Fosmidomycin uptake into Plasmodium and Babesia-infected erythrocytes is facilitated by parasite-induced new permeability pathways

Abstract

Background: Highly charged compounds typically suffer from low membrane permeability and thus are generally regarded as sub-optimal drug candidates. Nonetheless, the highly charged drug fosmidomycin and its more active methyl-derivative FR900098 have proven parasiticidal activity against erythrocytic stages of the malaria parasite Plasmodium falciparum. Both compounds target the isoprenoid biosynthesis pathway present in bacteria and plastid-bearing organisms, like apicomplexan parasites. Surprisingly, the compounds are inactive against a range of apicomplexans replicating in nucleated cells, including Toxoplasma gondii.

Methodology/principal findings: Since non-infected erythrocytes are impermeable for FR90098, we hypothesized that these drugs are taken up only by erythrocytes infected with Plasmodium. We provide evidence that radiolabeled FR900098 accumulates in theses cells as a consequence of parasite-induced new properties of the host cell, which coincide with an increased permeability of the erythrocyte membrane. Babesia divergens, a related parasite that also infects human erythrocytes and is also known to induce an increase in membrane permeability, displays a similar susceptibility and uptake behavior with regard to the drug. In contrast, Toxoplasma gondii-infected cells do apparently not take up the compounds, and the drugs are inactive against the liver stages of Plasmodium berghei, a mouse malaria parasite.

Conclusions/significance: Our findings provide an explanation for the observed differences in activity of fosmidomycin and FR900098 against different Apicomplexa. These results have important implications for future screens aimed at finding new and safe molecular entities active against P. falciparum and related parasites. Our data provide further evidence that parasite-induced new permeability pathways may be exploited as routes for drug delivery.

Figure 1. Fosmidomycin-sensitive Dxr activity in T. gondii.

A Principle of the coupled radiometric assay for Dxr activity measurement. MEP formed by Dxr from DOXP and NADPH is converted into [³²P]CDP-ME in a second reaction using recombinant E. coli YgbP (IspD) enzyme and [α-³²P]CTP. B Analysis of the Dxr assay with T. gondii lysate in the presence of different concentrations of Fos (lanes 2–4) and without drug (lane 1) by autoradiography of the TLC plate after separation of the reaction products. The appearance of [³²P]CDP-ME is indicative of Dxr activity (arrowhead). The smear at the bottom is [α-³²P]CTP (*), the one above (**) is [α-³²P]CDP whereas the prominent spot below [³²P]CDP-ME is caused by [α-³²P]CMP (***). The production of CDP and CMP from CTP is presumably due to the presence of phosphatases in the whole cell lysates, which explains the weaker corresponding signals with the recombinant E. coli Dxr protein (lane 6). As controls, either host cell lysate (negative control, lane 5) or 10 pg of purified recombinant E. coli Dxr protein (41 U/mg; positive control, lane 6) was used. C Densitometric evaluation of the [³²P]CDP-ME signal from B. AU, arbitrary units. D Comparison of the structures of the Dxr substrate DOXP with of Fos and FR.

Figure 2. Intracellular localization of Dxr in T. gondii tachyzoites and P. falciparum blood stages.

Antibody staining was performed on fixed parasites with a polyclonal antibody raised against recombinant P. falciparum Dxr. A Discrete anti-PfDxr reactivity (green) can be detected in T. gondii tachyzoites in the apicoplast (I), co-localizing with organellar DNA (blue; arrowheads in II; overlaid in III). Co-localization is visualized in IV using the ‘Colocalization’ plugin from the ImageJ software suite. White pixels indicate the summed-up overlapping co-localizing signals from III, superimposed onto the phase contrast image of the parasites within the vacuole. B Schizont stage of P. falciparum: (I) phase contrast of iRBC; (II) anti-PfACP (red); (III) anti-PfDxr (green); (IV) DAPI stain (blue); (V) merged images of II-IV (co-localization of anti-PfACP and anti-PfDxr (yellow); (VI) merged images of I and of co-localized pixels (using the ‘Colocalization’ plugin applied to II and III). Co-localization of anti-PfACP and anti-PfDxr (white). Scale bars = 5 µm.

Figure 3. Uptake studies of [¹⁴C]FR and [³H]Glu into human fibroblasts.

Duplicates of cells (HFF, non-infected; TgHFF, >50% infected with tachyzoites) were incubated in parallel in phosphate-free “extracellular buffer” (see Material and Methods) for 15 min at 37°C in the presence of either [¹⁴C]FR or [³H]Glu, respectively, of the same specific activity (1.45 µCi/ml, equaling 25 µM drug concentration per assay). Cell-associated radioactivity was determined by scintillation counting of an aliquot of lysed cells and dpm for each compound were determined according to the manufacturer's instructions (shown as dpm±SD). In some assays cells were preincubated for 30 min in 1 mM unlabelled FR or L-Glu to test for uptake specificity (indicated by ‘+FR’ and ‘+Glu’). This is one representative of three similar experiments.

Figure 4. Infection-induced, time-dependent increase of uptake of [¹⁴C]FR into P. falciparum parasitized erythrocytes.

Non-infected (RBC) and red blood cells infected with P. falciparum (Pf-iRBC), respectively, were incubated in RPMI-1640 medium for 60 min containing [¹⁴C]FR (A) or [¹⁴C]Glu (B) of the same specific activity. At the indicated time points aliquots were taken and the radiolabeled tracer in the cellular fraction was quantified (see Material and Methods). Uptake of labeled compounds was also evaluated in the presence of the NPP-inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) at 10 µM, or by pre-treatment of Pf-iRBC with 1 mg/ml chymotrypsin for 1 h at 37°C. Similarly treated RBC served as controls. Cpm±SD for three experiments are shown.

Figure 5. Increased uptake of [¹⁴C]FR into erythrocytes infected with Babesia divergens (Bd-iRBC).

Bd-iRBC were incubated in the presence of [¹⁴C]FR at 37°C for 20 min before the cells were harvested and the amount of intracellular [¹⁴C]FR determined. Non-infected RBC and Bd-iRBC treated with 100 µM furosemide (Furo) served as controls. Shown are the mean values of three independent experiments (±SD). The data represent the amount of intracellular [¹⁴C]FR in relation to the amount quantified for RBC (control). * indicates a p-value of 0.011.

Figure 6. Effect of FR900098 on P. berghei liver stage parasites (exoerythrocytic forms, EEF) in vitro and in vivo.

Human hepatoma cells infected with 10,000 GFP-expressing sporozoites and treated with 100 µM, 10 µM and 1 µM FR, respectively, were fixed 48 h p.i. Parasites were visualized with a monoclonal antibody directed against HSP70 and Alexa488-labelled secondary antibody (*), and Hoechst 33342 to stain the nuclei. A Immunofluorescence images of untreated and 100 µM-treated EEFs, respectively, showing no changes in morphological appearance upon FR treatment. Bar represents 10 µm. B Quantitative assessment of drug effects on parasite growth at the three different FR concentrations. Given are numbers of EEFs/well (mean value±SEM). C Quantitation of in vivo effect of FR treatment by real time PCR to assess parasite load in livers of infected C57BL/6 mice either untreated, treated with FR or, as a positive control, treated with primaquine. Parasite load on the y-axis is given as the percentage of the mean parasite load (±SEM) in PBS-treated control mice. D Effect of the batch of FR used in A-C on blood stage P. berghei in infected C57BL/6 mice. Parasitemia was determined by means of Giemsa-stained thin blood smears at the indicated time points after infection.