Benzimidazole-resistant beta-tubulin alleles in a population of parasitic nematodes (Cooperia oncophora) of cattle.

Three anthelmintic classes with distinct mechanisms of action are commercially available. Selection of nematode populations resistant to all these drugs has occurred, particularly in trichostrongyloid parasites of sheep. Anthelmintic resistance in cattle parasites has only recently been recognized and appears to be less pronounced, even though very similar species infect both hosts. To understand the bases for differences in the rate of resistance development in sheep versus cattle parasites, it is important to first demonstrate that the same kinds of resistance alleles exist in both. The benzimidazoles (BZ), which have been used for more than 40 years, were chosen as an example. BZ-sensitive (BZ(S)) and BZ-resistant (BZ(R)) nematodes that parasitize sheep have been distinguished at the molecular level by a single nucleotide change in the codon for amino acid 200 of a beta-tubulin gene, a switch from TTC (phenylalanine) to TAC (tyrosine). PCR primers were designed to completely conserved regions of trichostrongyloid beta-tubulin genes and were used to amplify DNA fragments from Haemonchus contortus (cDNA from a BZ(S) and a BZ(R) library) as positive controls. The technique was then extended to the cattle parasites, Cooperia oncophora and Ostertagia ostertagi (from genomic DNA). Sequence analysis proved the presence of amplified BZ(S) alleles in all three species and BZ(R) alleles in the BZ(R) population of H. contortus. Based on these data, nested PCR primers using the diagnostic T or A as the most 3' nucleotide were designed for each species. Conditions for selective PCR were determined. To demonstrate feasibility, genomic DNA was recovered from individual H. contortus L(3) larvae from both BZ(S) and BZ(R) populations. Genomic DNA was also isolated from >70 individual adult male C. oncophora collected from a cattle farm in New Zealand with reported BZ resistance. Allele-specific PCR discriminated among heterozygotes and homozygotes in both species. This method could find utility in studying the molecular epidemiology of BZ resistance in cattle parasites and for defining the variables that limit the development and spread of anthelmintic resistance in this host.

Individual expression of recombinant alpha- and beta-tubulin from Haemonchus contortus: polymerization and drug effects.

Three tubulin isotypes from the parasitic nematode Haemonchus contortus were individually expressed in Escherichia coli, purified, and induced to polymerize into microtubules in the absence of microtubule-associated proteins. The effect of different conditions on the rate of polymerization of pure tubulin was assessed. This is the first time that recombinant alpha-tubulin has been shown to be capable of polymerization into microtubule-like structures when incubated with recombinant beta-tubulin. In addition, the present study has shown that: (1) microtubule-associated proteins are not required for tubulin polymerization; and (2) pure beta-tubulin isotype, beta12-16, alone was capable of forming microtubule-like structures in the absence of alpha-tubulin. Polymerization of the recombinant invertebrate tubulin, as measured by a spectrophotometric assay, was found to be enhanced by a concentration of tubulin >0.25 mg/mL; temperature > or =20 degrees C; 2 mM GTP; glycerol; EGTA; and Mg(2+). Polymerization was inhibited by GTP (>2 mM) and albendazole. Calcium ions and a pH range of 6 to 8.5 had no measurable effect on polymerization. Individual isotypes of tubulin polymerized to approximately the same extent as an alpha-/beta-tubulin mixture. Samples of tubulin assembled under the above conditions for 60 min were also examined under a transmission electron microscope. Although the spectrophotometric assay indicated polymerization, it did not predict the structure of the polymer. In many cases tubulin sheets, folded sheets, and rings were observed in addition to, or instead of, microtubule-like structures.

Reconstitution of a bacterial/plant polyamine biosynthesis pathway in Saccharomyces cerevisiae.

Polyamine synthesis in most organisms is initiated by the decarboxylation of ornithine to form putrescine via ornithine decarboxylase (ODC). Plants, some bacteria and some fungi and protozoa generate putrescine from arginine, via arginine decarboxylase (ADC) and agmatine ureohydrolase (AUH) or agmatine iminohydrolase. A polyamine-requiring strain of Saccharomyces cerevisiae with a mutation in the gene encoding ODC was transformed with plasmids bearing genes encoding Escherichia coli ADC and AUH. Transformants regained the ability to grow in the absence of exogenous polyamines and contained enzyme activities consistent with the presence of both prokaryotic enzymes. Similar results were obtained when a plasmid containing a gene encoding oat (Avena sativa L.) ADC was substituted for the E. coli gene. These data demonstrate the successful complementation of a yeast biosynthetic polyamine synthesis defect by genes encoding an alternative pathway found in bacteria; they also show that plant ADC can substitute for the bacterial enzyme in this pathway. The recombinant yeast provides a tool for the study of the functional properties of these enzymes and for discovery of compounds that specifically inhibit this pathway.

Haemonchus contortus: cloning and functional expression of a cDNA encoding ornithine decarboxylase and development of a screen for inhibitors.

Polyamines (PA) are essential for viability and replication of all cells; organisms either synthesize PA or acquire them from the environment. How nematodes that parasitize the gut satisfy their PA requirement has not been resolved. The primary regulatory enzyme in PA biosynthesis in most animals is ornithine decarboxylase (ODC). This enzyme has recently been characterized in free-living nematodes and in the parasitic species. Haemonchus contortus. Nematode and mammalian ODC are reported to differ in subcellular localization, kinetics, and sensitivity to inhibitors. We cloned an H. contortus cDNA that encodes a full-length ODC (sequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AF016538 and AF016891). This cDNA was functionally expressed in strains of Escherichia coli and Saccharomyces cerevisiae that lack ODC and are dependent upon exogenous PA for survival. Expression of nematode ODC reversed the PA-dependence phenotype of both microorganisms. The complemented yeast strain was used to develop a nutrient-dependent viability screen for selective inhibitors of nematode ODC. The antiprotozoal drug stilbamidine isethionate was identified as active in this screen, but biochemical characterization revealed that this compound did not inhibit ODC. Instead, like other cationic diamidines, stilbamidine probably inhibits yeast S-adenosylmethionine decarboxylase. Nonetheless, the activity in the screen of the known ODC inhibitor difluoromethylornithine (DFMO) validates the concept that specific recombinant microorganisms can serve as the basis for extremely selective and facile screens.

The pharmacology of FMRFamide-related neuropeptides in nematodes: new opportunities for rational anthelmintic discovery?

The chemotherapeutic control of helminth parasites is compromised by the limited number of classes of anthelmintic drugs. Discovery of novel anthelmintics is impeded by the lack of novel screening technologies that overcome the difficulties inherent in screens based on whole organism toxicity. The development and implementation of mechanism-based screens for new anthelmintics offers great promise for the revitalization of antiparasitic drug discovery. However, mechanism-based screens must be based on a thorough understanding of the proteins or processes that offer the best chance for selective chemotherapeutic intervention. Basic research on the characterization of nematode FMRFamide-related peptides (FaRPs) has revealed that these peptides are ubiquitously distributed in helminths. Chemical identification of a number of nematode FaRPs has been achieved, and these peptides have potent and profound effects on the nematode neuromuscular system. Physiological processes mediated by nematode FaRPs (and other helminth neuropeptides) offer potential targets for the discovery of novel anthelmintics.

Nitric oxide mediates the inhibitory effects of SDPNFLRFamide, a nematode FMRFamide-related neuropeptide, in Ascaris suum.

1. The physiological effects of two Phe-Met-Arg-Phe-NH2 (FMRFamide)-related neuropeptides isolated from the free-living nematode Panagrellus redivivus, SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2), were examined using neuromuscular preparations from the parasitic nematode Ascaris suum. 2. PF1 and PF2 hyperpolarized muscle membrane and induced sustained flaccid paralysis, independent of external Cl-, in both innervated and denervated preparations. 3. PF1 reversed spastic contractions induced by the cholinomimetic levamisole, elevated K+, or the excitatory nematode FMRFamide-related neuropeptides KNEFIRFamide or KHEYLRFamide. 4. PF1 reversal of levamisole contraction was blocked by pretreatment with agents that interfere with nitric oxide (NO) synthesis (e.g., N-nitro-L-arginine), whereas sodium nitroprusside, which releases NO in solution, mimicked PF1 and PF2. 5. NO synthase activity, monitored by the conversion of [3H]arginine to [3H]citrulline, was twice as abundant in A. suum hypodermis as in muscle, but was not present in reproductive tissue. The relative abundance of NO synthase activity in these tissues was similar to the observed specific binding of [3H]PF1. 6. These results suggest that the inhibitory effects of PF1 and PF2 on nematode somatic muscle are mediated by NO, and that the hypodermis may serve a role in this process analogous to that of the endothelium in vertebrate vasculature.

Ascaris suum: cloning of a cDNA encoding phosphoenolpyruvate carboxykinase.

A cDNA encoding phosphoenolpyruvate carboxykinase (PEPCK) from Ascaris suum was cloned by complementation of a strain of Escherichia coli deficient in PEPCK and malic enzyme. The product of this cDNA was enzymatically similar to a recombinant PEPCK obtained from Haemonchus contortus by the same method. Comparison of the predicted amino acid sequence of A. suum PEPCK with other PEPCKs showed that this enzyme is most closely related to the H. contortus enzyme. The two nematode enzymes share considerable homology in regions thought to be functionally involved in substrate binding and catalysis, some of which distinguish the nematode enzymes from PEPCKs from other organisms. This analysis suggests a structural explanation for the kinetic differences seen between nematode and vertebrate PEPCKs and supports the hypothesis that nematode PEPCK is a target for selective inhibition.

Haemonchus contortus: ivermectin-induced paralysis of the pharynx.

How the avermectins cause the elimination of gastrointestinal nematodes from host animals has not yet been clearly identified. Using visual and radiometric parameters to measure oral ingestion in Haemonchus contortus, we showed that ivermectin (IVM) rapidly inhibited ingestion at concentrations > or = 10(-10) M. Motility, monitored quantitatively with an automated motility meter, was unaffected by IVM at concentrations < or = 10(-8) M, while ATP levels were unaffected at concentrations < or = 10(-6) M. Since motility and ATP levels, independent measures of short-term viability, are unaffected by concentrations of IVM that effectively block oral ingestion, the drug can be used as a chemical ligature. Although H. contortus was shown to be dependent upon an exogenous supply of glucose for survival in culture, IVM (10(-9) and 10(-7) M) altered neither the uptake of 3-O-[3H]methylglucose nor the metabolism of [13C]glucose by the parasite. These data suggest that H. contortus depends upon the transcuticular uptake of glucose in culture. If oral ingestion of other nutrients is essential for long-term survival in vivo, disruption of this process may represent the primary mechanism of IVM action.

Two FMRFamide-like peptides from the free-living nematode Panagrellus redivivus.

Peptides of the FXRFamide family, where X = M, I or L, are broadly distributed among invertebrates. Two such peptides were purified and sequenced from the free-living nematode, Panagrellus redivivus. Immunohistochemical techniques localized FMRFamide-like material in several regions of these organisms, including the nerve cords and, most prominently, in paired groups of cells located caudally to the base of the pharynx. RIA determinations gave an estimate of 2.8 nmol immunoreactive peptide/g of an acetone extract of P. redivivus. Four sequential HPLC purification steps, followed by sequencing by automated Edman degradation and FAB-MS, led to the identification of Ser-Asp-Pro-Asn-Phe-Leu-Arg-Phe-amide (SDPNFLRFamide) and Ser-Ala-Asp-Pro-Asn-Phe-Leu-Arg-Phe-amide (SADPNFLRFamide) as members of the FXRFamide family in this nematode.

Cloning of a cDNA encoding phosphoenolpyruvate carboxykinase from Haemonchus contortus.

Biochemical and metabolic data have led to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. To facilitate the determination of the molecular basis for host vs parasite differences in PEPCK, we have cloned a cDNA encoding this enzyme from a parasitic nematode of ruminants, Haemonchus contortus. H. contortus PEPCK was cloned by functional complementation of a PEPCK-, malic enzyme- strain of Escherichia coli (E1786) using an egg stage H. contortus cDNA library in lambda ZAPII. Selection was for growth on malate as the sole carbon source (malate+ phenotype). We isolated a plasmid, pPEPCK, which reproducibly confers a malate+ phenotype in E1786. The sequence of the 2.0-kb EcoRI insert of pPEPCK predicts a 612-amino acid protein which shows about 74% similarity to Drosophila melanogaster and chicken PEPCK. Extracts of E1786[pPEPCK], but not E1786, contain IDP- or GDP-dependent PEPCK enzyme activity. Sequence analysis revealed that the open reading frame (ORF) in pPEPCK lacked a 5' initiation codon and was probably expressed as an in-frame fusion protein with beta-galactosidase. A strategy combining library screening with PCR analysis of positive clones led to the identification of a clone encoding 6 additional NH2-terminal amino acids, including a Met, which, by comparison with known PEPCK amino acid sequences, is likely to be the translation initiation site.

Cloning of a cDNA encoding phosphofructokinase from Haemonchus contortus.

Phosphofructokinase (PFK), the key regulatory enzyme in glycolysis, has been cloned from the pathogenic parasitic nematode Haemonchus contortus by functional complementation in Escherichia coli. An E. coli strain deleted for both PFK loci (strain DF1020) was transformed with plasmid DNA from a lambda ZAP II H. contortus cDNA library. Two out of 3 x 10(7) transformants were able to grow on minimal medium with mannitol as the sole carbon source. A plasmid, pPFK, containing a 2.7-kb insert, was isolated from one of these transformants and conferred on DF1020 the ability to grow on mannitol (the PFK phenotype). The complemented cells contain PFK enzyme activity, absent in the E. coli mutant, at levels considerably higher than in wild type E. coli. Sequence analysis of the 2.7-kb insert shows an open reading frame that predicts a 789-amino acid protein that has approximately 70% similarity to mammalian PFKs. The amino acid sequence around asp182, thought to be the catalytic site, is completely conserved from nematodes to mammals.

Utility of a Haemonchus contortus/jird (Meriones unguiculatus) model for studying resistance to levamisole.

Trichostrongylid nematodes of sheep commonly are identified as exhibiting resistance to levamisole. In vitro assays have been developed to study levamisole resistance for Haemonchus contortus, but no in vivo model has been identified for this species. To determine the utility of a H. contortus/jird (Meriones unguiculatus) model for examining levamisole resistance, immunosuppressed jirds were inoculated with approximately 1,000 exsheathed infective larvae of H. contortus (resistant or susceptible to levamisole), treated per os on day 10 postinoculation (PI) with levamisole hydrochloride or analogs of the drug, and killed on day 13 PI. Stomachs were removed, opened longitudinally, incubated in distilled water at 37 C for 5 hr, fixed in formaldehyde solution, and stored for subsequent microscopic examination. Doses of levamisole and its analogs, which elicited percentage clearances of greater than or equal to 93.5 for the susceptible strain, cleared less than or equal to 68.9% of the resistant worms. These data are consistent with activities for the drugs against wild-type and levamisole-resistant strains of Caenorhabditis elegans. Thus, the H. contortus/jird model provides a useful in vivo tool to study resistance to levamisole and possibly other anthelmintics.

Effects of bile salt-stimulated lipase-treated triglycerides and free fatty acids on extracellular stages of Eimeria tenella.

Normal human milk (NHM) has antiprotozoal activity unrelated to immunological components; this activity extends to sporozoites of Eimeria tenella. This activity may be due to free fatty acids (FFA) enzymatically hydrolyzed from triacyl glycerols by a bile salt-stimulated lipase (BSSL) found in NHM. Sporozoites were therefore incubated in the presence of several saturated and unsaturated FFA. Anticoccidial activity was observed for many unsaturated fatty acids and for some saturated fatty acids. In addition, sporozoites were added to solutions of triglycerides (trilinolein, triolein and trilinolenin) preincubated with BSSL and sodium cholate, which resulted in killing of the parasites. Triglycerides alone showed no anticoccidial activity. These results were duplicated with first generation merozoites. Intracellular stages of E. tenella were affected by FFA only at concentrations that inhibited host cells.

Anti-coccidial activity of human milk.

Sporozoites of Eimeria tenella, an important pathogen of poultry, were killed in vitro in a time- and concentration-dependent manner by exposure to diluted concentrations of normal human milk. Sodium cholate (bile salt stimulator) potentiated the anti-coccidial activity. The anti-coccidial activity was not found in the milk of lower mammals (cow, sheep, goat, dog). The component in human milk showing the activity was initially hypothesized to be a bile salt-stimulated lipase. However, testing of purified lipase (with or without sodium cholate) indicated no anti-coccidial activity. Consequently, we theorize that the active component may be a free fatty acid.