Stage independent chloroquine resistance and chloroquine toxicity revealed via spinning disk confocal microscopy,☆☆

https://doi.org/10.1016/j.molbiopara.2007.12.014Get rights and content

Abstract

We previously customized a Nipkow spinning disk confocal microscope (SDCM) to acquire 4D data for live, intraerythrocytic malarial parasites [Gligorijevic B, McAllister R, Urbach JS, Roepe, PD. Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria. Biochemistry 2006;45:12400–10]. We reported that chloroquine (CQ) treatment did not appear to affect progress through the cell cycle, and suggested that toxicity may be manifested post-schizogony. We now use SDCM, synchronized cell culture and continuous vs. bolus drug dosing to investigate stage specific CQ effects in detail. We develop a novel, extremely rapid method for counting schizont nuclei in 3D. We then quantify schizont nuclei and hemozoin (Hz) production for live parasite cultures pulsed with CQ at different stages in the cell cycle and find that bolus treatment of rings affects the multiplicity of nuclear division. We quantify parasitemia and merozoite development in subsequent cycles following bolus CQ exposure and find that a portion of CQ toxicity is manifested post-schizogony as “delayed death”. Using these methods and others we compare CQ sensitive (CQS) vs. resistant (CQR) strains as well as transfectants that are CQR via introduction of mutant PfCRT. Surprisingly, we find that PfCRT confers resistance to CQ administered at the very early ring stage of development, wherein a digestive vacuole is not yet formed, as well as at the schizont stage, wherein Hz production is thought to plateau. Taken together, these data force a rethinking of CQ pharmacology and the mechanism of CQR.

Introduction

P. falciparum malarial parasites are now largely resistant to several key antimalarial drugs. Until recently chloroquine (CQ) was the drug of choice but globally >50% of P. falciparum infections are now chloroquine resistant (CQR). Elucidating the molecular mechanism of CQR is essential to the ongoing design of second line antimalarial drugs and new therapies [1]. In addition, the mechanism of action of CQ is not fully understood but is obviously also relevant for drug design.

Early on, parasite DNA was postulated to be the CQ target [2], [3]. Cohen and Yielding further proposed that DNA synthesis is inhibited through an effect on DNA and RNA polymerase, due to the binding of CQ to DNA primer [4]. Hahn and co-workers investigated aminoquinoline action vs. nucleic acids and proposed that CQ stabilizes double-stranded DNA [5], [6]. Meshnick and co-workers have also proposed that a portion of the CQ mechanism comes from binding to DNA, possibly by preventing formation of Z-DNA [7], [8]. However, none of these models easily explained much lower inhibitory concentrations of CQ for P. falciparum vs. mammalian cells (∼1000–10,000 fold lower). Thus the nucleic acid hypotheses have been largely discounted, particularly after the discovery that the P. falciparum genome was highly AT-rich whereas CQ preferentially interacts with GC-rich DNA [9].

When CQR parasites were found to accumulate substantially less CQ vs. CQ sensitive (CQS) [10], and a lysosomal-like organelle called the digestive vacuole (DV) was found to be the principle site of CQ accumulation [11], [12], focus began to shift towards investigation of DV processes. Importantly, Orjih and Fitch found that mid-stage trophozoites with fully formed DVs accumulate more CQ than any other stage [13]. Morever, recently, CQR was shown to segregate with mutations in the pfcrt gene which encodes a protein found in the DV membrane, which is only fully developed during the early trophozoite stage [14]. Also much physical–chemical data support direct interaction between CQ and various forms of non-crystalline heme released in the DV upon hemoglobin (Hb) digestion (e.g. [15], [16]). These interactions inhibit conversion of toxic heme to non-toxic crystalline hemozoin (Hz) and are thus believed to be central to CQ (and related quinoline antimalarial drug) pharmacology [17], [18]. Taken together, this is why it is now widely assumed that CQ acts primarily on the trophozoite stage of intraerythrocytic parasites, and that this is the stage wherein the CQR mechanism must operate. Indeed, mutant PfCRT causes CQR, directly binds CQ [19] and is postulated to be a transporter that either directly or indirectly alters CQ–heme interaction within the DV during the trophozoite stage [19], [20].

Further testing of this model and possibly others would benefit from additional close inspection of the stage specificity of CQ effects, using recently perfected imaging tools. Only a handful of studies report on the stage-specificity of CQ and related aminoquinolines, and many questions remain. Early on Peters demonstrated that CQ is not active against liver stages of infection, but is active vs. the erythrocytic stages that actively degrade Hb [21]. While describing gametocytogenesis, Smalley et al. found that early gametocytes are sensitive to CQ along with trophozoites that are actively digesting Hb [22]. Slater later summarized these results and others, noting that “… early rings, late schizonts and late gametocytes are innately resistant to concentrations of CQ that would kill trophozoites and rings on the border of trophozoite stage …” [23].

However, Zhang et al. [24] performed a detailed study on stage-specificity of CQ and reported that ring stages are the most sensitive, a conclusion also consistent with the work of Orjih [25], but in direct contrast to other studies by Yayon et al. [26] as well as others summarized in [23]. Interestingly, Zhang et al. also proposed that the effect of CQ on trophozoites and schizonts is not seen until the cycle following exposure to drug (after reinvasion and development of new rings; a phenomenon referred to as “delayed death”), a conclusion also reached by Krishna and co-workers [27]. Perhaps disagreement in the literature regarding stage specific CQ toxicity is due in part to measurement of survival at different endpoints (wherein different degrees of delayed death have occurred). In any case, collectively, these studies typically only report data for one parasite strain, do not quantify precise IC50 values for various dose schedules (i.e. continuous vs. bolus pulse for various time), and were performed well before our current level of molecular understanding of CQR and so do not compare CQS vs. CQR parasites.

In this study we investigate the stage specificity of CQ in detail using recently developed imaging methods [28], [29] to measure parameters that are central to various models for toxicity, but that have not previously been measurable for live parasites. Our results are in agreement with previous observations that the effect of CQ is both schedule and dose-dependent, but also clearly show that ring stage parasites are affected by CQ long before the mature DV is formed, and that schizont stage parasites are similarly sensitive to CQ long after Hz formation has plateaued. In addition, quite surprisingly, we find that the CQR mechanism mediated by mutant PfCRT is equally active in all stages of intraerythrocytic growth. Our results are consistent with the Hz crystallization process being an important, but likely not the only, target of CQ. We propose that both CQ toxicity and the CQR mechanism controlled by mutant PfCRT involve multiple stage-specific molecular pathways.

Section snippets

Materials

Fresh stocks of P. falciparum strains Dd2, HB3 and GCO3 were obtained from the Malaria Research and Reference Reagent Resource Center (MR4). The allelic exchange lines C4Dd2 and C2GCO3 were kindly provided by Dr. David Fidock (Albert Einstein College of Medicine, Bronx, NY). Custom 5% O2/5% CO2/90% N2 gas blends were purchased from Robert's Oxygen (Rockville, MD). Off-the-clot, heat-inactivated, pooled O+ human serum and pooled O+ human whole blood were purchased from Biochemed Services

Results

As described above, relatively few studies have probed the stage specificity of CQ toxicity. These report contrasting results, do not compute bolus dose IC50 and do not quantitatively compare CQS vs. CQR parasites. Data below are obtained using improved synchronization techniques, well characterized CQS vs. CQR parasites, and novel imaging methods for quantifying cellular effects of CQ.

Fig. 1 presents survival (parasitemia for drug treated/vehicle treated cultures) at 63 h vs. absolute dose of

Discussion

Relatively few studies have quantified the stage specificity of CQ action, and none have directly compared CQS vs. CQR parasites to test whether drug resistance is stage specific. In this study, for the first time to our knowledge, precise CQ IC50 vs. consistent stage-specific dosing schedules were quantified for both laboratory and transfectant model CQR vs. CQS malarial parasites. This is necessary if effects dependent on the dosing schedule are to be compared for multiple strains with

Acknowledgements

We thank Drs. Jeff Urbach and Ryan McAllister (Georgetown University) for helpful conversations and help with SDCM measurements, and Dr. David Fidock (Columbia University) for transfectants. This paper is dedicated to the memory of Dr. J. Dvorak.

References (55)

  • C.J. Janse et al.

    DNA synthesis in Plasmodium berghei during asexual and sexual development

    Mol Biochem Parasitol

    (1986)
  • B. Gligorijevic et al.

    Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria

    Biochemistry

    (2006)
  • J.L. Irvin et al.

    The interaction of antimalarials with nucleic acids

    Science

    (1949)
  • S.N. Cohen et al.

    Inhibition of DNA and RNA polymerase reactions by chloroquine

    Proc Natl Acad Sci USA

    (1965)
  • J. Ciak et al.

    Chloroquine: mode of action

    Science

    (1966)
  • R.L. O’Brien et al.

    Reactions of quinine, chloroquine, and quinacrine with DNA and their effects on the DNA and RNA polymerase reactions

    Proc Natl Acad Sci USA

    (1966)
  • J.L. Allison et al.

    DNA: reaction with chloroquine

    Science

    (1965)
  • C.D. Fitch

    Chloroquine resistance in malaria: a deficiency of chloroquine binding

    Proc Natl Acad Sci USA

    (1969)
  • M. Aikawa

    High-resolution autoradiography of malarial parasites treated with 3 H-chloroquine

    Am J Pathol

    (1972)
  • A. Yayon et al.

    Identification of the acidic compartment of Plasmodium falciparum-infected human erythrocytes as the target of the antimalarial drug chloroquine

    EMBO J

    (1984)
  • A. Leed et al.

    Solution structures of antimalarial drug–heme complexes

    Biochemistry

    (2002)
  • A.C. de Dios et al.

    Structure of the amodiaquine-FPIX mu oxo dimer solution complex at atomic resolution

    Inorg Chem

    (2004)
  • D.J. Sullivan et al.

    On the molecular mechanism of chloroquine's antimalarial action

    Proc Natl Acad Sci USA

    (1996)
  • D.E. Goldberg

    Hemoglobin degradation

    Curr Top Microbiol Immunol

    (2005)
  • H. Zhang et al.

    The antimalarial drug resistance protein Plasmodium falciparum chloroquine resistance transporter binds chloroquine

    Biochemistry

    (2004)
  • P.G. Bray et al.

    PfCRT and the trans-vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX

    Mol Microbiol

    (2006)
  • W. Peters

    The chemotherapy of rodent malaria. X. Dynamics of drug resistance. II. Acquisition and loss of chloroquine resistance in Plasmodium berghei observed by continuous bioassay

    Ann Trop Med Parasitol

    (1970)
  • Cited by (45)

    • Chloroquine analogs as antimalarial candidates with potent in vitro and in vivo activity

      2018, International Journal for Parasitology: Drugs and Drug Resistance
    • To kill or not to kill, that is the question: Cytocidal antimalarial drug resistance

      2014, Trends in Parasitology
      Citation Excerpt :

      Meaning, effects of CQ seen at concentrations closer to μM peak plasma [CQ]. For example, CQ DNA intercalation [38], particularly potent induction of oxidative damage [39], accumulation of odd Hb-containing vesicles [40], altered multiplicity of schizogony [41], and changes in additional important proteins for CQR parasites (e.g., glutathione [42] and PfATG8 [28]). These phenomena have been seen in a variety of experiments but are often not emphasized with respect to CQR or CQ pharmacology, presumably because they do not necessarily correlate with CQ IC50, DV CQ accumulation, or PfCRT status.

    View all citing articles on Scopus

    Supported by NIH grants AI045957 and AI052312 to P.D.R.

    ☆☆

    Quicktime movies that rotate 3D data sets of the SYBR Green I labeled schizonts on the x, y, and z axes are available as supplemental data from the authors.

    View full text