PCRCR complex is essential for invasion of human erythrocytes by Plasmodium falciparum

Abstract

The most severe form of malaria is caused by Plasmodium falciparum. These parasites invade human erythrocytes, and an essential step in this process involves the ligand PfRh5, which forms a complex with cysteine-rich protective antigen (CyRPA) and PfRh5-interacting protein (PfRipr) (RCR complex) and binds basigin on the host cell. We identified a heteromeric disulfide-linked complex consisting of P. falciparum Plasmodium thrombospondin-related apical merozoite protein (PfPTRAMP) and P. falciparum cysteine-rich small secreted protein (PfCSS) and have shown that it binds RCR to form a pentameric complex, PCRCR. Using P. falciparum lines with conditional knockouts, invasion inhibitory nanobodies to both PfPTRAMP and PfCSS, and lattice light-sheet microscopy, we show that they are essential for merozoite invasion. The PCRCR complex functions to anchor the contact between merozoite and erythrocyte membranes brought together by strong parasite deformations. We solved the structure of nanobody–PfCSS complexes to identify an inhibitory epitope. Our results define the function of the PCRCR complex and identify invasion neutralizing epitopes providing a roadmap for structure-guided development of these proteins for a blood stage malaria vaccine.

Plasmepsin X activates function of the PCRCR complex in P. falciparum by processing PfRh5 for binding to basigin and invasion of human erythrocytes

“…To determine if Rh5 proc and Rh5 unproc were able to bind to basigin on the surface of human erythrocytes, we used uorescence-activated cell sorting (FACS) and detected binding using anti-PfRh5 antibodies. This showed that Rh5 proc bound to human erythrocytes at levels like that described previously 23,30 , however, the full-length Rh5 unproc did not show any apparent binding (Fig. 5c).…”

“…Additionally, the ability of Rh5 proc and Rh5 unproc to participate in formation of the PCRCR complex was compared by rstly, testing their ability to bind the preformed PfRipr-CyRPA complex. Both proteins bound to PfRipr-CyRPA equally well and secondly, to the cysteine-linked PTRAMP-CSS hetero-dimer 23 . These showed that both Rh5 proc and Rh5 unproc were able to form the PCRCR complex equally well consistent with their normal function.…”

“…PCRCR proteins transit through micronemes where they are proteolytically processed by PMX. Previously, we showed the PTRAMP-CSS-PfRipr complex was pre-formed within the merozoite before surface relocation during erythrocyte invasion 23 . Consequently, we sought to determine the point at which PCRCR was PMX-processed and formed as a functional complex at the apical end of the merozoite surface.…”

“…PMX processing of PfRipr is not essential. PfRipr is part of the PCRCR complex with PfRh5 and it is essential for merozoite invasion 23,28 and processed by PMX 11 . While PMX processing of PfRh5 appears to be essential we sought to determine if this proteolytic processing of PfRipr was also required.…”

“…Functional PCRCR requires proteolytic processing of PfRh5 by PMX. To further investigate the function of the N-terminal domain of PfRh5 that is proteolytically removed by PMX both full-length (Rh5 unproc ) and the equivalent processed form lacking the N-terminal domain (Rh5 proc ) were expressed and puri ed from insect cells 23 . The Rh5 proc and Rh5 unproc forms have previously been shown to bind basigin on the erythrocyte membrane surface as well as in solution 23,30 .…”