Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors

Abstract

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad-selectivity channel known as the plasmodial surface anion channel, increased Ca⁺⁺ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca⁺⁺ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N-hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca⁺⁺ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca⁺⁺ permeability, suggesting involvement of parasite-encoded proteins trafficked to the host membrane. A high-throughput chemical screen identified the first Ca⁺⁺ transport inhibitors active against Plasmodium-infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca⁺⁺ ] is consistent with parasite killing specifically via action on one or more Ca⁺⁺ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca⁺⁺ transport and may be starting points for new antimalarial drugs.

Keywords: Plasmodium falciparum; calcium transport; high-throughput screen; inhibitors; malaria.

Fig 1

Surface labeling reveals differing mechanisms of Ca⁺⁺ uptake by infected and uninfected cells. (A) Ca⁺⁺ uptake kinetics for infected and uninfected cells in the presence of 1 mM external Ca⁺⁺ or 1 mM EGTA, as indicated. Symbols represent mean ± S.E.M. of replicate wells. Notice the greater permeability of infected cells. (B) Uptake kinetics with and without pre-labeling of cells with sulfo-NHS-LC-LC-biotin or sulfo-NHS-biotin (LC-LC and SNB); LC-LC labeling in the presence of 125 nM ISG-21 is also shown to evaluate the effect of NHS ester uptake. (C) Mean ± S.E.M. Ca⁺⁺ uptake after labeling with SNB or LC-LC (black and red bars, respectively), calculated as the change in fluorescence over 12 h measurements as shown in panels B and C followed by normalization to 100% for matched untreated cells. Dd2- and HB3-infected cells are shown. *, P < 0.05. (D) Preserved block of infected cell Ca⁺⁺ transport by LC-LC despite inhibition of NHS ester uptake by 125 nM ISG-21, shown as the mean ± S.E.M. of 100*(% block with ISG-21)/(% block in matched controls without ISG-21). (E) Mean ± S.E.M. % block of Ca⁺⁺ uptake by 125 nM ISG-21 alone. Values in D and E were calculated using change in fluorescence over 12 h.

Fig 2

Conditional knockdown of parasite protein export aborts development of host cell permeability. (A) Ca⁺⁺ uptake kinetics in the 13F10 HSP101-DDD clone after cultivation for 48 h with or without 10 μM TMP (black and red symbols, respectively). Note, the slower development of Ca⁺⁺-dependent fluorescence in the absence of TMP. (B) PSAC-mediated osmotic lysis kinetics in sorbitol, measured in 13F10 cultivated with and without TMP (black and red traces, respectively). (C) Mean ± S.E.M. residual transport activities, tallied from experiments as in panels (A) and (B).

Fig 3

Design and execution of a chemical screen for inhibitors of Ca⁺⁺ transport into infected cells. (A) Mean ± S.E.M. fluorescence signal from kinetic studies with infected and uninfected cells loaded with either Fluo-8 or Cal-520 indicator dyes at a 2.5 μM concentration. Pr represents 5 mM probenecid, added as an inhibitor of organic anion transporters. (B) Mean ± S.E.M. progression to ring stage parasites after Cal-520 loading and return to culture, normalized to a matched DMSO control. Progression was evaluated with Giemsa-stained slides; n = 3. (C) Z′ factor, calculated from positive and negative control wells in 384-well microplates at indicated timepoints after Ca⁺⁺ addition. The accepted minimum value for single-well screens, Z′ = 0.5, is shown (dashed line). (D and E) All points histogram of results from a screen of 52,478 compounds, normalized to 0% and 100% block for in-plate negative and positive controls (Ca⁺⁺ and EGTA, respectively). Panels D and E represent readings taken 15 min and 15 h after initiating Ca⁺⁺ uptake in screening plates. Hits were defined as compounds that produced block greater than the mean plus 3*standard deviation of the negative control wells.

Fig 4

Novel Ca⁺⁺ uptake inhibitors confirmed through secondary studies. (A) Chemical structures of six hits selected for detailed study. (B – C) Ca⁺⁺ uptake fluorescence kinetics with 0, 5, and 10 μM CA-5 (black, blue, and red symbols, respectively). Uptake was measured in HBS with Ca⁺⁺ buffered with EGTA to 8 μM and 1 mM free Ca⁺⁺ in panels B and C, respectively, using http://www.maxchelator.stanford.edu. (D) Mean ± S.E.M. half-maximal inhibitory concentrations for indicated blockers in 1 mM and 8 μM free Ca⁺⁺ (black and grey bars, respectively). Values were calculated from dose response measurements at the 330 min timepoint in kinetic experiments as in panels B and C. *, P < 0.05 for comparisons between the two buffers, n= 3 independent trials each, 2-way ANOVA with post-test. (E) Ca⁺⁺ dose responses for Cal-520 in HBS with indicated hits. Compounds were tested at a 10 μM concentration and compared to a matched DMSO only control. Symbols represent the mean ± S.E.M. of up to 3 independent measurements with fluorescence normalized to the DMSO control. Solid line represents the best fit to y = F_max*x/(K_d + x), yielding an estimated K_D of 450 nM for Cal-520 binding to Ca⁺⁺. (F) Confocal images of infected cells showing that CA-5 inhibits Ca⁺⁺ uptake and fluorescence development, in contrast to DMSO or the PSAC inhibitor furosemide. Dye loading and 12 h incubation with inhibitors (25 μM) were performed under standard culture conditions. Scale bar, 3 μm.

Fig 5

Ca⁺⁺ uptake inhibitors do not inhibit initial uptake kinetics at the host membrane, suggesting action at one or more intracellular sites. (A) Representative ⁴⁵Ca uptake kinetics for enriched trophozoite-stage infected cells using the Dd2 parasite line; [Ca⁺⁺] = 0.1 mM. Symbols represent ⁴⁵Ca⁺⁺ accumulation with either 10 μM CA-5 or a DMSO control. Solid lines represent the best fit y = m*x + y_o and reveal that uptake is linear over the measured duration. (B) Mean ± S.E.M. slopes of ⁴⁵Ca⁺⁺ uptake for indicated inhibitors, normalized to matched DMSO controls, as in panel (A). None of these inhibitors significantly alters initial Ca⁺⁺ uptake kinetics.

Fig 6

Ca⁺⁺ transport into freed parasites. (A) Fluorescence kinetic measurements using saponin-freed trophozoites in HBS with 1 mm free Ca⁺⁺ or 0.5 mM EGTA, as indicated. Arrow indicates addition of SDS to lyse cells and release intracellular Cal-520. Notice negligible Ca⁺⁺ uptake on this time scale. (B) Mean ± S.E.M. increase in fluorescence over a12 h incubation for freed parasites and intact infected cells suspended in HBS with buffering to maintain either an 8 μM or a 1 mM free Ca⁺⁺ (grey and black bars, respectively; n = 4-5 matched independent trials). *, P < 10⁻³.

Fig 7

Growth inhibitory effects of inhibitors are consistent with action on Ca⁺⁺ transport mechanisms. (A) Normalized parasite growth over 72 h with indicated concentrations of CA-5 in RPMI 1640-based medium without or with 0.44 mM EGTA added to limit availability of extracellular Ca⁺⁺. Symbols represent mean ± S.E.M. of 3 replicate measurements; solid lines represent best fits to y = a/(1 + (x/b)^c). Note that CA-5 reduces parasite growth at lower concentrations when extracellular Ca⁺⁺ is reduced by addition of 0.44 mM EGTA. (B) Mean ± S.E.M. IC₅₀ values for indicated inhibitors or antimalarial drugs in media without or with 0.44 mM EGTA, determined from experiments as in panel (A). *, P < 0.05, 2-way ANOVA with post-test.