Conditional permeabilization of the P. falciparum plasma membrane in infected cells links cation influx to reduced membrane integrity

Abstract

The intracellular human malaria parasite, Plasmodium falciparum, uses the PfATP4 cation pump to maintain Na+ and H+ homeostasis in parasite cytosol. PfATP4 is the target of advanced antimalarial leads, which produce many poorly understood metabolic disturbances within infected erythrocytes. Here, we expressed the mammalian ligand-gated TRPV1 ion channel at the parasite plasma membrane to study ion regulation and examine the effects of cation leak. TRPV1 expression was well-tolerated, consistent with negligible ion flux through the nonactivated channel. TRPV1 ligands produced rapid parasite death in the transfectant line at their activating concentrations, but were harmless to the wild-type parent. Activation triggered cholesterol redistribution at the parasite plasma membrane, reproducing effects of PfATP4 inhibitors and directly implicating cation dysregulation in this process. In contrast to predictions, TRPV1 activation in low Na+ media accentuated parasite killing but a PfATP4 inhibitor had unchanged efficacy. Selection of a ligand-resistant mutant revealed a previously uncharacterized G683V mutation in TRPV1 that occludes the lower channel gate, implicating reduced permeability as a mechanism for parasite resistance to antimalarials targeting ion homeostasis. Our findings provide key insights into malaria parasite ion regulation and will guide mechanism-of-action studies for advanced antimalarial leads that act at the host-pathogen interface.

Fig 1. Expression and localization of the rat TRPV1 channel protein in P. falciparum.

(A) Schematic showing piggyBac transfection strategy for stable expression of the rat trpv1 gene product (rTRPV1) with a C-terminal HA epitope tag upon integration in the Dd2-TRP parasite clone. This strategy integrates the sequence between two inverted terminal repeats (ITR1 and ITR2) into the genome at one or more sites; this region includes the hDHFR selection cassette. Primers to confirm retention of the channel gene and the site recognized by the Southern blotting probe (red bar) are shown. (B) Immunoblot of total cell lysates from wild-type (WT) and Dd2-TRP lines, probed with mouse anti-HA tag antibody. Ponceau S staining of hemoglobin on the membrane is shown at the bottom as a loading control. (C) Anti-HA immunoblot of match-loaded samples from Dd2-TRP after hypotonic lysis and fractionation into soluble, carbonate extractable, and integral membrane pools (sol, CO₃ ⁼, and membr, respectively). The expressed TRPV1 protein is integral to parasite membranes. (D) Immunofluorescence antibody (IFA) images of trophozoite-infected cells from indicated parasites, showing labeling with mouse anti-EXP2 and rabbit anti-HA antibodies. TRPV1 localizes within the intracellular parasite and is not exported into the host cell. Scale bar, 5 μm. (E, F) Immunoelectron microscopy images using rabbit anti-HA and a secondary goat anti-rabbit antibody conjugated with 18 nm colloidal gold particles, suggesting TRPV1 trafficking via the parasite ER to the parasite plasma membrane. N, parasite nucleus; DV, digestive vacuole; RBC, red blood cell cytosol; box in panel F, area of zoom in image to right. Scale bars, 500 nm.

Fig 2. Conditional permeation of the parasite plasma membrane is lethal.

(A) Structures of indicated antimalarial lead compounds targeting PfATP4. (B) Structure of capsaicin. Bottom panel shows mean ± S.E.M. parasite growth at indicated [capsaicin] for Dd2-TRP and wild-type Dd2 (red and black symbols, respectively), determined by combining results from 6–7 independent trials each; Dd2-TRP is killed by capsaicin. (C) Structure of arvanil. Plot shows mean ± S.E.M. parasite growth at indicated [arvanil] for Dd2-TRP and wild-type Dd2 (red and black symbols, respectively), determined from 3 independent trials each. (D) Mean ± S.E.M. parasite growth over 72 h with a pulse-chase application of 10 μM capsaicin for indicated durations, determined from 3–5 independent trails with normalization to 100% growth for DMSO control and 0% for matched cultures treated with 20 μM chloroquine for 72 h. Reduced growth relative to DMSO control indicates rapid killing with short applications of capsaicin.

Fig 3. TRPV1 activation renders the parasite plasma membrane sensitive to saponin, phenocopying PfATP4 block.

Immunoblot showing aldolase retention in trophozoite-stage freed parasites after pretreatment with 10 μM capsaicin, 200 nM PA21A092, or DMSO (control) and exposure to indicated saponin concentrations. Capsaicin pretreatment followed by 1% saponin exposure leads to aldolase release from Dd2-TRP, but not the Dd2 wild type parent. Notice that PA21A092-mediated PfATP4 block renders parasite membranes sensitive to lower concentrations of saponin than seen after capsaicin treatment.

Fig 4. Growth inhibition studies using low Na⁺ media.

(A) Schematic showing ion flux when infected erythrocytes are cultivated in media based on the standard RPMI 1640 and modified 4Suc:6KCl formulations. Erythrocyte [Na⁺] increases in RPMI 1640 medium due to PSAC-mediated influx at the host membrane but remains low in 4Suc:6KCl, yielding a negligible gradient for uptake at the parasite plasma membrane. Arrow lengths on TRPV1 indicate Na⁺ gradient; those on PfATP4 indicate expected requirement for pump-mediated extrusion. Values at bottom tabulated from [14] with similar increases in erythrocyte Na⁺ reported previously [29]. (B) Capsaicin dose response for Dd2-TRP killing in RPMI 1640 medium and 4Suc:6KCl (black and red symbols, mean ± S.E.M. of triplicate measurements from a matched growth experiment). Note the unexpected improvement in killing when Na⁺ is reduced. (C) Mean ± S.E.M. IC₅₀ values from growth inhibition experiments using indicated TRPV1 ligands and PA21A092 in RPMI 1640 and 4Suc:6KCl media (black and red bars, respectively). Only capsaicin produced a significant difference in IC₅₀ values for the two media. *, P = 0.05 (one-way ANOVA with Tukey’s multiple comparisons); n = 3–7 independent matched trials each.

Fig 5. Rapid selection of a mutant that avoids cation leak at the PPM.

(A, B) Mean ± S.E.M. growth of Dd2-CapR at indicated [capsaicin] and [arvanil] (red triangles), determined by combining results from 3 independent trials each. In each panel, black lines represent best fits for dose-response data in matched experiments using wild-type Dd2 and Dd2-TRP (top and bottom) to single sigmoidal decay. Note that Dd2-CapR sensitivity to both ligands is indistinguishable from that of the wild-type parent, indicating a quantitatively complete loss of efficacy against the engineered transfectant. (C) Mean ± S.E.M. half-maximal growth inhibition concentrations (IC₅₀) for indicated parasite lines by the PfATP4 inhibitor PA21A092. Selection did not alter the parasite’s susceptibility to PfATP4 block. (D) Anti-HA immunoblot showing TRPV1 expression in Dd2-CapR. Bottom, Ponceau S loading control. (E) Sequence chromatograms from the Dd2-TRP transfectant and the selected Dd2-CapR clone, showing the single nucleotide polymorphism that alters G683 (rat TRPV1 numbering) to valine. (F) Modeled cartoon structure of rat TRPV1 with the G683V mutation, based on the cryo-EM structure [15]. This mutation (red) is at the lower TRPV1 gate and lines the ion conduction pore; side and top views of the membrane-embedded channel are shown (top and bottom images). (G) Cutaway side views of wild-type and mutant channel pores. Blue sticks represent the sidechain of I679, which forms the lower gate in the wild-type channel [43]; the position of G683 and the larger valine sidechain in the mutant are shown in red cartoons, sidechain sticks, and associated surface patches. The solvent-accessible pathway was mapped using the HOLE program and its surface is shown in orange. Note the narrowed pore in the mutant (bottom), suggesting that cation flux is prevented by an occluded pore.