PfCERLI1, a conserved rhoptry associated protein essential for invasion byPlasmodium falciparummerozoites

Abstract

The disease-causing blood stage of the Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we identify an essential role for the conserved protein P. falciparumCytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. We show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, biochemical techniques and semi-quantitative super-resolution microscopy show that PfCERLI1 knockdown prevents secretion of key rhoptry antigens that coordinate merozoite invasion. PfCERLI1 is the first rhoptry associated protein identified to have a direct role in function of this essential malaria invasion organelle which has broader implications for understanding apicomplexan invasion biology.

Expansion microscopy of apicomplexan parasites

“…Anti-centrin antibodies, and likely all four centrin proteins encoded in Plasmodium, however, have recently been shown to localize exclusively to the cytoplasmic extension of the bipartite nuclear MTOC (Liffner et al, 2023;Simon et al, 2021;Voß, Klaus, Lichti, et al, 2023).…”

“…Across various stages of the Plasmodium lifecycle, NHS ester has been used to visualize the rhoptries, some cytoskeletal elements, basal complex, cytostomes, apical polar ring (APR) and nuclear microtubule organizing centre (MTOC) (Bertiaux et al, 2021;Liffner & Absalon, 2021;Liffner et al, 2023;Rashpa & Brochet, 2022;Rashpa et al, 2023;Simon et al, 2021;Yang, Cai, et al, 2023); highlighting it is a powerful tool for generally observing changes within the parasite. Following the publication of the first study using U-ExM in Plasmodium, further significant developments were made when the protocol was optimized for the most common Plasmodium fixative, paraformaldehyde (Simon et al, 2021), followed by the first U-ExM experiments using P. falciparum (Simon et al, 2021).…”

“…The ability to specifically detect SPMTs has subsequently been utilized in other studies to distinguish intranuclear microtubules from SPMTs (Liffner et al, 2023).…”

“…Similar to the use of NHS ester to identify the conoid of ookinetes, the coupling of U-ExM and NHS ester was used to visualize the bipartite nuclear MTOC of Plasmodium (Liffner & Absalon, 2021;Simon et al, 2021). Using U-ExM along with other advanced imaging modalities, it has recently been shown in asexual blood-stage parasites (Liffner & Absalon, 2021;Liffner et al, 2023;Simon et al, 2021) and gametocytes (Li, Shami, et al, 2022;Rashpa & Brochet, 2022) that the nuclear MTOC comprised an intranuclear region and a cytoplasmic extension(s). Until the advent of U-ExM, the only marker of the nuclear MTOC for light microscopy was anti-centrin antibodies.…”

“…This suggested that centrin was not required for nuclear microtubule polymerization, but raised the question of when the nuclear MTOC was formed. A study that tracked a number of P. falciparum markers across the asexual blood stage of the lifecycle, which used NHS ester and centrin as markers for the MTOC, subsequently showed that the intranuclear portion of the MTOC was built before the cytoplasmic extensions (Liffner et al, 2023), and that only the intranuclear portion is necessary for the nucleation of intranuclear microtubules. Additionally, it was shown that the MTOC is not observable in fully formed merozoites or newly invaded ring-stage parasites (Liffner et al, 2023), suggesting that the MTOC disassembles prior to egress and forms de novo following reinvasion.…”