LONDON – The first detailed map of gene expression in the malaria parasite Plasmodium shows exactly which genes are active at each stage of the complex life cycle of the single cell organism in the mosquito vector and the human host.

The Malaria Cell Atlas also provides new insights into the roles of the 40% of Plasmodium genes whose function is unknown and shows clusters of genes are expressed across the life span, pointing to a number of new therapeutic and diagnostic targets.

In addition, the transcriptome data open the way to increased understanding of the development of resistance to anti-malaria drugs and provide a grounding for strategies to limit transmission of the disease.

Timing of the publication of the map, in Science, coincides with a fresh plea by the World Health Organization (WHO) for a re-energized R&D push to develop the knowledge base, strategy, tools and vaccines needed to get malaria eradication efforts back on track.

The plea comes from WHO's strategic advisory group on malaria, as it publishes the results of a three-year investigation into how to permanently interrupt transmission of the disease at a global level.

The WHO analysis reaffirms there are no barriers to the vision of complete eradication, according to Pedro Alonso, director of WHO's global malaria program, launching the report. "It's biologically feasible," he said, "It's a question of how and when."

Despite significant progress since WHO launched its first eradication plan in 1955, there are still 200 million cases of malaria per annum. With the tools currently on hand and backed by favorable megatrends, in particular increased economic development in Africa, it will be possible to bring that down to 11 million cases per annum, by 2050.

"We can go a long way; we can get to within sight of eradication, but we won't reach it," Alonso said.

More work needed

There also is concern that the steady progress made since 1955 is slowing. To get back on track and lay foundations for future eradication efforts, there must be further improvements in the quality and use of data to detect changes in malaria transmission, and more investment in research.

"What I would particularly like to underscore, is the need for a revitalized R&D effort if we want to eradicate malaria," Alonso said.

Scientists at the Wellcome Sanger Institute in Cambridge, U.K., who compiled the Malaria Cell Atlas, said new insights it provides into gene function across the life cycle of the Plasmodium parasite will advance study of the pathology and transmission of malaria, and support development of much needed new drugs.

"This is the first atlas of its kind for a single-celled organism," said lead author Virginia Howick. "The [Plasmodium] life cycle is key to research into this disease and the atlas will help us to truly understand the parasite," she said.

To compile the atlas the researchers profiled the complete transcriptomes of 1,787 individual specimens of the rodent laboratory model, P. berghei, at 10 different time points, covering all its life cycle stages in the mosquito and the host.

Unscrambling bulk data

Establishing gene expression profiles for different stages of development is an important step forward, because transcriptomic studies of Plasmodium often are confounded by multiple life stages in a single sample. The atlas can now be used as a cross reference to unscramble bulk transcriptome data.

Each P. berghei specimen contained around 1,527 genes. From the transcriptome it was possible to understand when and where each gene is active across development, and to identify genes associated with each of the different phases.

For example, in the mosquito stages, the researchers observed a clear and abrupt change in which genes were expressed in the 48 hours following an infectious blood meal, as the parasite developed from an ookinete (mobile zygote) to a static oocyte embedded in the mosquito gut.

Further along the development pathway, when sporozoites are released from the salivary glands of the mosquito into the host, twice as many genes are expressed and genes necessary for host invasion are upregulated.

Comparing expression patterns across the life cycle highlighted groups of genes that behave similarly. Most of these clusters were highly expressed at single stage, meaning they could be used to infer the possible function of the 40% of Plasmodium genes whose role has not been characterized.

"We've inferred the roles of parasite genes that until now were entirely unknown," said Andrew Russell, co-lead author. "We do this through guilt-by-association. By looking at functions of previously studied genes, we can predict the roles of unknown genes, if they show similar activity patterns," he said.

The authors also analyzed the transcriptome of the rodent host, confirming at a single cell level that the development of P. berghei is independent of the cell cycle state of the host.

Using the technique of droplet sequencing, the researchers then sequenced thousands more single Plasmodium cells from the red blood cell stages of three species of the parasite that infect mice, monkeys and humans.

That showed similar gene activity across the three species, even though they infect such different hosts.

In addition, the atlas includes the transcriptomes of P. falciparum taken directly from human patients. That is important because in vitro systems are unlikely to fully capture the breadth of variation in gene expression that occurs in parasites circulating in naturally infected carriers.

The inclusion of wild-type transcriptomes provides the foundation for studying the biology of individual parasites taken directly from their natural environment, making possible to characterize phenotypes that confer drug resistance, pathogenicity and transmissibility.

In total, the Malaria Cell Atlas comprises 15,858 transcriptomes covering every life stage of Plasmodium at a single cell resolution. The data is freely available, providing an important resource to accelerate the research WHO says is necessary to eradicate malaria.

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