Malaria 2014: Advances in Pathophysiology, Biology, and Drug Development

Journal Articles

Amaratunga C, Witkowski B, Khim N, et al. Artemisinin resistance in Plasmodium falciparum. Lancet Infect Dis. 2014;14(6):449-50.

Ariey F, Witkowski B, Amaratunga C, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2014;505(7481):50-5.

Carlton JM, Adams JH, Silva JC, et al. Comparative genomics of the neglected human malaria parasite Plasmodium vivax. Nature. 2008;455:757-63.

Carlton JM, Das A, Escalante AA. Genomics, population genetics and evolutionary history of Plasmodium vivax. Adv Parasitol. 2013;81:203-22.

Carlton JM, Escalante AA, Neafsey D, et al. Comparative evolutionary genomics of human malaria parasites. Trends Parasitol. 2008;24(12):545-50.

Cassera MB, Hazleton KZ, Riegelhaupt PM, et al. Erythrocytic adenosine monophosphate as an alternative purine source in Plasmodium falciparum. J Biol Chem. 2008;283(47):32889-99.

Cui L, Fan Q, Li J. The malaria parasite Plasmodium falciparum encodes members of the Puf RNA-binding protein family with conserved RNA binding activity. Nucleic Acids Res. 2002;30(21):4607-17.

Dai M, Freeman B, Bruno FP, et al. The novel ETA receptor antagonist HJP-272 prevents cerebral microvascular hemorrhage in cerebral malaria and synergistically improves survival in combination with an artemisinin derivative. Life Sci. 2012;91(13-14):687-92.

Deponte M, Hoppe HC, Lee MC, et al. Wherever I may roam: protein and membrane trafficking in P. falciparum-infected red blood cells. Mol Biochem Parasitol. 2012;186(2):95-116.

Desruisseaux MS, Gulinello M, Smith DN, et al. Cognitive dysfunction in mice infected with Plasmodium berghei strain ANKA. J Infect Dis. 2008;197(11):1621-7.

Dorovini-Zis K, Schmidt K, Huynh H, et al. The neuropathology of fatal cerebral malaria in malawian children. Am J Pathol. 2011;178(5):2146-58.

Ejigiri I, Ragheb DR, Pino P, et al. Shedding of TRAP by a rhomboid protease from the malaria sporozoite surface is essential for gliding motility and sporozoite infectivity. PLoS Pathog. 2012;8(7):e1002725.

Ejigiri I, Sinnis P. Plasmodium sporozoite-host interactions from the dermis to the hepatocyte. Curr Opin Microbiol. 2009;12(4):401-7.

Fan Q, Li J, Kariuki M, et al. Characterization of PfPuf2, member of the Puf family RNA-binding proteins from the malaria parasite Plasmodium falciparum. DNA Cell Biol. 2004;23(11):753-60.

Flegg JA, Guerin PJ, Nosten F, et al. Optimal sampling designs for estimation of Plasmodium falciparum clearance rates in patients treated with artemisinin derivatives. Malar J. 2013;12:411.

Frame IJ, Merino EF, Schramm VL, et al. Malaria parasite type 4 equilibrative nucleoside transporters (ENT4) are purine transporters with distinct substrate specificity. Biochem J. 2012;446(2):179-90.

Frank M, Kirkman L, Costantini D, et al. Frequent recombination events generate diversity within the multi-copy variant antigen gene families of Plasmodium falciparum. Int J Parasitol. 2008;38(10):1099-109.

Freeman BD, Machado FS, Tanowitz HB, et al. Endothelin-1 and its role in the pathogenesis of infectious diseases. Life Sci. 2014. [Epub ahead of print]

Frosch AE, Laufer MK, Mathanga DP, et al. Return of widespread chloroquine-sensitive Plasmodium falciparum to Malawi. J Infect Dis. 2014. [Epub ahead of print]

Frosch AE, Venkatesan M, Laufer MK. Patterns of chloroquine use and resistance in sub-Saharan Africa: a systematic review of household survey and molecular data. Malar J. 2011;10:116.

Heinberg A, Siu E, Stern C, et al. Direct evidence for the adaptive role of copy number variation on antifolate susceptibility in Plasmodium falciparum. Mol Microbiol. 2013;88(4):702-12.

Jebiwott S, Govindaswamy K, Mbugua A, et al. Plasmodium berghei calcium dependent protein kinase 1 is not required for host cell invasion. PLoS One. 2013;8(11):e79171.

Kirkman LA, Deitsch KW. Antigenic variation and the generation of diversity in malaria parasites. Curr Opin Microbiol. 2012;15(4):456-62.

Kirkman LA, Lawrence EA, Deitsch KW. Malaria parasites utilize both homologous recombination and alternative end joining pathways to maintain genome integrity. Nucleic Acids Res. 2014;42(1):370-9.

Kumpornsin K, Modchang C, Heinberg A, et al. Origin of robustness in generating drug-resistant malaria parasites. Mol Biol Evol. 2014. [Epub ahead of print]

Lacroix C, Giovannini D, Combe A, et al. FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei. Nat Protoc. 2011;6(9):1412-28.

Laufer MK, Thesing PC, Dzinjalamala FK, et al. A longitudinal trial comparing chloroquine as monotherapy or in combination with artesunate, azithromycin or atovaquone-proguanil to treat malaria. PLoS One. 2012;7(8):e42284.

Laufer MK, Thesing PC, Eddington ND, et al. Return of chloroquine antimalarial efficacy in Malawi. N Engl J Med. 2006;355(19):1959-66.

Laufer MK, van Oosterhout JJ, Thesing PC, et al. Impact of HIV-associated immunosuppression on malaria infection and disease in Malawi. J Infect Dis. 2006;193(6):872-8.

Lim P, Dek D, Try V, et al. Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in western, northern, and eastern Cambodia, 2011-2012: association with molecular markers. Antimicrob Agents Chemother. 2013;57(11):5277-83.

Machado FS, Desruisseaux MS, Nagajyothi, et al. Endothelin in a murine model of cerebral malaria. Exp Biol Med (Maywood). 2006;231(6):1176-81.

malERA Consultative Group on Basic Science and Enabling Technologies. A research agenda for malaria eradication: basic science and enabling technologies. PLoS Med. 2011;8(1):e1000399.

Mathanga DP, Walker ED, Wilson ML, et al. Malaria control in Malawi: current status and directions for the future. Acta Trop. 2012;121(3):212-7.

McNamara CW, Lee MC, Lim CS, et al. Targeting Plasmodium PI(4)K to eliminate malaria. Nature. 2013;504(7479):248-53.

Miao J, Fan Q, Parker D, et al. Puf mediates translation repression of transmission-blocking vaccine candidates in malaria parasites. PLoS Pathog. 2013;9(4):e1003268.

Miao J, Li J, Fan Q, et al. The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum. J Cell Sci. 2010;123(Pt 7):1039-49.

Miotto O, Almagro-Garcia J, Manske M, et al. Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia. Nat Genet. 2013;45(6):648-55.

Neafsey DE, Galinsky K, Jiang RH, et al. The malaria parasite Plasmodium vivax exhibits greater genetic diversity than Plasmodium falciparum. Nat Genet. 2012;44(9):1046-50.

Panchal D, Bhanot P. Activity of a trisubstituted pyrrole in inhibiting sporozoite invasion and blocking malaria infection. Antimicrob Agents Chemother. 2010;54(10):4269-74.

Panchal D, Govindasamy K, Rana A, et al. Improved Plasmodium berghei lines for conditional mutagenesis. Mol Biochem Parasitol. 2012;184(1):52-4.

Potchen MJ, Kampondeni SD, Seydel KB, et al. Acute brain MRI findings in 120 Malawian children with cerebral malaria: new insights into an ancient disease. AJNR Am J Neuroradiol. 2012;33(9):1740-6.

Preston MD, Assefa SA, Ocholla H, et al. PlasmoView: A Web-based resource to visualise global Plasmodium falciparum genomic variation. J Infect Dis. 2014;209(11):1808-15.

Riegelhaupt PM, Cassera MB, Frohlich RF, et al. Transport of purines and purine salvage pathway inhibitors by the Plasmodium falciparum equilibrative nucleoside transporter PfENT1. Mol Biochem Parasitol. 2010;169(1):40-9.

Riegelhaupt PM, Frame IJ, Akabas MH. Transmembrane segment 11 appears to line the purine permeation pathway of the Plasmodium falciparum equilibrative nucleoside transporter 1 (PfENT1). J Biol Chem. 2010;285(22):17001-10.

Seydel KB, Fox LL, Glover SJ, et al. Plasma concentrations of parasite histidine-rich protein 2 distinguish between retinopathy-positive and retinopathy-negative cerebral malaria in Malawian children. J Infect Dis. 2012;206(3):309-18.

Shaukat AM, Gilliams EA, Kenefic LJ, et al. Clinical manifestations of new versus recrudescent malaria infections following anti-malarial drug treatment. Malar J. 2012;11:207.

Sheahan T, Jones CT, Ploss A. Advances and challenges in studying hepatitis C virus in its native environment. Expert Rev Gastroenterol Hepatol. 2010;4(5):541-50.

Silva JC, Egan A, Friedman R, et al. Genome sequences reveal divergence times of malaria parasite lineages. Parasitology. 2011;138(13):1737-49.

Sinnis P, De La Vega P, Coppi A, et al. Quantification of sporozoite invasion, migration, and development by microscopy and flow cytometry. Methods Mol Biol. 2013;923:385-400.

Sinnis P, Zavala F. The skin: where malaria infection and the host immune response begin. Semin Immunopathol. 2012;34(6):787-92.

Tachibana S, Sullivan SA, Kawai S, et al. Plasmodium cynomolgi genome sequences provide insight into Plasmodium vivax and the monkey malaria clade. Nat Genet. 2012;44(9):1051-5.

Towle T, Chang I, Kerns RJ, et al. Chemical probes of a trisubstituted pyrrole to identify its protein target(s) in Plasmodium sporozoites. Bioorg Med Chem Lett. 2013;23(6):1874-7.

Vaughan AM, Kappe SH, Ploss A, et al. Development of humanized mouse models to study human malaria parasite infection. Future Microbiol. 2012;7(5):657-65.

Vaughan AM, Mikolajczak SA, Wilson EM, et al. Complete Plasmodium falciparum liver-stage development in liver-chimeric mice. J Clin Invest. 2012;122(10):3618-28.

Voza T, Miller JL, Kappe SH, et al. Extrahepatic exoerythrocytic forms of rodent malaria parasites at the site of inoculation: clearance after immunization, susceptibility to primaquine, and contribution to blood-stage infection. Infect Immun. 2012;80(6):2158-64.

Whitten R, Milner DA Jr., Yeh MM, et al. Liver pathology in Malawian children with fatal encephalopathy. Hum Pathol. 2011;42(9):1230-9.

Witkowski B, Amaratunga C, Khim N, et al. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in vitro and ex vivo drug-response studies. Lancet Infect Dis. 2013;13(12):1043-9.

Woodrow CJ, Dahlstrom S, Cooksey R, et al. High-throughput analysis of antimalarial susceptibility data by the WorldWide Antimalarial Resistance Network (WWARN) in vitro analysis and reporting tool. Antimicrob Agents Chemother. 2013;57(7):3121-30.

Websites

Blantyre Malaria Project
University of Malawi College of Medicine malaria center that fosters collaborations with U.S. researchers.

Cellular and Molecular Biology of Plasmodium
Educational page developed and maintained by Mark Wiser, a malaria researcher at Tulane University.

Center for the Study of Complex Malaria in India
Collaboration between scientists at New York University and the National Institute of Malaria Research of India, aimed at addressing major gaps in understanding of malaria in India.

Centers for Disease Control and Prevention Malaria homepage
Comprehensive source of information on malaria, including statistics, prevention, and treatment.

National Institute of Allergy and Infectious Diseases (NIAID)
U.S. national research on understanding, treatment, and prevention of infectious, immunologic, and allergic diseases.

NIAID Malaria Research Program
Malaria research priorities for NIAID.

Roll Back Malaria
Global framework to facilitate coordinated action against malaria, comprised of more than 500 partners, including malaria endemic countries, their bilateral and multilateral development partners, the private sector, non-governmental and community-based organizations, foundations, and research and academic institutions.

Worldwide Antimalarial Resistance Network
Interactive maps and other resources for tracking the emergence and spread of resistance to antimalarial drugs.

Sponsors

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According to the latest World Health Organization (WHO) report, there were 207 million cases of malaria in 2012, resulting in 627 000 deaths, 77% among children under the age of five. These figures represent an improvement: as a result of programs such as the WHO's Roll Back Malaria initiative, both malaria incidence and mortality have decreased by 25% since 2000. However, 3.4 billion people, over half the world's population, remain at risk of morbidity and mortality from malaria and its complications.

Malaria is caused by intracellular parasites belonging to the genus Plasmodium. In sub-Saharan Africa, where the malaria disease burden is highest, most cases are caused by Plasmodium falciparum. In other regions where malaria is endemic, including Southeast Asia and South America, Plasmodium vivax is more prevalent. Over the years, P. falciparum has been the most intense subject of research, but P. vivax is gaining greater attention as its prevalence has increased over the past few decades. On World Malaria Day 2014, researchers gathered at the New York Academy of Sciences to present their latest findings on malaria epidemiology, genetics, and clinical manifestations, as well as to discuss treatment options to eradicate these parasites.

The malaria parasite carries out a complex life cycle as it is transmitted from mosquito to human host and back again. The parasite is injected into the human host from an infected mosquito in a form known as the sporozoite, which travels from the mosquito's salivary glands into the human skin and then through the bloodstream to reach its target, the liver. In the liver, sporozoites invade liver cells and differentiate into another form called the schizont. The number and types of liver stages vary among malaria species: some types, including P. vivax but not P. falciparum, can produce a dormant form known as the hypnozoite that resides in the liver for months before reappearing as an active infection. This characteristic creates a hidden reservoir of disease that can be difficult to detect and eradicate.

Plasmodium vivax life cycle. (Image courtesy of Alexander Ploss)

After the liver stages are completed, the schizont ruptures and releases another form, the merozoite, back into the bloodstream. Merozoites invade red blood cells and form more schizonts, which again rupture to release merozoites. These blood-stage forms are responsible for the uncomfortable symptoms and negative clinical consequences of malaria. Some merozoites differentiate into gametocytes, which are taken up when the infected host is bitten by another mosquito. In the mosquito, the gametocytes undergo further transformation and make their way from the mosquito's midgut back into the salivary glands, forming sporozoites that can be injected into another host and completing the cycle.

The conference included speakers whose work shed light on many stages of the parasite's life cycle and ranged from clinical to genetic to epidemiologic. Terrie E. Taylor and Mahalia S. Desruisseaux discussed fatal cerebral malaria, a rare and severe complication that kills mostly children in sub-Saharan Africa. Jane M. Carlton described the genetic diversity of P. vivax, highlighting the challenges likely to be involved in eradicating this pathogen. Liwang Cui discussed the genetic mechanisms that allow malaria parasites to lie dormant and then quickly transition to their next developmental stage. Photini Sinnis and Purnima Bhanot described the biochemical and physiological mechanisms used by malaria parasites to move from skin to blood to liver stages.

The WHO's goal of reducing and eventually eradicating the malaria parasite requires preventive methods as well as effective antimalarial drugs. Several speakers described their work to discover novel drug targets, to develop new experimental models for testing drug candidates, and to identify parasite resistance to current drugs. Rick M. Fairhurst discussed the epidemiology of artemisinin resistance in Southeast Asia, including the development and use of a new molecular marker. Laura Kirkman and Miriam K. Laufer respectively described the mechanisms and epidemiology of antifolate and chloroquine resistance. Promising new therapeutic targets were described by Marcus Lee and Myles Akabas. Alexander Ploss rounded out the discussion of drug development by presenting his work developing chimeric human–mouse models for malaria parasite liver stages, which are potential systems for antimalarial drug discovery and testing.

Keynote Speaker:
Terrie E. Taylor, Michigan State University
 
Speaker:
Mahalia S. Desruisseaux, Albert Einstein College of Medicine

Highlights

• New diagnostic techniques, including the use of magnetic resonance imaging, are improving management of children with cerebral malaria.
• Increased activity of endothelin-1, a vasoactive peptide, appears to be at the root of mortality and long-term neurological deficits among children who develop cerebral malaria.

A rare but severe malaria complication

A small proportion, about 0.1%–1%, of children who contract malaria in endemic regions such as sub-Saharan Africa develop cerebral malaria, a severe complication with a fatality rate of 15%–25%. Terrie E. Taylor of Michigan State University described her work to characterize and treat this deadly complication among young children in Malawi. In this area of Africa, the burden of disease of malaria falls largely on children between the ages of one and three. Children who develop cerebral malaria experience seizures and often become comatose. Death is usually related to respiratory arrest, although the exact process that leads to death in these children has until recently been somewhat of a mystery.

Over the past 30 years, Taylor and her colleagues have characterized hundreds of cases of cerebral malaria, studying clinical signs and symptoms and performing autopsies on children who die. The previous standard case definition of cerebral malaria, developed by the WHO, led to substantial misclassification of patients, such that one-quarter to one-third who were included in the cerebral malaria caseload did not have this complication. In response, the group developed a new case definition that relies on several novel parameters, including a group of ophthalmological abnormalities known collectively as malarial retinopathy, and rapid increases in brain volume (brain swelling) that can be visualized with magnetic resonance imaging (MRI).

MR imaging showed that 37% of children who experienced brain swelling died, compared to only 4% of those without swelling. Brain swelling increases intracranial pressure, pushing down on and impairing the function of the brain stem, which may account for the respiratory arrest observed in children who die. Children who survive cerebral malaria tend to experience reductions in brain volume, suggesting that therapeutic interventions that reduce brain swelling could improve mortality rates. Taylor's group is now working to identify the mechanisms by which malarial infection leads to brain swelling. Their diagnostic techniques, including the use of MRI, can also be used to identify children with cerebral malaria who are at a higher risk of dying, which may allow hospital personnel to manage these patients differently to reduce that risk.

Toward an understanding of molecular mechanisms

Mahalia S. Desruisseaux of the Albert Einstein College of Medicine is using an experimental mouse model to better understand the pathophysiological mechanisms of cerebral malaria. Thirty percent of children who survive cerebral malaria go on to experience neurological and cognitive deficits later in life, so in addition to mortality, her group is investigating the origins of these long-term effects. Deficits can include hearing and vision loss, memory and attention problems, and behavior and motor coordination difficulties.

Desruisseaux and her group have characterized specific adverse changes that occur in the brains of mice with experimentally induced cerebral malaria, including reduced cerebral blood flow, inflammation, leukocyte infiltration, disruption of the blood–brain barrier, and damage to neurons and glial cells. These mice also show deficits in behavioral testing that suggest problems with visual and working memory.

Potential roles for endothelin-1 (ET-1) in the pathophysiology of cerebral malaria. (Image courtesy of Mahalia S. Desruisseaux)

Many of the observed effects of cerebral malaria appear to be mediated by endothelin-1 (ET-1), a vasoactive peptide that is involved in inflammation, vascular tone, and control of blood–brain barrier permeability. ET-1 is increased in cerebral malaria both in humans and in experimental models. Desruisseaux's work in mice has shown that the axonal damage found in cerebral malaria may be related to changes in ET-1 signaling. Taken together, her findings suggest that both long-term cognitive deficits and mortality in children with cerebral malaria may be related to ET-1 levels. She plans to continue studying ET-1 in cerebral malaria to further elucidate the molecular mechanisms involved in these effects, with the goal of identifying targets for therapeutic intervention.

Keynote Speaker:
Jane M. Carlton, New York University
 
Speaker:
Liwang Cui, Pennsylvania State University

Highlights

• Genomic studies of P. vivax show a high degree of genetic diversity that may impede efforts to eradicate this pathogen.
• Puf proteins, a family of evolutionarily conserved translational regulators, are master regulators of the transition from human host to mosquito.

Getting a handle on P. vivax diversity

Jane M. Carlton of New York University is using advanced genomic studies to investigate the differences between P. falciparum and P. vivax, as well as variation in P. vivax isolates from different countries. P. vivax is the more challenging parasite to study because P. vivax infections involve much lower levels of parasite and P. vivax can form the dormant hypnozoite stage that lies hidden in the liver.

Carlton and her group completed the first genome sequence for P. vivax in 2008, using a strain isolated in El Salvador. Analysis of this sequence showed high diversity in many gene families involved in virulence, including a complicated set of genes needed for the invasion of red blood cells. Carlton has since analyzed and compared P. vivax genomes from Brazil, Mauritania, India, and North Korea, finding about twice as much genetic diversity as is observed among a comparable collection of P. falciparum isolates. The high genetic diversity and large degree of variability among P. vivax gene families suggest that this parasite is highly adaptable and likely to be even more difficult to eradicate than previously suspected.

Mapping the genetic diversity of P. vivax. (Image courtesy of Jane M. Carlton)

Carlton has also sequenced the genomes of several strains of Plasmodium cynomolgi, a malaria parasite that infects monkeys and is closely related to P. vivax. Studies in P. cynomolgi will help researchers develop experimental models of malaria and provide information about the evolutionary history of the many forms of malaria parasites and their interactions with human and monkey hosts. Her group has also begun to sequence 181 additional P. vivax patient isolates from around the world in collaboration with the Broad Institute. Carlton is the principal investigator of the Center for the Study of Complex Malaria in India, which will use next-generation sequencing methods to study genetic determinants of antimalarial drug resistance. The molecular insights gained from these studies will enhance public health efforts to detect, treat, and eradicate malaria in countries where P. vivax is endemic.

Rapid developmental transitions

During their life cycle, malaria parasites undergo periods of quiescence followed by quick developmental transitions to various life-cycle forms. These transitions occur too rapidly to allow time for gene transcription and are instead controlled at the translational level by the activation of large storage depots of mRNA. To function properly, this stored mRNA must be located in specific areas of the cell and protected from premature translation. Liwang Cui of Pennsylvania State University is studying the mechanisms of spatial and temporal regulation of mRNA translation in the P. falciparum gametocyte, the form that is transmitted from humans back to mosquitoes.

Cui and his group have focused on Puf proteins, a family of evolutionarily conserved translational regulators found in many eukaryotes. P. falciparum has two Puf proteins, PfPuf1 and PfPuf2, that share 25% homology in their RNA binding domains. Many translationally regulated genes have been identified in P. falciparum, including Pfs25 and Pfs28, which encode membrane surface proteins involved in adherence of the gametocyte to the mosquito gut wall. Cui's work shows that expression of these proteins, considered prime candidates as vaccine targets to block human-to-mosquito transmission, is regulated at the translational level by PfPuf2. A better understanding of this regulation could lead to the development of therapies that block expression of these proteins, thus blocking gut adherence and preventing transmission.

Cui's work on PfPuf1 and PfPuf2 sheds light on the mechanisms of translational control in P. falciparum, suggesting these proteins are important master regulators of a large number of proteins involved in the parasite's transition from human host to mosquito vector. His work also shows PfPuf2 regulates sexual development and sex differentiation in P. falciparum. Future work will elucidate the molecular details of the mechanisms by which the malaria parasite makes its transition from human-infecting to mosquito-infecting forms.

Speakers:
Photini Sinnis, Johns Hopkins University
Purnima Bhanot, University of Medicine and Dentistry of New Jersey

Highlights

• Malaria parasite transition from the skin to the bloodstream after a mosquito bite represents a life-cycle bottleneck that may be a useful therapeutic target.
• Agents that target cGMP-dependent protein kinase show promise as antimalarials, acting at the transition from human bloodstream to liver and at later developmental stages within the liver.

Traveling from skin to bloodstream

Photini Sinnis of Johns Hopkins University is using in vivo studies and rodent models to elucidate the molecular details of mosquito-to-mammal transmission. In particular, her group has identified exit from the skin into the bloodstream as an important bottleneck in the infection process, thus representing a useful target for therapeutic intervention.

Tracking the movements of sporozoites (small bright green objects) in the skin of the mouse ear (purple objects are blood vessels). (Image courtesy of Photini Sinnis)

Sinnis has developed a method for visualizing sporozoite migration in the ears of infected mice, using the rodent malaria parasite Plasmodium berghei. Videos capture the speed and displacement patterns of sporozoites as they move away from the site of injection. Mutant sporozoites that move more slowly than wild-type are used to identify proteins involved in migration from skin to bloodstream. A transmembrane protein called TRAP, with extracellular adhesive domains and a cytoplasmic tail that is linked to the actomyosin motor, appears to be central to the unique gliding motility exhibited by sporozoites. Sinnis is studying the function and regulation of this protein.

Sinnis is also investigating host responses to malaria parasite injection in mosquito-to-human transmission. Neutrophils are recruited into the site of injection after bites from both infected and uninfected mosquitoes, but neutrophil infiltration does not appear to affect infectivity of the P. berghei sporozoite. Instead, her studies suggest sporozoites avoid destruction by exiting the skin before neutrophil infiltration. Because only a small number of parasites are injected with each bite, and only a small percentage of those parasites reach the bloodstream, the injection and migration process represents a potential target for vaccines that could disrupt the parasite's life cycle and prevent mosquito-to-human transmission.

From bloodstream to liver, and beyond

Liver stages, also known as pre-erythrocytic stages, are necessary for the parasite to establish a niche in the human host, and thus represent another opportunity for intervention. Reducing the parasite burden during early stages reduces the severity of disease. Disruption of the life cycle at liver stages could be particularly useful in combating P. vivax infection if the treatment could prevent the formation of hypnozoites, the dormant liver stage that forms a cryptic disease reservoir for this species.

Purnima Bhanot of the University of Medicine and Dentistry of New Jersey is studying the mechanisms by which sporozoites infect liver cells, develop into the trophozoite form, and infect red blood cells. As part of this work, Bhanot and her group are studying the effects of a tri-substituted pyrolle (TSP) compound that is a potent inhibitor of both sporozoite infection of liver cells and liver-stage development. TSP blocks sporozoite invasion of the liver, affects sporozoite motility, and reduces the number of liver-stage parasites that develop in tissue culture. In mice, treatment with TSP leads to significantly reduced parasite burden in the liver and in the blood at later stages of infection.

TSP blocks parasite entry into cultured liver cells as well as subsequent intracellular development. (Image courtesy of Purnima Bhanot)

TSP is also effective against the parasite Toxoplasma gondii, where it has been shown to target cGMP-dependent protein kinase (PKG). Bhanot is investigating the role of PKG in Plasmodium infection using conditional knockouts to control the parasite's expression of the kinase. Her studies show that cGMP signaling mediated by PKG plays a key role in the invasion of host cells by sporozoites, as well as in the parasite's development within the liver. Her team is working to identify the downstream targets of cGMP signaling as potential drug targets for intervention in malaria infection. This research has shown that TSP inhibits Plasmodium PKG in vitro, which taken together with TSP's effects in mice suggests that targeting PKG and its downstream targets will be a useful antimalarial strategy.

Keynote Speaker:
Rick M. Fairhurst, National Institute of Allergy and Infectious Diseases, NIH
 
Speakers:
Laura Kirkman, Weill Cornell Medical College
Miriam K. Laufer, University of Maryland School of Medicine

Highlights

• The epidemiology of artemisinin resistance can be studied more precisely since the identification of mutations in the Kelch13 propeller domain as markers of resistance.
• Antifolate resistance may be caused or supported by amplification of the gene that encodes GTP-cyclohydrolase 1, the first step in the folate biosynthetic pathway, as well as by N-terminal mutations in this enzyme.
• When chloroquine use is stopped, susceptible strains become more prevalent, suggesting that antimalarial drug resistance can be managed by more judicious use of available drugs.

Tracking and measuring the spread of resistance

Resistance to the original antimalarial drug chloroquine was first detected at the border between Cambodia and Thailand in the 1960s. Since then, chloroquine has been largely replaced as the front-line treatment for P. falciparum malaria by artemisinin (ART), a highly effective antimalarial drug with a number of related derivatives. In 2009 the first case of ART resistance was detected in Pailin Province in western Cambodia, and resistance has since spread to many other regions of Southeast Asia. These circumstances led to the current WHO recommendation that cases of P. falciparum malaria found anywhere in the world be treated with ART combination therapy (ACT) rather than ART alone to reduce the chances of creating resistance. ART and its derivatives, which are short-acting antimalarial drugs, should now be used in combination with longer-acting partner drugs with different mechanisms of action to ensure that the parasites are completely eliminated with treatment, leaving behind no drug-resistant survivors.

Rick M. Fairhurst of the National Institute of Allergy and Infectious Diseases, NIH, reported current efforts to understand and combat the spread of resistant malaria parasites. International research collaborations have rapidly increased knowledge of the epidemiology of ART resistance, defining and mapping its spread across Southeast Asia. Many of these studies were carried out by measuring slow parasite clearance rates in patients treated with ART or ACT as a marker of resistance, a method that is highly labor and resource intensive.

Recently, however, researchers have identified specific mutations in the propeller domain of the Kelch13 (K13) protein of P. falciparum as a marker of ART resistance in patients in Southeast Asia. K13 is a member of a widespread protein family characterized by the presence of beta-pleated sheets that form a propeller-like structure thought to mediate protein–protein interactions in diverse settings. Use of this molecular marker has already begun to simplify and improve the accuracy of epidemiological studies of ART resistance, by providing a more direct measure of resistant infections.

Fairhurst said future work will investigate whether K13 propeller mutations are the cause of ART resistance and, if so, study the mechanism. The ACT combination dihydroartemisinin–piperaquine (DHA–PPQ) has also begun to fail in Southeast Asia, and Fairhurst is involved in efforts to map and better understand this type of resistance as well. Such studies are crucial steps toward eliminating ART-resistant Plasmodium species from Southeast Asia and preventing their spread to the rest of the world.

The K13 propeller domain. (Image courtesy of Rick M. Fairhurst)

Understanding resistance to antifolates

Many important antimalarial drugs, including pyrimethamine, cycloguanil, and trimethoprim, target the folate biosynthesis pathway, a series of metabolic enzymes that plays an important role in malaria parasite survival. Resistance to these drugs, collectively known as antifolates, has been linked to the presence of point mutations in dihydropteroate synthase (DHPS) and dihydrofolate synthase (DHFS), two enzymes that carry out later steps in the folate synthetic pathway. Laura Kirkman of Weill Cornell Medical College is studying additional genetic changes in this pathway that also appear to contribute to the rise and spread of antifolate resistance.

Kirkman is studying copy number amplification of the gene that encodes the first enzyme in the folate biosynthetic pathway, GTP-cyclohydrolase 1 (GCH1). She and her colleagues have found extensive copy number variation in this gene in malaria parasites isolated from areas with a long history of antifolate use. Although the effect is not entirely linear, the presence of extra copies of the gene leads to increased levels of GCH1 protein, which as the first and rate-limiting enzyme in the pathway allows for increased folate production, helping the parasite overcome antifolate inhibition of later steps in the pathway. Kirkman's studies have shown that increased GCH1 expression increases resistance to pyrimethamine in Plasmodium strains that are fully sensitive to antifolates and in strains that already have low-level resistance conferred by other mutations.

The folate biosynthetic pathway with points of genetic variation that contribute to antifolate resistance. (Image courtesy of Laura Kirkman)

The catalytic region of GCH1 is well conserved from bacteria through mammals, but the amino terminus of this protein varies considerably. The amino terminus of Plasmodium GCH1 is particularly lengthy, suggesting that it may play an important regulatory role. In further studies, Kirkman and her group are investigating the functions of this part of the protein and whether it may be involved in creating or modulating antifolate resistance.

Taking the long view of chloroquine resistance

Historically, resistance to antimalarial drugs has emerged in Southeast Asia and then spread to Africa. However, because malaria transmission is higher in Africa, antimalarial drug resistance poses a larger burden there in terms of increased disease severity and death. Miriam K. Laufer of the University of Maryland School of Medicine is studying human, vector, and parasite factors that contribute to antimalarial drug resistance.

Laufer and her coworkers are studying the molecular epidemiology, genetics, and clinical implications of drug resistance in Malawi, one of the highest malaria transmission regions in sub-Saharan Africa. Chloroquine resistance was widespread in Malawi in the early 1990s, and in 1993 the country became the first in Africa to switch from chloroquine to sulfadoxine-pyrimethamine (SP) as first-line treatment. By 2003, molecular genetics studies showed the prevalence of chloroquine-resistant strains had decreased considerably, while SP-resistant strains had increased. In a randomized clinical trial in children with malaria, Laufer and her group found chloroquine had regained its efficacy while SP had become much less effective. These studies suggest that chloroquine-susceptible strains of malaria are likely to return to other parts of sub-Saharan Africa, particularly as artemisinin combination therapy is more widely used in its place.

Laufer is also studying the spread of SP resistance, including the role of intermittent preventive treatment with the SP combination in pregnancy and the widespread use of antifolates as antibacterial treatments in Malawi. These studies will offer insights into the future spread of ART resistance, which also first arose in Southeast Asia. Laufer noted that in the era of combination therapy, parasite resistance to the drug that is partnered with ART will become increasingly important, because it may allow ART-resistant parasites to escape treatment and spread. Her findings also suggest it will be important for countries in Africa to adopt more homogeneous antimalarial drug-use policies to minimize resistance and maximize the effectiveness of available treatments.

Speakers:
Marcus Lee, Columbia University
Myles Akabas, Albert Einstein College of Medicine
Alexander Ploss, Princeton University

Highlights

• Imidazopyrazines are new candidates for antimalarial drug development targeting phosphatidylinositol 4-kinase, an enzyme involved in intracellular signaling and protein trafficking pathways.
• New compounds that inhibit purine uptake via equilibrative nucleoside transporter 1 of Plasmodium are also in development as potential antimalarial drugs.
• Chimeric mice that harbor human liver cells are facilitating the study of parasite–host interactions in the liver and may be useful as drug development models.

Meeting the need for new therapeutic targets

The continued rise of resistance to antimalarial drugs highlights the importance of discovering new compounds that use different mechanisms from current drugs. Marcus Lee of Columbia University is investigating the mechanism of imidazopyrazines, a new class of antimalarial drug candidates that are particularly promising because they act across all stages of infection against multiple Plasmodium species, including P. falciparum and P. vivax. These compounds inhibit the growth of hypnozoites formed by P. cynomolgi in monkey liver, potentially reducing this cryptic, difficult-to-reach reservoir of infection.

Lee and his group used molecular genetics and biochemical methods to identify the molecular target of imidazopyrazines as phosphatidylinositol 4-kinase (PI4-kinase), a highly conserved enzyme found in eukaryotes that modifies lipid molecules as part of the regulation of intracellular signaling and protein trafficking. The group has determined that imidazopyrazines block the ATP-binding site of the enzyme, inhibiting parasite development at later life-cycle stages by preventing the formation of new membranes needed for developing daughter parasites. Molecular-level knowledge of these mechanisms will enhance the development of imidazopyrazines as drug candidates and may also lead to the identification of new targets in related signaling pathways.

Targeting purine uptake

Plasmodium species are purine auxotrophs: unable to synthesize these important components of nucleic acids, the parasites must import purines from the external environment to survive. Purine import is accomplished via a family of membrane proteins known as equilibrative nucleoside transporters (ENTs), which carry out the passive transport of a broad array of purines and other similar substrates. Myles Akabas and his coworkers at Albert Einstein College of Medicine are investigating potential antimalarial drugs that target this family of transporters.

Purine uptake and interconversion pathways in P. falciparum and red blood cells. (Image courtesy of Myles Akabas)

P. falciparum strains that lack the ENT1 subtype (PfENT1) are unable to survive in the purine concentrations found in human serum. This and other lines of evidence suggest that this particular transporter is the primary purine transporter in P. falciparum, and thus would be an effective target for inhibitory drugs. Akabas searched for inhibitors of PfENT1 using a novel yeast-based high-throughput system to screen about 64 000 compounds, ultimately identifying nine compounds with the ability to inhibit PfENT1 at relatively low concentrations. All nine compounds also inhibit the growth of P. falciparum in human red blood cells in culture. Ongoing studies will be needed to develop these candidate compounds into antimalarial drugs for use in humans.

New animal models of human malaria

Small animal models play important roles in testing drug candidates for use in humans. Although some Plasmodium species infect rodents and can be used to model human disease, these species are not genetically or biochemically identical to the human pathogens P. falciparum and P. vivax, limiting their predictive value. Alexander Ploss of Princeton University described his group's work to produce humanized mice that can be infected by P. falciparum. Such models can replicate important physiological aspects of parasite–host interaction that two-dimensional tissue culture systems are unable to recapitulate.

To create new mouse models, Ploss and his colleagues injected human liver cells into immunodeficient mice that had sustained a liver injury, resulting in human cell engraftment within the mouse liver. The chimeric mice thus produced can be infected with P. falciparum sporozoites, leading to faithful reproduction of human liver stages of parasite development in the mouse liver. Ploss is using transcriptomic and proteomic profiling to investigate the molecular aspects of liver-stage development in the infected human liver cells and in the parasite, as a means to better understand the physiological processes involved in liver-stage infection and development.

Use of chimeric mice for molecular characterization of plasmodial liver stages. (Image courtesy of Alexander Ploss)

Ploss is working to produce a similar rodent model for P. vivax infection, which would be particularly useful for understanding the development of the dormant hypnozoite stage. Another alternative for this type of work would be to make chimeric mice with liver cells from monkeys that can be infected with P. cynomolgi, which might be advantageous because this parasite is more readily available and has less genetic heterogeneity. In addition, P. cynomolgi is not a human pathogen, so fewer precautions need to be taken when working with it. In addition to uses in basic biological studies of the parasite's life cycle, chimeric human–mouse models could be used to test drug candidates and drug dosing schedules, to elucidate the mechanisms of antimalarial drug resistance, and to test antimalarial vaccines.