Skip to main content
Premium Trial:

Request an Annual Quote

Loss-of-function Mutations Lead to Drug Resistance in Blood Flukes

NEW YORK (GenomeWeb News) – Researchers in Texas uncovered loss-of-function mutations that lead the human blood fluke to be resistant to the antihelminthic oxamniquine.

As they reported in Science today, the investigators crossed resistant and sensitive helminthes and examined the genomes of the parental, offspring, and grand-offspring for clues that led them to a causal gene, dubbed SmSULT-OR. In addition, they used X-ray crystallography and computational methods to visualize how the drug interacts with the protein this gene encodes and how changes to the gene affect that interaction.

"We were able to identify the critical gene by crossing resistant and sensitive worms in the laboratory and then analyzing the genomes of the progeny," first author Claudia Valentim from the University of Texas Health Sciences Center and the Texas Biomedical Research Institute said in a statement. "This method is commonly used for fruit flies and other laboratory organisms, but has not previously been possible for schistosome parasites."

Schistosomes such as Schistosoma mansoni, S. haematobium, and S. japonicum are thought to infect 200 million people in Africa, Asia, and South America, leading to portal hypertension, liver failure, and bladder cancer, according to the World Health Organization. Some 200,000 people die of schistosomiasis each year.

The researchers noted that there is no vaccine for the disease, and there are a limited number of drugs to treat the condition. Oxamniquine, the drug Valentim and her colleagues focused on, is effective only against one species, in this case S. mansoni, and beginning in the 1970s, S. mansoni in Brazil began exhibiting resistance to oxamniquine, which was a first-line treatment for the disease until the 1990s, though it remains in use.

The researchers added that their work on drug resistance may help inform the development of drugs to treat schistosomiasis more widely.

To search for genes linked to oxamniquine resistance, the researchers crossed OXA-sensitive and OXA-resistant parasites and their progeny and grew them up in the lab. They determined resistance by exposing the worms to the drug. The F1 generation and nearly 75 percent of the F2 generation were sensitive to the drug, suggesting a recessive inheritance.

By genotyping the different generations using some 62 microsatellite markers, the researchers homed in on a quantitative trait locus on chromosome 6.

To fine-map the QTL regions, the researchers sequenced two parents and two F1s to between 11x and 29x coverage, and identified more than 558,000 SNPs that differed between the sensitive and resistance parents. Then by drawing on the F2 generation, they narrowed the number of genes down to 16.

They prioritized those candidate genes based on whether they contained fixed non-synonymous differences between the parents, whether there was a difference in transcript abundance, and whether the size of the gene products matched predictions. Just one gene — Smp_119060 — met all three criteria.

Using recombinant assays, the Texas investigators looked into the function of that gene as well as five others that met two of their three criteria. Again, only Smp_119060 expressed a protein that activated OXA and, further, dsRNA targeting Smp_119060 in sensitive parasites led to resistance.

The Smp_119060 gene in resistant worms differed from the sensitive ones at two spots: at a L256W substitution and a 142E deletion. Through a complementation assay, the researchers determined that proteins with the substitution could activate OXA while the deletion did not, indicating that it was the cause of resistance.

However, researchers pointed out that a parasite sample collected in Brazil in the 1970s had yet another Smp_119060 mutation. This one, C35R, was also unable to activate OXA.

"Hence, mutations causing loss-of-function in Smp_089320 can be independently derived in field- and laboratory-derived OXA-resistant parasites," the researchers said.

In schistosomes, OXA is activated by sulfotransferases that transfer sulfate groups from the universal sulfate donor 3'-phosphoadenosine-5'-phosphosulfate to the drug. Through a sulfonation assay, the researchers confirmed that Smp_119060 is a sulfotransferase and that proteins encoded by the sensitive Smp_119060 could transfer the sulfate group while the proteins encoded by Smp_119060 with the 142E deletion or C35R substitution had lost that function.

Then by examining the crystal structure of Smp_119060 with PAP and OXA bound, the researchers noted that a 142E deletion or C35R substitution would affect OXA binding and/or limit sulfonation.

OXA, the researchers noted, is only effective in treating one schistosome species. By drawing up a phylogenetic tree based on genes with homology to Smp_119060 from other schistosomes, they found that one S. haematobium residue that would interact with OXA contains a substitution that is predicted to result in a polarity change as well as a change to protein shape that would disallow OXA binding.

This, they added, may provide a framework to develop OXA-related drugs effective against both S. mansoni and S. haematobium.

"By using X-ray crystallography and computational methods, we were able to precisely pinpoint how the drug interacts with the critical protein in one schistosome species and to identify the key differences in this protein in the related parasite species," said UTHSC's John Hart. "With some targeted chemical modification, we think it will be possible to make a drug that kills both major schistosome species. This is what we are working on now."

Filed under

The Scan

Driving Malaria-Carrying Mosquitoes Down

Researchers from the UK and Italy have tested a gene drive for mosquitoes to limit the spread of malaria, NPR reports.

Office Space to Lab Space

The New York Times writes that some empty office spaces are transforming into lab spaces.

Prion Pause to Investigate

Science reports that a moratorium on prion research has been imposed at French public research institutions.

Genome Research Papers on Gut Microbe Antibiotic Response, Single-Cell RNA-Seq Clues to Metabolism, More

In Genome Research this week: gut microbial response to antibiotic treatment, approach to gauge metabolic features from single-cell RNA sequencing, and more.