Rhomboids in action : Tracking rhomboid proteases as regulators of Plasmodium falciparum blood stage development

Anagha Rajesh Salvi1, Marie-Charlotte Domart2, Lucy Collinson2, Michael J Blackman2, Abhinay Ramaprasad1 Rhomboids in action: Tracking rhomboid proteases as regulators of Plasmodium falciparum blood stage development 1. Parasite Functional Genetics Laboratory, School of Infection and Immunity, University of Glasgow, G12 8TA 2. Malaria Biochemistry Laboratory, The Francis Crick Institute, 1 Midland Road, NW1 1AT London, UK. Rhomboid proteases Growth profiles across three erythrocytic cycles show ROM4, 6 and 8-null mutants fail to grow. In ROM6’s case, uninduced line grew slower than wildtype, possibly due to partial functional disruption by the introduced C-terminal HA tag. ROM4, 6 & 8 essential for blood stage growth Inducible knockout screen Frameshift-based trackable inducible knockout (SHIFTiKO) Rapamycin added at ring stages Gene excision verified by PCR SHIFTiKO used for all targets DiCre for ROM6 & ROM7 Phenotyping in same and subsequent cycles Dimerisable Cre-lox system (DiCre) Aim: To identify rhomboid proteases essential for blood stage survival and characterise their stage-specific functions. Acknowledgements •HORIZON EUROPE Marie Sklodowska-Curie Actions (751865), ESCMID Research Grant (2023) and MRC Career Development Award (MR/Z504762/1) to A.R.; Wellcome Trust (220318/A/20/Z) to M.J.B.; Wellcome Trust (CC2129), Cancer Research UK (CC2129) and UK Medical Research Council (CC2129). •MVLS Cellular Analysis Facility at University of Glasgow •Electron Microscopy facility at Francis Crick Institute. ROM4 is required for invasion ROM4-null parasites show reduced rates of invasion, producing less rings compared to untreated controls. ROM4-null parasites unable to invade RBCs. Scan to see ROM4-null parasites struggle to invade! Further work •Generate ROM6 mutant without HA tag and further phenotypic characterisation. •Elucidate substrate specificity and molecular mechanisms underlying ROM4,6 and 8 roles by proteomics. ROM6 is required for trophozoite-schizont transition ROM6-null parasites arrested at late trophozoite/early schizont stages. Serial block-face scanning electron microscopy show incomplete segmentation in ROM8-null parasites compared to untreated controls. ROM8 is required for schizont development Ultrastructure expansion microscopy confirms PPM localisation of 3HA-ROM8 and its loss upon RAP treatment. Fewer, clumped merozoites in ROM8-null parasites. 3HA-ROM8 localises to the parasite plasma membrane across schizogony. MSP1, PPM marker; GAP45, IMC marker. Rhomboid proteases, a set of intramembrane serine proteases conserved across apicomplexans. Eight rhomboid proteases known but are yet to be systematically characterised. Important events in Plasmodium falciparum blood stage replication cycle driven by proteases. ROM8-null parasites exhibit aberrant schizogony. Undergo schizogony without arrest at any stage but producing fewer merozoites compared to untreated controls. Merozoites appear clumped.