- Open Access
Growth of Plasmodium falciparum in response to a rotating magnetic field
© The Author(s) 2018
- Received: 8 February 2018
- Accepted: 25 April 2018
- Published: 3 May 2018
Plasmodium falciparum is the deadliest strain of malaria and the mortality rate is increasing because of pathogen drug resistance. Increasing knowledge of the parasite life cycle and mechanism of infection may provide new models for improved treatment paradigms. This study sought to investigate the paramagnetic nature of the parasite’s haemozoin to inhibit parasite viability.
Paramagnetic haemozoin crystals, a byproduct of the parasite’s haemoglobin digestion, interact with a rotating magnetic field, which prevents their complete formation, causing the accumulation of free haem, which is lethal to the parasites. Plasmodium falciparum cultures of different stages of intraerythrocytic growth (rings, trophozoites, and schizonts) were exposed to a magnetic field of 0.46 T at frequencies of 0 Hz (static), 1, 5, and 10 Hz for 48 h. The numbers of parasites were counted over the course of one intraerythrocytic life cycle via flow cytometry. At 10 Hz the schizont life stage was most affected by the rotating magnetic fields (p = 0.0075) as compared to a static magnetic field of the same strength. Parasite growth in the presence of a static magnetic field appears to aid parasite growth.
Sequestration of the toxic haem resulting from haemoglobin digestion is key for the parasites’ survival and the focus of almost all existing anti-malarial drugs. Understanding how the parasites create the haemozoin molecule and the disruption of its creation aids in the development of drugs to combat this disease.
- Magnetic field
Currently, malaria is the second leading cause of death in the tropical and subtropical regions of the world and, among the five species of malaria, Plasmodium falciparum is the deadliest . The parasite matures through three life stages when in human blood: ring, trophozoite, and schizont. At the beginning of the ring stage, the parasite uptakes the cytoplasm of the red blood cells (RBC), called the “Big Gulp” . The haemoglobin is transported to a vacuole where it is digested into peptides that are degraded into amino acids. This process leaves free haem, which consists of an iron atom bound to four nitrogen atoms of the pyrrole ring of protoporphyrin IX with two carboxylic side chains . Free haem is toxic to the parasite because the iron at the centre can alternate between its + 2 and + 3 states, leading to the generation of free radicals, the destabilization of the cell membrane, and the disruption of protein structures . To detoxify the haem, the parasite dimerizes it to form β-haematin through a bond between the iron of the first haem and an oxygen of the carboxylic side chain of the second haem . In addition, β-haematins form hydrogen bonds between the oxygens of their second carboxylic side chains yielding a haemozoin crystal. The haemozoin crystal consists of layers of these long chains, held together by π–π stacking forces . The iron bond in the β-haematin causes the whole haemozoin crystal to be paramagnetic [6–8]. One can, therefore, study the effects of the associated net magnetization of a crystal sample arising from an exposure to an external magnetic field [9–13]. This project sought to exploit the paramagnetic nature of the parasite’s haemozoin to inhibit parasite viability.
Plasmodium falciparum HB3 strain (MR4-155, contributed by T.E. Wellems, NIAID) was obtained from the Malaria Research and Reference Reagent Resource (ATCC, Manassas, Virginia). The parasite cultures were maintained at 4% haematocrit, in RPMI 1640 supplemented with 0.5% Albumax, 80 ng/mL gentamicin, and 8200 ng/mL hypoxanthine, at 37 °C in an incubator containing 5% CO2, 1% O2, and 94% N2 and the media was changed daily . The parasitaemia of the culture was checked daily by microscopy and the parasitaemia was kept under 6%. To synchronize cultures the standard procedures for Plasmagel® were used to collect late stage parasites, and sorbitol, to collect ring-stage parasites .
Magnetic field exposure
Data acquisition/flow cytometry
Standard light microscopy was used to observe parasitized RBCs blood smears as described previously [18, 20]. In brief, thin smears were prepared by spreading 5 µL of blood from cultures with a glass slide, fixed in 100% methanol, stained in 4% Giemsa (Sigma-Aldrich, St. Louis, MO), and examined by oil immersion LM (1000×).
Theory and calculations
As discussed, haemozoin leads to a net magnetization associated with paramagnetism when exposed to a magnetic field. In particular, the iron atom at the centre of each haem has five unpaired electrons. According to Hund’s rule, energy is minimized when the total spin is maximized; therefore, the iron is in a spin S = 5/2 state. Since the haemozoin crystal is anisotropic, the magnetic susceptibility is a tensor. Therefore, with the application of a magnetic field, the induced magnetization is not parallel to the magnetic field, and the crystal will undergo a torque. An analysis of the susceptibility tensor shows that the long axis of the crystal will tend to align perpendicularly to the magnetic field .
Assuming crystal dimensions of 600 nm by 200 nm by 200 nm, a field magnitude of 0.46 T, and a temperature of 295 K, and noting that μ0 = 4π × 10−7 Tm/A the ratio of the magnetic to thermal energy is found to be 39. Since the magnetic energy is more than a magnitude greater than the thermal energy, the rotation of a haemozoin crystal is seen to be quite reasonable.
These results demonstrate that rotational frequency is proportional to parasite death. The increased growth observed in the presence of a static field may indicate that aligned crystals process waste faster allowing for improved growth rate, compared with the random motion in the absence of a static field at higher frequency. In the presence of a static field, the magnetic field is aligning (perpendicularly) with the haemozoin crystals, thereby reducing the effects of random thermal motion and parasitic digestion. In this case, the free haem molecules will more easily bind to the ends of the haemozoin crystal, reduce the energy expenditure of the parasite, and allow reduced energy utilization for replication. A similar mechanism could be used to explain the growth of the 1 Hz culture. The schizont culture was the only one that grew when exposed to a static magnetic field. Schizonts have fully formed haemozoin crystals, unlike trophozoites. This supports the hypothesis that the field helped align the haemozoin crystals, reducing the energy needed to process free haem and, therefore, aided the growth of the parasites. At 5 Hz the number of schizonts were suppressed by a factor of 30, while the trophozoites were suppressed by a factor of 5. At 10 Hz the schizonts were down by a factor of 120, while the trophozoites were down by a factor of 7. The increase in schizont death reveals that the higher rotating magnetic field is influencing the fully formed haemozoin crystals and not their formation. The decrease in m-value for the ring culture most likely occurs when they are cycling through the trophozoite and schizont stage; however, it is unclear why the ring culture decreases. It may be because the ring is forming haemozoin under the influence of the magnetic field, while the schizonts have fully formed haemozoin.
This study is the first to show the effect of a rotating magnetic field on the growth of Plasmodium falciparum, to construct a way of quantifying not only the growth of the parasites but also their death, and to show that a static field aids parasite growth. The rotating magnetic field appears to be affecting fully formed haemozoin crystals, not the formation of them.
In the future, the experiment will be repeated at 1 Hz in order to reduce the error in the results and to see if the parasites grow in a magnetic field for a longer time period, particularly the ring culture, as those parasites experience less mortality. In addition, an asymmetric rotating magnetic field could provide two mechanisms for disrupting haemozoin: the first is the rotation of the field and the second is the attractive force between the haemozoin and a gradient magnetic field. This does not occur in the current apparatus because the magnetic field is symmetric. Disruptive measures can be added, such as ferromagnetic particles and antimalarial drugs, to infected cultures to determine what effects they have on parasite growth with and without a rotating magnetic field.
In addition to changes in the nature of the magnetic field, it would be interesting to see the effect of the field on different strains of malaria that form different shapes. For example, Plasmodium knowlesi has diffuse haemozoin throughout the parasite instead of being constrained to the lipid body.
One can also consider the possible treatment applications for malaria patients. However, the magnetic field strength and frequency would first have to be optimized to cause the greatest malaria infected cell death in the shortest amount of time. Instead of applying a magnetic field to the whole body of the patient, a smaller apparatus could be constructed into which a patient’s extremity is placed. As the blood circulates throughout the body each parasite would be exposed to the magnetic field. Depending on the time needed to kill all parasites, patients could either receive one long exposure or a series of shorter exposures over a period of time. This new form of treatment would be beneficial as it would reduce the increasing number of new drug resistant parasites .
RCG analysed and interpreted data and wrote the first draft. RJD, RFB, WCC, MET, DB, KOG, RWB, and BTG, conceived of and analysed the experiments. All authors contributed to, read, and approved the final manuscript.
This work was conducted at CWRU, RCG’s current affiliation is in Small Molecule Sciences, School of Medical Sciences, University of New South Wales, Sydney, Australia. This research was supported by the Cytometry & Imaging Microscopy Shared Resource of the Case Comprehensive Cancer Center (P30CA043703). We are also grateful to the Ohio Third Frontier for support throughout this research.
The authors declare that they have no competing interests.
Availability of data and materials
Consent for publish
Ethics approval and consent to participate
Patient blood for the culture of malaria parasites was obtained from University Hospitals under an Institutional Review Board-approved protocol (Approval #12-09-07).
The authors would like to thank SOURCE for funding this research over the summer, the Case Alumni Association program, the Ohio Third Frontier, and the SAGES Senior Capstone Resource Grant. This work was also supported by the NIH (AI079388, AI116709), Case Western Reserve University School of Medicine Vision Fund, and the Cytometry and Imaging Microscopy Shared Resource of the Case Comprehensive Cancer Center (P30 CA43703).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- WHO. World malaria report, 2015. Geneva: World Health Organization; 2015.Google Scholar
- Elliott DA, McIntosh MT, Hosgood HD 3rd, Chen S, Zhang G, Baevova P, et al. Four distinct pathways of hemoglobin uptake in the malaria parasite Plasmodium falciparum. Proc Natl Acad Sci USA. 2008;105:2463–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Klonis N, Dilanian R, Hanssen E, Darmanin C, Streltsov V, Deed S, et al. Hematin-hematin self-association states involved in the formation and reactivity of the malaria parasite pigment, hemozoin. Biochemistry. 2010;49:6804–11.View ArticlePubMedGoogle Scholar
- Toh SQ, Glanfield A, Gobert GN, Jones MK. Heme and blood-feeding parasites: friends or foes? Parasit Vectors. 2010;3:108.View ArticlePubMedPubMed CentralGoogle Scholar
- Feagin JE, Wurscher MA, Ramon C, Lai HC. Magnetic fields and malaria. In: Biologic effects of light: proceedings of the biologic effects of light symposium. Hingham: Kluwer Academic Publishers; 1999.Google Scholar
- Casabianca LB, An D, Natarajan JK, Alumasa JN, Roepe PD, Wolf C, et al. Quinine and chloroquine differentially perturb heme monomer-dimer equilibrium. Inorg Chem. 2008;47:6077–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Uhlemann A, Staalsoe T, Klinkert M, Hviid L. Analysis of Plasmodium falciparum-infected red blood cells. MACS & more. 2000;4:7–8.Google Scholar
- Moore LR, Fujioka H, Williams PS, Chalmers JJ, Grimberg B, Zimmerman PA, et al. Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis. FASEB J. 2006;20:747–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Griffiths D, editor. Introduction to electrodynamics. 3rd ed. Upper Saddle River: Prentice Hall; 1999.Google Scholar
- Butykai A, Orban A, Kocsis V, Szaller D, Bordacs S, Tatrai-Szekeres E, et al. Malaria pigment crystals as magnetic micro-rotors: key for high-sensitivity diagnosis. Sci Rep. 2013;3:1431.View ArticlePubMedPubMed CentralGoogle Scholar
- Orban A, Butykai A, Molnar A, Prohle Z, Fulop G, Zelles T, et al. Evaluation of a novel magneto-optical method for the detection of malaria parasites. PLoS ONE. 2014;9:e96981.View ArticlePubMedPubMed CentralGoogle Scholar
- Orban A, Rebelo M, Molnar P, Albuquerque IS, Butykai A, Kezsmarki I. Efficient monitoring of the blood-stage infection in a malaria rodent model by the rotating-crystal magneto-optical method. Sci Rep. 2016;6:23218.View ArticlePubMedPubMed CentralGoogle Scholar
- Grimberg BT, Grimberg KO. Hemozoin detection may provide an inexpensive, sensitive, 1-minute malaria test that could revolutionize malaria screening. Expert Rev Anti Infect Ther. 2016;14:879–83.View ArticlePubMedPubMed CentralGoogle Scholar
- Grimberg BT, Jaworska MM, Hough LB, Zimmerman PA, Phillips JG. Addressing the malaria drug resistance challenge using flow cytometry to discover new antimalarials. Bioorg Med Chem Lett. 2009;19:5452–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Ljungström I, Perlmann H, Schlichtherle M, Scherf A, Wahlgren M, editors. Methods in Malaria Research. 4th ed. Manassas: Malaria Research and Reference Reagent Resource Center, American Type Culture Collection; 2004.Google Scholar
- Newman DM, Heptinstall J, Matelon RJ, Savage L, Wears ML, Beddow J, et al. A magneto-optic route toward the in vivo diagnosis of malaria: preliminary results and preclinical trial data. Biophys J. 2008;95:994–1000.View ArticlePubMedPubMed CentralGoogle Scholar
- Grimberg BT. Methodology and application of flow cytometry for investigation of human malaria parasites. J Immunol Methods. 2011;367:1–16.View ArticlePubMedPubMed CentralGoogle Scholar
- Grimberg BT, Erickson JJ, Sramkoski RM, Jacobberger JW, Zimmerman PA. Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy. Cytometry A. 2008;73:546–54.View ArticlePubMedPubMed CentralGoogle Scholar
- Shapiro HM, Apte SH, Chojnowski GM, Hanscheid T, Rebelo M, Grimberg BT. Cytometry in malaria–a practical replacement for microscopy? Curr Protoc Cytom. 2013;11–20.Google Scholar
- McNamara DT, Kasehagen LJ, Grimberg BT, Cole-Tobian J, Collins WE, Zimmerman PA. Diagnosing infection levels of four human malaria parasite species by a polymerase chain reaction/ligase detection reaction fluorescent microsphere-based assay. Am J Trop Med Hyg. 2006;74:413–21.PubMedPubMed CentralGoogle Scholar
- Coronado LM, Nadovich CT, Spadafora C. Malarial hemozoin: from target to tool. Biochim Biophys Acta. 2014;1840:2032–41.View ArticlePubMedPubMed CentralGoogle Scholar
- Grimberg BT, Mehlotra RK. Expanding the antimalarial drug arsenal-now, but how? Pharmaceuticals (Basel). 2011;4:681–712.View ArticleGoogle Scholar