Date Published: September 6, 2019
Publisher: Public Library of Science
Author(s): Emma S. Sherling, Abigail J. Perrin, Ellen Knuepfer, Matthew R. G. Russell, Lucy M. Collinson, Louis H. Miller, Michael J. Blackman, Oliver Billker.
The malaria parasite Plasmodium falciparum invades, replicates within and destroys red blood cells in an asexual blood stage life cycle that is responsible for clinical disease and crucial for parasite propagation. Invasive malaria merozoites possess a characteristic apical complex of secretory organelles that are discharged in a tightly controlled and highly regulated order during merozoite egress and host cell invasion. The most prominent of these organelles, the rhoptries, are twinned, club-shaped structures with a body or bulb region that tapers to a narrow neck as it meets the apical prominence of the merozoite. Different protein populations localise to the rhoptry bulb and neck, but the function of many of these proteins and how they are spatially segregated within the rhoptries is unknown. Using conditional disruption of the gene encoding the only known glycolipid-anchored malarial rhoptry bulb protein, rhoptry-associated membrane antigen (RAMA), we demonstrate that RAMA is indispensable for blood stage parasite survival. Contrary to previous suggestions, RAMA is not required for trafficking of all rhoptry bulb proteins. Instead, RAMA-null parasites display selective mislocalisation of a subset of rhoptry bulb and neck proteins (RONs) and produce dysmorphic rhoptries that lack a distinct neck region. The mutant parasites undergo normal intracellular development and egress but display a fatal defect in invasion and do not induce echinocytosis in target red blood cells. Our results indicate that distinct pathways regulate biogenesis of the two main rhoptry sub-compartments in the malaria parasite.
Malaria is a devastating disease of tropical and subtropical regions. Requiring a mammalian host and a mosquito vector for transmission, at least six species of the genus Plasmodium cause disease in humans, with Plasmodium falciparum being responsible for the great majority of mortality. All the manifestations of clinical disease result from repeated cycles of invasion, replication within and lytic egress from red blood cells (RBC). Invasion is an orchestrated process, comprising several steps including merozoite attachment, deformation of the RBC membrane, merozoite reorientation, formation of a high affinity interaction between the apical zone of the merozoite and the RBC surface, active entry, and finally sealing of the RBC membrane behind the intracellular parasite [1–3]. Invasion is generally immediately followed by a period of transient RBC echinocytosis, a morphological transformation of the RBC surface into an undulated or ‘spiky’ appearance, although this can also be induced under certain conditions even in the absence of successful invasion [3–5]. Entry into the host cell occurs concomitantly with formation of a membrane-bound parasitophorous vacuole (PV) within which the invading parasite comes to rest. The parasite then transforms within minutes into a ‘ring’ stage form before initiating intracellular development, progressing through a mononuclear trophozoite stage to a multinucleated schizont which undergoes segmentation to form a new generation of daughter merozoites. Parasite-induced rupture of the PV membrane (PVM) and host RBC membrane eventually enables egress of the merozoites to initiate a fresh erythrocytic cycle.
The involvement of rhoptry proteins in host cell entry has long been proposed, supported by early observations that rhoptry discharge coincides temporally with invasion in several apicomplexan parasites, including Toxoplasma [53–56] and P. falciparum . Those studies have been burgeoned by recent demonstrations that correct positioning of rhoptries at the apical pole of the Toxoplasma tachyzoite is a prerequisite for invasion (but not egress)  whilst in both P. falciparum merozoites and Toxoplasma tachyzoites rhoptries appear to undergo fusion with each other during invasion [30, 57]. The discovery of the role of several RON proteins in TJ formation [15, 16] was consistent with this overall model for rhoptry function, but as discussed in the Introduction it is also now clear that rhoptry proteins play roles following completion of invasion in such diverse functions as PVM generation, subversion of host cell signalling, and nutrient acquisition. Here we have established a new role for a Plasmodium rhoptry protein in biogenesis of these important organelles.