Ronald D. Klein

Three β-tubulin cDNAs from the parasitic nematode Haemonchus contortus

Abstract Experimental evidence indicates that tubulin is the site of action of the anthelmintic benzimidazoles. Furthermore, certain residues of β-tubulin seem to be critical for this mechanism. Although the benzimidazoles selectively affect nematode vs. mammalian β-tubulin, the molecular basis for this differential action is not known. To enhance our understanding of this phenomenon, and to provide the basis for investigating benzimidazole resistance in parasitic nematodes, we undertook the cloning of β-tubulin cDNAs from the ruminant parasite, Haemonchus contortus . We have cloned and sequenced three β-tubulin cDNAs from this organism, β12–16, β12–164, and β8–9. The first 2 differ at only 23 nucleotides, which give rise to 4 amino acid changes, β8–9 represents a different isotype class from the other two, since it differs extensively in the carboxyterminus. By comparing the sequences of these and other nematode β-tubulins with mammalian β-tubulins, several regions of consistent difference can be recognized; the functional significance of these regional differences has not been defined. Sequences very similar or identical to β8–9 and β12–16 are present in both benzimidazole-sensitive and benzimidazole-resistant populations of H. contortus . However, it appears that drug-resistant organisms may differ in the presence of a gene product which is closely related to β8–9.

Mechanism-based screening: discovery of the next generation of anthelmintics depends upon more basic research.

The therapeutic arsenal for the control of helminth infections contains only a few chemical classes. The development and spread of resistance has eroded the utility of most currently available anthelmintics, at least for some indications, and is a constant threat to further reduce the options for treatment. Discovery and development of novel anthelmintic templates is strategically necessary to preserve the economic and health advantages now gained through chemotherapy. As the costs of development escalate, the question of how best to discover new drugs becomes paramount. Although random screening in infected animals led to the discovery of all currently available anthelmintics, cost constraints and a perception of diminishing returns require new approaches. Taking a cue from drug discovery programmes for human illnesses, we suggest that mechanism-based screening will provide the next generation of anthelmintic molecules. Critical to success in this venture will be the exploitation of the Caenorhabditis elegans genome through bioinformatics and genetic technologies. The greatest obstacle to success in this endeavour is the paucity of information available about the molecular physiology of helminths, making the choice of a discovery target a risky proposition.

THE NERVOUS SYSTEMS OF HELMINTHS AS TARGETS FOR DRUGS

Processes that critically differentiate parasitic helminths and their hosts are obvious candidates for chemotherapeutic intervention. The recognition that neurobiology distinguishes helminths from their vertebrate hosts is due in part to the fact that several efficacious anthelmintics, derived generally from empirical screening, have been found to act selectively on the neuromuscular system of these parasites. In addition, basic physiological and pharmacological research has revealed considerable differences in the ways in which helminths and their hosts transmit information in the nervous system and respond to it in innervated tissues. Unfortunately, most of these differences have yet to be exploited in chemotherapy. The topics for this review include an analysis of mechanistic aspects of the pharmacology of anthelmintics that act on neuromuscular systems and a consideration of the prospects for discovery of novel drugs that act on this system.

Haemonchus contortus: The role of two β-tubulin gene subfamilies in the resistance to benzimidazole anthelmintics

The role of beta-tubulin genes in benzimidazole (BZ) resistance was investigated using one susceptible (S) and two resistant (Rt and Rc) strains of Haemonchus contortus. The Rt strain was isolated from the field on the basis of thiabendazole resistance. The Rc strain was derived from the S strain by treatment with cambendazole. cDNAs, derived from the S strain, encoding two isoforms of beta-tubulin (beta 12-16 and beta 8-9), alpha-tubulin and phosphofructokinase (Pfk) were used as probes for Southern hybridization analysis of genomic DNA digested by restriction enzymes. Genomic DNA was isolated from a pool of worms or single worms. The restriction-enzyme fragment length polymorphism (RFLP) differences among these strains depended on the enzyme and the probe used. When digested with Stu I or Hpa I, and probed under stringent conditions with beta 8-9 or beta 12-16, fewer fragments were seen in the Rt and Rc strains than in the S strain. Different hybridizing fragments were found in different individuals. The frequency of individuals bearing certain fragments hybridizing to beta 12-16 or beta 8-9 in the susceptible population was reduced significantly in the resistant populations. Some differences in RFLP between these strains were observed when probed with alpha-tubulin or Pfk, but the changes were not consistent with fragments being lost from the resistant strains as observed for beta-tubulin probes. These changes in RFLP pattern correlate with changes in the binding profiles of BZs and isoelectric isoform patterns reported previously for these strains. The data confirm that reduced heterogeneity within the population is associated with BZ resistance. Our results show that both the beta 8-9 and the beta 12-16 subfamilies of beta-tubulin are affected to a similar extent by this reduction in heterogeneity in a resistant population.

Characterization of a β-tubulin gene and β-tubulin gene products of Brugia pahangi

Abstract A genomic clone containing a β-tubulin gene from the parasitic nematode Brugia pahangi was isolated. This gene was sequenced to determine its size, structural organization, and corresponding primary amino acid sequence. The coding sequence of the β-tubulin gene spans 3.8 kb, is organized into 9 exons and expresses an mRNA of 1.8 kb which codes for a protein of 448 amino acids. The predicted β-tubulin amino acid sequence is 89%, 94%, 90% and 88% identical to the chicken β2, and the Caenorhabditis elegans ben-1, tub-1 and mec-7 gene products, respectively. Southern hybridization analyses demonstrated that there is only one copy of this gene isotype but that other distinct β-tubulin genes may exist in the Brugia pahangi genome. A nematode specific antipeptide rabbit antiserum raised against the predicted amino acid sequence of the extreme carboxy-terminal region of the B. pahangi β-tubulin was used to identify β-tubulin isoforms in adult nematodes and microfilariae. Isoforms detected by this nematode-specific antipeptide antiserum were identical in both adult worms and microfilarine and did not differ from the isoform patterns detected by a monoclonal antibody recognizing a conserved β-tubulin epitope. This suggests that this carboxy-terminal peptide is highly represented in the β-tubulin isoforms of B. pahangi.

Expression of cloned β-tubulin genes of Haemonchus contortus in Escherichia coli: interaction of recombinant β-tubulin with native tubulin and mebendazole

Two distinct β-tubulin cDNA isotypes (β8–9 and β12–16) from Haemonchus contortus were expressed for the first time in Escherichia coli and characterised by their specific mebendazole (MBZ) binding and polymerization properties. Beta-tubulin was expressed without translational fusion to an E. coli sequence under the regulation of the tryptophan promoter in the pTrp2 vector. Beta-tubulin was produced in large amounts in insoluble ‘inclusion bodies’. The inclusion bodies were purified and solubilised and the β-tubulin renatured by treatment with urea followed by dilution with alkaline buffer and a shift to physiological pH. The yield was more than 10 mg of β-tubulin per litre of cell culture. The recombinant tubulin produced was recognized in Western blot by specific anti-β-tubulin antibodies. Tritiated MBZ binding to the recombinant H. contortus β-tubulin was measured in the presence or absence of whole, tubulin-free or tubulin-rich extracts of H. contortus. Some [3H]MBZ high-affinity binding (HB) to ‘pure’ (no other eukaryotic protein present) β8–9 or β12–16 was observed. Enhanced high-affinity binding was observed when recombinant β8–9 or β12–16 were mixed and pre-incubated with whole supernatants or tubulin-enriched extracts from H. contortus. The enhancement was more than additive, β12 16 bound more MBZ and caused a greater enhancement than β8–9. Mixing recombinant β8–9 or β12–16 with whole supernatants or tubulin-enriched fractions from H. contortus promoted polymerization at 37°C. Use of 3sS-labelled protein showed that the polymer contained recombinant tubulin. Western blot using specific anti-α-tubulin monoclonal antibodies showed that the polymer contained α-tubulin. Similarly the recombinant nematode β-tubulin co-polymerized with tubulin from chicken brain. Our data suggest that the recombinant β-tubulin can interact and copolymerize with parasite or chicken tubulin. Furthermore the interaction of recombinant nematode β-tubulin with native tubulin and/or microtubule associated proteins (MAPs) resulted in the formation of high-affinity MBZ-binding sites. However, interaction of recombinant β-tubulin with microtubule proteins from chicken brain did not result in the formation of high-affinity MBZ-binding sites.

Cloning and sequence analysis of the gene encoding invertase from the yeast Schwanniomyces occidentalis.

SummaryThe gene encoding invertase (INV) has been cloned from Schwanniomyces occidentalis. The enzyme consists of 533 amino acids, 8 potential glycosylation sites and has a 45 % identity with the invertase from Saccharomyces cerevisiae. The proenzyme has a 22 amino acid signal sequence that has a high α-helical transmembrane potential which differs significantly from that predicted for the Saccharomyces cerevisiae enzyme.

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.

Purification and Characterization of Invertase from a Novel Industrial Yeast, Schwanniomyces Occidentalis

The use of yeast as an expression system for heterologous proteins offers several potential advantages with respect to industrial scale-up and genetics over other expression systems, but suffers from several drawbacks. For example, the secreted proteins of S. cerevisiae, found in the periplasm, are hyperglycosylated and the organism has a limited range of usable substrates. Other yeasts have similar disadvantages in addition to producing a variety of proteases. We have investigated the use of Schwanniomyces occidentalis as a host for developing a gene expression system in which these and several disadvantages are minimized. The present paper describes the isolation and characterization of an invertase from cell free supernatants of the yeast Schwanniomyces occidentalis grown on lactose. The enzyme is a beta-D-fructofuranoside-fructohydrolyase, composed of two identical subunits of 76,000 to 78,000 da. with a native molecular mass of 125,000 +/- 25,000 da. of which approximately 17% can be attributed to N-linked carbohydrate. The enzyme has a Vmax of 0.49 +/- 0.025 units, a Km of 21 +/- 1.5 mM, and temperature and pH optima of 55 degrees C and 3.9-4.5, respectively. The amino acid sequences of the amino terminal region and an internal tryptic peptide support an 81% identity with the invertase from Saccharomyces cerevisiae. The enzyme is induced by low glucose and is catabolite repressed.

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.