Independent origins of loss-of-function mutations conferring oxamniquine resistance in a Brazilian schistosome population - PubMed (original) (raw)

Independent origins of loss-of-function mutations conferring oxamniquine resistance in a Brazilian schistosome population

Frédéric D Chevalier et al. Int J Parasitol. 2016 Jun.

Abstract

Molecular surveillance provides a powerful approach to monitoring the resistance status of parasite populations in the field and for understanding resistance evolution. Oxamniquine was used to treat Brazilian schistosomiasis patients (mid-1970s to mid-2000s) and several cases of parasite infections resistant to treatment were recorded. The gene underlying resistance (SmSULT-OR) encodes a sulfotransferase required for intracellular drug activation. Resistance has a recessive basis and occurs when both SmSULT-OR alleles encode for defective proteins. Here we examine SmSULT-OR sequence variation in a natural schistosome population in Brazil ∼40years after the first use of this drug. We sequenced SmSULT-OR from 189 individual miracidia (1-11 per patient) recovered from 49 patients, and tested proteins expressed from putative resistance alleles for their ability to activate oxamniquine. We found nine mutations (four non-synonymous single nucleotide polymorphisms, three non-coding single nucleotide polymorphisms and two indels). Both mutations (p.E142del and p.C35R) identified previously were recovered in this field population. We also found two additional mutations (a splice site variant and 1bp coding insertion) predicted to encode non-functional truncated proteins. Two additional substitutions (p.G206V, p.N215Y) tested had no impact on oxamniquine activation. Three results are of particular interest: (i) we recovered the p.E142del mutation from the field: this same deletion is responsible for resistance in an oxamniquine selected laboratory parasite population; (ii) frequencies of resistance alleles are extremely low (0.27-0.8%), perhaps due to fitness costs associated with carriage of these alleles; (iii) that four independent resistant alleles were found is consistent with the idea that multiple mutations can generate loss-of-function alleles.

Keywords: Biochemical assay; Loss-of-function; Oxamniquine resistance; Schistosoma mansoni; Soft selective event; Sulfotransferase.

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Figures

Fig. 1

Mapping of the mutations on the gene sequence and structure of Schistosoma mansoni SmSUTL-OR sulfotransferase. Exon 1 and exon 2 are represented in orange and beige, respectively. Single nucleotide polymorphisms and insertion/deletion events are represented in cyan and magenta, respectively. Loss-of-function mutations are highlighted in black. (A) Linear representation of the SmSULT-OR gene showing the relative position of the mutations and their translation in amino acid sequences. (B) Positions of mutations on the SmSULT-OR protein. Oxamniquine (OXA) is represented in yellow, 3′-phosphoadenosine-5′-phosphosulfate (PAPS) co-factor is represented in green, and spatial distortions are represented by red discs. For a more detailed view of the mutations on the structure and their functional impact, see Supplementary Move S1.

Fig. 2

Enzymatic activity of recombinant Schistosoma mansoni SmSULT-OR sulfotransferase expressed from different allelic variants. This in vitro oxamniquine OXA activation assay quantifies DNA-OXA complexes by scintillation (counts per min) (see section 2.9). Bars show the mean of three replicates, while error bars are S.E.M. Enzyme carrying loss-of-function mutations, such as p.C35 or p.E142del, showed no OXA activation, while two newly identified alleles (p.G206V and p.N215Y) did not impair OXA activation. * P < 0.05.

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Independent origins of loss-of-function mutations conferring oxamniquine resistance in a Brazilian schistosome population - PubMed (original) (raw)