Deletion of Plasmodium falciparum Protein RON3 Affects the Functional Translocation of Exported Proteins and Glucose Uptake

Abstract

The survival of Plasmodium spp. within the host red blood cell (RBC) depends on the function of a membrane protein complex, termed the Plasmodium translocon of exported proteins (PTEX), that exports certain parasite proteins, collectively referred to as the exportome, across the parasitophorous vacuolar membrane (PVM) that encases the parasite in the host RBC cytoplasm. The core of PTEX consists of three proteins: EXP2, PTEX150, and the HSP101 ATPase; of these three proteins, only EXP2 is a membrane protein. Studying the PTEX-dependent transport of members of the exportome, we discovered that exported proteins, such as ring-infected erythrocyte surface antigen (RESA), failed to be transported in parasites in which the parasite rhoptry protein RON3 was conditionally disrupted. RON3-deficient parasites also failed to develop beyond the ring stage, and glucose uptake was significantly decreased. These findings provide evidence that RON3 influences two translocation functions, namely, transport of the parasite exportome through PTEX and the transport of glucose from the RBC cytoplasm to the parasitophorous vacuolar (PV) space where it can enter the parasite via the hexose transporter (HT) in the parasite plasma membrane. IMPORTANCE The malarial parasite within the erythrocyte is surrounded by two membranes. Plasmodium translocon of exported proteins (PTEX) in the parasite vacuolar membrane critically transports proteins from the parasite to the erythrocytic cytosol and membrane to create protein infrastructure important for virulence. The components of PTEX are stored within the dense granule, which is secreted from the parasite during invasion. We now describe a protein, RON3, from another invasion organelle, the rhoptry, that is also secreted during invasion. We find that RON3 is required for the protein transport function of the PTEX and for glucose transport from the RBC cytoplasm to the parasite, a function thought to be mediated by PTEX component EXP2.

EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole

“…Known essential functions that manifest defects at the PVM include inactivation of protein export and small molecule transport following knockdown of PTEX components (Beck et al, 2014;Charnaud, Kumarasingha, Bullen, Crabb, & Gilson, 2018;Elsworth et al, 2014;Garten et al, 2018) and RON3 (Low et al, 2019) or a block in egress following knockdown of key players in the protease cascade that mediates PVM destruction at the end of the cycle (Nasamu et al, 2017;Pino et al, 2017;Thomas et al, 2018). Parasites depleted of EXP1 do not display an export defect and develop to a late stage where they arrest mainly as trophozoites, suggesting EXP1…”

Transport mechanisms at the malaria parasite-host cell interface

“…Membrane insertion and/or oligomerization might also be influenced by other factors such as a PVM insertase or interaction with PTEX150/HSP101 in the context of PTEX. Along these lines, rhoptry neck protein 3 (RON3), a multi-pass rhoptry membrane protein secreted into the PV during invasion, has recently been suggested to play a role in establishing proper EXP2/PTEX functionality [ 105 ]. Conditional deletion of RON3 leads to developmental arrest and death shortly after invasion that corresponds with defects in protein export and uptake of fluorescent glucose analogs, consistent with a function in establishment of transport activities at the PVM.…”

How Malaria Parasites Acquire Nutrients From Their Host

“…RON3, a protein secreted from the rhoptry bulb and that localizes to the parasite periphery following invasion, has also been linked to PVM nutrient uptake. Parasites deficient in RON3 exhibit a similar phenotype to parasites deficient in EXP2 and fail to progress beyond the ring stages ( Low et al, 2019 ). They are not only affected in their ability to import glucose, as they also fail to export proteins into the erythrocyte cytosol.…”