Conservation of red blood cell signalling in Plasmodium merozoite invasion

• Main Author: Yong, James Jia Ming
• Other Authors: Peter Preiser
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2023
• Subjects: Science::Biological sciences::Molecular biology; Science::Biological sciences::Microbiology::Microorganisms
• Online Access: https://hdl.handle.net/10356/169702
• Institution: Nanyang Technological University

Red blood cells (RBC) possess essential signalling cascades that are involved in Plasmodium merozoite invasion. While P. falciparum reticulocyte binding protein homologue 5 (RH5) binding to RBC surface receptor Basigin is crucial for invasion and induces calcium (Ca2+) influx in the RBC, the mechanisms governing these invasion events remains unclear. It is though clear that the parasite has exploited innate RBC signalling cascades to direct invasion. Here we observed that RH5 binding to Basigin promotes the increased assembly and organisation of a multimeric RBC membrane complex consisting of β2-adrenergic receptor (β2AR) and CD44, demonstrating that this RH5-induced oligomerisation of RBC proteins is necessary to promote downstream invasion events. Furthermore, we detected a sharp rise in RBC cytosolic cyclic adenosine monophosphate (cAMP) levels after RH5-Basigin binding that can be blocked by cAMP signalling protein inhibitors. This is consistent with the effective inhibition of merozoite invasion by cAMP signalling and Ca2+ channel inhibitors. We also provided further insights into an active host cAMP-Ca2+ signalling crosstalk that drives junction formation. Additionally, we showed that different combinations of signalling inhibitors drastically amplified invasion inhibition levels, further highlighting that cAMP signalling proteins are functioning in a linear signalling cascade to promote RBC L-type Ca2+ channels activation. Merozoite invasion by Plasmodium parasites is a key stage of the parasite life cycle and requires specific ligand and receptor interactions between the merozoite and the RBC. Here we demonstrated that Ca2+ influx in the RBC is critical for P. knowlesi and P. yoelii invasion, suggesting that host Ca2+ signalling is conserved in Plasmodium merozoite invasions. Furthermore, we provide novel evidence that host cAMP-Ca2+ signalling is required for RBC Ca2+ influx in both P. falciparum and P. knowlesi invasion, in which specific ligand-receptor interactions lead to β-adrenergic-receptor-mediated cAMP generation in the RBC that activates the host L-type Ca2+ channels. This is also evident when L-type Ca2+ channel inhibitor and cAMP signalling inhibitors block both P. falciparum and P. knowlesi invasion. Overall, our study presents clear evidence of the importance of a functionally conserved host cAMP-Ca2+ signalling crosstalk during merozoite invasion and creates new opportunities to tackle the invasion process. In addition, RH5 is refractory to genetic disruption and conserved in P. falciparum strains, highlighting its importance in parasite survival and disease transmission. Invasion inhibitory effects of antibodies against RH5 and Basigin have been shown to significantly block merozoite invasion, but an invasion-targeted drug intervention approach have not been well-understood. Here we present new data on small molecule compounds that are able to block Ca2+ influx in the RBC and invasion by hindering RH5-Basigin binding, thereby potentially serving as antimalarial drugs. This is consistent with past evidence that the interference with RH5-Basigin binding is highly effective in impeding merozoite invasion. Therefore, small molecule compounds that specifically target the RH5-Basigin binding opens up an attractive avenue for drug development against malaria.