Malaria parasites tolerate a broad range of ionic environments and do not require host cation remodelling

Abstract

Malaria parasites grow within erythrocytes, but are also free in host plasma between cycles of asexual replication. As a result, the parasite is exposed to fluctuating levels of Na(+) and K(+) , ions assumed to serve important roles for the human pathogen, Plasmodium falciparum. We examined these assumptions and the parasite's ionic requirements by establishing continuous culture in novel sucrose-based media. With sucrose as the primary osmoticant and K(+) and Cl(-) as the main extracellular ions, we obtained parasite growth and propagation at rates indistinguishable from those in physiological media. These conditions abolish long-known increases in intracellular Na(+) via parasite-induced channels, excluding a requirement for erythrocyte cation remodelling. We also dissected Na(+) , K(+) and Cl(-) requirements and found that unexpectedly low concentrations of each ion meet the parasite's demands. Surprisingly, growth was not adversely affected by up to 148 mM K(+) , suggesting that low extracellular K(+) is not an essential trigger for erythrocyte invasion. At the same time, merozoite egress and invasion required a threshold ionic strength, suggesting critical electrostatic interactions between macromolecules at these stages. These findings provide insights into transmembrane signalling in malaria and reveal fundamental differences between host and parasite ionic requirements.

Fig. 1

Parasite growth in low Na⁺-media. (A) P. falciparum growth over 5 days in media prepared with sucrose and KCl. Abscissa shows increasing mole fractions of KCl and decreasing fractions of sucrose to achieve a constant total osmolarity. Each medium was supplemented with 5% serum; growth is normalized to 100% for a control culture in RPMI 1640 medium with serum. Symbols represent mean ± S.E.M. from 4 experiments. Red and blue circles represent 4suc:6KCl and 7suc:3KCl media, respectively. (B) Identical rates of growth and expansion in 4suc:6KCl and RPMI (circles and triangles, respectively), determined by flow cytometry (n = 3 replicates each). (C) Photomicrographs of Giemsa-stained ring-, trophozoite-, and schizont-stage parasites in sucrose:KCl medium reveal unchanged parasite morphology (left to right, respectively). Scale bar, 5 μm. (D) Osmotic lysis kinetics for trophozoite-infected cells resuspended in sorbitol lysis solution (black trace), full-K⁺ medium with 5% serum (red), or full-K⁺ with 5% serum and 50 mM sucrose (blue). Infected cells undergo osmotic lysis in the full-K⁺ medium; lysis is prevented by addition of 50 mM sucrose. Lysis rates are inversely proportional to the PSAC permeabilities of sorbitol and K⁺ (black and red traces, respectively).

Fig. 2

Effects on erythrocyte cytosolic ion contents. (A) Measured Na⁺ concentrations in indicated media with or without 5% serum (“+ S” and “− S”, respectively). (B) Erythrocyte cytosolic Na⁺ content in uninfected erythrocytes or in enriched trophozoite-infected cells after culturing for 24 h in indicated media with 5% serum. (C) Erythrocyte cytosolic K⁺ content in uninfected erythrocytes or in enriched trophozoite-infected cells after culturing for 24 h in indicated media with 5% serum.

Fig. 3

Low Na⁺ concentrations are required. (A) Normalized parasite growth over 72 h in 4suc:6KCl with 10% dialyzed serum and indicated Na⁺ concentrations, revealing a sigmoidal dose response for Na⁺ requirement. Mean ± S.E.M. of 9 replicates from 3 experiments. (B) Photomicrograph of Giemsa-stained trophozoite-infected cells after 24 h cultivation in 4suc:6KCl with 10% dialyzed serum without Na⁺ supplementation. Two parasites are shown with a rim of blue stain and central clearing. (C) Absorbance scan showing reduced β-hematin production in trophozoites cultivated in Na⁺-deficient medium (red trace); supplementation with 7 mM Na⁺ restores β-hematin production (blue trace), yielding levels similar to those observed with standard RPMI-based medium (upper gray trace). Positive control, 20 μM chloroquine (lower gray trace). AU, arbitrary units. (D) Mean ± S.E.M. β-hematin production vs. external Na⁺ concentration, normalized to zero for control culture with 20 μM chloroquine. (E) Transmission electron micrographs showing trophozoite-infected erythrocytes cultivated in 4suc:6KCl with 10% dialyzed serum with or without Na⁺ supplementation to a final 7 mM concentration (left and right panels, respectively). Red arrowheads demarcate the parasite digestive vacuole; black arrows show electron dense spots in nucleus when Na⁺ is not added. N, nucleus; h, normal hemozoin crystals; scale bars, 0.5 μm. Images are representative of 89 cells from two separate experiments.

Fig. 4

Increased cytosolic Na⁺ does not promote phosphate acquisition. Phosphate dose responses for parasite growth over 5 days in RPMI vs. 4:6 sucrose:KCl with 5% serum (circles and triangles, respectively; mean of 3 measurements each). In contrast to the prediction for Na⁺-coupled uptake, the phosphate EC₅₀ is not increased when erythrocyte Na⁺_i is reduced.

Fig. 5

A requirement for low levels of external K⁺. (A) Parasite growth in zeroK medium with 5% dialyzed serum with or without 5 mM KCl supplementation (white and black symbols, respectively; mean ± S.E.M of 4 trials), evaluated by microscopy of Giemsa-stained smears. (B) 72 h growth rate vs. external [K⁺] in the same medium, measured using SYBR Green I. Mean ± S.E.M. of replicates from 4 experiments.

Fig. 6

Gluconate⁻ substitution and a Cl⁻ requirement. (A) Matched parasite growth over 5 days in NaCl:KGluc in comparison to standard RPMI medium, each supplemented with 10% serum. Bars represent mean ± S.E.M. SYBR Green I fluorescence in arbitrary units. (B) 72 h parasite growth as a function of external [Cl⁻] in 4suc:6KCl with 10% dialyzed serum. Reduced external Cl⁻ concentrations were achieved through isomolar substitution of KCl with K-gluconate. Mean ± S.E.M. of SYBR Green I fluorescence normalized to 100% for 4suc:6KCl (n = 6 trials).

Fig. 7

An ionic strength requirement for parasite cultivation. (A) Parasite growth in 4suc:3KCl:3Kgluc and 7suc:3KCl media (white and black symbols, respectively; mean ± S.E.M. of 5 replicates from two trials). Both media were supplemented with 7% human serum, which provides sufficient Na⁺ for cultivation. (B) Progression from late-stage schizonts to ring-stage infected erythrocytes in indicated media. (C) Number of merozoites released from late stage-schizont cultures over a 4 h incubation in each medium. (D) Erythrocyte invasion by mechanically freed merozoites resuspended in indicated media. In panels (B), (C), and (D), each bar represents the mean ± S.E.M. of up to 8 trials after normalization to 100% in matched controls using RPMI 1640 medium. The nominal K⁺ concentration and calculated ionic strength (I) of each medium prior to addition of serum is listed above the bars. Two media, zeroK with or without added K⁺, were supplemented with dialyzed serum to minimize contaminating K⁺, but all other media used undialyzed serum to provide Na⁺ at required levels.