Characterization Of The Beta-Tubulin Gene Family In Ascaris Lumbricoides And Ascaris Suum And Its Implication For The Molecular Detection Of Benzimidazole Resistance

Author summaryBenzimidazole (BZ) drugs remain the standard of treatment in large-scale deworming programs that aim to control the morbidity caused by intestinal worms. As these deworming programs are expanding world-wide, there is an increasing risk of worms becoming resistant to BZ drugs, highlighting the necessity for tools to detect gene mutations associated with drug resistance. However, the development of such tools is impeded by a lack of insights into the genes that are coding for beta-tubulin proteins, which are the principal targets of BZ drugs. The aim of this study was to comprehensively characterize these genes in the worm species Ascaris lumbricoides and Ascaris suum. The findings highlight that these species have at least seven beta-tubulin genes. Only two genes are highly expressed throughout the different life stages of the worm, and hence are more likely to be involved in the development of BZ resistance. No mutations that have previously been associated with BZ resistance in other intestinal worms were found. This study provides a baseline towards more efficient and accurate monitoring of drug resistance in large-scale deworming programs.

BackgroundThe treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding beta-tubulin proteins, the principal targets of BZ drugs.

Methodology and principal findingsFirst, the beta-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different beta-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations.

SignificanceThis study demonstrated the presence of at least seven beta-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the beta-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of beta-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.