oxidation of NADPH leads to the formation of the reduced form of thioredoxin (thioredoxin SH2), which has a dithiol. TrxR directly reduces not only Trx from different species but also many non-disulfide substrates, such as selenite lipid hydroperoxides, although not glutathione disulfide (GSSG).

The C. elegans genome encodes two thioredoxin reductases: thioredoxin reductase-1 (TRXR-1), which is the sole selenoprotein in C. elegans, with a UGA-encoded Sec in the C-terminal active site, and thioredoxin reductase (TRXR-2), a homolog of a UGU-encoded cysteine substitution for Sec. TRXR-1, the cytosolic TrxR, is required for the acidification of the lysosomal compartment in the intestine, whereas TRX-2, the mitochondrial TrxR, is critical for producing stress conditions. Interestingly, the gene expression of both TRX-1 and TRX-2 is induced by heat shock, which results in the production of ROS. Both observations suggest the involvement of these TrxRs in protection against micro pathogens found in the intestine of the worm [40].

Tioredoxin (Trx), the major TrxR substrate, as mentioned previously, is a disulfide reductase with a molecular weight of approximately 12 kDa and has two cysteine residues in its consensus sequence (CGPC motif). When chemically reduced, this allows for the transfer of reducing equivalents to a wide variety of substrates, such as H₂O₂. Thus, Trxs can, either directly or via 2-Cys peroxidases, catalyze the reduction of hydrogen peroxide (H₂O₂) to water and lipid hydroperoxides (R∙O∙O∙R) to alcohols in the cell. Trxs can also inhibit and/or activate transcription factors related to immune responses in mammals. For example, the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) is inhibited when TRX1 prevents the release of IkB, an inhibitor of NF-κB.

Although the thioredoxin and glutaredoxin systems are vital for aerobic organisms, in platyhelminths (flatworms), both GR and TrxR are missing in their tissues. Instead of these proteins, some platyhelminths have a GR and TrxR molecular link exhibiting the fusion of glutaredoxin (Grx) and thioredoxin reductase (TrxR) domains into a single protein, a selenocysteine-containing enzyme that acts as a thioredoxin glutathione reductase (TGR) [41, 42].

Thus, TGR plays a central role in thiol-disulfide redox reactions by providing electrons to essential detoxification enzymes, such as GR and Prx. GR reduces the tripeptide GSSG to GSH, which acts as the main reducing agent in the catalytic functions displayed by GSTs [43].

Because conventional TrxR and GR are functional in C. elegans, no TGR is found in this worm. However, in platyhelminths, TGRs exert an efficient antioxidant defense against lipid peroxidation metabolites. In addition to the essential detoxification function of TGRs in flatworm parasites, as described above, it is necessary to include the TGR activities associated with the Grx domain, such as the deglutathionylase activity of GSH-protein mixed disulfides (protein-S-SG). Protein glutathionylation is the mechanism by which protein-SH groups form mixed disulfides with glutathione to avoid protein-SH group oxidation. In addition, TGRs play essential roles in redox cell signaling and sensing. Cell signaling transduction is the mechanism by which external stimuli are transferred to their inner compartments, resulting in the activation or inhibition of genes; cell sensing is the oxidative modification of protein cysteines with consequent events, such as changes in its activities and interactions with other biomacromolecules, such as native immunity receptors.

Haemonchus contortus has been used as an excellent model to gain insights into the oxidative defenses of hosts against nematodes [44]. H. contortus is a blood-sucking nematode parasite of the abomasum of small ruminants that causes a disease known as haemonchosis, in which the abomasal epithelium and highly toxic heme molecules are released [45]. Free heme catalyzes the formation of cytotoxic lipid peroxides via lipid peroxidation using hydrogen peroxide (H₂O₂) as Fenton reaction.

The range of antioxidant enzyme systems available to H. contortus for the detoxification of H₂O₂ has been investigated using molecular biology tools. As a result, full-length sequences were obtained for a 2-Cys peroxiredoxin (Prx), a catalase, and a selenium-independent glutathione peroxidase (GPx), indicating that H. contortus expresses several antioxidant systems with the potential to detoxify peroxide, most of them within the host’s immune response. Other studies identified additional three thioredoxin reductases (TrxRs) (HcTrxR-1, HcTrxR-2, and HcTrxR-3), two mitochondrial thioredoxins (HcTrx-1, HcTrx-5), and one cytosolic (Trx-3) thioredoxin (Trx), increasing the possible mechanisms of H. contortus detoxification. All the abovementioned detoxification enzymes and proteins, except catalase, work closely with the two major detoxification and redox systems in animal cells: thioredoxin (Trx) and glutathione (GSH) [46].

Interestingly, for the first time in the study of the TGR system [47], HcTrxR3 was found to catalyze the direct reduction of GSSG, the specific substrate for GR, in the same catalytic range as that of any GR. Its affinity for GSSG, measured as Km value, was higher than that of the 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) substrate for TrxR, demonstrating its preference for the GSSG substrate. Until now, no TrxR has been identified that is able to directly reduce GSSG.

This GR activity from HcTrxR3 is important not only because the enzyme is a TrxR, but also because information on the presence of GR in the H. contortus tissues is lacking thus far Figure 3.

Figure 3.

Kinetic evidences of TR and GR activity from HcTrxR3. (A) Reduction of ebselen by NADPH catalyzed by HcTrxR3 produced ebselen diselenide and ebselen selenol. To 1 ml solutions containing 50 mM Tris-Cl, 1 mM EDTA, pH 7.5, 100 mM NADPH, and 0.1 mM ebselen, 2 μg (▪) or 4 μg (•) HcTrxR3, was added, and A₃₄₀ was measured against a blank without ebselen (∆). Ebselen reduction was shown when absorbance decreased followed by ebselen selenol formation in the highest enzyme concentration. (B) Effect of NADP⁺ on the glutathione reductase activities of HcTrxR3. IC₅₀ plots were obtained; an enzyme aliquot (about 2 μg) was pre-incubated at 25°C in the presence of 100 μM NADPH and different concentrations of NADP⁺. To start the reaction GSSG at a final concentration of 0.2 mM was added. (C) Show a competitive type inhibition where the 1/v versus 1/[NADPH⁺] plot of initial velocities HcTrxR3 activity in absence (◆) and the presence of 0.1 mM (•) and 0.5 mM (▪) of NADP⁺ with various concentrations of NADPH⁺ (0.01–10 μM). Inset shows secondary plot of the slope values derived from the primary 1/v versus 1/[NADPH⁺] plot versus NADP⁺ concentration for the determination of Ki [47].

13. The models of detoxified and innate immunity in pioneer earthworms

In addition to being essential for soil fertility, earthworms are also an excellent model for the study of the protection mechanisms used by helminths against micro pathogens [48], as in C. elegans.

Earthworms are terrestrial invertebrates belonging to the order Oligochaeta, class Chaetopoda, and phylum Annelidae. They range in size from a fraction of a centimeter to exceptional individuals of Megascolides australis, which can measure up to 2.75 m in length and 3 cm in diameter. Approximately 1800 species are distributed all over the world.

Earthworms became a model for comparative immunology in the early 1960s with the publication of results from transplantation experiments that proved the existence of self/non-self-recognition in earthworms. This initiated extensive studies on the immune mechanisms of earthworms, which evolved to prevent invasion by pathogens. In recent decades, important cellular and humoral pathways have been discovered, and numerous biologically active compounds have been characterized and cloned [49].

For example, earthworm coelomocytes (macrophage-like cells) are part of the cellular immune response and are both morphologically and functionally analogous to vertebrate phagocytes. Coelomocyte subpopulations (named as hyaline-, granular amoebocytes, and eleocytes) possess distinct functions, such as phagocytosis, encapsulation, and cellular cytotoxicity.

Additionally, phagocytic defense by the earthworm Eisenia foetida against certain pathogenic bacteria has been found to be aided by bacteriostatic or bactericidal substances, which may also play an opsonic role, as in vertebrates. Therefore, given the molecular tools of earthworm coelomocytes, there is a possibility that these organisms also use a functional NOX/DUOX system to eliminate invading micro pathogens via ROS production, as in C. elegans.

14. Conclusion

The invertebrate research model has been used to reveal the evolutive link between the oxygen atmosphere and the adaptability of helminths to aggressive environments. These organisms have been found to use oxygen molecules and redox reactions to exert protective effects against micro pathogens. Helminth models have also revealed similarities between the cells, molecules, and mechanisms of helminths and those of human components used against pathogens, highlighting the evolutionary success of these molecules, structures, and biological procedures. Thus, this review shows how understanding the mechanisms by which invertebrates manage their environmental adaptability can provide insights into how humans protect themselves against their own pathogens. Honey bees are another example of this idea, in which individuals are protected against micro pathogens via the concept of social immunity [50].

Acknowledgments

We thank Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT), UNAM, IN209819.