57
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Targeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.

          Abstract

          Resistance to artemisinin antimalarial drugs is jeopardizing malaria control. This study shows that proteasome-mediated stress responses can be targeted to overcome artemisinin resistance and suggests alternate therapeutic regimens and monitoring strategies.

          Author Summary

          Resistance to artemisinin antimalarials, some of the most effective antimalarial drugs, has emerged in Southeast Asia, jeopardizing malaria control. We have undertaken a detailed study of artemisinin-sensitive and-resistant strains of Plasmodium falciparum, the parasite responsible for malaria, taken directly from the field in a region where resistance is developing. We compared these strains to lab strains engineered with either mutant or wild-type resistance alleles. We demonstrate that in sensitive P. falciparum, artemisinin induces growth retardation and accumulation of ubiquitinated proteins, indicating that the drugs activate the cellular stress response. Resistant parasites, on the other hand, exhibit reduced protein ubiquitination and delayed onset of cell death following drug exposure. We show that proteasome inhibitors strongly synergize artemisinin activity, offering a means of overcoming artemisinin resistance. We have developed a detailed model of parasite responses and have modelled in vivo clearance profiles. Our data indicate that extending artemisinin treatment from the standard three-day treatment to a four-day treatment will clear resistant parasites, thus preserving the use of this critical therapy in areas experiencing artemisinin resistance.

          Related collections

          Most cited references47

          • Record: found
          • Abstract: found
          • Article: not found

          Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study

          Summary Background Artemisinin-resistant falciparum malaria has arisen in western Cambodia. A concerted international effort is underway to contain artemisinin-resistant Plasmodium falciparum, but containment strategies are dependent on whether resistance has emerged elsewhere. We aimed to establish whether artemisinin resistance has spread or emerged on the Thailand–Myanmar (Burma) border. Methods In malaria clinics located along the northwestern border of Thailand, we measured six hourly parasite counts in patients with uncomplicated hyperparasitaemic falciparum malaria (≥4% infected red blood cells) who had been given various oral artesunate-containing regimens since 2001. Parasite clearance half-lives were estimated and parasites were genotyped for 93 single nucleotide polymorphisms. Findings 3202 patients were studied between 2001 and 2010. Parasite clearance half-lives lengthened from a geometric mean of 2·6 h (95% CI 2·5–2·7) in 2001, to 3·7 h (3·6–3·8) in 2010, compared with a mean of 5·5 h (5·2–5·9) in 119 patients in western Cambodia measured between 2007 and 2010. The proportion of slow-clearing infections (half-life ≥6·2 h) increased from 0·6% in 2001, to 20% in 2010, compared with 42% in western Cambodia between 2007 and 2010. Of 1583 infections genotyped, 148 multilocus parasite genotypes were identified, each of which infected between two and 13 patients. The proportion of variation in parasite clearance attributable to parasite genetics increased from 30% between 2001 and 2004, to 66% between 2007 and 2010. Interpretation Genetically determined artemisinin resistance in P falciparum emerged along the Thailand–Myanmar border at least 8 years ago and has since increased substantially. At this rate of increase, resistance will reach rates reported in western Cambodia in 2–6 years. Funding The Wellcome Trust and National Institutes of Health.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study.

            Artemisinin-resistant Plasmodium falciparum has been reported in Pailin, western Cambodia, detected as a slow parasite clearance rate in vivo. Emergence of this phenotype in western Thailand and possibly elsewhere threatens to compromise the effectiveness of all artemisinin-based combination therapies. Parasite genetics is associated with parasite clearance rate but does not account for all variation. We investigated contributions of both parasite genetics and host factors to the artemisinin-resistance phenotype in Pursat, western Cambodia. Between June 19 and Nov 28, 2009, and June 26 and Dec 6, 2010, we enrolled patients aged 10 years or older with uncomplicated falciparum malaria, a density of asexual parasites of at least 10,000 per μL of whole blood, no symptoms or signs of severe malaria, no other cause of febrile illness, and no chronic illness. We gave participants 4 mg/kg artesunate at 0, 24, and 48 h, 15 mg/kg mefloquine at 72 h, and 10 mg/kg mefloquine at 96 h. We assessed parasite density on thick blood films every 6 h until undetectable. The parasite clearance half-life was calculated from the parasite clearance curve. We genotyped parasites with 18 microsatellite markers and patients for haemoglobin E, α-thalassaemia, and a mutation of G6PD, which encodes glucose-6-phosphate dehydrogenase. To account for the possible effects of acquired immunity on half-life, we used three surrogates for increased likelihood of exposure to P falciparum: age, sex, and place of residence. This study is registered with ClinicalTrials.gov, number NCT00341003. We assessed 3504 individuals from all six districts of Pursat province seeking treatment for malaria symptoms. We enrolled 168 patients with falciparum malaria who met inclusion criteria. The geometric mean half-life was 5·85 h (95% CI 5·54-6·18) in Pursat, similar to that reported in Pailin (p=0·109). We identified two genetically different parasite clone groups: parasite group 1 (PG1) and parasite group 2 (PG2). Non-significant increases in parasite clearance half-life were seen in patients with haemoglobin E (0·55 h; p=0·078), those of male sex (0·96 h; p=0·064), and in 2010 (0·68 h; p=0·068); PG1 was associated with a significant increase (0·79 h; p=0·033). The mean parasite heritability of half-life was 0·40 (SD 0·17). Heritable artemisinin resistance is established in a second Cambodian province. To accurately identify parasites that are intrinsically susceptible or resistant to artemisinins, future studies should explore the effect of erythrocyte polymorphisms and specific immune responses on half-life variation. Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Copyright © 2012 Elsevier Ltd. All rights reserved.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              A major genome region underlying artemisinin resistance in malaria.

              Evolving resistance to artemisinin-based compounds threatens to derail attempts to control malaria. Resistance has been confirmed in western Cambodia and has recently emerged in western Thailand, but is absent from neighboring Laos. Artemisinin resistance results in reduced parasite clearance rates (CRs) after treatment. We used a two-phase strategy to identify genome region(s) underlying this ongoing selective event. Geographical differentiation and haplotype structure at 6969 polymorphic single-nucleotide polymorphisms (SNPs) in 91 parasites from Cambodia, Thailand, and Laos identified 33 genome regions under strong selection. We screened SNPs and microsatellites within these regions in 715 parasites from Thailand, identifying a selective sweep on chromosome 13 that shows strong association (P = 10(-6) to 10(-12)) with slow CRs, illustrating the efficacy of targeted association for identifying the genetic basis of adaptive traits.
                Bookmark

                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS Biol
                PLoS Biol
                plos
                plosbiol
                PLoS Biology
                Public Library of Science (San Francisco, CA USA )
                1544-9173
                1545-7885
                22 April 2015
                April 2015
                : 13
                : 4
                : e1002132
                Affiliations
                [1 ]Department of Biochemistry and Molecular Biology and ARC Centre of Excellence for Coherent X-ray Science, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Victoria, Australia
                [2 ]John Curtin School of Medical Research, the Australian National University, Canberra, Australian Capital Territory, Australia
                [3 ]Australian School of Advanced Medicine, Macquarie University, Sydney, New South Wales, Australia
                [4 ]School of Biological Sciences, Nanyang Technological University, Singapore
                [5 ]Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, University of Melbourne, Parkville, Victoria, Australia
                [6 ]Murdoch Childrens Research Institute, Royal Childrens Hospital, Victoria, Australia
                [7 ]Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
                [8 ]Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, United States of America
                [9 ]Division of Infectious Diseases, Department of Medicine, Columbia University Medical Center, New York, New York, United States of America
                [10 ]Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, Oxford, United Kingdom
                Stanford University, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: CD SCX GB JB SM SF DAF NK LT. Performed the experiments: CD SCX GB JB SK NK. Analyzed the data: CD SCX GB JMM JB JAS SF NK LT. Contributed reagents/materials/analysis tools: KC NG JS AMD JMM JAS ZB DAF NK. Wrote the paper: CD SCX GB JMM KC JB AMD JAS SF DAF ZB NK LT.

                ‡ CD and SCX contributed equally to this work. NK and LT also contributed equally to this work.

                Article
                PBIOLOGY-D-14-03592
                10.1371/journal.pbio.1002132
                4406523
                25901609
                dc9a31e9-9ac2-4289-b41b-3aec9dcd50aa
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 13 October 2014
                : 16 March 2015
                Page count
                Figures: 8, Tables: 1, Pages: 26
                Funding
                This work was supported by the National Health & Medical Research Council of Australia (Grant#541904), the Australia Research Council (Grants: CE0561787 & DP110100624), the Singapore National Medical Council (NMRC/1265/2010), and National Institutes of Health, USA (R01 AI109023, to DAF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                All primary data are available by contacting the corresponding author. The Excel spreadsheet used for the modelling has been provided as Supporting Information. The entire genomes for the four Pailin strains have been made available through the European Nucleotide Archive (ENA) database under the accession number PRJEB8074.

                Life sciences
                Life sciences

                Comments

                Comment on this article