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Ascaris lumbricoides ? carbonic anhydrase: a potential target enzyme for treatment of ascariasis.

ABSTRACT: A parasitic roundworm, Ascaris lumbricoides, is the causative agent of ascariasis, with approximately 760 million cases around the world. Helminthic infections occur with a high prevalence mostly in tropical and developing xcountries. Therefore, design of affordable broad-spectrum anti-helminthic agents against a variety of pathogens, including not only A. lumbricoides but also hookworms and whipworms, is desirable. Beta carbonic anhydrases (?-CAs) are considered promising targets of novel anthelminthics because these enzymes are present in various parasites, while completely absent in vertebrates.In this study, we identified an A. lumbricoides ?-CA (AIBCA) protein from protein sequence data using bioinformatics tools. We used computational biology resources and methods (including InterPro, CATH/Gene3D, KEGG, and METACYC) to analyze AlBCA and define potential roles of this enzyme in biological pathways. The AlBCA gene was cloned into pFastBac1, and recombinant AIBCA was produced in sf-9 insect cells. Kinetics of AlBCA were analyzed by a stopped-flow method.Multiple sequence alignment revealed that AIBCA contains the two sequence motifs, CXDXR and HXXC, typical for ?-CAs. Recombinant AIBCA showed significant CA catalytic activity with kcat of 6.0?×?10(5) s(-1) and kcat/KM of 4.3?×?10(7) M(-1) s(-1). The classical CA inhibitor, acetazolamide, showed an inhibition constant of 84.1 nM. Computational modeling suggests that the molecular architecture of AIBCA is highly similar to several other known ?-CA structures. Functional predictions suggest that AIBCA might play a role in bicarbonate-mediated metabolic pathways, such as gluconeogenesis and removal of metabolically produced cyanate.These results open new avenues to further investigate the precise functions of ?-CAs in parasites and suggest that novel ?-CA specific inhibitors should be developed and tested against helminthic diseases.

SUBMITTER: Zolfaghari Emameh R

PROVIDER: S-EPMC4575479 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Ascaris lumbricoides β carbonic anhydrase: a potential target enzyme for treatment of ascariasis.

Zolfaghari Emameh Reza R Kuuslahti Marianne M Vullo Daniela D Barker Harlan R HR Supuran Claudiu T CT Parkkila Seppo S

Parasites & vectors 20150918

<h4>Background</h4>A parasitic roundworm, Ascaris lumbricoides, is the causative agent of ascariasis, with approximately 760 million cases around the world. Helminthic infections occur with a high prevalence mostly in tropical and developing xcountries. Therefore, design of affordable broad-spectrum anti-helminthic agents against a variety of pathogens, including not only A. lumbricoides but also hookworms and whipworms, is desirable. Beta carbonic anhydrases (β-CAs) are considered promising tar ...[more]

PMID: 26385556

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Project description:BackgroundThe soil-transmitted helminths (STH) Ascaris lumbricoides and Trichuris trichiura are gastrointestinal parasites causing many disabilities to humans, particularly children. The benzimidazole (BZ) drugs, albendazole (ALB) and mebendazole (MBZ), are commonly used for mass treatment for STH. Unfortunately, there is concern that increased use of anthelmintics could select for resistant populations of these human parasites. In veterinary parasites, and lately in filarial nematodes, a single amino acid substitution from phenylalanine to tyrosine, known to be associated with benzimidazole resistance, has been found in parasite beta-tubulin at position 200. We have developed pyrosequencer assays for codon 200 (TTC or TAC) in A. lumbricoides and T. trichiura to screen for this single nucleotide polymorphism (SNP).Method and findingsPyrosequencing assays were developed and evaluated for detecting the TTC or TAC SNP at codon 200 in beta-tubulin in A. lumbricoides and T. trichiura. Genomic DNA from individual worms, eggs isolated from individual adult worms or from fecal samples with known treatment history and origin, were sequenced at beta-tubulin by pyrosequencing, and genotypes were confirmed by conventional sequencing. The assays were applied to adult worms from a benzimidazole-naïve population in Kenya. Following this, these assays were applied to individual worms and pooled eggs from people in East Africa (Uganda and Zanzibar) and Central America (Panama) where mass anthelmintic drug programs had been implemented. All A. lumbricoides samples were TTC. However, we found 0.4% homozygous TAC/TAC in T. trichiura worms from non-treated people in Kenya, and 63% of T. trichiura egg pools from treated people in Panama contained only TAC.ConclusionAlthough the codon 200 TAC SNP was not found in any of the A. lumbricoides samples analyzed, a rapid genotyping assay has been developed that can be used to examine larger populations of this parasite and to monitor for possible benzimidazole resistance development. The TAC SNP at codon 200, associated with benzimidazole resistance in other nematodes, does occur in T. trichiura, and a rapid assay has been developed to allow populations of this parasite to be monitored for the frequency of this SNP. Sample sizes were small, anthelmintic efficacy was not assessed, and treated and non-treated samples were from different locations, so these frequencies cannot be extrapolated to other populations of T. trichiura or to a conclusion about resistance to treatment. The occurrence of the TAC SNP at codon 200 of beta-tubulin in T. trichiura may explain why benzimidazole anthelmintics are not always highly effective against this species of STH. These assays will be useful in assessing appropriate treatment in areas of high T. trichiura prevalence and in monitoring for possible resistance development in these STH.

Dataset Information

Ascaris lumbricoides ? carbonic anhydrase: a potential target enzyme for treatment of ascariasis.

Publications

Ascaris lumbricoides β carbonic anhydrase: a potential target enzyme for treatment of ascariasis.

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