Exploring how energy metabolism differs in male and female Plasmodium falciparum gametocytes

Malaria remains a devastating global disease, killing 619 000 people in 2021. The causative agent, Plasmodium, has a complex life cycle involving a human and mosquito host. Most antimalarials are designed to treat the disease-causing asexual blood stages. However, it is now generally accepted that drugs that block transmission between the human and mosquito are needed to achieve the goal of global malaria eradication. Sexual blood stages, gametocytes, are solely responsible for transmission of malaria from humans to mosquitoes. Therefore, gametocytes represent an attractive target for transmission-blocking drugs.

Gametocytes circulate in the peripheral blood until ingested by a female mosquito where they continue their development. Despite being essential for transmission, little is known about how gametocyte metabolism is regulated. In addition, gametocytes can be male or female. It has not been previously been investigated whether there are differences in energy metabolism between the sexes. Both male and female gametocytes grow a large, complex mitochondrion during development. However, only the female passes on its mitochondrion after sexual reproduction. This raises the question as to why male gametocytes also invest in growing a mitochondrion, only to shed it at the next developmental stage.

The aim of the project were to 1) identify and characterise proteins putatively important for gametocyte energy metabolism (possible regulators) and 2) develop methods to compare mitochondrial energy metabolism between males and female gametocytes.

The project fitted with the MRC strategic skill priorities. I have gained quantitive skills (image analysis, machine learning and their associated statistical analyses) and interdisciplinary skills (use of a variety of imaging techniques which will be useful in multiple disciplines outside parasitology.) I have investigated how transgenic deletion affects not only gametocyte development but their ability to continue their life cycle in the mosquito in vivo (whole organism biology, in vivo work in the natural host, phenotyping of mutant parasites.) The parasite species I worked on was Plasmodium falciparum, a human malaria parasite, so the data will be translational to human disease.

Aim 1: Data mining of published proteomic data and bioinformatics analysis identified 6 proteins putatively important for energy metabolism. Due to COVID-19 related delays, I focused on characterisation 2 of the genes, ACS ADP and PFK11. ACS ADP was tagged with a green fluorescent protein and was found to be localised to the mitochondrion and expressed throughout the life cycle. PFK11 was successfully knocked out and this was found to lead to a dramatic reduction of the number of oocysts when fed to mosquitoes - the first indication of a transmission-blocking phenotype.

Aim 2: To distinguish male and female gametocytes in fluorescence images, I generated an antibody to a male specific protein which I showed to be superior to other sex-specific markers. I developed an image analysis method to compare mitochondrial morphology and activity between male and female gametocytes. A machine learning algorithim was used as a secondary method to compare morphology. I also compare responses of male and female gametocytes to mitochondrial inhibitors and demonstrated for the first time a link between mitochondrial inhibition and ability to form gametes (gametogenesis) suggesting that mitochondrial respiration is important for gametogenesis. Parasites with a genetically encoded ATP sensor were also generated, which allowed measurement of ATP levels in gametocytes under different conditions (e.g. glucose availability and the presence of a mitochondrial inhibitor) which have laid the groundwork for future experiments.