Characterization of the β-tubulin gene family in Ascaris lumbricoides and Ascaris suum and its implication for the molecular detection of benzimidazole resistance

Abstract

Background: The 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 β-tubulin proteins, the principal targets of BZ drugs.

Methodology and principal findings: First, the β-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 β-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.

Significance: This study demonstrated the presence of at least seven β-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 β-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of β-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.

Fig 1. Schematic representation of the research workflow.

RNA-Seq: RNA sequencing, NCBI-SRA: NCBI Sequence Read Archive, cDNA: complementary DNA, L3: third larval stage, M: male, F: female, RT-PCR: reverse transcription PCR, SNPs: single nucleotide polymorphisms.

Fig 2. Phylogenetic tree of β-tubulin proteins of Ascaris species, other parasitic nematodes, and C. elegans.

Phylogenies were reconstructed with RAxML and MRBAYES. The topology shown is from RaxML. The node support values are percent bootstraps / posterior probabilities. The ‘-‘ indicates that a node was not present in the MRBAYES tree. The branches are equal length to show topology clearly; a tree with branch lengths showing divergence is available in supplementary materials (S14 Info).

Fig 3. Transcription profiles of the β-tubulins of Ascaris suum.

Panel A. Reverse transcription (RT)-PCR analyses were performed on cDNA samples with gene specific primer sets. The agarose gels show the transcription profiles of the β-tubulins in four developmental stages (egg, third larval stage (L3) in egg, L3 in liver, L3 in lung) and three tissue types of adult male and female worms (intestinal, reproductive, and cuticle). Panel B. RNA-seq analyses were performed using RNA-seq data obtained from the NCBI Sequence Read Archive. The stacked bar charts show the expression of each β-tubulin (relative to total β-tubulin expression) in different developmental stages (24h embryos to L4 stage larvae) and different tissue types of A. suum.

Fig 4. Allele counts for Alu/Asu-bt-A and Alu/Asu-bt-B in three adult Ascaris worm populations.

Ascaris suum worms were collected from the intestines of pigs slaughtered in Belgium. Ascaris lumbricoides worms were collected during two expulsion studies in Ethiopia and Tanzania. The total number of each amplicon sequence variant (ASV) per population is displayed (allele count) and the color indicates the proportion within the population (allele frequency). Heatmaps were created using the R package PopGenReport [65,66].