Periodic Reporting for period 4 - SEXinMALARIA (Sex in malaria parasites – from basic biology to targets for transmission blocking interventions.)
Reporting period: 2023-03-01 to 2024-08-31
Sexual development in malaria parasites is critical for parasite transmission between infected individuals, and is therefore a major target for the malaria elimination agenda. However, there are currently no effective drugs or vaccines that block parasite transmission to mosquitoes, and we currently do not understand the molecular mechanisms involved. This is primarily because Plasmodium genetics has been slow, with the majority of the genome unexplored. I here propose to conduct the first genome-scale screen for male and/or female fertility genes by leveraging a game-changing genetic system we have developed and recently validated through the first genome-scale in vivo gene KO screen in any parasite. Using simultaneous phenotyping of barcoded mutants, we will conduct the first genome-scale screen for male and/or female fertility genes. My team will systematically map specific biological roles for hundreds of parasite genes, ranging from sex determination to zygote differentiation. We will also overcome the next hurdle in Plasmodium genetics by developing a method for massive parallel phenotyping, using the power of single cell transcriptomics to validate the screen and reveal molecular mechanisms at previously intractable points in the Plasmodium life cycle. This approach has clear translational implications, as it will identify both drug and vaccine candidates.Sexual development in malaria parasites is critical for parasite transmission between infected individuals, and is therefore a major target for the malaria elimination agenda. However, there are currently no effective drugs or vaccines in use that block parasite transmission to mosquitoes, and we have a poor understanding of the molecular mechanisms involved. This is primarily because Plasmodium genetics has been slow, with the majority of the genome unexplored.
In this project we have devised a method to mutagenise parasite genes systematically and at genome scale in either the male or the female sexual precursor stage, to ask which parasite genes are needed for each sex to infect the mosquito. Through this approach we managed to identify 471 sex specific gene functions and evaluate their importance for male and female fertility separately.
We validate the data by looking in depth at a small number of selected gene functions and verify the robustness of the screen data. We also created a public database to integreat the screen data with phenotype and gene expression data from earlier genome scale experiments. We have stratified male fertility genes further to distinguish those acting before gametocyte egress from the erythrocyte and those that act after microgametes have become motile. The latter group contains potential new gamete fusion factors that may serve as transmission blocking vaccine candidates. Overall, the screens reveal deep insights into the metabolic reorganisation of a malaria parasite, identifies new regulatory and structural elements of the cell cycle and provide an improved understanding of the evolution of sexual processes in eukaryotes.
In this project we have devised a method to mutagenise parasite genes systematically and at genome scale in either the male or the female sexual precursor stage, to ask which parasite genes are needed for each sex to infect the mosquito. Through this approach we managed to identify 471 sex specific gene functions and evaluate their importance for male and female fertility separately.
We validate the data by looking in depth at a small number of selected gene functions and verify the robustness of the screen data. We also created a public database to integreat the screen data with phenotype and gene expression data from earlier genome scale experiments. We have stratified male fertility genes further to distinguish those acting before gametocyte egress from the erythrocyte and those that act after microgametes have become motile. The latter group contains potential new gamete fusion factors that may serve as transmission blocking vaccine candidates. Overall, the screens reveal deep insights into the metabolic reorganisation of a malaria parasite, identifies new regulatory and structural elements of the cell cycle and provide an improved understanding of the evolution of sexual processes in eukaryotes.
To identify P. berghei sterility genes through genome-scale screens, we first produced mutant lines of P. berghei which produced gametocytes of only one or the other sex. This allowed us to mutagenise this both lines with barcoded knockout vectors from our PlasmoGEM resource, which we than mated with wildtype gametocytes of the opposite sex to observe the development in the mosquito. Barcode counting at the oocyst stage revealed sex-specific gene functions (Aim 1). We than mapped the functions of >100 male genes further by looking specifically at gene functions before male gametocyte egress and in microgamete fertility (Aim 2). A database was produced to release data from the fertility screens and focussed sub-screens, into which we also included phenotypes and expression data from previous genome scale analysis (Aim 2). We used single cell analysis on a subset of developmental mutants to identify gene functions in greater depth (Aim 3). To expand the analysis of posttranscritionally regulated, female inherited genes further, we developed an improved screening protocal that exploits CRISPR homing after fertilisation to create homozygous mutations when pools of mutants are transmitted. We also explored an alternative methods to study such genes at scale, by developing a freezing protocol for homozygous knockout ookinetes. We then validated specific gene functions with cloned mutants (Aim 4). This resulted in the identification of SUN-domain protein of the nuclear envelop of the male gametocyte, where we show that it is part of acomplex with a moonlighting allantoicase protein, which during the rapid closed mitotic division that lead to male gamete formation is required for anchoring the cytosolic microtubule organising centre to the spindle microtubules inside the nucleus. In the absence of a canonical LINC complex, we speculate that the SUN-allantoicase complex of malaria parasites may be an ancient feature linking the eukaryotic axoneme to the nuclear envelope. Other validation experiments looked at the specific mechanisms through which the male development gene MD1 interacts with nucleic acids and other proteins to regulate male developments. We also studied posttranscriptional regulation during female development and how target genes regulate the maturation of the motile zygote (Aim 4). The main screen results were released through two publications and a public database hosted sustainably on a server provided by the Swedish research infrastructure, SciLifeLab. A CRISPR homing pilot screen and an ookinete freezing protocol to get at postzygotic gene functions at scale are either available as a pre-print or are in preparation for publication.
'The identification of 471 fertility genes in P. berghei increases the number of gene functions assigned at this life cycle stage by an order of magnitude. The data were released through a versatile database that allows visualisation and integration with other data for deeper functional analysis. The project reveals that sexual processes in a malaria parasite present as a mosaic of highly conserved and more recently evolved biological process. The data provide starting points for future work into the biology of a divergent eukaryote, which may look at the cell cycle, meiosis, replication or the axoneme. We also provide starting points for the systematic validation of transmission blocking vaccine targets and for a better understanding of the evolution of sexual reproduction of sexual processes in eukaryotes.