The advancing capabilities and reduced costs of sequencing technologies are enabling a deeper appreciation of the genotypic and phenotypic variability of infectious disease pathogens. Two new studies in Science survey diversity in malarial Plasmodium species, assessing genetic diversity across Africa, as well as transcriptomic diversity through the pathogen life cycle.
In their study, Amambua-Ngwa et al. carried out whole-genome sequencing of 2,263 isolates of Plasmodium falciparum, which is a single-celled protozoan that is the cause of the most lethal form of malaria. Samples were collected from 15 countries across sub-Saharan Africa, spanning the entire continent from west-to-east, as well as the island of Madagascar.
Analysis of genetic diversity was largely consistent with the model of human-infectious P. falciparum originating from a great-ape-tropic P. falciparum species that crossed into humans ~10,000 years ago in central Africa, as ancestral genome segments of likely central African origin were found to contribute to derived P. falciparum populations throughout the continent.
However, clustering analysis showed that populations were not continuous throughout Africa: six population subgroups localized to particular geographical regions. Reasons for this population stratification are likely to be multifactorial, including geographical barriers, different genetic ancestries of human hosts and different species of Anopheles mosquito vectors, as well as wider climate differences.
One major motivation for P. falciparum genomic analyses is to monitor the emergence of antimalarial drug resistance, especially in response to recent interventions with modern drugs such as artemisinin. The sequencing data enabled a genome-wide view of loci under recent selection. The authors searched for ‘identical-by-descent’ regions, which are regions showing a significant lack of recombination (which is otherwise rife throughout the P. falciparum genome) and is indicative of positive selection retaining intact alleles.
It is widely thought that the main threat for developing artemisinin resistance in Africa is through spread of existing resistance alleles from Southeast Asia. However, the authors found likely de novo artemisinin resistance alleles in the pfap2mu gene in Ghana and Malawi. This finding indicates that African-origin artemisinin resistance is possible and that the prevalence and spread of such alleles should be closely monitored.
In a separate study, Howick, Russell et al. used single-cell RNA sequencing (scRNA-seq) as part of a Malaria Cell Atlas project. Cell Atlas approaches have typically been applied to analyse intercellular phenotypic diversity across multicellular organisms, but here the authors applied it to characterize the diversity of Plasmodium parasites during the complex life cycle stages between mosquitoes and mammalian hosts.
They initially used a mosquito and mouse model of Plasmodium berghei infection sampled throughout the life cycle and generated 1,787 single-cell transcriptomes using a Smart-seq2 scRNA-seq protocol adapted for Plasmodium RNAs. Visualizing the data by clustering the cells according to transcriptome similarity showed clusters of distinct cell states representing the different life-cycle stages, as well as transitions between various stages. For example, the intra-erythrocytic developmental cycle (IDC) indeed formed a ring shape in the displayed data, representing all cell stages and their transition states.
Beyond illustrating the diversity of cell states, the transcriptome data expanded the range of genes that are known to be upregulated at key life-cycle stages, such as host liver cell invasion. As ~40% of Plasmodium genes are still of unknown function, these data may assist in assigning gene functions and increasing the number of potential targets for therapeutic intervention.
The authors further enhanced the cell atlas with data from additional models and RNA-seq technologies. They characterized P. falciparum and Plasmodium knowlesi cultured in human blood to capture the IDC stages. Overall, 15,858 cells were processed by droplet-based scRNA-seq technology, which offers lower transcriptomic depth but increased cellular throughput at lower cost than Smart-seq2.
Finally, as support that the atlas has wide applicability, including clinically, the team showed that samples from three infected humans in Kenya could be preserved locally, then subsequently processed and mapped onto the atlas to reveal the cellular transcriptional diversity in natural infections, including a mixed-species infection in one of the donors.
“de novo artemisinin resistance alleles in the pfap2mu gene in Ghana and Malawi”
These studies thus provide valuable resources for the malaria community to further understand pathogen diversity.
Amambua-Ngwa, A. et al. Major subpopulations of Plasmodium falciparum in sub-Saharan Africa. Science 365, 813–816 (2019)
Howick, V. M. et al. The Malaria Cell Atlas: single parasite transcriptomes across the complete Plasmodium life cycle. Science 365, eaaw2619 (2019)
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Burgess, D.J. Sequencing for malaria diversity. Nat Rev Genet 20, 629 (2019). https://doi.org/10.1038/s41576-019-0176-5