Extensive transmission and variation in a functional receptor for praziquantel resistance in endemic Schistosoma mansoni

Abstract

Mass-drug administration (MDA) of human populations using praziquantel monotherapy has become the primary strategy for controlling and potentially eliminating the major neglected tropical disease schistosomiasis. To understand how long-term MDA impacts schistosome populations, we analysed whole-genome sequence data of 570 Schistosoma mansoni samples (and the closely related outgroup species, S. rodhaini) from eight countries incorporating both publicly-available sequence data and new parasite material. This revealed broad-scale genetic structure across countries but with extensive transmission over hundreds of kilometres. We characterised variation across the transient receptor potential melastatin ion channel, TRPM_PZQ, a target of praziquantel, which has recently been found to influence praziquantel susceptibility. Functional profiling of TRPM_PZQ variants found in endemic populations identified four mutations that reduced channel sensitivity to praziquantel, indicating standing variation for resistance. Analysis of parasite infrapopulations sampled from individuals pre- and post-treatment identified instances of treatment failure, further indicative of potential praziquantel resistance. As schistosomiasis is targeted for elimination as a public health problem by 2030 in all currently endemic countries, and even interruption of transmission in selected African regions, we provide an in-depth genomic characterisation of endemic populations and an approach to identify emerging praziquantel resistance alleles.

Fig. 1:. Population structure of Schistosoma accessions.

A. Global distribution of the 574 Schistosoma accessions used in this study. Sampling locations are coloured by geographical divisions: Puerto Rico (PR; brown), Guadeloupe (GP; orange), Senegal (SN; dark purple), Cameroon (pink), Coastal Kenya (KE; teal), Lake Albert (dark blue), Eastern Uganda (red), Southern Uganda (light blue), Koome group islands (yellow), Northern Tanzania (dark green). Black points represent the sampling coordinates of S. rodhaini accessions. B. The maximum-likelihood phylogenetic tree was inferred using 188,923 autosomal single-nucleotide polymorphisms (SNPs) and all 574 accessions. Branches are coloured based on the subdivisions described in a), and the tree is rooted on S. rodhaini. Highlighted clade ‘C1’ represents all West African (Senegalese and Cameroonian), Caribbean (Puerto Rican and Guadeloupean) and Coastal Kenyan accessions. C. Principal component analysis (PCA) of genetic differentiation between 505 unrelated S. mansoni accessions using 214,445 autosomal SNPs. Points are coloured and shaped based on the groups described in (A). D. ADMIXTURE plots illustrating the inferred ancestry of 505 unrelated S. mansoni and four S. rodhaini accessions. Here, we assume five populations (K) are present, inferred using 10-fold cross-validation and a standard error estimation with 250 bootstraps. Y-axis values show the admixture proportions for each accession, and colours for each population were assigned based on the majority ancestry of each geographical division.

Fig. 2:. Genetic variation in the candidate mediator of praziquantel susceptibility, the transient potential receptor channel Sm.TRPM_PZQ.

Mutational frequency along the protein structure of Sm.TRPM_PZQ (Smp_246790). Frequencies of mutations are reported across 550 S. mansoni accessions (y-axis), representing samples from Eastern Uganda (n = 17), Southern Uganda (n = 328), the Koome islands (n = 174) and Northern Tanzania (n = 31). X-axis values represent the location of the mutations on the protein, and bars (and terminal points) are coloured by the predicted impact of each mutation. Structure of Sm.TRPM_PZQ: N-terminal TRPM homology region (MHR) domain, ankyrin-like repeat domain (AD), pre-S1 helix (shaded), TM-spanning helices (1-6), pore helices (PH), TRP domain (TRP), rib helices (R), pole helices (P), coiled-coil (CC) region and the COOH terminal NUDT9H domain (NUDT9H).

Fig. 3:. Functional profiling of variants of the transient potential receptor channel Sm.TRPM_PZQ.

Concentration-response relationships for consensus Sm.TRPM_PZQ sequence compared with twelve Sm.TRPM_PZQ variants, which exhibit an average EC₅₀ to ±PZQ of (A) <1 μM (green) or (B) >1 μM (orange). Constructs for which no praziquantel-evoked activity was observed (Y1554C, Q1670K) are shown in (b) in red. Results represent mean±sem from at least three independent transfections. C. Inset, homology model of the transmembrane spanning region (residues 1100 to 1800) of a Sm.TRPM_PZQ monomer from [2]. The enlarged view shows the location of the functionally profiled variants. Praziquantel and residues within 5Å of the praziquantel binding poise (white) are shown at the base of the voltage-sensor-like domain (VSLD) of the channel, with the S5 and S6 pore-forming helices to the left.

Fig. 4:. Relatedness between Schistosoma mansoni accessions from endemic regions.

A. Ternary plots representing pairwise relationships between accessions using the three relatedness coefficients K₀, K₁, and K₂, representing the probabilities that at a given locus, the two accessions shared zero, one or two-allele identity-by-descent, respectively. Each point represents a pairwise relationship between two accessions, showing whether each accession was from the same (triangles) or different (circles) donor. Points are coloured by the inferred relationship: first-degree (pink), second-degree (green), third-degree (orange) or unrelated (blue). Only accessions from three Southern Ugandan donors and all Koome Island donors are shown. B. Inferred relationships within and between donors. The outer lines represent each donor; within each line, individual accessions are shown coloured by whether they were sampled before (dark grey) or after (light grey) treatment with praziquantel. Coloured lines represent the same pairwise relationships as a). For donors Bb1 and Bu3, all relationships are shown below the circular plot and shown in boxes where they are grouped into pre-treatment samples, post-treatment samples, and pre- and post-treatment samples (spanning treatment). Within the boxes, dotted lines represent relationships inferred by NgsRelate but were found to have a lower degree or no-relation by Sequoia. C. The effect of praziquantel treatment on nucleotide diversity (π) was calculated for each donor infrapopulation (Bb1, Bu1 and Bu3) and all Koome group island samples. Nucleotide diversity was calculated in 5 kb non-overlapping windows across each autosome for each pre-treatment (purple) and post-treatment (yellow) population (including related samples). For all boxplots, the central line indicates the median, and the top and bottom edges of the box indicate the 25th and 75th percentiles, respectively. The maximum whisker lengths are specified as 1.5 times the interquartile range. D. Identification of a circulating lineage of S. mansoni on Damba Island (the northernmost island of the Koome group islands). We identified a cluster of nine accessions, all with third-degree identical by descent relationships, which clustered phylogenetically with accessions from Lake Albert.