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

Abstract

Merozoites formed after asexual division of the malaria parasite invade the host red blood cells (RBCs), which is critical for initiating malaria infection. The process of invasion involves specialized organelles like micronemes and rhoptries that discharge key proteins involved in interaction with host RBC receptors. RhopH complex comprises at least three proteins, which include RhopH3. RhopH3 is critical for the process of red blood cell (RBC) invasion as well as intraerythrocytic development of human malaria parasite Plasmodium falciparum. It is phosphorylated at serine 804 (S804) in the parasite; however, it is unclear if phosphorylation regulates its function. To address this, a CRISPR-CAS9-based approach was used to mutate S804 to alanine (A) in P. falciparum. Using this phosphomutant (R3_S804A) of RhopH3, we demonstrate that the phosphorylation of S804 is critical for host RBC invasion by the parasite but not for its intraerythrocytic development. Importantly, the phosphorylation of RhopH3 regulates its localization to the rhoptries and discharge from the parasite, which is critical for RBC invasion. We also identified P. falciparum CDPK1 (PfCDPK1) as a possible candidate kinase for RhopH3-S804 phosphorylation and found that it regulates RhopH3 secretion from the parasite. These findings provide novel insights into the role of phosphorylation in rhoptry release and invasion, which is poorly understood. IMPORTANCE Host cell invasion by the malaria parasite is critical for establishing infection in human host and is dependent on discharge of key ligands from organelles like rhoptry and microneme, and these ligands interact with host RBC receptors. In the present study, we demonstrate that phosphorylation of a key rhoptry protein, RhopH3, is critical for host invasion. Phosphorylation regulates its localization to rhoptries and discharge from the parasite.

Malaria parasites use a soluble RhopH complex for erythrocyte invasion and an integral form for nutrient uptake

“…Our structure similarity searches found that RhopH3 residues 434–665 align with domains 2 and 3 of SepL; because domain three mediates interaction with the Tir receptor ( Burkinshaw et al, 2015 ), one possibility is that RhopH3 interacts with an unidentified host cell receptor at this site. The RhopH3 C-terminus provides another surface for the presumed interactions, as suggested by site-directed mutagenesis of serine 804 and by studies with a monoclonal antibody against a 134 aa recombinant fragment ( Doury et al, 1994 ; Ekka et al, 2020 ). This entire region (residues 716–897) is not resolved in our structure and appears to be flexible.…”

Complementary crosstalk between palmitoylation and phosphorylation events in MTIP regulates its role during Plasmodium falciparum invasion

“…Accumulating evidence has shown that global phosphorylation of P. falciparum proteome governs many cellular processes including invasion ( Ekka et al., 2020 ; More et al., 2020 ; Perrin et al., 2020 ). In addition, there are scanty findings available that also suggest that global palmitoylation of glideosome-actinomycin motor assembly proteins in P. falciparum might impact the invasion phenotypes ( Jones et al., 2012 ).…”

CDPKs: The critical decoders of calcium signal at various stages of malaria parasite development

“…In addition, cross talk between CDPK1 and Protein Kinase A plays a critical role during the invasion process [65] . A recent study has demonstrated that phosphorylation of RhopH3 is critical for invasion of RBCs, correct localization in rhoptries and discharge during invasion [33] . It has been suggested that phosphorylation of RhopH3 is mediated by CDPK1 [33] .…”

“…A recent study has demonstrated that phosphorylation of RhopH3 is critical for invasion of RBCs, correct localization in rhoptries and discharge during invasion [33] . It has been suggested that phosphorylation of RhopH3 is mediated by CDPK1 [33] . A recent elegant study by More et al, demonstrates formation of a multimeric complex in merozoites involving phosphorylated CDPK1, regulatory subunit of PKA (PKAr) and Pf14-3-3I that plays a critical role in the invasion process and microneme discharge [79] .…”

“… CDPKs Expression & Localisation Function in Plasmodium spp. References Putative Orthologous AtCDPKs* (percentage identity) P. falciparum CDPK1 Asexual (Rings, Trophozoites, Schizonts, Merozoites) Sexual (Gametocytes, Gametes) Merozoite invasion, Schizont development and egress Male and Female gametogenesis, Mosquito transmission [84] , [9] , [59] , [5] , [2] , [8] , [55] , [37] , [33] , [79] [6] AtCDPK29 (37%) /23 (36%) /21 (36%) /9 (36%) /26 (36%) CDPK2 Asexual (Trophozoites, Schizonts) Sexual (Gametocytes) - Male Gametocyte Exflagellation and Mosquito infection [7] [7] AtCDPK29 (40%) /30 (39%) /4 (39%) /9 (39%) /5 (39%) CDPK3 Sexual (Gametocytes) – [69] AtCDPK33 (41%) /29 (40%) /9 (40%) /4 (40%) CDPK4 - Sexual (Gametocytes) Merozoite Invasion Male Gametocyte Exflagellation and mosquito transmission [35] [58] , [97] , [112] , [91] , [90] AtCDPK29 (40%) /21 (39%)/ 26 (37%)/ 34 (37%) CDPK5 Asexual (Schizonts, Merozoites) Merozoite egress (Release of Egress-Specific Organelles) [32] [1] AtCDPK25 (39%) /1 (38%) /21 (37%) /29 (37%) /23 (37%) …”