Haemonchus contortus: HcGluCla expressed in Xenopus oocytes forms a glutamate-gated ion channel that is activated by ibotenate and the antiparasitic drug ivermectin

Ion channels are targets for many drugs including insecticides and anthelminthic agents such as ivermectin (IVM) and moxidectin (MOX). IVM has been shown to activate glutamate-gated chloride channels (GluCls) from the free-living nematode, Caenorhabditis elegans. Haemonchus contortus is a parasitic nematode that is also extremely sensitive to IVM. The high sensitivity of H. contortus to IVM is probably the result of the fact that, like C. elegans, H. contortus also express GluCls. To investigate the potential physiological response to IVM in H. contortus we have expressed a GluCl from this parasite (H. contortus glutamate-gated chloride channel, HcGluCla) in Xenopus oocytes. HcGluCla expressed in oocytes formed a homomeric channel that responded to glutamate and ibotenate as well as the anthelmintics IVM and MOX. The response to glutamate and ibotenate was fast acting and reversible whereas the response to IVM and MOX was a slower activating channel that was essentially irreversible. These results suggest that IVM toxicity in H. contortus is the result of its irreversible activation of GluCls.     

Cytotoxicity of hydrogen peroxide produced by Enterococcus faecium

Although the opportunistic bacterial pathogen Enterococcus faecium is a leading source of nosocomial infections, it appears to lack many of the overt virulence factors produced by other bacterial pathogens, and the underlying mechanism of pathogenesis is not clear. Using E. faecium-mediated killing of the nematode worm Caenorhabditis elegans as an indicator of toxicity, we determined that E. faecium produces hydrogen peroxide at levels that cause cellular damage. We identified E. faecium transposon insertion mutants with altered C. elegans killing activity, and these mutants were altered in hydrogen peroxide production. Mutation of an NADH oxidase-encoding gene eliminated nearly all NADH oxidase activity and reduced hydrogen peroxide production. Mutation of an NADH peroxidase-encoding gene resulted in the enhanced accumulation of hydrogen peroxide. E. faecium is able to produce hydrogen peroxide by using glycerol-3-phosphate oxidase, and addition of glycerol to the culture medium enhanced the killing of C. elegans. Conversely, addition of glucose, which leads to the down-regulation of glycerol metabolism, prevented both C. elegans killing and hydrogen peroxide production. Lastly, detoxification of hydrogen peroxide either by exogenously added catalase or by a C. elegans transgenic strain overproducing catalase prevented E. faecium-mediated killing. These results suggest that hydrogen peroxide produced by E. faecium has cytotoxic effects and highlight the utility of C. elegans pathogenicity models for identifying bacterial virulence factors.     

Peroxides and peroxide-degrading enzymes in the thyroid

Iodination of thyroglobulin is the key step of thyroid hormone biosynthesis. It is catalyzed by thyroid peroxidase and occurs within the follicular space at the apical plasma membrane. Hydrogen peroxide produced by thyrocytes as an oxidant for iodide may compromise cellular and genomic integrity of the surrounding cells, unless these are sufficiently protected by peroxidases. Thus, peroxidases play two opposing roles in thyroid biology. Both aspects of peroxide biology in the thyroid are separated in space and time and respond to the different physiological states of the thyrocytes. Redox-protective peroxidases in the thyroid are peroxiredoxins, glutathione peroxidases, and catalase. Glutathione peroxidases are selenoenzymes, whereas selenium-independent peroxiredoxins are functionally linked to the selenoenzymes of the thioredoxin reductase family through their thioredoxin cofactors. Thus, selenium impacts directly and indirectly on protective enzymes in the thyroid, a link that has been supported by animal experiments and clinical observations. In view of this relationship, it is remarkable that rather little is known about selenoprotein expression and their potential functional roles in the thyroid. Moreover, selenium-dependent and -independent peroxidases have rarely been examined in the same studies. Therefore, we review the relevant literature and present expression data of both selenium-dependent and -independent peroxidases in the murine thyroid.     

Cuticle collagen genes of Haemonchus contortus and Caenorhabditis elegans are highly conserved

Several genes and partial cDNAs encoding cuticle collagens have been isolated from the sheep parasitic nematode Haemonchus contortus. DNA sequencing and Southern blot hybridization studies reveal that H. contortus collagens comprise a large family of related, but non-identical genes. The genes appear to be dispersed throughout the genome. The predominant size of collagen mRNA in molting worms was found to be between 1.0 and 1.2 kb. The one complete gene that was sequenced contains two short introns and encodes a protein of about 300 amino acids. The predicted protein sequence contain several (Gly-X-Y)n triple helix-coding domains that are interrupted by short stretches of non-helix-coding amino acids. The size of the predicted protein and the organization of the triple-helix coding domains are similar to that of Caenorhabditis elegans collagens. All the H. contortus genes studied show a striking homology to the C. elegans collagen gene subfamily represented by col-1. In particular, the amino acid sequence of the carboxy-terminal non-(Gly-X-Y)n region and the positions of cysteine residues flanking the (Gly-X-Y)n domains were found to be highly conserved in the collagens of these two nematodes.     

The Haemonchus contortus UNC-49B subunit possesses the residues required for GABA sensitivity in homomeric and heteromeric channels

Hco-UNC-49 is a GABA receptor from the parasitic nematode Haemonchus contortus that has a relatively low overall sequence similarity to vertebrate GABA receptors but is very similar to the UNC-49 receptor found in the free living nematode Caenorhabditis elegans. While the nematode receptors do share >80% sequence similarity they exhibit different sensitivities to GABA. In addition, the UNC-49C subunit appears to be a positive modulator of GABA sensitivity in the H. contortus heteromeric channel, but is a negative modulator in the C. elegans heteromeric channel. The cause(s) of these differences is currently unknown since the structural elements essential for GABA sensitivity in nematode receptors have been largely unexplored. Thus, the overall aim of this study was to investigate the residues that are important for UNC-49 receptor sensitivity through the use of homology modeling, site-directed mutagenesis, and two-electrode voltage clamp. This study revealed that Met(170) in Loop B of the GABA binding-site may partially account for the observed differences in GABA receptor sensitivity between the nematode species. Residues in Loops A-D that have been reported to form the GABA binding pocket in mammalian receptors, including those forming the conserved 'aromatic box', also appear to play analogous roles in Hco-UNC-49. In addition, the two mutations that produced the most significant reduction in GABA sensitivity were R66S and Y166S. Homology modeling indicates that these two residues share a hydrogen bond and are positioned close to the carboxyl end of the GABA molecule. However, of residues examined in this study, only those on the Hco-UNC-49B subunit and not its subunit partner, Hco-UNC-49C, appear important for GABA sensitivity. Overall, results from this study suggest that the binding site of the UNC-49 heteromeric GABA receptor exhibits some differences compared to classical vertebrate GABA(A) receptors.     

Thiol chemistry in peroxidase catalysis and redox signaling

The oxidation chemistry of thiols and disulfides of biologic relevance is described. The review focuses on the interaction and kinetics of hydrogen peroxide with low-molecular-weight thiols and protein thiols and, in particular, on sulfenic acid groups, which are recognized as key intermediates in several thiol oxidation processes. In particular, sulfenic and selenenic acids are formed during the catalytic cycle of peroxiredoxins and glutathione peroxidases, respectively. In turn, these enzymes are in close redox communication with the thioredoxin and glutathione systems, which are the major controllers of the thiol redox state. Oxidants formed in the cell originate from several different sources, but the major producers are NADPH oxidases and mitochondria. However, a different role of the oxygen species produced by these sources is apparent as oxidants derived from NADPH oxidase are involved mainly in signaling processes, whereas those produced by mitochondria induce cell death in pathways including also the thioredoxin system, presently considered an important target for cancer chemotherapy.     

Binding of hematin by a new class of glutathione transferase from the blood-feeding parasitic nematode Haemonchus contortus

The phase II detoxification system glutathione transferase (GST) is associated with the establishment of parasitic nematode infections within the gastrointestinal environment of the mammalian host. We report the functional analysis of a GST from an important worldwide parasitic nematode of small ruminants, Haemonchus contortus. This GST shows limited activity with a range of classical GST substrates but effectively binds hematin. The high-affinity binding site for hematin was not present in the GST showing the most identity, CE07055 from the free-living nematode Caenorhabditis elegans. This finding suggests that the high-affinity binding of hematin may represent a parasite adaptation to blood or tissue feeding from the host.     

Is overoxidation of peroxiredoxin physiologically significant?

Eukaryotic peroxiredoxins are highly susceptible to sulfinic acid formation. This overoxidation, which is thought to convert peroxiredoxins into chaperones, can be reversed by sulfiredoxins. Several organisms, including Caenorhabditis elegans, lack sulfiredoxins but encode sestrins, proteins proposed to be functionally equivalent. We induced peroxiredoxin overoxidation in C. elegans with a short peroxide pulse. We found that reduction of overoxidized peroxiredoxin 2 (PRDX-2) was extremely slow and sestrin-independent, strongly implying that worms lack an efficient repair system. Analysis of PRDX-2's overoxidation status during C. elegans lifespan revealed no accumulation of overoxidized PRDX-2 at any point, questioning whether PRDX-2 overoxidation in worms is physiologically relevant.     

Characterisation of a polymorphic Tc1-like transposable element of the parasitic nematode Haemonchus contortus.

Hctc1, a member of the Tc1-family of transposable elements was isolated from the parasitic nematode Haemonchus contortus. Hctc1 is 1590 bp long, is flanked by 55 bp inverted repeats and carries a single open reading frame of a 340 amino acid transposase-like protein. Hctc1 is similar to Tc1 of Caenorhabditis elegans and elements Tcb1 and Tcb2 of Caenorhabditis briggsae in the inverted terminal repeats, the open reading frame, as well as the target insertion sequence. Furthermore, the copy number of Hctc1 is comparable with the Tc1 copy number in low copy strains of C. elegans. The sequence of Hctc1 is highly variable in H. contortus due to deletions, insertions and point mutations, with at least five distinct length variants of Hctc1. Most of the Hctc1 variation was within rather than between H. contortus populations. The high level of sequence variation is probably due to variation generally found for members of the Tc1-family, as well as a high background level of genetic variation of H. contortus.     

A cathepsin L protease essential for Caenorhabditis elegans embryogenesis is functionally conserved in parasitic nematodes

Proteolytic enzymes are involved in processes important to development and survival of many organisms. Parasite proteases are considered potential targets of parasite control yet, for most, their precise physiological functions are unknown. Validation of potential targets requires analysis of function. We have recently identified a cathepsin L (CPL) cysteine protease, Ce-CPL-1, which is essential for embryonic development of the free-living nematode Caenorhabditis elegans. We now show that CPL genes closely related to Ce-cpl-1 are expressed in the animal parasitic nematodes Haemonchus contortus, Dictyocaulus viviparus, Teladorsagia circumcincta, Ancylostoma caninum and Ascaris suum, as well as in plant parasitic nematodes. The similarities in gene structure and encoded amino acid sequence indicate that the parasite and C. elegans CPLs are homologous enzymes. We demonstrate functional compensation of the loss of C. elegans cpl-1 by transgenic expression of the H. contortus cpl-1 gene, rescuing the embryonic lethality. These genes may therefore be orthologues, sharing the same function in both species. Targeting of this enzyme has potential in inhibiting development and transmission of parasitic nematodes. In addition, the role of CPL is important to our understanding of nematode development.     

Antioxidant enzyme families in parasitic nematodes

Parasitic nematodes, like all aerobic organisms, require antioxidant enzymes to cope with reactive oxygen species (ROS) generated during cellular metabolism. Additionally, they have to protect themselves against ROS produced by the host. Parasitic nematode enzymes that deal with the superoxide anion radical, the superoxide dismutases (SODs), have been described in every species examined, whereas enzymes that deal with hydrogen peroxide have been difficult to identify. A major family of enzymes in mammals, the selenium-containing glutathione peroxidases (GPXs), appears to be absent, although a selenium-independent GPX family exists. These enzymes demonstrate little or no activity with hydrogen peroxide. Catalase (CAT) activity has been detected, but sequences encoding a typical CAT polypeptide have only been identified in a few species, despite the active EST sequencing projects. However, a new family of enzymes has recently been described, the peroxiredoxins (PRXs), which are abundant in parasitic nematodes and have been shown to react with hydrogen peroxide. This review summarizes the major characteristics of each of these enzyme families in general and in parasitic nematodes, emphasizing and comparing the newer data on the family of PRXs.     

Peroxiredoxin 2 and peroxide metabolism in the erythrocyte

Peroxiredoxin 2 (Prx2) is an antioxidant enzyme that uses cysteine residues to decompose peroxides. Prx2 is the third most abundant protein in erythrocytes, and competes effectively with catalase and glutathione peroxidase to scavenge low levels of hydrogen peroxide, including that derived from hemoglobin autoxidation. Low thioredoxin reductase activity in the erythrocyte is able to keep up with this basal oxidation and maintain the Prx2 in its reduced form, but exposure to exogenous hydrogen peroxide causes accumulation of the disulfide-linked dimer. The high cellular concentration means that although turnover is slow, erythrocyte Prx2 can act as a noncatalytic scavenger of hydrogen peroxide and a sink for hydrogen peroxide before turnover becomes limiting. The consequences of Prx2 oxidation for the erythrocyte are not well characterized, but mice deficient in this protein develop severe hemolytic anemia associated with Heinz body formation. Prx2, also known as calpromotin, regulates ion transport by associating with the membrane and activating the Gárdos channel. How Prx2 redox transformations are linked to membrane association and channel activation is yet to be established. In this review, we discuss the functional properties of Prx2 and its role as a major component of the erythrocyte antioxidant system.     

A novel candidate cis-regulatory motif pair in the promoters of germline and oogenesis genes in C. elegans

In this study we report on a novel pair of cis-regulatory motifs in promoter sequences of the nematode Caenorhabditis elegans. The motif pair exhibits extraordinary genomic traits: The order and the orientation of the two motifs are highly specific, and the distance between them is almost always one of two frequent distances. In contrast, the sequence between the motifs is variable across occurrences. Thus, the motif pair constitutes a nearly combinatorial sequence configuration. We further show that this module is conserved among, and unique to, the entire Caenorhabditis genus. By analyzing several gene expression data sets, our data suggest that this motif pair may function in germline development, oogenesis, and early embryogenesis. Finally, we verify that the motifs are indeed functional cis-regulatory elements using reporter constructs in transgenic C. elegans.     

Molecular characterisation of mitochondrial and cytosolic trypanothione-dependent tryparedoxin peroxidases in Trypanosoma brucei

In trypanosomatids, removal of hydrogen peroxide and other aryl and alkyl peroxides is achieved by the NADPH-dependent trypanothione peroxidase system, whose components are trypanothione reductase (TRYR), trypanothione, tryparedoxin (TRYX) and tryparedoxin peroxidase (TRYP). Here, we report the cloning of a multi-copy tryparedoxin peroxidase gene (TRYP1) from Trypanosoma brucei brucei encoding a protein with two catalytic VCP motifs similar to the cytosolic TRYP from Crithidia fasciculata. In addition, we characterise a novel single copy gene encoding a second tryparedoxin peroxidase (TRYP2). TRYP2 shows 51% similarity to TRYP1, possesses a putative mitochondrial import sequence at its N-terminus and has a variant IPC motif replacing the second VCP motif implicated in catalysis in other 2-Cys peroxiredoxins. TRYP1 and TRYP2 were expressed in Escherichia coli, and the purified recombinant proteins shown to utilise hydrogen peroxide in the presence of NADPH, trypanothione, TRYR and TRYX from T. brucei, similar to the C. fasciculata cytoplasmic system. Western blots showed that TRYX, TRYP1 and TRYP2 are expressed in both bloodstream and procyclic forms of the life cycle. To determine the precise localisation of TRYX, TRYP1 and TRYP2 in the parasite, polyclonal antibodies to purified recombinant TRYX and TRYP1 and monoclonal antibody to TRYP2 were generated in mice. In-situ immunofluorescence and immunoelectron microscopy revealed a colocalisation of TRYX and TRYP1 in the cytosol, whereas TRYP2 was principally localised in the mitochondrion.     

Caenorhabditis elegans ivermectin receptors regulate locomotor behaviour and are functional orthologues of Haemonchus contortus receptors

The target site for the anthelmintic action of ivermectin is a family of nematode glutamate-gated chloride channel alpha subunits (GluClalpha) that bind the drug with high affinity and mediate its potent paralytic action. Whilst the action of ivermectin on the pharyngeal muscle of nematodes is relatively well understood, its effect on locomotor activity is less clear. Here we use RNAi and gene knockouts to show that four GluClalpha subunits are involved in regulating the pattern of locomotor activity in Caenorhabditis elegans. A Haemonchus contortus orthologue of these subunits, HcGluClalpha3, has been shown to be expressed in the motor nervous system and here we have shown that it is a functional, as well as a structural, orthologue by virtue of the observation that it can restore normal motor movement in the C. elegans GluClalpha mutant, avr-14(ad1032), when expressed under the control of the avr-14 promoter. This supports the contention that ivermectin exerts its paralytic action on parasitic nematodes through activation of GluCl channels in the motor nervous system. Furthermore, functional complementation in C. elegans provides a method to further the understanding of this important class of anthelmintic targets.