Conformational flexibility, lipid binding, and regulatory domains in CelTOS

CelTOS is an essential Plasmodium traversal protein and conserved in apicomplexan parasites. We showed that CelTOS forms pores in cell membranes to enable traversal of parasites through cells (Jimah et al., 2016). Here, we establish roles for the distinct regions of CelTOS, examine the mechanism of pore formation and evaluate the immunogenicity of engineered CelTOS variants. CelTOS dimer dissociation is required for pore formation as disulfide bridging between monomers inhibits pore formation and this inhibition is rescued by disulfide-bridge reduction. A helix destabilizing Pro127 allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C-terminus of CelTOS is a negative regulator that limits pore formation. Lipid binding is a pre-requisite for pore assembly as mutation of a phospholipid binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. The disulfide-locked and N-terminal deletion mutants showed improved immunogenicity relative to wild-type CelTOS in mice. These findings have implications for pore-forming proteins that are essential for diverse functions, identify critical regions in CelTOS, and will guide the design of effective CelTOS-targeting vaccines to combat infection and transmission of malaria and apicomplexan parasites. ### Competing Interest Statement The authors, N.H.T., H.K., J.R.J., J.H.A., F.B.N. and S.J.B. are listed as co-inventors on a provisional patent application related to this work.