Identification of Key Interactions of Benzimidazole Resistance-Associated Amino Acid Mutations in Ascaris β-Tubulins By Molecular Docking Simulations

Ascaris species are soil-transmitted helminths that infect humans and livestock mainly in low and middle-income countries. Benzimidazole (BZ) class drugs have predominated for many years in the treatment of Ascaris infections, but persistent use of BZs has already led to widespread resistance in other nematodes, and treatment failure is emerging for Ascaris. Benzimidazoles act by binding to β-tubulin proteins and destabilising microtubules. Three mutations in the β-tubulin protein family are associated with BZ resistance. Seven shared β-tubulin isotypes were identified in Ascaris lumbricoides and A. suum genomes. Benzimidazoles were predicted to bind to all β-tubulin isotypes using in silico docking, demonstrating that the selectivity of BZs to interact with one or two β-tubulin isotypes is likely the result of isotype expression levels affecting the frequency of interaction. Ascaris β-tubulin isotype A clusters with helminth β-tubulins previously shown to interact with BZ. Molecular dynamics simulations using β-tubulin isotype A highlighted the key role of amino acid E198 in BZ-β-tubulin interactions. Simulations indicated that mutations at amino acids E198A and F200Y alter binding of BZ, whereas there was no obvious effect of the F167Y mutation. In conclusion, the key interactions vital for BZ binding with β-tubulins have been identified and show how mutations can lead to resistance in nematodes.

The Beta-Tubulin Isotype TUBB6 Controls Microtubule and Actin Dynamics in Osteoclasts

Osteoclasts are bone resorbing cells that participate in the maintenance of bone health. Pathological increase in osteoclast activity causes bone loss, eventually resulting in osteoporosis. Actin cytoskeleton of osteoclasts organizes into a belt of podosomes, which sustains the bone resorption apparatus and is maintained by microtubules. Better understanding of the molecular mechanisms regulating osteoclast cytoskeleton is key to understand the mechanisms of bone resorption, in particular to propose new strategies against osteoporosis. We reported recently that β-tubulin isotype TUBB6 is key for cytoskeleton organization in osteoclasts and for bone resorption. Here, using an osteoclast model CRISPR/Cas9 KO for Tubb6, we show that TUBB6 controls both microtubule and actin dynamics in osteoclasts. Osteoclasts KO for Tubb6 have reduced microtubule growth speed with longer growth life time, higher levels of acetylation, and smaller EB1-caps. On the other hand, lack of TUBB6 increases podosome life time while the belt of podosomes is destabilized. Finally, we performed proteomic analyses of osteoclast microtubule-associated protein enriched fractions. This highlighted ARHGAP10 as a new microtubule-associated protein, which binding to microtubules appears to be negatively regulated by TUBB6. ARHGAP10 is a negative regulator of CDC42 activity, which participates in actin organization in osteoclasts. Our results suggest that TUBB6 plays a key role in the control of microtubule and actin cytoskeleton dynamics in osteoclasts. Moreover, by controlling ARHGAP10 association with microtubules, TUBB6 may participate in the local control of CDC42 activity to ensure efficient bone resorption.

Identification of key interactions of benzimidazole resistance-associated amino acid mutations in Ascaris β-tubulins by molecular docking simulations

Ascaris species are soil-transmitted helminths that infect humans and livestock mainly in low and middle-income countries. Benzimidazole (BZ) class drugs have predominated for many years in the treatment of Ascaris infections, but persistent use of BZs has already led to widespread resistance in other nematodes, and treatment failure is emerging for Ascaris. Benzimidazoles act by binding to β-tubulin proteins and destabilising microtubules. Three mutations in the β-tubulin protein family are associated with BZ resistance. Seven shared β-tubulin isotypes were identified in Ascaris lumbricoides and A. suum genomes. Benzimidazoles were predicted to bind to all β-tubulin isotypes using in silico docking, demonstrating that the selectivity of BZs to interact with one or two β-tubulin isotypes is likely the result of isotype expression levels affecting the frequency of interaction. Ascaris β-tubulin isotype A clusters with helminth β-tubulins previously shown to interact with BZ. Molecular dynamics simulations using β-tubulin isotype A highlighted the key role of amino acid E198 in BZ-β-tubulin interactions. Simulations indicated that mutations at amino acids E198A and F200Y alter binding of BZ, whereas there was no obvious effect of the F167Y mutation. In conclusion, the key interactions vital for BZ binding with β-tubulins have been identified and show how mutations can lead to resistance in nematodes.

The beta-tubulin isotype TUBB6 controls microtubule and actin dynamics in osteoclasts.

Osteoclasts are bone resorbing cells that participate in the maintenance of bone health. Pathological increase in osteoclast activity causes bone loss eventually resulting in osteoporosis. Actin cytoskeleton of osteoclasts organizes into a belt of podosomes, which sustains the bone resorption apparatus and is maintained by microtubules. Better understanding of the molecular mechanisms regulating osteoclast cytoskeleton is key to understand the mechanisms of bone resorption, in particular to propose new strategies against osteoporosis. We reported recently that β-tubulin isotype TUBB6 is key for cytoskeleton organization in osteoclasts and for bone resorption. Here, using an osteoclast model CRISPR/Cas9 KO for Tubb6, we show that TUBB6 controls both microtubule and actin dynamics in osteoclasts. Osteoclasts KO for Tubb6 have reduced microtubule growth speed with longer growth life time, higher levels of acetylation and smaller EB1-caps. On the other hand, lack of TUBB6 increases podosome life time while the belt of podosomes is destabilized. Finally, we performed proteomic analyses of osteoclast microtubule-associated protein enriched fractions. This highlighted ARHGAP10 as a new microtubule-associated protein, which binding to microtubules appears to be negatively regulated by TUBB6. ARHGAP10 is a negative regulator of CDC42 activity, which participates in actin organization in osteoclasts. Our results suggest that TUBB6 plays a key role in the control of microtubule and actin cytoskeleton dynamics in osteoclasts. Moreover, by controlling ARHGAP10 association with microtubules, TUBB6 may participate in the local control CDC42 activity to ensure efficient bone resorption.

Modeling a disease-correlated tubulin mutation in budding yeast reveals insight into MAP-mediated dynein function

Mutations in the genes that encode α- and β-tubulin underlie many neurological diseases, most notably malformations in cortical development (MCD). In addition to revealing the molecular basis for disease etiology, studying such mutations can provide insight into microtubule function, and the role of the large family of microtubule effectors. In this study, we use budding yeast to model one such mutation – Gly436Arg in α-tubulin, which is causative of MCD – in order to understand how it impacts microtubule function in a simple eukaryotic system. Using a combination of in vitro and in vivo methodologies, including live cell imaging and electron tomography, we find that the mutant tubulin incorporates into microtubules, causes a shift in α-tubulin isotype usage, and dramatically enhances dynein activity, which leads to spindle positioning defects. We find that the basis for this latter phenotype is an impaired interaction between She1 – a dynein inhibitor – and the mutant microtubules. In addition to revealing the natural balance of α-tubulin isotype utilization in cells, our results provide evidence of an impaired interaction between microtubules and a dynein regulator as a consequence of a tubulin mutation, and sheds light on a mechanism that may be causative of neurodevelopmental diseases. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

The first molecular identification of benzimidazole resistance in Haemonchus contortus from goats in Thailand

Background and Aim: Haemonchus contortus is one of the major trichostrongyloid nematodes affecting small ruminant production worldwide, especially in tropical and subtropical regions. Adult H. contortus suck the blood from the host abomasum leading to anemia and often death in heavily infected animals. The mainstay of parasitic control is an anthelmintic drug, but long-term drug use may cause drug resistance. The aim of this study was to examine benzimidazole resistance in H. contortus of goats from different regions in Thailand by detecting the frequency of the F200Y polymorphism in the β-tubulin isotype 1 gene. Materials and Methods: A total of 121 H. contortus adults were obtained from 31 naturally infected out of 37 slaughtered goats from city abattoirs in five regions of Thailand. The frequency of the F200Y polymorphism in the β-tubulin isotype 1 gene was detected following the allele-specific polymerase chain reaction protocol. Results: The overall genotype frequencies in Thailand were homozygous resistant (RR: 24%), heterozygous (SR: 44.6%), and homozygous susceptible (SS: 31.4%). The allele frequencies were resistant allele (R: 46%) and susceptible allele (S: 54%). The R allele frequency and the RR genotype varied from 30% to 65% and 0% to 43.9%, respectively. The frequency of R alleles was significantly higher in the southern region (0.65) as compared to northern (0.30, p=0.001), western (0.38, p=0.04), and central regions (0.30, p=0.03). The RR genotype was also significantly higher in the southern region (43.9%) versus the northern (0 %, p=0.001), western (11.8%, p=0.012), and central regions (17.4%, p=0.001). Conclusion: This is the first study of the detection of single-nucleotide polymorphisms in codon 200 of the β-tubulin isotype 1 gene of H. contortus from goats in Thailand. These findings are essential and imply that an integrated approach is needed for issues such as drug treatment, farm management, prevention, and control strategies. This is of interest to farmers, veterinarians, and the department of livestock.

Does the in vitro egg hatch test predict the failure of benzimidazole treatment in Haemonchus contortus?

Considerable research has been directed towards optimising in vitro tests that can diagnose resistance in pre-parasitic stages of parasites. The objective of this study was to compare the in vivo faecal egg count reduction test (FECRT), the in vitro egg hatch test (EHT), and the molecular determination of the frequency of a codon 200 allele of β-tubulin isotype 1 associated with benzimidazole resistance in larval stages of Haemonchus contortus obtained from infected goats. Animals were infected with composite infective doses representing 10, 20, 30, 40, 60, and 80% resistant alleles. Faecal samples for the EHT were collected on 28, 33, and 35 days post-infection. The results of the in vivo FECRT indicated that albendazole treatment reduced infections consisting of composite doses of 10, 20, 30, 40, 60, and 80% larvae of the resistant isolate by 91.3, 78.0, 63.3, 48.4, 36.5, and 41.4%, respectively. The drug concentration at which 50% of the eggs were prevented from developing hatching larvae (ED50) in the in vitro EHT varied from 0.09 ± 0.01 to 15.63 ± 12.10 μg/mL thiabendazole. The results of the in vitro EHT indicated that the test could estimate in vivo resistance well. The EHT could thus accurately estimate the in vivo efficacy of the drug and percentage of the resistance allele in the population using hatching parameters in delineation doses. This finding was also supported by comparing the FECRT data to the hatching percentages in the EHT on 30 goat farms in Slovakia with natural mixed infections of gastrointestinal parasites.

Modeling disease-correlated TUBA1A mutation in budding yeast reveals a molecular basis for tubulin dysfunction

Malformations of cortical development (MCD) of the human brain are a likely consequence of defective neuronal migration, and/or proliferation of neuronal progenitor cells, both of which are dictated in part by microtubule-dependent transport of various cargoes, including the mitotic spindle. Throughout the evolutionary spectrum, proper spindle positioning depends on cortically anchored dynein motors that exert forces on astral microtubules emanating from spindle poles. A single heterozygous amino acid change, G436R, in the conserved TUBA1A α-tubulin gene was reported to account for MCD in patients. The mechanism by which this mutation disrupts microtubule function in the developing cerebral cortex is not understood. Studying the consequence of tubulin mutations in mammalian cells is challenging partly because of the large number of α-tubulin isotypes expressed. To overcome this challenge, we have generated a budding yeast strain expressing the mutated tubulin (Tub1G437R in yeast) as one of the main sources of α-tubulin (in addition to Tub3, another α-tubulin isotype in this organism). Although viability of the yeast was unimpaired by this mutation, they became reliant on Tub3, as was apparent by the synthetic lethality of this mutant in combination with tub3Δ. We find that Tub1G437R assembles into microtubules that support normal G1 activity, but lead to enhanced dynein-dependent nuclear migration phenotypes during G2/M, and a consequential disruption of spindle positioning. We find that this mutation impairs the interaction between She1 – a negative regulator of dynein – and microtubules, as was apparent from a yeast two-hybrid assay, a co-sedimentation assay, and from live cell imaging. We conclude that a weaker interaction between She1 and Tub1G437R-containing microtubules results in enhanced dynein activity, ultimately leading to the spindle positioning defect. Our results provide the first evidence of an impaired interaction between microtubules and a dynein regulator as a consequence of a tubulin mutation, and sheds light on a mechanism that may be causative of neurodevelopmental diseases.