Malaria is a parasitic infectious disease, which is caused by parasites of the Plasmodium genus which are transmitted to vertebrate hosts by mosquito bite. Despite major intervention and control efforts, malaria is still responsible for almost half a million deaths every year, primarily in sub-Saharan Africa. All malaria symptoms are caused by the blood stages of the parasite's complex life cycle, during which they invade red blood cells (RBC), mature, multiply, and egress, before the cycle begins again in a new 24, 48 or 72 hour cycle, depending on the species. RBC invasion is therefore essential for parasite replication and malaria pathology, and a process of particular interest for vaccine development. Whilst P. falciparum is responsible for the majority of cases and fatalities, P. knowlesi is the only zoonotic malaria parasite, infecting both humans and macaques. It is also the closest relative of human-infecting P. vivax, which is currently not possible to continuously culture in vitro. Therefore, as well as being an interesting disease-causing parasite in itself, P. knowlesi acts as an excellent model for P. vivax.

The work described in this thesis begins by using real-time live microscopy to characterise the morphology and RBC invasion kinetics of P. knowlesi, and compare it to the more widely studied P. falciparum. Wild-type P. knowlesi was shown to have similar pre-invasion timings to P. falciparum in human red blood cells, however with a reduced merozoite-erythrocyte attachment force. P. knowlesi had a longer duration internalisation and echinocytosis, which may be due to the larger size of merozoites in this species.

Next, host cell physical parameters were assessed to investigate whether different experimental conditions have an effect on the biophysical properties, and whether different host cell types have different properties. First, flickering spectroscopy was used to assess plasma membrane fluctuations, and measure the bending modulus and membrane tension in human and macaque red blood cells. Optical tweezers were then used to measure the stiffness of human normocytes, reticulocytes, and macaque RBCs. It was found that erythrocyte membrane biophysics do not significantly change with surface adherence, and that membrane tension and bending modulus could be artificially altered using glutaraldehyde, however not reliably enough to yield further experimentation. The two types of red blood cells that P. knowlesi preferentially invades, macaque RBCs and human reticulocytes, had different biophysical properties to human normocytes and each other, suggesting different contributing factors to merozoite preference.

To investigate the mechanisms behind the preference of P. knowlesi for specific red blood cell types, flow cytometry, live video microscopy and optical tweezer traps were employed. P. knowlesi preferentially invades both macaque RBCs and human reticulocytes. It also relies heavily on the interaction between the parasite DARC (Duffy antigen receptor for chemokines) and human Duffy proteins for invasion, and the parasites are also unable to invade human RBCs without the parasite microneme protein NBPXa. I used these different cell types and invasion preferences to explore the steps during the invasion process where RBC selectivity occurs. My findings led me to propose that P. knowlesi selectivity occurs primarily during the pre-invasion stage, during which merozoites make fewer separate contacts and induce greater deformation in preferred RBC types, as compared to more individual contacts in which deformation and invasion capacity decrease over time where the RBCs are non-optimal or invasion is inhibited.

Finally, a genetic engineering project using Cas9-enhanced homologous recombination was begun, which aimed to fluorescently tag P. knowlesi apex and membrane proteins for use in later live video experiments. Target genes were identified, template and guide plasmids produced and transfections undertaken, with one of the targets showing initial promise for future modifications and experiments. In summary, the work in this thesis highlights the importance of using biophysical approaches to the study of parasite invasion, and provides new insights into the mechanics of erythrocyte preference in P. knowlesi.