Periodic Reporting for period 1 - ARISE (mAlaRIa Sex dEtermination)
Période du rapport: 2020-10-01 au 2022-09-30
In malaria parasites, while the clinical symptoms are caused by asexual parasites that replicate within the red blood cells, sexual forms are the only ones capable of transmitting the infection to mosquitoes. Targeting this small population of sexually differentiated parasites would provide an extremely powerful malaria control method overcoming limitations in vector control and antimalarials.
During gametocyte developmental pathway, there are two distinct cellular decisions at molecular level: first, commitment to sexual differentiation and, then, determination of the parasite into one sex or the other. Although several genes have been found to have sex-specific roles, the molecular basis of the sex-determining mechanisms of Plasmodium and the relation of this decision with the sexual commitment pathway is completely unknown. It has also been suggested that sex-determination is a flexible phenotype that can be influenced by environmental variation to maximise parasite fitness and, consequently, transmission. However, the mechanisms underlying on the process of environment sensing and the molecular players involved remain unclear.
Malaria is the most lethal parasitic disease in the world causing more than 600000 deaths of children under 5 years old in 2021, with approximately half of the human population exposed to be infected every day by one of the five Plasmodium species able to affect humans. The current scenario to combat this health and social burden is that the deadliest parasite responsible for this disease, P. falciparum, has developed resistance to all antimalarials individually, so the most potent treatment is based in combination of two different drugs while there are no promising alternatives in last phases of clinical trials. Therefore, searching for novel alternatives to tackle malaria infections is clearly needed. We have focused on using cutting-edge technologies, such as single-cell transcriptomics (scRNAseq), to expand and unravel sexual determination and differentiation, two essential steps for the parasite life cycle that are still largely unknown. That could lead to discover attractive drug targets that could block transmission to mosquitoes and, then, alleviate the burden of this disease.
In this proposal, we have applied innovative approaches to uncover the pathway of sex-determination in P. falciparum, in both lab-adapted parasites and wild parasite populations. Specifically to:
- Determine the earliest events in sex determination using scRNAseq in a lab-adapted P. falciparum strain (NF54)
- Understand determinants of malaria sexual determination in wild parasites by harnessing the power of scRNAseq
- Identify factors that influence determination into male or female gametocytes in natural parasite populations obtained from infected carriers.
During gametocyte developmental pathway, there are two distinct cellular decisions at molecular level: first, commitment to sexual differentiation and, then, determination of the parasite into one sex or the other. Although several genes have been found to have sex-specific roles, the molecular basis of the sex-determining mechanisms of Plasmodium and the relation of this decision with the sexual commitment pathway is completely unknown. It has also been suggested that sex-determination is a flexible phenotype that can be influenced by environmental variation to maximise parasite fitness and, consequently, transmission. However, the mechanisms underlying on the process of environment sensing and the molecular players involved remain unclear.
Malaria is the most lethal parasitic disease in the world causing more than 600000 deaths of children under 5 years old in 2021, with approximately half of the human population exposed to be infected every day by one of the five Plasmodium species able to affect humans. The current scenario to combat this health and social burden is that the deadliest parasite responsible for this disease, P. falciparum, has developed resistance to all antimalarials individually, so the most potent treatment is based in combination of two different drugs while there are no promising alternatives in last phases of clinical trials. Therefore, searching for novel alternatives to tackle malaria infections is clearly needed. We have focused on using cutting-edge technologies, such as single-cell transcriptomics (scRNAseq), to expand and unravel sexual determination and differentiation, two essential steps for the parasite life cycle that are still largely unknown. That could lead to discover attractive drug targets that could block transmission to mosquitoes and, then, alleviate the burden of this disease.
In this proposal, we have applied innovative approaches to uncover the pathway of sex-determination in P. falciparum, in both lab-adapted parasites and wild parasite populations. Specifically to:
- Determine the earliest events in sex determination using scRNAseq in a lab-adapted P. falciparum strain (NF54)
- Understand determinants of malaria sexual determination in wild parasites by harnessing the power of scRNAseq
- Identify factors that influence determination into male or female gametocytes in natural parasite populations obtained from infected carriers.
To cover the relevant part of the differentiation pathway, we have analysed the transcriptomes at single cell level of P. falciparum cultures in vitro at different time points post-red blood cell invasion of committed parasites. We have then establish the transcriptional hierarchy of events in the sexual bifurcation of P. falciparum and identify early markers of male and female lineage in P. falciparum.
We have then dissected the function of the main candidate sex determining gene, md1, and its phenotypic effects by CRISPR-mediated generation of parasite mutant lines and multiple molecular tools as immunofluorescence assays, mass spectrometry and gene expression quantification. We have described at an unparalleled molecular level the mechanism of the sex-determining process in human malaria parasites, showing that it requires a robust regulatory machinery around master regulators.
However, in vitro culture systems cannot fully capture the natural environment in which sex determination takes place. Therefore, we have studied complex parasite phenotypes shortly after the sex-determining decision and uncovering biological mechanisms directly in populations of wild Plasmodium parasites.
We have demonstrated that P. falciparum preferentially invades and resides in reticulocytes (younger red blood cells in circulation). We have also used scRNAseq of parasites from infected individuals to decipher how the host cell influences parasite decision making at transcriptional level. We have described that commitment to sex occurs in both reticulocytes and normocytes but commitment is much more probable in reticulocytes, highlighting that the host cell plays a critical factor on the parasite’s developmental decisions.
We have then dissected the function of the main candidate sex determining gene, md1, and its phenotypic effects by CRISPR-mediated generation of parasite mutant lines and multiple molecular tools as immunofluorescence assays, mass spectrometry and gene expression quantification. We have described at an unparalleled molecular level the mechanism of the sex-determining process in human malaria parasites, showing that it requires a robust regulatory machinery around master regulators.
However, in vitro culture systems cannot fully capture the natural environment in which sex determination takes place. Therefore, we have studied complex parasite phenotypes shortly after the sex-determining decision and uncovering biological mechanisms directly in populations of wild Plasmodium parasites.
We have demonstrated that P. falciparum preferentially invades and resides in reticulocytes (younger red blood cells in circulation). We have also used scRNAseq of parasites from infected individuals to decipher how the host cell influences parasite decision making at transcriptional level. We have described that commitment to sex occurs in both reticulocytes and normocytes but commitment is much more probable in reticulocytes, highlighting that the host cell plays a critical factor on the parasite’s developmental decisions.
This has been a productive, enriching and rewarding project for all the people involved. First, scientifically, as we have used cutting-edge technology as single-cell transcriptomics to dissect into an unparalleled detail the role of a sex-master regulator in malaria parasites. This has been recognised with a publication in Nature where we have showed convincingly that Md1 is both necessary and sufficient for male fate determination in the human malaria parasite Plasmodium falciparum. We have also described one of only a few known non-genetic mechanisms of sex determination in a eukaryote and highlight Md1 as a potential target for interventions blocking malaria transmission.
Furthermore, we have exploited this technology to assess gene expression in natural parasite populations. Surprisingly, we have observed a much higher number of committed cells than frequently described with lower resolution technologies. This opens up a novel avenue to further investigate the fate of sexually committed parasites as there might be important losses of maturing gametocytes or even sequestered gametocyte maturation may actually be longer than previously thought.
The experience gained during this fellowship has contributed to the awardee to secure a permanent position at the host institution and has allow him to apply for a young researcher grant (JCJC) from the French National Research Agency (ANR). If successful, it will provide the opportunity to harness cutting-edge technologies to dissect and tackle severe malaria. It has also allowed three Scicling actions (www.scicling.org) where 24 high schools in Asturias and Tenerife (Spain) have been visited, engaging with over 1200 students, 35 teachers and their educational communities
Furthermore, we have exploited this technology to assess gene expression in natural parasite populations. Surprisingly, we have observed a much higher number of committed cells than frequently described with lower resolution technologies. This opens up a novel avenue to further investigate the fate of sexually committed parasites as there might be important losses of maturing gametocytes or even sequestered gametocyte maturation may actually be longer than previously thought.
The experience gained during this fellowship has contributed to the awardee to secure a permanent position at the host institution and has allow him to apply for a young researcher grant (JCJC) from the French National Research Agency (ANR). If successful, it will provide the opportunity to harness cutting-edge technologies to dissect and tackle severe malaria. It has also allowed three Scicling actions (www.scicling.org) where 24 high schools in Asturias and Tenerife (Spain) have been visited, engaging with over 1200 students, 35 teachers and their educational communities