Background
The inhibition of the haem detoxification pathway in the malaria parasite,
Plasmodium falciparum remains an attractive drug target with several important anti-malarials, primarily quinolines proposed to target it [
1]. During its asexual erythrocytic cycle, free haem, derived from the digestion of host red blood cell haemoglobin (62 ± 13 − %) is autoxidized to poisonous haematin (Fe(III)PPIX), predominantly in the metabolically active trophozoite [
2]. In an early study Ginsburg et al. detected an increase in membrane associated haem upon CQ and amodiaquine (AQ) treatment of cultured
P. falciparum, but recovery of haemozoin in drug-free controls in this study was very low, with only 20–30 % detected [
3]. In a subsequent study it was shown that the overwhelming majority of this released haem (up to 95 %) is efficiently sequestered into inert crystalline haemozoin (Hz) within the parasite’s acidic digestive vacuole. Transmission electron microscopy (TEM) with electron energy loss spectroscopy (EELS) used to map the distribution of elemental Fe in mature trophozoite-infected erythrocytes (chloroquine (CQ) sensitive, D10 strain), confirmed Hz associated Fe is concentrated in the acidic digestive vacuole with very little Fe in the cytosol [
2]. Hz formation has been shown to be closely associated with lipids [
4]. Recently, neutral lipids associated with Hz were shown to mediate β-haematin (BH, synthetic Hz) formation under physiological conditions at rates analogous to parasite Hz formation [
5]. This process of cellular lipid mediated Hz formation is mimicked synthetically in a detergent-mediated assay which substitutes neutral lipids for the lipophilic detergent NP-40 [
6]. This assay has been applied as a high throughput screening tool and identified several novel scaffolds capable of inhibiting Hz formation [
7,
8]. The Hz inhibition pathway remains a favourable drug target despite resistance to known quinolines and related drugs which have been attributed to mutations in the parasite’s ‘chloroquine resistance transporter’ (PfCRT) and other digestive vacuole membrane proteins [
9]. In comparison to CQ-sensitive strains (CQS), resistant parasites have been shown to accumulate up to ten times less CQ in the digestive vacuole, falling outside the effective therapeutic range for CQ [
10]. The formation of Hz is unaffected by PfCRT mutations and so Hz inhibitors remain one of the most successful classes of anti-malarials to date.
The use of detergent-based screening methods as a synthetic measure of the ability of a compound to inhibit the formation of BH is routinely used to identify BH inhibitors [
8,
11]. This method has been translated into an analogous process in malaria parasites cultured in vitro, the haem fractionation assay, a method capable of directly measuring haem species in CQS
P. falciparum [
12]. Originally applied to CQ, this cellular fractionation technique colorimetrically measures haem as Fe(III)haem-pyridine complex based on a previously published method by Ncokazi and Egan in which it was shown that in a mixture of BH and haematin, the latter forms a low-spin complex with aqueous pyridine (5 % v/v, pH 7.5) without disturbing BH [
13]. Using the cellular haem fractionation assay, CQ was shown to cause a dose-dependent increase in ‘free’ haem (i.e., labile haem that can be solubilized with detergent), along with a decrease in Hz correlated to the survival of
P. falciparum cells. TEM with EELS of CQ-treated cells showed a redistribution of Fe from the digestive vacuole into the parasite cytoplasm [
12]. The method provided valuable information, successfully demonstrating the ability of CQ to inhibit cellular Hz formation. Due to its protracted nature, however, it could not easily be extended to other drugs. Here, a modified technique designed to increase output and reduce material costs is described in detail. Performed in 24-well plates, multiple drug concentrations can be evaluated in one session with relatively low amounts of parasite starting material. At minimum results can be obtained for two compounds per week, a significant improvement in output over the previous method, which produced results for one compound every 2 months. The method was successfully validated against the original haem fractionation assay performed in flasks using CQ. This validated method was applied to three clinically relevant anti-malarials covering a broad spectrum of mechanisms of actions: the 4-aminoquinoline AQ which like CQ has been shown to inhibit BH formation, the non-BH inhibiting antifolate pyrimethamine (PYR) [
7] and finally the non-BH inhibiting anti-malarial atovaquone (Atov). While initial results for Atov showed an unanticipated increase in the percentage of free haem with increasing Atov concentration, the total amount of haem iron per cell was found to be significantly less in cells treated with Atov. This corresponds to unchanged free haem per cell with increasing Atov concentration. This example with Atov demonstrates the importance of establishing the total amount of haem iron per cell, a calculation which requires the determination of the cell count in each well of the 24-well plate. Cell counts were determined using a haemocytometer and flow cytometry based method with fluorescent cell staining [
14‐
19]. Acquiring cell counts for a large number of samples with a haemocytometer proved a tedious process, as a result counting was also performed with a novel flow cytometry method using SYBR green I fluorescent staining of isolated cells. Over the course of the 48 h lifecycle of the parasite the amount of DNA increases as the parasite matures from early rings to multi-nucleated schizonts, translating to an increase in the SYBR green I fluorescence signal as a result of binding to double stranded parasite DNA [
19]. SYBR green I was specifically chosen due to its low binding affinity for RNA and preference for double stranded DNA over single stranded DNA [
20]. In addition to relieving the burden of manual cell counting, this flow cytometry method can also provide important information about morphological changes in the presence of cells treated with anti-malarials.
The method described here is a modified version of an established technique which details the increased output of a previously published assay allowing for the determination of haem species spectroscopically in isolated P. falciparum trophozoites.
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Competing interests
The authors declare they have no competing interests.
Authors’ contributions
JC designed and carried out laboratory experiments, analysed and interpreted data and results and wrote the manuscript. KYF performed experiments on pyrimethamine, analysed the data and reviewed and edited the manuscript. LG assisted in the design and interpretation of all flow cytometry experiments and analysis. PJS co-supervised all the research and reviewed, edited and commented on the manuscript. DWW supervised all research performed by KYF and reviewed, edited and commented on the manuscript. TJE supervised and contributed to all the research, data analysis and interpretation of results, reviewed, edited and commented on the manuscript. All the authors have contributed to, seen and approved the final manuscript.