CLAG3 Self-Associates in Malaria Parasites and Quantitatively Determines Nutrient Uptake Channels at the Host Membrane

Abstract

Malaria parasites increase host erythrocyte permeability to ions and nutrients via a broad-selectivity channel known as the plasmodial surface anion channel (PSAC), linked to parasite-encoded CLAG3 and two associated proteins. These proteins lack the multiple transmembrane domains typically present in channel-forming proteins, raising doubts about their precise roles. Using the virulent human parasite, we report that CLAG3 undergoes self-association and that this protein's expression determines channel phenotype quantitatively. We overcame epigenetic silencing of paralogs and engineered parasites that express two CLAG3 isoforms simultaneously. Stoichiometric expression of these isoforms yielded intermediate channel phenotypes, in agreement with observed trafficking of both proteins to the host membrane. Coimmunoprecipitation and surface labeling revealed formation of CLAG3 oligomers. selections applied to these transfectant lines yielded distinct mutants with correlated changes in channel activity. These findings support involvement of the identified oligomers in PSAC formation and parasite nutrient acquisition. Malaria parasites are globally important pathogens that evade host immunity by replicating within circulating erythrocytes. To facilitate intracellular growth, these parasites increase erythrocyte nutrient uptake through an unusual ion channel. The parasite CLAG3 protein is a key determinant of this channel, but its lack of homology to known ion channels has raised questions about possible mechanisms. Using a new method that allows simultaneous expression of two different CLAG3 proteins, we identify self-association of CLAG3. The two expressed isoforms faithfully traffic to and insert in the host membrane, while remaining associated with two unrelated parasite proteins. Both the channel phenotypes and molecular changes produced upon selections with a highly specific channel inhibitor are consistent with a multiprotein complex that forms the nutrient pore. These studies support direct involvement of the CLAG3 protein in channel formation and are relevant to antimalarial drug discovery projects targeting parasite nutrient acquisition.

Complex nutrient channel phenotypes despite Mendelian inheritance in a Plasmodium falciparum genetic cross

“…The proteins encoded by these clag3 alleles are more than 90% identical, with most of the variation present in a small hypervariable region near the C-terminus (HVR, S1A Fig). The unique HVR sequence of the Dd2 CLAG3.1 protein is responsible for PSAC block by ISPA-28 in this laboratory clone and lack of activity against channels from other parasite lines [15,37]. In keeping with this, we found that ISPA-28 is ineffective against 7G8 and GB4 channels ( Fig 1B and S1B Fig).…”

“…PSAC activity is essential for intracellular parasite growth and replication as it functions in nutrient uptake from host plasma [14,20]. Although originally proposed to result from upregulation of transporters endogenous to the erythrocyte, many studies have now linked PSAC activity to a parasite-encoded RhopH protein complex consisting of CLAG3, RhopH2, and RhopH3 [12,20,21,37]. The present studies provide three new insights into the role of CLAG3 in PSAC formation and intracellular growth of malaria parasites.…”

“…Larger complexes are also supported by the recognition that a 1:1:1 complex of CLAG3, RhopH2, and RhopH3, as suggested by immunoprecipitation studies [66], lacks the number of predicted transmembrane domains generally needed to form a stable aqueous pore in biological membranes [16,26]. Anomalous migration of these membrane proteins in native PAGE experiments, as often seen for integral proteins [67], has prevented an accurate size determination for this complex [37]. While coimmunoprecipitation has detected associations between two CLAG3 isoforms in a transfected parasite [37], our transport measurements provide the first experimental evidence for functional oligomerization to form the nutrient channel at the host membrane.…”

“…The pBAC-Dd2C3-TetR-DOZI plasmid carries a 3.2 kB fragment from the end of the Dd2 clag3.1 gene with an in-frame C-terminal HA epitope tag and a 10x aptamer sequence in the 3'UTR for recruitment of TetR-DOZI. We selected KC5, a progeny clone from the GB4 x 7G8 cross, for transfection because it carries a single clag3h gene and has been successfully used in prior studies [37]; a single clag3h gene avoids epigenetic switching between the desired integrated gene and a second unmodified clag3 gene [12].…”

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

“…Similarly, a rhoptry bub location does not preclude roles during invasion, since the rhoptry bulb protein RhopH3, a component of the high molecular weight (HMW) RhopH complex, is important for RBC entry [22]. In contrast, RhopH3 and its partner proteins RhopH1/Clag and RhopH2 have additionally been shown to be involved in nutrient uptake during development of the intracellular parasite [22][23][24][25], whilst the rhoptry bulb low molecular weight (LMW) RAP1/RAP2 complex has been implicated in PVM formation [26]. In Toxoplasma (but not in Plasmodium) several rhoptry bulb proteins are enzymes, including proteases, phosphatases and kinases or pseudokinases, the latter two groups of which dramatically modulate host cell STAT signalling and the immunity-related GTPase (IRG) pathway involved in controlling parasite replication, and play key roles in virulence [27,28].…”