The beta-tubulin genes of two Strongyloides species

The World Health Organization is sponsoring major treatment programs with the aim of controlling helminth infection throughout the tropical world. Prominent among the anthelmintics recommended for use in these programs are drugs in the benzimidazole (BZ) class. Resistance to these drugs has been associated with polymorphisms in the beta-tubulin gene. We have cloned and sequenced the beta-tubulin genes of Strongyloides stercoralis and Strongyloides ratti and have proceeded to develop a protocol for genotyping single worms for polymorphisms in beta-tubulin. Our findings indicate that S. ratti has a single beta-tubulin gene, making DNA sequence analysis of a single larva PCR product a feasible means of studying BZ resistance in these species. Our genotyping test allows the identification of polymorphisms at codons 167, 198, and 200 in the Strongyloides beta-tubulin gene, thus enabling survey for BZ resistant genotypes.     

Allele-specific PCR for the beta-tubulin codon 200 TTC/TAC polymorphism using single adult and larval small strongyle (Cyathostominae) stages

It has been shown that benzimidazole (BZ) resistance in sheep gastrointestinal nematodes is linked with an increase in beta-tubulin codon 200 tyrosine-expressing alleles in the resistant parasite populations. Here, an allele-specific PCR has been developed for the discrimination of the TAC/TTC polymorphism in the beta-tubulin 200 codon of small strongyles. One reverse primer was used in 2 separate amplifications with 1 of 2 forward primers that differed only in their final 3' nucleotide. The primers flank a facultative intron/exon. Therefore, the amplified fragments are either 251 or 308 bp in size, depending on the presence or absence of the intron in individual worms. Amplification of genomic DNA isolated from single adult small strongyles from a set of 7 species consistently generated allele-specific products. Three worms each of the following species were used: Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus elongatus, Cylicocyclus radiatus, Cyathostomum pateratum, Cyathostomum catinatum, and Cyathostomum coronatum. PCR with DNA isolated from single larvae also reproducibly generated specific fragments. This method might be applied for the future assessment of allele frequencies in susceptible and resistant populations to further investigate the mechanism of BZ-resistance in small strongyles.     

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.     

Non-classic characteristics define prominent DNase activities from the intestine and other tissues of Haemonchus contortus

The anthelmintic fenbendazole (FBZ) induces nuclear DNA fragmentation (DF) in intestinal cells of Haemonchus contortus. The DNA fragments had 3'-OH, which suggests involvement of a neutral DNase. To identify candidate DNase(s) involved, DNase activity in H. contortus intestine and other worm fractions was characterized relative to classic DNases I (neutral) and II (acidic). Seven distinct DNase activities were identified and had Mrs of 34, 36, 37 or 38.5 kDa on zymographic analysis. The different activities were distinguished according to pH requirement, sensitivity to 10 mM EDTA and worm compartment. Activities of intestinal DNases at 34, 36 and 38.5 kDa were sensitive to EDTA at pH 5.0 and 7.0. Sensitivity to EDTA at pH 5.0 was unexpected compared to classic acidic DNase II activity, suggesting unusual properties of these DNases. In whole worms, however, the activities at 36 and 38.5 kDa were relatively insensitive to EDTA, indicating predominance of DNases that are distinct from the intestine. The activity at 37 kDa in excretory/secretory products had an acidic pH requirement and was insensitive to EDTA, resembling classic acidic DNase activity. Under conditions of pH 5.0 and 7.0, intestinal DNases produced 3'-ends that could be labeled by terminal deoxynucleotidyl transferase, indicating presence of 3'-OH. The labeling of 3'-ends at pH 5.0, again, was unexpected for acidic DNase activity. These results and several other activities suggest that multiple H. contortus DNases have characteristics distinct from the classic mammalian DNases I and II. Treatment of H. contortus with FBZ did not induce any detectable DNase activities distinct from normal intestine, although relative activities of intestinal DNases appear to have been altered by this treatment.     

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.     

Amplified fragment length polymorphism analysis of genetic diversity of Haemonchus contortus during selection for drug resistance.

For the first time we used amplified fragment length polymorphism on individual nematode parasites to analyse the genetic diversity between and within isolates during consecutive stages of increased benzimidazole resistance and of increased levamisole resistance of Haemonchus contortus. The genetic diversity of the H. contortus genome turned out to be unusually high, within and between the isolates. The difference between individuals of an isolate could be as high as between individuals of two different mammalian species that do not interbreed. During benzimidazole selection the genetic constitution of the population was changed, but did not lead to a decrease in the genetic diversity. The selection for levamisole resistance resulted in a limited reduction of the genetic diversity only after the first selection step. The extensive genetic diversity apparently has allowed a fast and flexible response of H. contortus to drug selection as shown by the appearance of drug resistant isolates. This selection however has little or no effect on the extent of the genetic diversity of these resistant isolates. Implications for more sustainable control methods are discussed.     

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.     

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.     

Distinct cytoskeletal tracks direct individual vesicle populations to the apical membrane of epithelial cells

A key aspect in the structure of epithelial and neuronal cells is the maintenance of a polarized organization based on highly specific sorting machinery at the exit site of the trans Golgi network (TGN). Epithelial cells sort protein and lipid components into different sets of carriers for the apical or basolateral plasma membrane. The two intestinal proteins lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are delivered to the apical plasma membrane of epithelial cells with high fidelity but differ in their affinity to detergent-insoluble, glycolipid-enriched complexes (DIGs). Using a two-color labeling technique, we have recently characterized two post-Golgi vesicle populations that direct LPH and SI separately to the apical cell surface. Here, we investigated the structure and identification of protein components in these vesicle populations and assessed the role of cytoskeletal post-Golgi transport routes for apical cargo. Apart from the central role of microtubules in vesicle transport, we demonstrate that the transport of SI-carrying apical vesicles (SAVs) occurs along actin tracks in the cellular periphery, whereas LPH-carrying apical vesicles (LAVs) are transferred in an actin-independent fashion to the apical membrane. Our data further indicate that myosin 1A is the actin-associated motor protein that drives SAVs along actin filaments to the apical cell surface.     

Subcellular distribution of CFTR in rat intestine supports a physiologic role for CFTR regulation by vesicle traffic

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel critical to intestinal anion secretion. In addition to phosphorylation, vesicle traffic regulates CFTR in some epithelial cells. Studies of cultured intestinal cells are conflicting regarding the role of cAMP-dependent vesicle traffic in regulating chloride transport. Whether CFTR is present in vesicular compartments within chloride secretory cells in the intestine is unknown and the role of cAMP-dependent vesicle insertion in regulating CFTR and intestinal fluid secretion remains unclear. The purpose of this study was to: (1) examine and quantify the subcellular distribution for CFTR in rat intestine, (2) further define the ultrastructure of the previously identified CFTR High Expresser (CHE) cell, and (3) examine the cellular distribution of CFTR following cAMP stimulation in vivo. Using the sensitive techniques of cryoimmunogold electron microscopy we identified CFTR in subapical vesicles and on the apical plasma membrane in crypt, Brunner glands, and CHE cells. cAMP stimulation in rat proximal small intestine produced a fluid secretory response and was associated with an apical redistribution of CFTR, supporting a physiologic role for cAMP-dependent CFTR vesicle insertion in regulating CFTR in the intestine.     

Vasoactive intestinal peptide stimulates apical secretion in hamster seminal vesicle epithelial cells in culture

In this study, we investigated the presence of vasoactive intestinal peptide (VIP) receptors in the epithelial cells of the hamster seminal vesicle, by using cell clusters isolated from the gland and cultivated in a serum-free bicameral culture system. An immunocytochemical approach and autoradiographic and biochemical binding experiments with 125I-VIP as radioligand were performed. The effect of this neuropeptide on cultured cell proliferation and protein secretory activity was also analysed. The release of trichloroacetic-acid-precipitable radioactive material by epithelial cells to the apical and basal compartments of the bi-chamber was estimated in absolute and relative terms. The results of this work indicate that: (1) VIP receptors are present in the membranes of clusters of epithelial cells from seminal vesicles and are further maintained in cultured cells; (2) VIP does not exert any mitogenic effect in these cells; (3) VIP affects the directionality of secretion, favouring the absolute and relative amounts of protein released to the apical compartment of the bi-chamber. The expression of VIP receptors in the epithelial cells of the hamster seminal vesicle and the direct secretagogue-like activity of this neuropeptide in these cells might be affected by other factors, namely, androgens.     

Effect of benzimidazole drugs on tubulin in benzimidazole resistant and susceptible strains of Caenorhabditis elegans

An in vitro assay was used to determine efficacy and if side resistance was present to benzimidazole anthelmintics tested against Caenorhabditis elegans after selection with albendazole. Side resistance was present to all the benzimidazoles tested, except for oxibendazole and parbendazole. At a concentration of 1 mM, all of the drugs, except thiabendazole, were effective in killing 100% of the albendazole susceptible worms. Tubulin from albendazole resistant and susceptible C. elegans was isolated and run on polyacrylamide gels. Western blots with anti-tubulin antibody showed that the albendazole resistant strain had an altered tubulin. Electron microscopy of albendazole-treated drug resistant worms showed microtubules throughout the intestinal cells. Microtubules were not observed in albendazole-treated drug susceptible worms.     

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.     

The distribution of MUC1, an apical membrane glycoprotein, in mammary epithelial cells at the resolution of the electron microscope: implications for the mechanism of milk secretion

The distribution of the glycoprotein, mucin 1 (MUC1), was determined in lactating guinea-pig mammary tissue at the resolution of the electron microscope. MUC1 was detected on the apical plasma membrane of secretory epithelial cells, the surface of secreted milk-fat globules, the limiting membranes of secretory vesicles containing casein micelles and in small vesicles and tubules in the apical cytoplasm. Some of the small MUC1-containing vesicles were associated with the surfaces of secretory vesicles and fat droplets in the cytoplasm. MUC1 was detected in much lower amounts on basal and lateral plasma membranes. By quantitative immunocytochemistry, the ratio of MUC1 on apical membranes and milk-fat globules to that on secretory vesicle membranes was estimated to be 9.2:1 (density of colloidal gold particles/microm membrane length). The ratio of MUC1 on apical membranes compared with basal/lateral membranes was approximately 99:1. The data are consistent with a mechanism for milk-fat secretion in which lipid globules acquire an envelope of membrane from the apical surface and possibly from small vesicles containing MUC1 in the cytoplasm. During established lactation, secretory vesicle membrane does not appear to contribute substantially to the milk-fat globule membrane, or to give rise in toto to the apical plasma membrane.     

Analysis of the role of p200-containing vesicles in post-Golgi traffic.

p200 is a cytoplasmic protein that associates with vesicles budding from the trans-golgi network (TGN). The protein was identified by a monoclonal antibody AD7. We have used this antibody to analyze whether p200 functions in exocytic transport from the TGN to the apical or basolateral plasma membrane in Madin-Darby canine kidney cells. We found that transport of the viral marker proteins, influenza hemagglutinin (HA) to the apical surface or vesicular stomatitis virus glycoprotein (VSV G) to the basolateral surface in streptolysin O-permeabilized cells was not affected when p200 was depleted from both the membranes and the cytosol. When vesicles isolated from perforated cells were analyzed by equilibrium density gradient centrifugation, the p200 immunoreactive membranes did not comigrate with either the apical vesicle marker HA or the basolateral vesicle marker VSV G. Immunoelectron microscopy of perforated and double-labeled cells showed that the p200 positive vesicular profiles were not labeled by antibodies to HA or VSV G when the viral proteins were accumulated in the TGN. Furthermore, the p200-decorated vesicles were more electron dense than those labeled with the viral antibodies. Together, these results suggest that p200 does not function in the transport pathways that carry HA from the TGN to the apical surface or VSV G from the TGN to the basolateral surface.     

Effects of cholinergic drugs on longitudinal contraction in levamisole-susceptible and -resistant Haemonchus contortus.

A novel force transducer was used to measure the effects of cholinergic agonists on longitudinal contraction in Haemonchus contortus. Drugs were applied to whole worms or injected via a cannula in the pseudocoelomic cavity. A number of agonists, including nicotine and the anthelmintics m-aminolevamisole, levamisole and morantel, caused contractions in whole worms. Four- to 25-fold increases in concentration of the active compounds were required to cause contractions in each of two levamisole-resistant strains of H. contortus. Of the other compounds tested, bephenium had equivalent activity against susceptible and resistant strains. Anticholinesterase compounds caused contractions after a slight delay in susceptible, but not resistant worms. Numerous cholinergic agonists and other compounds did not cause contraction when applied to whole worms. One of these, acetylcholine, caused contractions in cannulated worms. Compared with the susceptible strain, five- to six-fold higher concentrations of acetylcholine were required to cause equivalent contractions in the resistant strains. Levamisole resistance in adult H. contortus is likely to be due to a change in the characteristics of the cholinergic receptor(s).     

Isolation and characterization of the Fc receptor from the fetal yolk sac of the rat

The yolk sac of the fetal rat and the proximal small intestine of the neonatal rat selectively transport maternal IgG. IgG-Fc receptors are thought to mediate transport across the epithelium of both tissues. We used a mouse mAb (MC-39) against the 45-54-kD component of the Fc receptor of the neonatal intestine to find an antigenically related protein that might function as an Fc receptor in fetal yolk sac. In immunoblots of yolk sac, MC-39 recognized a protein band with apparent molecular mass of 54-58 kD. MC-39 bound to the endoderm of yolk sac in immunofluorescence studies. In immunogold-labeling experiments MC-39 was associated mainly with small vesicles in the apical cytoplasm and in the region near the basolateral membrane of endodermal cells. The MC- 39 cross-reactive protein and beta 2-microglobulin, a component of the intestinal Fc receptor, were copurified from detergent-solubilized yolk sac by an affinity purification that selected for proteins which, like the intestinal receptor, bound to IgG at pH 6.0 and eluted at pH 8.0. In summary, the data suggest that we have isolated the Fc receptor of the yolk sac and that this receptor is structurally and functionally related to the Fc receptor of the neonatal intestine. An unexpected finding is that, unlike the intestinal receptor which binds maternal IgG on the apical cell surface, the yolk sac receptor appears to bind IgG only within apical compartments which we suggest represent the endosomal complex.