NEW YORK (GenomeWeb) – Researchers from the Infectious Disease Research Center at Université Laval in Quebec have developed a method that combines high-throughput functional screening with next-generation sequencing to quickly identify drug targets and resistance genes for the parasite Leishmania, which causes the tropical disease known as sleeping sickness.
The method, which they described in a proof-of-concept study in the Proceedings of the National Academy of Sciences this week, could also be applied to other pathogens, senior author Marc Oullette told GenomeWeb.
Ouellette said that the team decided to focus on Leishmania because traditional functional screening methods like RNA interference do not work on Leishmania since it lacks the RNAi machinery.
Cosmids have previously been used for functional screening, Ouellette said, so the researchers decided to try to pair that with NGS to do it in a more high-throughput fashion. Cosmids are a type of hybrid plasmid that contain a cos sequence and a lambda phage. While plasmids tend to be short fragments of DNA, cosmids are longer, stretching to between 35 kilobases and 50 kilobases of DNA, Ouellette said. The lambda phage "facilitates entry and the cloning of the large fragments," he added.
In the study, the researchers digested a Leishmania infantum genome, creating cosmid inserts averaging 40 kilobases that covered the more than 8,000 genes within the L. infantum genome. Next, they inserted the various cosmids into wild-type L. infantum and applied drug pressure to the parasites. They then extracted cosmids from the populations that survived and sequenced them. The researchers did this multiple times, incrementally increasing the concentrations of antileishmanials to enrich for subpopulations with drug resistance. To control for cosmids that contained genes that contributed to the overall fitness and survival of the organism regardless of drugs, they performed the same steps in the absence of drugs.
They then used bioinformatics tools to analyze which genes were enriched as concentrations of drugs increased, generating a map of the genomic loci that were enriched by drug selection.
Once those drug resistance genes and gene clusters were identified, they validated them by isolating them and transfecting them into a wild-type organism.
To demonstrate that the approach could identify resistance mechanisms, the team performed Cos-Seq using a well-studied drug, methotrexate, even though it is not typically used to treat sleeping sickness.
When they performed Cos-Seq, the researchers identified four loci with enriched genes on chromosomes 6, 21, 23, and 34. Several of those regions were covered by overlapping cosmids, and the loci that were enriched on chromosomes 6 and 23 included the known primary and secondary drug targets of methotrexate and known mechanisms of resistance.
The researchers were also able to characterize two genomic regions that had not been previously associated with methotrexate resistance. To validate those regions, they isolated the genes, grew them in Escherichia coli and then transfected them back into wild-type L. infantum and showed that they did indeed confer resistance and that the level of resistance correlated with the level of enrichment of the cosmid.
Next, the researchers did the experiment for five different antileishmanial drugs — antimony, miltefosine, paromomycin, amphotericin B, and pentamidine.
"These are molecules that we know are active against the parasite but we don't know how they function," Oullette said.
After running the Cos-Seq screening method on all five drugs, the researchers identified 64 loci enriched for resistance, 12 or which were common to at least two different drugs.
"The high number of drug-enriched loci suggests that the five antileishmanials studied might act via different targets and/or trigger distinct resistance mechanisms, whereas the 12 shared loci may correspond to more universal mechanisms as part of a global stress response," the authors wrote.
The authors then picked a few of the identified novel genomic targets to study further. For instance, looking at the PTD screen, the group identified an enriched cosmid that contained the gene encoding the ABC transporter pentamidine resistance protein 1, which is known to confer PTD resistance. They also identified overlapping cosmids that contained five genes that were enriched in PTD resistance as well as PMM and AMB. When looking at expression of the five genes independently, they were able to pinpoint one that was responsible for both PTD and PMM resistance. That gene is novel and encodes a protein of unknown function.
The authors noted that the drugs they investigated are older drugs that may act on many different targets. "Additional work is needed to determine whether some of these genes are genuine drug targets," they wrote. In addition, there are now several newer candidate compounds that might be more specific and the Cos-Seq method could be applied to those to "pinpoint their putative targets, which could be helpful for further drug development."
Ouellette added that this technique could complement drug development efforts to identify mechanisms of action and resistance. He added, however, that the technique is only suitable for identifying protein targets. "It's very powerful for isolating drug targets when they are protein based," he said, but will not work for lipid or RNA targets.
Oullette said that the investigators are now following up on this proof of concept in several ways. They are continuing to study the genomic loci that they identified here. In addition, they are working with clinical isolates from the field, including isolates that are resistant as well as sensitive to drugs to "step-by-step isolate drug-resistance markers."
Once those markers are identified and validated, he said, they could be used to develop better diagnostic tools.
And finally, he said, aside from Leishmaniasis, the researchers are looking to apply a similar technique to other types of parasites and pathogens. Already, Ouellete said, they have begun to work on E. coli, except that instead of using cosmids they modified the technique to use plasmids. Plasmids consist of shorter fragments of DNA, which he said was better for organisms like E. coli that have smaller genomes.