Sequence variation in the Trichuris trichiura beta-tubulin locus: implications for the development of benzimidazole resistance

Benzimidazole resistance has evolved in a variety of organisms and typically results from mutations in the beta-tubulin locus at specific amino acid sites. Despite widespread treatment of human intestinal nematodes with benzimidazole drugs, there have been no unambiguous reports of resistance. However, since beta-tubulin mutations conferring resistance are generally recessive, frequencies of resistance alleles less than 30% would be difficult to detect on the basis of drug treatment failures. Here we investigate sequence variation in a 1079 bp segment of the beta-tubulin locus in the human whipworm Trichuris trichiura from 72 individual nematodes from seven countries. We did not observe any alleles with amino acid mutations indicative of resistance, and of 40 point mutations there were only four non-synonymous mutations all of which were singletons. Estimated effective population sizes are an order of magnitude lower than those from another nematode species in which benzimidazole resistance has developed (Haemonchus contortus). Both the lower diversity and reduced population sizes suggest that benzimidazole resistance is likely to evolve less rapidly in Trichuris than in trichostrongyle parasites of livestock. We observed moderate levels of population subdivision (Phi(ST)=0.26) comparable with that previously observed in Ascaris lumbricoides, and identical alleles were frequently found in parasites from different continents, suggestive of recent admixture. A particularly interesting feature of the data is the high nucleotide diversities observed in nematodes from the Caribbean. This genetic complexity may be a direct result of extensive admixture and complex history of human populations in this region of the world. These data should encourage (but not make complacent) those involved in large-scale benzimidazole treatment of human intestinal nematodes.     

Systematic mutational analysis of the yeast beta-tubulin gene.

A systematic strategy was used to create a synoptic set of mutations that are distributed throughout the single beta-tubulin gene of Saccharomyces cerevisiae. Clusters of charged amino acids were targeted for mutagenesis and converted to alanine to maximize alterations on the protein's surface and minimize alterations that affect protein folding. Of the 55 mutations we constructed, three confer dominant-lethality, 11 confer recessive-lethality, 10 confer cold-sensitivity, one confers heat-sensitivity, and 27 confer altered resistance to benomyl. Only 11 alleles give no discernible phenotype. In spite of the fact that beta-tubulin is a highly conserved protein, three-fourths of the mutations do not destroy the ability of the protein to support the growth of yeast at 30 degrees C. The lethal substitutions are primarily located in three regions of the protein and presumably identify domains most critical for beta-tubulin function. Interestingly, most of the conditional-lethal alleles produce specific defects in spindle assembly at their restrictive temperature; cytoplasmic microtubules are relatively unaffected. The exceptions are two mutants that contain abnormally long cytoplasmic microtubules. Mutants with specific spindle defects were not observed in our previous collection of beta-tubulin mutants and should be valuable in dissecting spindle function.     

Genetics of the Tubulin Gene Families of Physarum

The organization of the alpha- and beta-tubulin gene families in Physarum was investigated by Mendelian analysis. Restriction endonuclease-generated DNA fragments homologous to alpha- and beta-tubulin show length polymorphisms that can be used as markers for genetic mapping. Analysis of meiotic assortment among progeny of heterozygotes allowed alpha- and beta-tubulin sequence loci to be defined. There are four unlinked alpha-tubulin sequence loci (altA, altB, altC and altD) and at least three unlinked beta-tubulin sequence loci (betA, betB and betC). The alpha-tubulin loci are not linked to the beta-tubulin loci. --Segregation of tubulin sequence loci with respect to ben mutations that confer resistance to antitubulin benzimidazole drugs was used to investigate whether any members of the alpha- or beta-tubulin gene families are allelic to ben loci. The beta-tubulin sequence locus betB is allelic to the resistance locus benD, the betA locus is probably allelic to benA and the alpha-tubulin sequence locus altC may be allelic to benC. The molecular implications of benzimidazole resistance phenotypes when only one of the expressed beta-tubulin gene family members mutates to drug resistance are discussed in relation to tubulin function.     

Paclitaxel resistance in cells with reduced beta-tubulin

We previously described the isolation of colcemid resistant Chinese hamster ovary cell lines containing alpha- and beta-tubulin mutations that increase microtubule assembly and stability. By analyzing colcemid sensitive revertants from one of the beta-tubulin mutants, we now find that loss or inactivation of the mutant allele represents the most common mechanism of reversion. Consistent with this loss, the revertants have 35% less tubulin at steady state, no evidence for the presence of a mutant polypeptide, and a normal extent of tubulin polymerization. In addition to the loss of colcemid resistance, the revertant cells exhibit increased resistance to paclitaxel relative to wild-type cells. This paclitaxel resistance can be suppressed by transfecting the revertant cells with a cDNA for wild-type beta-tubulin, indicating that the reduction in tubulin in the revertant cells is responsible for the resistance phenotype. We propose that reducing tubulin levels may represent a novel mechanism of paclitaxel resistance.     

Diversity of benzimidazole-resistance alleles in populations of small ruminant parasites

The resistance of gastro-intestinal nematodes of small ruminants (sheep and goat) to benzimidazole anthelmintic drugs seems to be linked primarily to a single mutation in the isotype 1 beta-tubulin gene. This study was carried out to investigate the origin and diversity of benzimidazole-resistance alleles in trichostrongylid nematodes. We sequenced a 550 bp fragment of the isotype 1 beta-tubulin gene from several benzimidazole-resistant Teladorsagia circumcincta populations isolated from dairy goat farms in the central and south-western France. We also sequenced the same beta-tubulin fragment from Trichostrongylus colubriformis and Haemonchus contortus populations in south-western France. We found eight benzimidazole-resistance alleles in all T. circumcincta populations studied, six in H. contortus populations, and only one in T. colubriformis populations. In most cases, only one benzimidazole-resistance allele was present in T. circumcincta and H. contortus populations, but two alleles were found in a fewer number of them. Some T. circumcincta populations shared the same benzimidazole-resistance allele whereas some others had a specific benzimidazole-resistance allele. Similar findings were obtained for H. contortus. As no parasites are introduced once the flock of dairy goat farms has been constituted, these data indicate for the three studied species that rare pre-existing benzimidazole-resistance alleles already present before the isolation of populations had been selected. On the other hand, the fact that some benzimidazole-resistance alleles were specific to one population of T. circumcincta or H. contortus, seems to be in agreement with the hypothesis of the selection of spontaneous mutations. Thus, the origin of benzimidazole-resistance alleles in trichostrongylid nematodes seems to involve primarily the selection of rare alleles and possibly of spontaneous mutations.     

Multi-sited mutations of beta-tubulin are involved in benzimidazole resistance and thermotolerance of fungal biocontrol agent Beauveria bassiana

Fungicide resistance and thermotolerance of biocontrol agents in mitosporic fungi are of merits for enhancing fungal formulations against insect pests in the field. Among 20 wild strains of Beauveria bassiana (a well-known fungal biocontrol agent) tested in this study, 19 were sensitive or highly sensitive to carbendazim (methyl 2-benzimidazole carbamate), a typical benzimidazole fungicide, despite low resistance found in one strain. Sequential mutagenesis of a carbendazim-sensitive wild strain [minimal inhibitory concentration (MIC) = 1.32 microg ml(-1)] under artificial selection pressure generated 11 mutants sharing a common MIC of > 1000 microg ml(-1) without visible variation in colony growth and conidiation capacity. This represents at least 758-fold enhancement of the resistance among the mutants. However, accompanied with the enhanced resistance, all the mutants became less thermotolerable. Stressed at 48 degrees C, conidial LT(50)s of the mutants varied from 1.8 to 9.6 min and were lower than the parental LT(50) (36 min). Moreover, the contents of hydrophobin-like proteins in conidial walls declined significantly among the mutants compared with that of the wild parent. Mutations commonly relating to benzimidazole resistance in fungi were located at Q134, F167 and/or E198 around the taxol-binding site of beta-tubulin by sequencing the beta-tubulin of the mutants. Also, mutations of other 37 amino acid residues in the sequences (each having one to five residues mutated) were found for the first time and they were diverse in spatial structure. All mutations restricted to the half of beta-tubulin close to alpha-tubulin were likely involved in variation in each of the traits concerned but their interactions were complicated.     

Isolation of mip (microtubule-interacting protein) mutations of Aspergillus nidulans.

We identified four mutations in two previously undescribed loci involved in microtubule function in Aspergillus nidulans as extragenic suppressors of benA33, a heat-sensitive beta-tubulin mutation. Three of the four mutations map to a locus closely linked to riboB on linkage group VIII; we designated this locus mipA (for microtubule-interacting protein). We were not able to map the remaining suppressor because of chromosomal rearrangements. However, since it recombines with riboB at a significantly higher frequency than the mipA alleles, it is unlikely to be in mipA; thus, we designated it mipB1. The mip mutations are not allelic to the previously identified loci that encode alpha- and beta-tubulin, and it is likely that mipA and mipB encode previously unidentified nontubulin proteins involved in microtubule function. Each of the mip mutations suppresses the heat sensitivity conferred by benA33 and suppresses the blockage of nuclear division and movement conferred by this mutation at high temperatures. Interactions between mipA and benA are allele specific. All of the mipA mutations are cryptic in a wild-type benA background but cause cold sensitivity in combination with benA33. These mutations also confer cold sensitivity in combination with benA31 and benA32 and reduce the resistance conferred by these mutations to the antimicrotubule agent benomyl but do not suppress the heat sensitivity conferred by these alleles. Finally, the mipA alleles suppress the heat sensitivity conferred by benA11, benA17, and benA21 but do not confer cold sensitivity in combination with these alleles.     

Genetic and molecular analysis of a Caenorhabditis elegans beta-tubulin that conveys benzimidazole sensitivity

Benzimidazole anti-microtubule drugs, such as benomyl, induce paralysis and slow the growth of the nematode Caenorhabditis elegans. We have identified 28 mutations in C. elegans that confer resistance to benzimidazoles. All resistant mutations map to a single locus, ben-1. Virtually all these mutations are genetically dominant. Molecular cloning and DNA sequence analysis established that ben-1 encodes a beta- tubulin. Some resistant mutants are completely deleted for the ben-1 gene. Since the deletion strains appear to be fully resistant to the drugs, the ben-1 product appears to be the only benzimidazole-sensitive beta-tubulin in C. elegans. Furthermore, since animals lacking ben-1 are viable and coordinated, the ben-1 beta-tubulin appears to be nonessential for growth and movement. The ben-1 function is likely to be redundant in the nematode genome.     

A beta-tubulin leucine cluster involved in microtubule assembly and paclitaxel resistance.

Analysis of beta-tubulin alleles from nine paclitaxel-resistant Chinese hamster ovary cell lines revealed an unexpected cluster of mutations affecting Leu-215, Leu-217, and Leu-228. Six of the mutant alleles encode a His, Arg, or Phe substitution at Leu-215; another mutant allele has an Arg substitution at Leu-217; and the final two mutant alleles have substitutions of His or Phe at Leu-228. Using plasmids that allow tetracycline regulated expression, the L215H, L217R, and L228F mutations were introduced into a hemagglutinin antigen-tagged beta-tubulin cDNA and transfected into wild-type Chinese hamster ovary cells. In all three cases, low to moderate expression of the transfected mutant gene conferred paclitaxel resistance. Higher levels of expression caused disruption of microtubule assembly, cell cycle arrest at mitosis, and failure to proliferate. Consistent with reduced microtubule stability, cells expressing mutant hemagglutinin beta-tubulin had fewer acetylated microtubules than nonexpressing cells in the same population. These data, together with previous studies showing that the paclitaxel-resistant mutant cell lines have less stable microtubules, indicate that the leucine cluster represents an important structural motif for microtubule assembly.     

Identification and characterization of benzimidazole resistance in Monilinia fructicola from stone fruit orchards in California

Low and high levels of resistance to the benzimidazole fungicides benomyl and thiophanate-methyl were observed in field isolates of Monilinia fructicola, which is the causative agent of brown rot of stone fruit. Isolates that had low levels of resistance (hereafter referred to as LR isolates) and high levels of resistance (hereafter referred to as HR isolates) were also cold and heat sensitive, respectively. Results from microsatellite DNA fingerprints showed that genetic identities among the populations of sensitive (S), LR, and HR isolates were very high (>0.96). Analysis of DNA sequences of the beta-tubulin gene showed that the LR isolates had a point mutation at codon 6, causing a replacement of the amino acid histidine by tyrosine. Codon 198, which encodes a glutamic acid in S and LR isolates, was converted to a codon for alanine in HR isolates. Based on these point mutations in the beta-tubulin gene, allele-specific PCR assays were developed for rapid detection of benzimidazole-resistant isolates of M. fructicola from stone fruit.     

Molecular characterization of benzimidazole-resistant isolates of Cladosporium fulvum

The benzimidazole fungicide thiophanate-methyl is commonly applied to control leaf mould of tomato caused by Cladosporium fulvum in China. In this study, 32 isolates of C. fulvum were examined for their sensitivities to thiophanate-methyl, and two benzimidazole-resistant (BenR) phenotypes BenR1 and BenR2 were identified. The BenR1 isolates were resistant to thiophanate-methyl, but were more sensitive to the phenylcarbamate fungicide diethofencarb than the wild-type isolates. The BenR2 isolates resistant to thiophanate-methyl were insensitive to diethofencarb. All tested isolates were sensitive to the dicarboximide fungicide iprodione. The complete beta-tubulin gene was isolated from this fungus to study its potential role in benzimidazole resistance. Analysis of the DNA sequence of the beta-tubulin gene showed that the BenR1 isolates had a point mutation at codon 198, causing a substitution of glutamic acid to alanine. In the BenR2 isolates, a point mutation at codon 200 causing a substitution of phenylalanine to tyrosine was detected. Based on these point mutations, a multiplex allele-specific PCR method was developed successfully for the first time to detect two point mutations at the beta-tubulin gene simultaneously in single PCR amplifications.     

A molecular tool for species identification and benzimidazole resistance diagnosis in larval communities of small ruminant parasites

This report describes a molecular method for determining in a first step the generic composition of a nematode community and in a second step, the resistance of each species to benzimidazole (BZ). We first established a polymerase chain reaction (PCR) linked to a restriction fragment length polymorphism strategy using the isotype 1 beta-tubulin gene. This method overcame the limitations of morphological identification of larval stages of trichostrongylid nematode species. Geographically distant isolates from the three main gastrointestinal species in temperate zones, Teladorsagia circumcincta, Haemonchus contortus, and Trichostrongylus colubriformis, were distinguished using this method. We then used an allele-specific PCR (AS-PCR) to detect mutations of residue 200 of the beta-tubulin, which is implicated in BZ resistance. The sequences of several samples confirmed the BZ-resistance genotype determined by AS-PCR. The ability to process large numbers of samples simultaneously makes this PCR-based strategy particularly suitable for epidemiological studies. It may also be useful for monitoring the emergence of resistant alleles in nematode communities.     

Real-time PCR assays for monitoring benzimidazole resistance-associated mutations in Ancylostoma caninum

Frequent and broad application of anthelmintic drugs for treatment of intestinal parasite infection has led to drug resistance that often renders whole populations of livestock unresponsive to treatment. Therefore, it is important to detect mutations associated with drug resistance before it becomes clinically manifest. To monitor developing drug resistance against benzimidazoles (BZ), we developed real-time PCR assays and applied them to analyse the beta-tubulin isotype-1 gene of the hookworm Ancylostoma caninum, an important parasite of dogs. Previously, we developed PCR assays to monitor codon positions 167 and 200. Here, we describe an assay which is able to detect resistance alleles in codon 198. These real-time PCR assays were subsequently applied to screen hookworm specimens recovered from dogs in Georgia. No elevated levels of polymorphisms at the investigated loci were found, suggesting that selection for resistance in the tested samples did not occur.     

Beta-tubulin complementary DNA sequence variations observed between cyathostomins from benzimidazole-susceptible and -resistant populations

The molecular mechanism of benzimidazole (BZ) resistance in cyathostomins of horses is still unclear. Previous studies revealed that the TTC or TAC polymorphism in codon 200 of the beta-tubulin isotype 1 gene is not as strictly correlated with BZ resistance as in trichostrongyles in sheep. To identify further sites of polymorphism within the beta-tubulin gene related to BZ resistance, complete complementary DNAs (cDNAs) encoding beta-tubulin of adult worms of Cylicocyclus nassatus, Cyathostomum pateratum, Cyathostomum coronatum, Cyathostomum catinatum, Cylicostephanus longibursatus, and Cylicostephanus goldi of a BZ-resistant cyathostomin population were characterized using specific primers. The cDNA sequence of each species spans 1,429 bp, encoding a protein of 448 amino acids. The interspecific identities are 95.2-99.6% at the nucleotide and 98.7-100.0% at the peptide level. The comparison of the amino acid sequences of individuals isolated from the BZ-resistant cyathostomin population with those from individuals of Cc. nassatus, Cy. coronatum, Cy. pateratum, and Cy. catinatum of a BZ-susceptible one showed differing amino acids in 11 positions. The commonness of a phenylalanine to tyrosine mutation at position 167 in all the 6 cyathostomin species isolated from a BZ-resistant population suggests its involvement in the molecular mechanism in BZ resistance.     

Tumor cells resistant to a microtubule-depolymerizing hemiasterlin analogue, HTI-286, have mutations in alpha- or beta-tubulin and increased microtubule stability

Hemiasterlins are sponge-derived tripeptides that inhibit cell growth by depolymerizing existing microtubules and inhibiting microtubule assembly. Since hemiasterlins are poor substrates for P-glycoprotein, they are attractive candidates for cancer therapy and have been undergoing clinical trials. The basis of resistance to a synthetic analogue of hemiasterlin, HTI-286 (HTI), was examined in cell populations derived from ovarian carcinoma (A2780/1A9) cells selected in HTI-286. 1A9-HTI-resistant cells (1A9-HTI(R) series) were 57-89-fold resistant to HTI. Cross-resistance (3-186-fold) was observed to other tubulin depolymerizing drugs, with collateral sensitivity (2-14-fold) to tubulin polymerizing agents. Evaluation of the percentage of polymerized and soluble tubulin in 1A9 parental and 1A9-HTI(R) cells corroborated the HTI cytotoxicity data. At 22 degrees C or 37 degrees C, in the absence of any drug, the percentage of polymerized microtubules for each of the 1A9-HTI(R) populations was greater than that in the 1A9 parental cells, consistent with more stable microtubules. Furthermore, microtubules in the 1A9-HTI(R) populations were also more resistant to depolymerization at 4 degrees C and had more acetylated and detyrosinated (Glu-tubulin) alpha-tubulin, all characteristic of more stable microtubules. The 1A9-HTI(R) cell populations exhibited either a single nucleotide change in the M40 beta-tubulin isotype, S172A, or in two cell populations where no beta-tubulin mutation was detected, mutations in the Kalpha-1 alpha-tubulin isotype, S165P and R221H in one resistant cell population and I384V in another. Unlike reports of mutations resulting in reduced drug affinity, the experimental data and location of mutations are consistent with resistance to HTI-286 mediated by microtubule-stabilizing mutations in beta- or alpha-tubulin.     

Unlinked Noncomplementation: Isolation of New Conditional-Lethal Mutations in Each of the Tubulin Genes of Saccharomyces Cerevisiae

Mutations in genes of Saccharomyces cerevisiae that code for proteins that interact with beta-tubulin were sought by screening for unlinked mutations that fail to complement mutations in the single beta-tubulin-encoding gene (TUB2). Among the first three noncomplementing mutations examined, two are linked to TUB2 while one is unlinked. The unlinked mutation was shown to be a conditional-lethal allele of the major alpha-tubulin-encoding gene (TUB1) and represents the first such mutation in that gene. The tub1-1 mutation itself causes a cold-sensitive cell-cycle arrest, and confers supersensitivity to the antimicrotubule drug benomyl. These phenotypes occur in the presence of a wild-type copy of the minor alpha-tubulin-encoding gene, TUB3; the combination of tub1-1 and a tub3 null mutation is inviable in haploids. Through further application of this method, new mutations in TUB2 and TUB3 were isolated as unlinked noncomplementers of tub1-1. The noncomplementation between tub1 and tub2 mutations is gene specific and allele specific, suggesting that the phenotype is due to an interaction at the protein level. We conclude that isolation of unlinked noncomplementing mutations is likely to be a generally useful method for isolating mutations in interacting gene products.     

Mutations at leucine 215 of beta-tubulin affect paclitaxel sensitivity by two distinct mechanisms

Paclitaxel resistance mutations in Chinese hamster ovary cells frequently alter a cluster of leucine residues in the H6-H7 loop region of beta-tubulin. To gain further insight into the role of this region in microtubule assembly and drug resistance, site-directed mutagenesis was used to systematically change amino acid L215. The mutated genes were cloned into a tetracycline-regulated expression vector and transfected into wild-type cells. Most of the mutations destabilized microtubule assembly, causing a decreased fraction of tubulin to appear in the microtubule cytoskeleton. In each case, the decreased level of assembly was associated with paclitaxel resistance and increased colcemid sensitivity. In two cases, however, the alteration did not significantly perturb the level of assembled tubulin or confer resistance to paclitaxel. One of these, L215V, produced little or no detectable phenotype, while the other, L215I, conferred increased sensitivity to paclitaxel. The increased drug sensitivity did not extend to epothilone A, a drug that binds to the same site and has a mechanism of action similar to that of paclitaxel, or colcemid, a drug with an opposing mechanism of action and a distinct binding site. Moreover, L215I conferred enhanced paclitaxel sensitivity at very low levels of expression, and sensitivity was not further enhanced in cells with higher levels of expression, implying that paclitaxel acts substoichiometrically. These properties, along with the proximity of L215 to the drug binding site, suggests that the L215I substitution may enhance the binding or effectiveness of paclitaxel. Our studies confirm the importance of the H6-H7 loop of beta-tubulin in microtubule assembly and resistance to antimitotic drugs. They also identify the first mammalian mutation shown to specifically increase sensitivity to paclitaxel.     

Revertants of a Chinese hamster ovary cell mutant with an altered beta-tubulin: evidence that the altered tubulin confers both colcemid resistance and temperature sensitivity on the cell

We recently described the isolation of a mutant Chinese hamster ovary cell (Cmd 4) resistant to the cytotoxic effects of colcemid (Cabral et al., Cell 20:29-36, 1980). This mutant carries an altered beta-tubulin but still grows normally at 37 degrees C. In the present study we found that Cmd 4 is temperature sensitive for growth at 40.3 degrees C. A class of revertants selected for temperature resistance had simultaneously lost colcemid resistance and the altered beta-tubulin. In addition, we isolated a temperature-resistant revertant which carries a further alteration in the mutant beta-tubulin polypeptide. This second alteration appears to make the mutant beta-tubulin incompetent to assemble into microtubules, resulting in a strain which is again colcemid sensitive. These revertant cell lines provide strong evidence that a mutation in beta-tubulin can confer both colcemid resistance and temperature sensitivity on a mammalian cell line. Cellular microtubules studied by indirect immunofluorescence in both mutant and revertant cell lines had an apparently normal distribution at permissive and nonpermissive temperatures, yet mitosis appears to be abnormal in the mutant cell line. We conclude from these studies that incorporation of the altered beta-tubulin into microtubules does not affect their distribution but may affect their function during mitosis.     

Molecular bases for sensitivity to tubulin-binding herbicides in green foxtail

We investigated the molecular bases for resistance to several classes of herbicides that bind tubulins in green foxtail (Setaria viridis L. Beauv.). We identified two alpha- and two beta-tubulin genes in green foxtail. Sequence comparison between resistant and sensitive plants revealed two mutations, a leucine-to-phenylalanine change at position 136 and a threonine-to-isoleucine change at position 239, in the gene encoding alpha2-tubulin. Association of mutation at position 239 with herbicide resistance was demonstrated using near-isogenic lines derived from interspecific pairings between green foxtail and foxtail millet (Setaria italica L. Beauv.), and herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Association of mutation at position 136 with herbicide resistance was demonstrated using herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Both mutations were associated with recessive cross resistance to dinitroanilines and benzoic acids, no change in sensitivity to benzamides, and hypersensitivity to carbamates. Using three-dimensional modeling, we found that the two mutations are adjacent and located into a region involved in tubulin dimer-dimer contact. Comparison of three-dimensional alpha-tubulin models for organisms with contrasted sensitivity to tubulin-binding herbicides enabled us to propose that residue 253 and the vicinity of the side chain of residue 251 are critical determinants for the differences in herbicide sensitivity observed between organisms, and that positions 16, 24, 136, 239, 252, and 268 are involved in modulating sensitivity to these herbicides.