Mapping the sex lives of malaria parasites at single-cell resolution reveals the genetics underlying transmission

Malaria is caused by a eukaryotic microbe of the genus Plasmodium and is responsible for more deaths than all other parasitic diseases combined. To be transmitted from the human host to the mosquito vector, the parasite must differentiate into its sexual stage, called the gametocyte stage.

Unlike primary sex determination in mammals, which occurs at the chromosomal level, it is not known what drives this single-celled parasite to form males and females. New research at Stockholm University has applied high-resolution genomic tools to map the global repertoire of gametocyte development genes according to male or female sexual fate.

The study, published in Nature Communicationsreveals the genes expressed in Plasmodium falciparum, the deadliest malaria parasite, from the very beginning of sexual development to maturity. At this point, male and female gametocytes are ready to be taken up by the female Anopheles mosquito to initiate the relentless cycle of transmission.

“We combined cutting-edge single-cell genomics with a novel computational approach to define the expression of several important genetic regulators along the developmental trajectory of male and female gametocytes,” says Johan Ankarklev, associate professor in the Department of Molecular Biosciences at the Wenner Gren Institute and lead author of the study.

The research conducted by Stockholm University in collaboration with Dr Johan Henriksson from MIMS at Umeå University and the Single Cell Microbial Genomics Facility at SciLifeLab is important for improving our understanding of the genetics underlying malaria transmission.

A widely conserved family of transcription factors, termed ApiAP2, has emerged as a key regulator of gene expression during the Plasmodium life cycle and development.

“Our high-resolution data allowed us to computationally link the expression of several of these ApiAP2 genes to the male or female lineage, implying their involvement in determining sex cell fate. Importantly, we have also established a large set of novel candidate “driver” genes for male and female cell fate, which we are currently exploring in more detail in the laboratory using CRISPR technology,” Ankarklev continues.

The study brings important results to the malaria community, but also to the scientific community as a whole:

• From a clinical perspective, treatment strategies have historically targeted the asexual and highly symptomatic blood stage of infection, with varying degrees of success. Importantly, current treatment strategies do not inhibit malaria transmission. This study provides important new genetic markers for the future development of transmission-blocking therapies, which are the only way to inhibit the spread of malaria.
• From an evolutionary perspective, considering that Plasmodium is an ancient microbial eukaryote that produces males and females, the new data and analyses provide new information and clues regarding the evolution of sex in eukaryotes.

There is currently little knowledge about the sexual reproduction of malaria.

Most eukaryotes reproduce sexually to ensure diversity and fitness selection. In animals, sex determination most often involves males and females. However, among the great diversity of organisms that make up eukaryotic microbes, the systems by which sex is defined are very diverse and often cryptic.

The malaria parasite, Plasmodium spp., belongs to the phylum Apicomplexa, a group of obligate invasive single-celled parasites that form both male and female gametes. The crescent-shaped malaria gametocyte was first described by French scientist Alphonse Laveran in 1880. Two decades later, British physician Robert Ross discovered that malaria was transmitted by mosquitoes.

Despite these important discoveries, it is only in recent years that significant progress has been made in improving our understanding of the biology of malaria transmission stages, thanks to new and revolutionary biotechnology.

How new genomic tools are advancing malaria research

Single-cell transcriptome profiling provides a snapshot of a broad range of genes expressed in a cell, in this case a malaria parasite, at a single point in development. By adding thousands of single-cell transcriptomes to the analysis, it becomes a powerful tool for determining genetic pathways and developmental bifurcations, which is essential for lineage tracing.

“By combining Pseudotime and RNA Velocity, two recently developed computational tools, we aligned several thousand cells along a branched pseudo-time axis. Then, we used RNA velocity estimates to define the splicing kinetics among transcripts on the developmental axes. This in turn allowed us to predict a large panel of putative ‘driver genes’ for male and female sexual fates, and interestingly, many of these genes had not been annotated before,” says Mubasher Mohammed, a former PhD student in Ankarklev’s lab and lead author of the study.

Mohammed grew up in Sudan, where he experienced the devastating effects of malaria up close.

“It’s an exciting time to be a scientist, where new technologies are allowing us to make huge advances in our understanding of the different types of diseases that afflict humanity,” says Mohammed.

The transmission phase of malaria marks a dramatic decline in the parasite population, making it an attractive target for malaria control efforts.

“When such a bottleneck occurs in the population, it becomes more vulnerable to drugs and environmental factors. By describing the molecular mechanisms of gametocyte development, we can target these pathways to develop effective transmission-blocking strategies, vital for malaria eradication efforts,” says Alexis Dziedziech, a former postdoctoral fellow at AnkarklevLab and co-author of the study.