RESUMO
Nematode parasites infect approximately 1.5 billion people globally and are a significant public health concern. There is an accepted need for new, more effective anthelmintic drugs. Nicotinic acetylcholine receptors on parasite nerve and somatic muscle are targets of the cholinomimetic anthelmintics, while glutamate-gated chloride channels in the pharynx of the nematode are affected by the avermectins. Here we describe a novel nicotinic acetylcholine receptor on the nematode pharynx that is a potential new drug target. This homomeric receptor is comprised of five non-α EAT-2 subunits and is not sensitive to existing cholinomimetic anthelmintics. We found that EAT-18, a novel auxiliary subunit protein, is essential for functional expression of the receptor. EAT-18 directly interacts with the mature receptor, and different homologs alter the pharmacological properties. Thus we have described not only a novel potential drug target but also a new type of obligate auxiliary protein for nAChRs.
Assuntos
Antinematódeos/farmacologia , Ascaris suum/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Helminto/metabolismo , Faringe/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Ascaris suum/efeitos dos fármacos , Ascaris suum/genética , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Helminto/genética , Faringe/efeitos dos fármacos , Receptores Nicotínicos/genéticaRESUMO
Glutamate-gated chloride channels are the most important target of ivermectin and related compounds in parasitic nematodes. A small family of genes encode subunits of these channels, allowing the assembly of multiple channel subtypes; the subunit composition of most of the native receptors is unknown. The members of the gene family vary between species, making extrapolation from C. elegans to parasites difficult. Expression of recombinant receptors in Xenopus oocytes can identify subunits that have the ability to co-assemble into novel channels, but localisation data, ideally at the single-cell level, is required to confirm that these subunits are expressed in the same cells and tissues. Fortunately, recent advances in this area are starting to make this information available; this information is adding to our understanding of how the drugs act and of the possible subunit combinations that create their targets in vivo.
Assuntos
Caenorhabditis elegans , Ivermectina , Animais , Canais de Cloreto/genética , Ivermectina/análogos & derivados , Ivermectina/farmacologia , MacrolídeosRESUMO
Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode Caenorhabditis elegans, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: unc-38, unc-63, unc-29, lev-1 and lev-8. These subunits form a functional L-AChR when heterologously expressed in Xenopus laevis oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to C. elegans lev-8. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the C. elegans L-AChR when expressed in Xenopus oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a 'model hopping' approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode Haemonchus contortus to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of C. elegans lev-8 null mutants. The critical role of acr-8 to in vivo drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for Hco-acr-8 which reduced the sensitivity of H. contortus larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus C. elegans. More broadly, this highlights the limits of C. elegans as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds.
Assuntos
Anti-Helmínticos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Agonistas Colinérgicos/farmacologia , Animais , Animais Geneticamente Modificados , Antinematódeos/farmacologia , Caenorhabditis elegans/genética , Feminino , Inativação Gênica , Genes de Helmintos , Haemonchus/efeitos dos fármacos , Haemonchus/genética , Haemonchus/patogenicidade , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Levamisol/farmacologia , Nematoides/classificação , Nematoides/genética , Infecções por Nematoides/tratamento farmacológico , Infecções por Nematoides/parasitologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Filogenia , Subunidades Proteicas , Pirantel/farmacologia , Receptores Colinérgicos/química , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
Acetylcholine receptors are pentameric ligand-gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR.
Assuntos
Proteínas de Helminto/metabolismo , Nematoides/metabolismo , Receptores Colinérgicos/metabolismo , Animais , Anti-Helmínticos/farmacologia , Ascaridoidea/genética , Ascaridoidea/metabolismo , Sequência de Bases , Haemonchus/genética , Haemonchus/metabolismo , Proteínas de Helminto/genética , Hibridização In Situ , Dados de Sequência Molecular , Morantel/farmacologia , Nematoides/genética , Técnicas de Patch-Clamp , Filogenia , Reação em Cadeia da Polimerase , Receptores Colinérgicos/genéticaRESUMO
Prevention of heartworm disease caused by Dirofilaria immitis in domestic dogs and cats relies on a single drug class, the macrocyclic lactones (MLs). Recently, it has been demonstrated that ML-resistant D. immitis are circulating in the Mississippi Delta region of the USA, but the prevalence and impact of these resistant parasites remains unknown. We review published studies that demonstrated resistance in D.immitis, along with our current understanding of its mechanisms. Efforts to develop in vitro tests for resistance have not yet yielded a suitable assay, so testing infected animals for microfilariae that persist in the face of ML treatment may be the best current option. Since the vast majority of D. immitis populations continue to be drug-sensitive, protected dogs are likely to be infected with only a few parasites and experience relatively mild disease. In cats, infection with small numbers of worms can cause severe disease and so the clinical consequences of drug resistance may be more severe. Since melarsomine dihydrochloride, the drug used to remove adult worms, is not an ML, the ML-resistance should have no impact on our ability to treat diseased animals. A large refugium of heartworms that are not exposed to drugs exists in unprotected dogs and in wild canids, which may limit the development and spread of resistance alleles.
Assuntos
Dirofilaria immitis/fisiologia , Dirofilariose/parasitologia , Doenças do Cão/parasitologia , Resistência a Medicamentos , Animais , Antiparasitários/farmacologia , Antiparasitários/uso terapêutico , Dirofilaria immitis/efeitos dos fármacos , Dirofilariose/tratamento farmacológico , Dirofilariose/prevenção & controle , Doenças do Cão/tratamento farmacológico , Doenças do Cão/prevenção & controle , Cães , Lactonas/farmacologia , Lactonas/uso terapêuticoRESUMO
Caenorhabditis elegans is widely used as a model for investigation of the relationships between aging, nutrient restriction and signalling via the DAF-2 (abnormal dauer formation 2) receptor for insulin-like peptides and AGE-1 [ageing alteration 1; orthologue of PI3K (phosphoinositide 3-kinase)], but the identity of the glucose transporters that may link these processes is unknown. We unexpectedly find that of the eight putative GLUT (glucose transporter)-like genes only the two splice variants of one gene have a glucose transport function in an oocyte expression system. We have named this gene fgt-1 (facilitated glucose transporter, isoform 1). We show that knockdown of fgt-1 RNA leads to loss of glucose transport and reduced glucose metabolism in wild-type worms. The FGT-1 glucose transporters of C. elegans thus play a key role in glucose energy supply to C. elegans. Importantly, knockdown of fgt-1 leads to an extension of lifespan equivalent, but not additive, to that observed in daf-2 and age-1 mutant worms. The results of the present study are consistent with DAF-2 and AGE-1 signalling stimulating glucose transport in C. elegans and this process being associated with the longevity phenotype in daf-2 and age-1 mutant worms. We propose that fgt-1 constitutes a common axis for the lifespan extending effects of nutrient restriction and reduced insulin-like peptide signalling.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Envelhecimento , Sequência de Aminoácidos , Animais , Transporte Biológico , Proteínas de Caenorhabditis elegans/química , Células Cultivadas , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/química , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , XenopusRESUMO
The heartworm, Dirofilaria immitis, is a filarial parasitic nematode responsible for significant morbidity and mortality in wild and domesticated canids. Resistance to macrocyclic lactone drug prevention represents a significant threat to parasite control and has prompted investigations to understand the genetic determinants of resistance. This study aimed to improve the genomic resources of D. immitis to enable a more precise understanding of how genetic variation is distributed within and between parasite populations worldwide, which will inform the likelihood and rate by which parasites, and in turn, resistant alleles, might spread. We have guided the scaffolding of a recently published genome assembly for D. immitis (ICBAS_JMDir_1.0) using the chromosomal-scale reference genomes of Brugia malayi and Onchocerca volvulus, resulting in an 89.5 Mb assembly composed of four autosomal- and one sex-linked chromosomal-scale scaffolds representing 99.7% of the genome. Publicly available and new whole-genome sequencing data from 32 D. immitis samples from Australia, Italy and the USA were assessed using principal component analysis, nucleotide diversity (Pi) and absolute genetic divergence (Dxy) to characterise the global genetic structure and measure within- and between-population diversity. These population genetic analyses revealed broad-scale genetic structure among globally diverse samples and differences in genetic diversity between populations; however, fine-scale subpopulation analysis was limited and biased by differences between sample types. Finally, we mapped single nucleotide polymorphisms previously associated with macrocyclic lactone resistance in the new genome assembly, revealing the physical linkage of high-priority variants on chromosome 3, and determined their frequency in the studied populations. This new chromosomal assembly for D. immitis now allows for a more precise investigation of selection on genome-wide genetic variation and will enhance our understanding of parasite transmission and the spread of genetic variants responsible for resistance to treatment.
Assuntos
Dirofilaria immitis , Dirofilariose , Doenças do Cão , Cães , Animais , Dirofilaria immitis/genética , Metagenômica , Genoma Helmíntico , Lactonas , Austrália , Dirofilariose/epidemiologia , Doenças do Cão/parasitologiaRESUMO
The ascarids are a large group of parasitic nematodes that infect a wide range of animal species. In humans, they cause neglected diseases of poverty; many animal parasites also cause zoonotic infections in people. Control measures include hygiene and anthelmintic treatments, but they are not always appropriate or effective and this creates a continuing need to search for better ways to reduce the human, welfare and economic costs of these infections. To this end, Le Studium Institute of Advanced Studies organized a two-day conference to identify major gaps in our understanding of ascarid parasites with a view to setting research priorities that would allow for improved control. The participants identified several key areas for future focus, comprising of advances in genomic analysis and the use of model organisms, especially Caenorhabditis elegans, a more thorough appreciation of the complexity of host-parasite (and parasite-parasite) communications, a search for novel anthelmintic drugs and the development of effective vaccines. The participants agreed to try and maintain informal links in the future that could form the basis for collaborative projects, and to co-operate to organize future meetings and workshops to promote ascarid research.
Assuntos
Anti-Helmínticos , Zoonoses , Animais , Humanos , Zoonoses/prevenção & controle , Caenorhabditis elegans , Academias e Institutos , Pesquisa , Anti-Helmínticos/uso terapêuticoRESUMO
Glutamate-gated chloride channels (GluCls) are found only in protostome invertebrate phyla but are closely related to mammalian glycine receptors. They have a number of roles in these animals, controlling locomotion and feeding and mediating sensory inputs into behavior. In nematodes and arthropods, they are targeted by the macrocyclic lactone family of anthelmintics and pesticides, making the GluCls of considerable medical and economic importance. Recently, the three-dimensional structure of a GluCl was solved, the first for any eukaryotic ligand-gated anion channel, revealing a macrocyclic lactone-binding site between the channel domains of adjacent subunits. This minireview will highlight some unique features of the GluCls and illustrate their contribution to our knowledge of the entire Cys loop ligand-gated ion channel superfamily.
Assuntos
Canais de Cloreto/metabolismo , Proteínas de Helminto/metabolismo , Proteínas de Insetos/metabolismo , Insetos/metabolismo , Nematoides/metabolismo , Animais , Canais de Cloreto/química , Canais de Cloreto/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Insetos/química , Proteínas de Insetos/genética , Insetos/química , Insetos/genética , Nematoides/química , Nematoides/genética , Conformação ProteicaRESUMO
The heartworm Dirofilaria immitis is an important parasite of dogs. Transmitted by mosquitoes in warmer climatic zones, it is spreading across southern Europe and the Americas at an alarming pace. There is no vaccine, and chemotherapy is prone to complications. To learn more about this parasite, we have sequenced the genomes of D. immitis and its endosymbiont Wolbachia. We predict 10,179 protein coding genes in the 84.2 Mb of the nuclear genome, and 823 genes in the 0.9-Mb Wolbachia genome. The D. immitis genome harbors neither DNA transposons nor active retrotransposons, and there is very little genetic variation between two sequenced isolates from Europe and the United States. The differential presence of anabolic pathways such as heme and nucleotide biosynthesis hints at the intricate metabolic interrelationship between the heartworm and Wolbachia. Comparing the proteome of D. immitis with other nematodes and with mammalian hosts, we identify families of potential drug targets, immune modulators, and vaccine candidates. This genome sequence will support the development of new tools against dirofilariasis and aid efforts to combat related human pathogens, the causative agents of lymphatic filariasis and river blindness.
Assuntos
Anti-Helmínticos/farmacologia , Dirofilaria immitis/genética , Dirofilariose/parasitologia , Doenças do Cão/parasitologia , Genoma Helmíntico , Vacinas/imunologia , Animais , Anti-Helmínticos/uso terapêutico , Dirofilaria immitis/efeitos dos fármacos , Dirofilaria immitis/imunologia , Dirofilaria immitis/microbiologia , Dirofilariose/tratamento farmacológico , Dirofilariose/prevenção & controle , Doenças do Cão/tratamento farmacológico , Doenças do Cão/prevenção & controle , Cães , Feminino , Variação Genética , Genoma Bacteriano , Masculino , Filogenia , Proteoma , RNA de Helmintos/química , Simbiose , Transcriptoma/genética , Wolbachia/genética , Wolbachia/fisiologiaRESUMO
Levamisole is a broad-spectrum anthelmintic which permanently activates cholinergic receptors from nematodes, inducing a spastic paralysis of the worms. Whereas this molecule is widely used to control parasitic nematodes impacting livestock, its efficacy is compromised by the emergence of drug-resistant parasites. In that respect, there is an urgent need to identify and validate molecular markers associated with resistance. Previous transcriptomic analyses revealed truncated cholinergic receptor subunits as potential levamisole resistance markers in the trichostrongylid nematodes Haemonchus contortus, Telodorsagia circumcincta and Trichostrongylus colubriformis. In the present study we used the Xenopus oocyte, as well as the free-living model nematode Caenorhabditis elegans, as heterologous expression systems to functionally investigate truncated isoforms of the levamisole-sensitive acetylcholine receptor (L-AChR) UNC-63 subunit. In the Xenopus oocyte, we report that truncated UNC-63 from C. elegans has a strong dominant negative effect on the expression of the recombinant C. elegans L-AChRs. In addition, we show that when expressed in C. elegans muscle cells, truncated UNC-63 induces a drastic reduction in levamisole susceptibility in transgenic worms, thus providing the first known functional validation for this molecular marker in vivo.
Assuntos
Anti-Helmínticos , Haemonchus , Nematoides , Animais , Levamisol/farmacologia , Levamisol/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Caenorhabditis elegans , Anti-Helmínticos/farmacologiaRESUMO
RIC-3 enhances the functional expression of certain nicotinic acetylcholine receptors (nAChRs) in vertebrates and invertebrates and increases the availability of functional receptors in cultured cells and Xenopus laevis oocytes. Maximal activity of RIC-3 may be cell-type dependent, so neither mammalian nor invertebrate proteins is optimal in amphibian oocytes. We cloned the X. laevis ric-3 cDNA and tested the frog protein in oocyte expression studies. X. laevis RIC-3 shares 52% amino acid identity with human RIC-3 and only 17% with that of Caenorhabditis elegans. We used the C. elegans nicotinic receptor, ACR-16, to compare the ability of RIC-3 from three species to enhance receptor expression. In the absence of RIC-3, the proportion of oocytes expressing detectable nAChRs was greatly reduced. Varying the ratio of acr-16 to X. laevis ric-3 cRNAs injected into oocytes had little impact on the total cell current. When X. laevis, human or C. elegans ric-3 cRNAs were co-injected with acr-16 cRNA (1 : 1 ratio), 100 µM acetylcholine induced larger currents in oocytes expressing X. laevis RIC-3 compared with its orthologues. This provides further evidence for a species-specific component of RIC-3 activity, and suggests that X. laevis RIC-3 is useful for enhancing the expression of invertebrate nAChRs in X. laevis oocytes.
Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Chaperonas Moleculares/fisiologia , Oócitos/metabolismo , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Regulação para Cima/genética , Proteínas de Xenopus/fisiologia , Sequência de Aminoácidos , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Dados de Sequência Molecular , Oócitos/fisiologia , Receptores Nicotínicos/fisiologia , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect approximately 1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs) on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5:1 (Asu-unc-38ratioAsu-unc-29), nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1:5 Asu-unc-38ratioAsu-unc-29), levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the development of parasite-specific screens for future anthelmintics.
Assuntos
Ascaris suum/metabolismo , Proteínas de Helminto/metabolismo , Receptores Nicotínicos/metabolismo , Sequência de Aminoácidos , Animais , Antinematódeos/farmacocinética , Ascaris suum/citologia , Ascaris suum/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Expressão Gênica , Proteínas de Helminto/química , Proteínas de Helminto/genética , Imuno-Histoquímica , Microscopia de Fluorescência , Dados de Sequência Molecular , Nicotina/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp , Multimerização Proteica , Subunidades Proteicas , RNA Complementar/metabolismo , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
A wide range of single- and multi-cellular parasites infect humans and other animals, causing some of the most prevalent and debilitating diseases on the planet. There have been virtually no published studies on the TRP channels of this diverse group of organisms. However, since many parasite genomes have been sequenced, it is simple to demonstrate that they are present in all parasitic metazoans and that sequences related to the yeast trp are present in many protozoans, including all the kinetoplastids. We compared the TRP genes of three species of animal and plant parasitic nematode to those of C. elegans and found that the parasitic species all had fewer such genes. These differences may reflect the phylogenetic distance between the species studied, or may be due to loss of specific gene functions following the evolution of the parasitic lifestyle. Other helminth groups, the trematodes and cestodes, seem to possess many TRPC and TRPM genes, but lack TRPV and TRPN. Most ectoparasites are insects or arachnids. We compared the TRP genes of a plant parasitic aphid and an animal parasite louse and tick with those of Drosophila. Again, all the parasitic species seemed to have fewer types of TRP channel, though the difference was less marked than for the nematodes. The aphid lacks TRPP and TRPML channel genes, whereas the tick lacked those encoding TRPVs. Again, these differences may reflect adaptation to parasitism, and could enable TRP channels to be targeted in the development of novel antiparasitic drugs.
Assuntos
Parasitos/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Canais de Potencial de Receptor Transitório/genéticaRESUMO
BACKGROUND: Ivermectin is widely used in human and animal medicine to treat and prevent parasite nematode infections. It has been suggested that its mode of action requires the host immune system, as it is difficult to reproduce its clinical efficacy in vitro. We therefore studied the effects of a single dose of ivermectin (Stromectol®-0.15 mg/kg) on cytokine levels and immune cell gene expression in human volunteers. This dose reduces bloodstream microfilariae rapidly and for several months when given in mass drug administration programmes. METHODS: Healthy volunteers with no travel history to endemic regions were given 3-4 tablets, depending on their weight, of either ivermectin or a placebo. Blood samples were drawn immediately prior to administration, 4 h and 24 h afterwards, and complete blood counts performed. Serum levels of 41 cytokines and chemokines were measured using Luminex® and expression levels of 770 myeloid-cell-related genes determined using the NanoString nCounter®. Cytokine levels at 4 h and 24 h post-treatment were compared to the levels pre-treatment using simple t tests to determine if any individual results required further investigation, taking p = < 0.05 as the level of significance. NanoString data were analysed on the proprietary software, nSolver™. RESULTS: No significant differences were observed in complete blood counts or cytokine levels at either time point between people given ivermectin versus placebo. Only three genes showed a significant change in expression in peripheral blood mononuclear cells 4 h after ivermectin was given; there were no significant changes 24 h after drug administration or in polymorphonuclear cells at either time point. Leukocytes isolated from those participants given ivermectin showed no difference in their ability to kill Brugia malayi microfilariae in vitro. CONCLUSIONS: Overall, our data do not support a direct effect of ivermectin, when given at the dose used in current filarial elimination programmes, on the human immune system. Trial registration ClinicalTrials.gov NCT03459794 Registered 9th March 2018, Retrospectively registered https://clinicaltrials.gov/ct2/show/NCT03459794?term=NCT03459794&draw=2&rank=1 .
Assuntos
Antiparasitários/administração & dosagem , Antiparasitários/imunologia , Citocinas/sangue , Imunidade Inata/efeitos dos fármacos , Ivermectina/administração & dosagem , Ivermectina/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Adolescente , Adulto , Idoso , Animais , Brugia Malayi/efeitos dos fármacos , Citocinas/imunologia , Expressão Gênica/efeitos dos fármacos , Experimentação Humana , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Pessoa de Meia-Idade , Neutrófilos/imunologia , Neutrófilos/parasitologia , Adulto JovemRESUMO
A population of Haemonchus contortus that was highly resistant to benzimidazoles and avermectin/milbemycins with a subpopulation that was resistant to levamisole, was replaced with a susceptible laboratory isolate of H. contortus in a flock of sheep. The anthelmintic susceptibility and population genetics of the newly established population were evaluated for 3.5 years using in vivo, in vitro, and molecular methods. Successful replacement of the resistant population with a susceptible population was confirmed using phenotypic and genotypic measurements; larval development assay indicated full anthelmintic susceptibility; albendazole treatment yielded 98.7% fecal egg count reduction; pyrosequence genotyping of single nucleotide polymorphisms in positions 167 and 200 of the isotype-1 beta tubulin gene were present at 0.0 and 1.7%, respectively; microsatellite genotyping indicated the background haplotype was similar to the susceptible isolate; and haplotypes of the isotype-1 beta tubulin gene were similar to the susceptible isolate. To sustain the susceptibility of the new population, targeted selective treatment was implemented using albendazole. Surprisingly, within 1.5 years post-replacement, the population reverted to a resistant phenotype. Resistance to albendazole, ivermectin, and moxidectin was confirmed via fecal egg count reduction test, larval development assay, and pyrosequencing-based genotyping. Targeted selective treatment was then carried out using levamisole. However, within one year, resistance was detected to levamisole. Population genetics demonstrated a gradual change in the genetic structure of the population until the final population was similar to the initial resistant population. Genetic analyses showed a lack of diversity in the susceptible isolate, suggesting the susceptible isolate had reduced environmental fitness compared to the resistant population, providing a possible explanation for the rapid reversion to resistance. This work demonstrates the power of combining molecular, in vitro, and in vivo assays to study phenotypic and genotypic changes in a field population of nematodes, enabling improved insights into the epidemiology of anthelmintic resistance.
Assuntos
Anti-Helmínticos , Hemoncose , Haemonchus , Preparações Farmacêuticas , Doenças dos Ovinos , Animais , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Resistência a Medicamentos/genética , Fazendas , Estruturas Genéticas , Hemoncose/tratamento farmacológico , Hemoncose/veterinária , Haemonchus/genética , Contagem de Ovos de Parasitas/veterinária , Ovinos , Doenças dos Ovinos/tratamento farmacológico , Doenças dos Ovinos/epidemiologiaRESUMO
Filariae are vector-borne nematodes responsible for an enormous burden of disease. Human lymphatic filariasis, caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori, and onchocerciasis (caused by Onchocerca volvulus) are neglected parasitic diseases of major public health significance in tropical regions. To date, therapeutic efforts to eliminate human filariasis have been hampered by the lack of a drug with sufficient macrofilaricidal and/or long-term sterilizing effects that is suitable for use in mass drug administration (MDA) programs, particularly in areas co-endemic with Loa loa, the causative agent of loiasis. Emodepside, a semi-synthetic cyclooctadepsipeptide, has been shown to have broad-spectrum efficacy against gastrointestinal nematodes in a variety of mammalian hosts, and has been approved as an active ingredient in dewormers for cats and dogs. This paper evaluates, compares (where appropriate) and summarizes the in vitro effects of emodepside against a range of filarial nematodes at various developmental stages. Emodepside inhibited the motility of all tested stages of filariae frequently used as surrogate species for preclinical investigations (Acanthocheilonema viteae, Brugia pahangi, Litomosoides sigmodontis, Onchocerca gutturosa, and Onchocerca lienalis), human-pathogenic filariae (B. malayi) and filariae of veterinary importance (Dirofilaria immitis) in a concentration-dependent manner. While motility of all filariae was inhibited, both stage- and species-specific differences were observed. However, whether these differences were detected because of stage- and/or species-specific factors or as a consequence of variations in protocol parameters among the participating laboratories (such as purification of the parasites, read-out units, composition of media, incubation conditions, duration of incubation etc.) remains unclear. This study, however, clearly shows that emodepside demonstrates broad-spectrum in vitro activity against filarial nematode species across different genera and can therefore be validated as a promising candidate for the treatment of human filariases, including onchocerciasis and lymphatic filariasis.
Assuntos
Brugia Malayi , Depsipeptídeos , Filariose Linfática , Loíase , Animais , Gatos , CãesRESUMO
The recent identification of isolates of D. immitis with confirmed resistance to the macrocyclic lactone preventatives presents an opportunity for comparative genomic studies using these isolates, and examining the genetic diversity within and between them. We studied the genomes of Wolbachia endosymbionts of five isolates of D. immitis maintained at the University of Georgia. Missouri and Georgia-2 are maintained as drug susceptible isolates, and JYD-27, Yazoo-2013 and Metairie-2014 are resistant to the macrocyclic lactone preventatives. We used whole genome amplification followed by Illumina-based sequencing from 8 to 12 individual microfilariae from each of the five isolates, obtaining a depth of coverage of approximately 40-75 fold for each. The Illumina sequences were used to create new genome assemblies for all the Wolbachia isolates studied. Comparisons of the Wolbachia sequences revealed more than 3000 sequence variations in each isolate. We identified 67 loci specific in resistant isolates but not in susceptible isolates, including 18 genes affected.Phylogenetic analysis suggested that the endosymbionts of the drug-susceptible isolates are more closely related to each other than to those from any of the resistant parasites. This level of variation in the Wolbachia endosymbionts of D. immitis isolates suggests a potential for selection for resistance against drugs targeting them.
Assuntos
Dirofilaria immitis/efeitos dos fármacos , Resistência a Medicamentos , Variação Genética , Genoma Bacteriano , Lactonas/farmacologia , Wolbachia/genética , Animais , Dirofilaria immitis/microbiologia , Compostos Macrocíclicos/farmacologiaRESUMO
Prevention of infection with canine heartworm (Dirofilaria immitis) is based on the compliant administration of macrocyclic lactone (ML) drugs. Resistance to ML drugs is well documented in D. immitis; however, there remains a paucity of information on the spatial distribution and prevalence of resistant isolates. This project aims to improve understanding of ML-resistance by using a population genetic approach. We developed a large panel of microsatellite loci and identified 12 novel highly polymorphic markers. These 12, and five previously published markers were used to screen pools of microfilariae from 16 confirmed drug-susceptible, 25 confirmed drug-resistant, and from 10 suspected drug-resistant field isolates. In isolates where microfilarial suppression testing indicated resistance, Spatial Principal Component Analysis (sPCoA), Neighbor Joining Trees and Bayesian clustering all revealed high genetic similarity between pre- and post-treatment samples. Somewhat surprisingly, the Neighbor Joining tree and sPCoA generated using pairwise Nei's distances did not reveal clustering for resistant isolates, nor did it reveal state-level geographic clustering from samples collected in Georgia, Louisiana or Mississippi. In contrast, Discriminant Analysis of Principle Components was able to discriminate between susceptible, suspected-resistant and resistant samples. However, no resistance-associated markers were detected, and this clustering was driven by the combined effects of multiple alleles across multiple loci. Additionally, we measured unexpectedly large genetic distances between different passages of laboratory strains that originated from the same source infection. This finding strongly suggests that the genetic makeup of laboratory isolates can change substantially with each passage, likely due to genetic bottlenecking. Taken together, these data suggest greater than expected genetic variability in the resistant isolates, and in D. immitis overall. Our results also suggest that microsatellite genotyping lacks the sensitivity to detect a specific genetic signature for resistance. Future investigations using genomic analyses will be required to elucidate the genetic relationships of ML-resistant isolates.
Assuntos
Dirofilaria immitis/genética , Resistência a Medicamentos/genética , Filaricidas/farmacologia , Lactonas/farmacologia , Repetições de Microssatélites , Animais , Dirofilaria immitis/efeitos dos fármacos , Dirofilaria immitis/crescimento & desenvolvimento , Marcadores Genéticos , Geografia , Compostos Macrocíclicos/farmacologia , Microfilárias/efeitos dos fármacos , Microfilárias/genética , Microfilárias/crescimento & desenvolvimento , Estados UnidosRESUMO
Nematode glutamate-gated chloride channels are targets of the macrocyclic lactones, the most important group of anthelmintics available. In Xenopus laevis oocytes, channels formed by the GluClalpha3B subunit from the parasite Haemonchus contortus were more sensitive to l-glutamate (EC(50) = 27.6 +/- 2.7 microM) than those formed by the homologous subunit from Caenorhabditis elegans (EC(50) = 2.2 +/- 0.12 mM). Ibotenate was a partial agonist (EC(50) = 87.7 +/- 3.5 microM). The H. contortus channels responded to low concentrations of ivermectin (estimated EC(50) = approximately 0.1 +/- 1.0 nM), opening slowly and irreversibly in a highly cooperative manner: the rate of channel opening was concentration-dependent. Responses to glutamate and ivermectin were inhibited by picrotoxinin and fipronil. Mutating an N-terminal domain amino acid, leucine 256, to phenylalanine increased the EC(50) for l-glutamate to 92.2 +/- 3.5 microM, and reduced the Hill number from 1.89 +/- 0.35 to 1.09 +/- 0.16. It increased the K(d) for radiolabeled ivermectin binding from 0.35 +/- 0.1 to 2.26 +/- 0.78 nM. Two other mutations (E114G and V235A) had no effect on l-glutamate activation or ivermectin binding: one (T300S) produced no detectable channel activity, but ivermectin binding was similar to wild-type. The substitution of any aromatic amino acid for Leu256 had similar effects in the radioligand binding assay. Molecular modeling studies suggested that the GluCl subunits have a fold similar to that of other Cys-loop ligand-gated ion channels and that amino acid 256 was unlikely to play a direct role in ligand binding but may be involved in mediating the allosteric properties of the receptor.