The Plasmodium falciparum rhoptry bulb protein RAMA plays an essential role in rhoptry neck morphogenesis and host red blood cell invasion

Abstract

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. Author summaryDespite improved control measures over recent decades, malaria is still a considerable health burden across much of the globe. The disease is caused by a single-celled parasite PLOS Pathogens | https://doi.

Phosphorylation of Rhoptry Protein RhopH3 Is Critical for Host Cell Invasion by the Malaria Parasite

“…Since there was a defect in the discharge of the S804A mutant of RhopH3, we investigated if its localization was dependent on the phosphorylation of this site. To this end, immunofluorescence assays (IFAs) were performed on late schizonts/segmenters as RhopH3 is positioned in the rhoptries of merozoites at this stage for the release (8,19). RhopH3 was localized as puncta adjacent to rhoptry bulb protein RAP1 as expected in 3D7 parasites (Fig.…”

“…One is low-molecular-weight (LMW) complex, comprising RAMA, RAP1, RAP2, and RAP3. RAMA was recently demonstrated to play a key role in invasion (8), and RAP1 seems to be involved in trafficking of RAP2, which is essential for parasite survival (9). The other, high-molecular-weight (HMW) complex, consists of RhopH1/Clag, RhopH2, and RhopH3 (6,10,11).…”

Identification of a Novel RAMA/RON3 Rhoptry Protein Complex in Plasmodium falciparum Merozoites

“…In addition, we previously reported that PfRON3 interacts with PfRON2 and PfRON4, but not with PfAMA1 ( Ito et al, 2011 ), suggesting that a portion of PfRON3 is involved in the formation of a RON complex (PfRON2, 3, and 4), but not in the moving junction complex (PfRON2, 4, 5, and PfAMA1) ( Ito et al, 2011 ). Taken together, the absence of PfRAMA affects the trafficking of its associated RONs, and this could potentially explain the abnormal rhoptry neck biogenesis as observed by Sherling et al, (2019) .…”

“…Recently the PfRAMA-PfRAP1 complex was also suggested as a cargo for the Plasmodium orthologue of sortilin ( Hallee et al, 2018b ). To further investigate the role of PfRAMA, Sherling et al, (2019) generated a PfRAMA conditional knockdown parasite line. Contrary to previous findings ( Topolska et al, 2004 ; Richard et al, 2009 ; Hallee et al, 2018b ), the PfRAMA knockdown parasites presented correct trafficking of PfRAP1 and PfRhopH3.…”

“…Therefore, their findings were inconsistent with the proposed rhoptry bulb-specific protein escorter role of PfRAMA ( Hallee et al, 2018b ). Furthermore, although the knockdown parasites showed that some RON proteins—PfRON2, PfRON3, and PfRON4—were diminished in mature schizonts, the rhoptry neck proteins PfRON12 and Rh2b were normally localized in the rhoptry ( Sherling et al, 2019 ). While PfRON3 is now known as a rhoptry body protein ( Ito et al, 2011 ) ( Figure 3C ), they suggested that the mislocalization of the above RON proteins may be due to abnormal rhoptry neck biogenesis.…”