It is very difficult to understand how malaria parasites evolve after a human has been bitten by an infected mosquito. There can be billions of individual parasites in a patient’s bloodstream, and traditional genetic sequencing techniques cannot identify the raw material of evolution: new mutations.
If you want to understand if the parasites are related to each other, if they all come from one mosquito or multiple mosquito bites, and what new mutations emerge in an infection, then you have to bring them down to the individual genome level. “
Ian Cheeseman, Ph.D., Assistant Professor and Co-Head of the Host-Pathogen Interaction Program at the Texas Biomedical Research Institute
Through a combination of advanced techniques, Cheeseman and colleagues are now able to sequence the genomes of individual parasites found in the blood of infected patients. Notably, they can now do this even when the infection burden is very low, which can occur with asymptomatic infections. They describe their approach this month in the newspaper Cellular host and microbe. Obtaining this incredibly detailed view of the genetics and evolution of malaria parasites should give researchers and pharmaceutical companies ammunition to develop more effective treatments, vaccines or therapies.
Malaria infects more than 200 million people a year, killing more than 400,000 people in 2019 – most of them young children. Of the five species of malaria parasites that infect humans, two are the most common: Plasmodium falciparum, which is the deadliest; and Plasmodium vivax, which is the main cause of recurrent malaria infections because it can lie dormant in the liver and come back later.
“We were really excited to understand how this dormant stage of the liver could impact genetic variation and evolution in a P.vivax infection, ”says Aliou Dia, Ph.D., co-author of the first article, a postdoctoral researcher in Cheeseman’s lab who is now in the University of Maryland School of Medicine.
The challenge is that when P. vivax emerges, it infects only very young red blood cells, so parasites are rare in the blood. Analyzing these low levels of infection is the microbiological equivalent of looking for a needle in a haystack.
Scientists start with red blood cells, which become slightly magnetic when infected with malaria parasites. They used a strong magnet to separate infected red blood cells from uninfected cells. The infected cells were then passed through a machine called a flow cytometer, which uses a laser and fluorescent markers to detect if there is indeed stray DNA present. Cells containing parasite DNA are deposited one by one into test wells and finally passed through a genetic sequencing machine to decode each individual parasite genome.
Single-cell sequencing allows scientists to precisely compare the genomes of individual parasites to each other to determine their relationship. They can also really dig in and identify unique differences in the genetic code – say an A is changed to a T – to see what has happened since the parasite infected that patient.
“We would expect these brand new mutations to be scattered randomly throughout the genome,” Cheeseman said. “Instead, we find that they often target a family of genes that controls transcription in malaria.”
But that’s not the only thing noticeable about the results. What really turns Cheeseman on is that when the team compared sequencing data from a single cell for P.vivax and P. falciparum, the same family of transcription genes contained the majority of the new mutations for both species.
“We have two different species of malaria in two different parts of the world, Thailand and Malawi,” he says. “When we see the same thing happening independently in different species, that’s an example of convergent evolution.”
In other words, similar processes could shape similar mutation patterns in the two species, even though their last common ancestor dates back millions of years.
The team does not yet know what impact the mutations have on the parasite and its ability to persist and cause damage in human hosts. Mutations may be critical for survival, or something like drug resistance, or may reveal that these genes are unimportant.
“We don’t know what these mutations do,” Cheeseman says. “But the fact that they are targeting what is considered a fairly fundamental part of the parasite’s life cycle is interesting and deserves a lot of follow-up.”