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

Abstract

Malaria parasites use the RhopH complex for erythrocyte invasion and channel-mediated nutrient uptake. As the member proteins are unique to Plasmodium spp., how they interact and traffic through subcellular sites to serve these essential functions is unknown. We show that RhopH is synthesized as a soluble complex of CLAG3, RhopH2, and RhopH3 with 1:1:1 stoichiometry. After transfer to a new host cell, the complex crosses a vacuolar membrane surrounding the intracellular parasite and becomes integral to the erythrocyte membrane through a PTEX translocon-dependent process. We present a 2.9 Å single-particle cryo-electron microscopy structure of the trafficking complex, revealing that CLAG3 interacts with the other subunits over large surface areas. This soluble complex is tightly assembled with extensive disulfide bonding and predicted transmembrane helices shielded. We propose a large protein complex stabilized for trafficking but poised for host membrane insertion through large-scale rearrangements, paralleling smaller two-state pore-forming proteins in other organisms.

Defining the Essential Exportome of the Malaria Parasite

“…In contrast to the CLAGs, two of PSAC's other main constituents, RhopH2 (PF3D7_0929400) and RhopH3 (PF3D7_0905400), are essential for parasite proliferation under normal culturing conditions, supporting the notion that nutrient acquisition is indeed essential for in vitro growth [50][51][52]. The precise roles that these two RhopH proteins play in the formation of a functional PSAC is currently not clear, but a Förster resonant energy transfer (FRET)-based study, and the recent structural elucidation of the RhopH2-RhopH3-CLAG3 complex, indicate a possible role for RhopH2 and RhopH3 in escorting a soluble form of CLAG3 to the RBC surface whereupon CLAG3 can then insert into the RBC membrane (Figure 1C) [53,54].…”

Kinetic tracking of Plasmodium falciparum antigen presentation reveals determinants of protein export and membrane insertion

“…At merozoite egress and reinvasion, rhoptry proteins are secreted into the next erythrocyte and deposited into the parasitophorous vacuole [29]. From there, through an incompletely understood interaction with the PTEX translocon, CLAG3 is exported into host cytosol for trafficking to the host membrane [28,30]. Imaging revealed that the tandem-tagged CLAG3 protein in 8-1HA trafficked as expected and colocalized with RhopH3 at the host cell surface (Fig 1D, lower group); an antibody specific for the CLAG3 c-terminus further confirmed this localization (S1B Fig).…”

“…CLAG3 was recovered from 8-1HA and 8-1HAtrunc lysates but not from negative control 8-1 and 8-1trunc lines (bands labeled “1”), confirming specific pull-down. RhopH2 and RhopH3, unrelated proteins that interact with CLAG3 [24], were recovered from 8-1HA (“2” and “3”), albeit with lower efficiency than in experiments using CLAG3-tv2, an engineered control parasite that a full-length CLAG3 with a C-terminal HA epitope tag (Fig 1A, ref # [30]). This reduced yield may result from compromised binding and recovery with an internal HA epitope tag when compared to the C-terminal tag in CLAG3-tv2.…”

“…This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted September 6, 2021. ; https://doi.org/10.1101/2021.09.06.459085 doi: bioRxiv preprint trafficking to the host membrane [28,30]. Imaging revealed that the tandem-tagged CLAG3 protein in 8-1HA trafficked as expected and colocalized with RhopH3 at the host cell surface Stage-specific immunoblotting using 8-1HA parasites revealed increases in CLAG3 abundance upon parasite maturation to the schizont stage (Fig 1E), consistent with synthesis of this and other RhopH proteins predominantly in the late-stage parasites [31].…”

Non‐ribosomal insights into ribosomal P2 protein in Plasmodium falciparum‐infected erythrocytes

“…While the enhanced permeability of iRBCs has been experimented out for decades, the molecular identity of PSAC was largely unknown until recently, when a cytoadherence‐linked antigen 3 (Clag3) has been shown to be the key player of the channel for nutrient uptake (Alkhalil et al, 2009 ; Desai, 2012 , 2014 ; Gupta et al, 2018 ; Nguitragool et al, 2011 ). In the formation of PSAC, Clag3 protein forms a homodimer, and RhopH2 and RhopH3 do associate with the dimer for the construction of functional channel (Counihan et al, 2017 ; Gupta et al, 2018 ; Ito et al, 2017 ; Kaneko et al, 2005 ; Nguitragool et al, 2011 ; Schureck et al, 2021 ; Sherling et al, 2017 ). Under PTEX suppressed condition, Clag3 still translocate into the host cell suggesting an alternative mechanism of Clag3 export but in the PTEX suppressed parasite lines, the transport of solutes by PSAC was diminished (Beck et al, 2014 ; Comeaux et al, 2011 ) indicating that other exported proteins are required for channel formation either in association with or independent of Clag3.…”