For the first time the developmental stages of the deadliest human malaria parasite, Plasmodium falciparum, have been mapped in high resolution, allowing scientists to better understand this constantly adapting parasite in greater detail than has previously been possible. The researchers, headed by a team at the Wellcome Sanger Institute, together with scientists at the Malaria Research and Training Center (MRTC) in Mali, and collaborators, used single-cell RNA sequencing to generate datasets of both laboratory strains of the parasite, and parasites from natural infections, and create a high-resolution reference atlas that details the critical sexual developmental stages of the parasite.

The results offer up detailed information on genetic and transcriptional variation at the different life stages of P. falciparum as the organism matures, changing from an asexual state to a sexual state, which is necessary before the parasite can be transmitted to mosquitoes.

The work adds to the freely available Malaria Cell Atlas, which provides information for researchers worldwide to investigate and generate tools to track malaria. The research provides more in depth insight into the variation of individual malaria parasites, compared with grouping samples together, and bulk sequencing. The Wellcome Sanger team suggests the novel insights accessible through the Malaria Cell Atlas may also help to identify new drug or vaccine-based strategies for blocking the parasite’s development and so prevent transmission.

Abdoulaye Djimdé, PhD, at the Malaria Research and Training Center, University of Bamako, and Honorary Faculty at the Wellcome Sanger Institute, said, “Malaria has a huge global impact, affecting millions of people each year, and attempts to control and treat the disease are quickly overcome by the parasite. Understanding more about the parasite’s life cycle, the genes involved, and the factors that control these, can be vital to ongoing malaria research. Our research highlights key points in the sexual development of the parasite, which if targeted in future drug development could break the cycle of transmission and help minimise the spread.”

Mara Lawniczak, PhD, at the Wellcome Sanger Institute, further commented, “This new focus of the Malaria Cell Atlas project on natural infections coincides with malaria vaccines being used for the first time and a continued rise of drug resistance. Single-cell RNA sequencing gives us a window into parasite gene usage that is not possible with any other approach, while also providing a much clearer understanding of just how genetically diverse parasites are, even within the same person. The Malaria Cell Atlas is a resource we hope will be increasingly useful on the path to malaria elimination.”

Lawniczak is co-senior author of the team’s published paper in Science, titled “A single-cell atlas of sexual development in Plasmodium falciparum.” In their research summary the team concluded, “Single cell evaluations of malaria parasites from natural infections will enhance our understanding of malaria parasite persistence, pathology, and transmission dynamics, and this atlas will be a key resource underpinning future work.”

Malaria is a life-threatening disease caused by species of the Plasmodium parasite. P. falciparum is the deadliest type and the most prevalent on the African continent, the authors suggested, citing WHO figures indicating that there were an estimated 249 million malaria cases and 608,000 related deaths globally in 2022.

Plasmodium falciparum is a single-celled parasite that evolves quickly, making it difficult for scientists to develop long-lasting and effective diagnostics, drugs and protective vaccines. Malaria parasites have a huge amount of genetic diversity and people are frequently infected with multiple different strains. “The extensive spatiotemporal genetic diversity of P. falciparum presents a challenge to the development of effective diagnostics, drugs, and vaccines,” the authors wrote. “Coinfection with different P. falciparum strains occurs in more than 70% of infections in malaria-endemic populations.”

Malaria parasites are found in either an asexual or sexually developed form in the human host. Asexual replication in humans is what causes the symptoms of malaria, but to transmit, parasites have to develop and become either a male or female reproductive cell, known as a gametocyte.  “… transmission from human to human can only occur when parasites successfully reproduce sexually in the mosquito vector”, the team continued. Sexual commitment and development are controlled by transcription factors, which are proteins that regulate gene activity. The mature sexual forms of the parasite circulate in the bloodstream until they are taken up by mosquitoes. “Preventing sexual reproduction by stopping the parasite’s development at any point during its sexual cycle breaks the cycle of transmission and will contribute to malaria control,” the scientists noted.

For their newly reported research, teams from the Wellcome Sanger Institute and the MRTC in Mali used both long-read and short-read single-cell RNA sequencing (scRNA-seq) to map the sexual development stages of P. falciparum in the laboratory. This allowed them to track the gene expression levels and highlight which genes are involved in each stage of the process. “We generated both short- and long-read scRNA-seq data from ~37,000 laboratory malaria parasite cells covering the asexual and sexual developmental stages,” the team explained in the research article summary. “We characterized the intraerythrocytic cycle in lab strains with a focus on sexual development, exploring distinct expression modules underlying gametocyte development … Cell and gene clustering revealed a topology reflecting the intraerythrocytic stages, with sexual developmental stages branching off from the asexual replication cycle, progressing to form female and male gametocytes.”

The team then applied the same approach to parasites from blood samples collected from four people naturally infected with malaria in Mali. “We also profiled and investigated ~8000 parasites obtained from four naturally infected malaria carriers, each of whom carried multiple genotypic strains …” This is the first time that these technologies have been applied to real-time infection strains at such a high resolution. By comparing the laboratory data with the natural infection data, the researchers found parasite cell types not previously seen in laboratory strains, highlighting the importance of real-world data.

The team compared different natural P. falciparum strains within each donor to identify genes of interest. “Investigating natural infections at single-cell resolution enabled strain and stage assignment for each parasite and revealed unexpected transcriptomic clusters and differential expression between strains even within the same host,” the team stated. The next step will be to assess the impact these genes have on transmission.

Wellcome Sanger Institute co-first author Jesse Rop, said, “This is the first time that we have been able to map the sexual development stages of malaria parasites in both laboratory and natural strains, allowing us to gain deeper insight into the similarities and differences. Our research uncovered new biology present in the naturally occurring strains that are not seen in laboratory strains, improving our understanding of how malaria develops and spreads.”

The authors further commented in their research article summary, “Single cell evaluations of malaria parasites from natural infections will enhance our understanding of malaria parasite persistence, pathology, and transmission dynamics … The integrated dataset, comprising cells from laboratory strains and natural infections, spanning asexual and sexual development is presented as a new chapter in the interactive Malaria Cell Atlas data resource.”

Co-first author Sunil Dogga, PhD, at the Wellcome Sanger Institute, added, “Our research adds to the growing Malaria Cell Atlas, giving a high-quality, open-access genomic resource for researchers worldwide. This high-resolution atlas can be used by scientists to gain a clear understanding of the genes they are investigating, combine research efforts, and help us more effectively prevent, control, and treat malaria. Working together as a scientific community is the only way we are going to successfully control and treat malaria.” In their main paper, the team further noted, “Single-cell approaches now enable us to study strain composition and expression behavior including differential expression and isoform usage in natural infections, and have enormous potential toward improving our understanding of malaria parasite biology and transmission in natural infections.”

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