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- Materials and methods
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- Supporting Information
Iron is a critical and tightly regulated nutrient for both the malaria parasite and its human host. The importance of the relationship between host iron and the parasite has been underscored recently by studies showing that host iron supplementation may increase the risk of falciparum malaria. It is unclear what host iron sources the parasite is able to access. We developed a flow cytometry-based method for measuring the labile iron pool (LIP) of parasitized erythrocytes using the nucleic acid dye STYO 61 and the iron sensitive dye, calcein acetoxymethyl ester (CA-AM). This new approach enabled us to measure the LIP of P. falciparum through the course of its erythrocytic life cycle and in response to the addition of host serum iron sources. We found that the LIP increases as the malaria parasite develops from early ring to late schizont stage, and that the addition of either transferrin or ferric citrate to culture media increases the LIP of trophozoites. Our method for detecting the LIP within malaria parasitized RBCs provides evidence that the parasite is able to access serum iron sources as part of the host vs. parasite arms race for iron.
Each year up to 250 million clinical cases of malaria and nearly 1 million deaths from malaria are reported in official statistics (WHO, 2011). Plasmodium falciparum malaria is the most deadly of all the species of malaria that infect humans. The malaria parasite has a complex life cycle in the human host. Anopheles mosquitoes inject sporozoite stage P. falciparum parasites during a blood meal; sporozoites then migrate to the liver where they infect hepatocytes and multiply over a clinically silent 7–10 d period (Sinnis et al, 1996). During the asexual erythrocyte stage of the parasite, merozoites invade red blood cells (RBCs) and progress from the metabolically inactive ring stage to the metabolically active trophozoite stage to the schizont stage. DNA replication is initiated during the schizont stage and results in the production of new merozoites that burst from the host RBC into the blood stream and invade new RBCs. The RBC stage of the malaria parasite is responsible for the morbidity and mortality associated with P. falciparum infection and is exquisitely sensitive to iron chelators (Ferrer et al, 2012).
Despite the essential role of iron in parasite development, it is unknown what host iron sources P. falciparum utilizes during any stage of the human infection. The two principal sources of host iron available to the parasite during the RBC stage are extra-erythrocytic (serum) iron and intra-erythrocytic iron. The intra-erythrocytic iron pool amounts to 100 fg (20 mmol/l) iron, partitioned into haemoglobin, ferritin, and the cytoplasmic labile iron pool (LIP). The erythrocytic LIP consists of residual cytoplasmic bioavailable iron that was not incorporated into haemoglobin or stored within ferritin during the maturation of erythrocyte precursors (Prus & Fibach, 2008a). The majority of host serum iron is bound to host protein transferrin (0·6–1·5 g/l), with a residual amount of iron, non-transferrin-bound iron (NTBI), circulating in the serum chelated by low molecular weight molecules such as citrate (Cook & Skikne, 1989). To date there is no evidence that P. falciparum is able to release iron from haem or host ferritin (Sigala & Goldberg, 2012). The relationship between the host erythrocyte LIP and the malaria parasite infectivity and maturation is unknown (Scholl et al, 2005). It is unknown how the host LIP impacts the malaria parasite's infectivity and maturation. The ability of the parasite to access serum iron is unclear, and data are conflicting (Pollack & Fleming, 1984; Haldar et al, 1986; Rodriguez & Jungery, 1986; Sanchez-Lopez & Haldar, 1992).
Calcein acetoxymethyl ester (CA-AM) has been widely used to examine the cytoplasmic LIP of mammalian cells (Breuer et al, 1995a,b, 1996; Epsztejn et al, 1997; Tenopoulou et al, 2007). CA-AM is non-fluorescent, non-iron binding, neutrally charged, and easily permeates cell membranes. Upon cellular entry, intracellular esterases cleave CA-AM into the green-fluorescent molecule calcein, which is then trapped within the cell. Calcein fluorescence is quenched by 1:1 stoichiometric binding of iron in pH range of 7–7·5 (Breuer et al, 1995a, 1996). The addition of non-fluorescent, high affinity iron chelators removes iron from calcein and consequently increases calcein fluorescence, providing an effective method for assessing the labile iron content of cells. Alternatively the addition of iron, capable of being incorporated by a cell, quenches calcein fluorescence (Tsien, 1989; Breuer et al, 1995b). Previous investigators have utilized a microscopy-based approach for the measurements of calcein fluorescence, to investigate the site of action of anti-malarial iron chelators and gain preliminary insight into the LIP of parasitized human erythrocytes (Loyevsky et al, 1999). More recently, CA-AM has been utilized to assess the LIP of the heterogeneous cell populations of peripheral blood and bone marrow by flow cytometry (Prus & Fibach, 2008b).
In the present study, we adapted the CA-AM flow cytometry method in order to assess the LIP of P. falciparum -infected erythrocytes (Prus & Fibach, 2008a,b). We combined the technique of identifying parasitized erythrocytes with the fluorescent DNA dye, SYTO 61 (Fu et al, 2010), with the CA-AM method for assessing cellular labile iron to determine the LIP of P. falciparum during asexual maturation by flow cytometry. This flow cytometry approach allows for the analysis of the LIP of a mixed population of uninfected and P. falciparum -infected erythrocytes. Furthermore, we utilized this approach to investigate the effect of extracellular iron sources, transferrin and ferric citrate, on the LIP of the erythrocytic stage of P. falciparum.
- Top of page
- Materials and methods
- Author Contributions
- Supporting Information
During the course of microbial infections, there is an arms race between the pathogen and host for iron. In the course of this arms race pathogens have evolved sophisticated methods of scavenging host iron while the host acute activation of the nutritional immune response effectively limits the availability of iron to invading pathogens (Skaar, 2010). Iron chelating agents suppress the growth of P. falciparum in vitro and in vivo (Hershko & Peto, 1988; Gordeuk et al, 1992). In addition, iron chelators also bolster the host innate immune response by synergistically acting with cytokines to increase stimulation of NO production, which is protective against severe malaria infection (Weiss et al, 1997; Fritsche et al, 2001). The importance of iron to malaria is additionally demonstrated by clinical studies that have documented an increased susceptibility to malaria infection in individuals given high doses of iron supplementation (Murray et al, 1975; Smith et al, 1989; Oppenheimer, 2001; Sazawal et al, 2006). The sources of host iron used by P. falciparum and the strategies used by the parasite to evade host nutritional immunity have remained elusive.
The labile iron pool represents the transition zone for iron between import, cellular utilization and storage and it is thought to change in response to the metabolic needs of the cell. As a cells' metabolic demand for iron increases, it will increase the amount of iron in the labile iron pool. The CA-AM LIP assay measures the LIP present in uninfected RBCs and pRBCs. Calcein fluorescence is sensitive to iron at physiological pH 7·2–7·4 (Tenopoulou et al, 2007). As RBC precursors mature, all their organelles are lost, producing an anucleate mature erythrocyte with a cytoplasm of pH 7·2–7·4 (Tenopoulou et al, 2007). At this pH, calcein is sensitive to iron and is able to detect the entire LIP. Upon infection of the RBC, P. falciparum introduces new organelles and structures including: a nucleus, mitochondria, apicoplast, endoplasmic reticulum (ER) and golgi apparatus as well as a parasitophorous vacuole and a food vacuole with pH 3·7–6·5 (Hayward et al, 2006).
The LIP, which is detected within pRBCs using the CA-AM method, is the bioavailable/labile iron present in the neutral pH regions of the residual RBC cytoplasm, parasitophorous vacuole, and parasite cytoplasm. We defined LIP as the ΔMFI of calcein that occurred with the addition of an iron chelator or iron source. We observed that the basal calcein fluorescence was greater within pRBCs than within uninfected RBCs and that fluorescence increased with parasite maturation. The addition of iron chelators to calcein-loaded uninfected and pRBCs resulted in greater ΔMFI within pRBCs than uninfected RBCs, and the ΔMFI further increased with increasing parasite maturation. Interestingly, the extra-erythrocytic merozoite stage of P. falciparum had no detectable LIP. Our data indicate that the parasite may be able to access both intra-erythrocytic and serum iron. Given that total iron does not differ between uninfected and pRBCs (Marvin et al, 2012), our observation that LIP increases with parasite maturation when it is grown in very low (1·79–2·685 μmol/l) iron media, suggests that the parasite may be able to release iron from either RBC haemoglobin or ferritin, redistributing but not altering the total cellular iron. Increasing LIP with parasite maturation is consistent with the increasing iron demands of the parasite during the trophozoite and schizont stage as the parasite's metabolic activity dramatically increases and commences DNA replication. Alternatively, changes in intracellular iron levels may not only reflect iron consumption by the parasite but may be due to regulation of iron import/export in infected cells, as has been shown in macrophages targeted by intracellular bacteria (Nairz et al, 2007; Paradkar et al, 2008).
To provide new insight into the potential ability of P. falciparum to access serum iron, either transferrin or non-transferrin bound iron (ferric citrate), we measured the impact of holo-transferrin and ferric citrate on the LIP of uninfected and pRBCs. We observed that the addition of increasing physiological concentrations of either ferric citrate or holo-transferrin increased the LIP of trophozoite pRBCs to a significantly greater degree than of ring pRBCs and uninfected RBC. This provides evidence that trophozoite stage pRBC can access serum iron sources. These results do not address whether pRBCs specifically bind or internalize transferrin. Rodriguez and Jungery (1986) and Haldar et al (1986) independently postulated the existence of a P. falciparum transferrin receptor, however such a receptor has yet to be isolated and cloned. Alternatively, it is well established that pRBCs are able to non-specifically incorporate both micro- and macro-molecules from the serum (Pouvelle et al, 1991; Nguitragool et al, 2011). Human transferrin, like other abundant serum proteins, such as albumin, may be non-specifically internalized into pRBCs (El Tahir et al, 2003).
Our results are relevant to the clinical question of whether host iron status and host iron supplementation affects risk of malarial infection. The relationship between host iron and the malaria parasite is complex and is tightly regulated by both host and parasite. A study published in 2006 that was conducted in Pemba, Zanzibar, involving more than 24,000 children in a setting where anti-malarial treatment was not readily available, showed that routine supplementation with iron and folic acid increased the rates of severe illness and death from malaria in iron-replete children who took iron supplements (Sazawal et al, 2006). Because non-transferrin bound serum iron transiently increases in iron-replete individuals who are given oral iron supplementation (Schümann et al, 2012), we speculated that P. falciparum may scavenge serum iron in order to augment intraerythrocytic growth and thereby potentiate the risk of malaria.
Our application of the flow cytometry based CA-AM LIP assay has revealed that the LIP content of infected RBCs steadily increases with increasing maturation of the intra-erythrocytic stage of the parasite. Additionally, we demonstrated that the LIP content of late stage trophozoite pRBCs is increased in the presence of extracellular transferrin and ferric citrate. Further studies are needed to elucidate the mechanisms by which the malaria parasite senses, acquires, utilizes, regulates, and stores iron during the erythrocytic stage of its life cycle and the impact of host serum iron on these processes. Elucidation of parasite iron biology will provide therapeutic insights into how to augment the host innate immune response and may reveal targets for anti-malarial drug development.