Malaria biology and disease pathogenesis: insights for new treatments

Abstract

Plasmodium falciparum malaria, an infectious disease caused by a parasitic protozoan, claims the lives of nearly a million children each year in Africa alone and is a top public health concern. Evidence is accumulating that resistance to artemisinin derivatives, the frontline therapy for the asexual blood stage of the infection, is developing in southeast Asia. Renewed initiatives to eliminate malaria will benefit from an expanded repertoire of antimalarials, including new drugs that kill circulating P. falciparum gametocytes, thereby preventing transmission. Our current understanding of the biology of asexual blood-stage parasites and gametocytes and the ability to culture them in vitro lends optimism that high-throughput screenings of large chemical libraries will produce a new generation of antimalarial drugs. There is also a need for new therapies to reduce the high mortality of severe malaria. An understanding of the pathophysiology of severe disease may identify rational targets for drugs that improve survival.

Figure 1

Severe malaria in children. (a) Life cycle and pathogenesis of malaria. Malaria infections begin with the injection of parasite sporozoites by infected mosquitoes during a blood meal. Sporozoites invade hepatocytes and proliferate into merozoites. One P. falciparum sporozoite develops into 40,000 merozoites per liver cell over 6 d. During P. vivax and Plasmodium ovale infection, some sporozoites also differentiate into hypnozoites that remain dormant in the liver for months to years before undergoing division and development into merozoites. Only one drug family, the 8-aminoquinolines such as primaquine, kills hypnozoites. However, the 8-aminoquinolines are toxic in glucose-6-phosphate dehydrogenase (G6PD)-deficient humans, a common deficiency in malaria-endemic regions of the world. Consequently, elimination of P. vivax and P. ovale may require new antihypnozoite drugs that can be safely administered to a population in which G6PD deficiency is prevalent. The blood stage of malaria begins when hepatic merozoites invade erythrocytes. Within 12 h of invasion, the parasite remodels the red blood cell (RBC), facilitating the growth of the parasite and transporting PfEMP1 to the erythrocyte membrane. Infected RBCs (iRBCs) bind to endothelium through PfEMP1 primarily to avoid clearance by the spleen. Sequestration of infected RBCs injures endothelial cells (ECs) and disrupts blood flow, causing tissue hypoxia and lactic acidosis. These mechanisms contribute to organ-specific syndromes such as cerebral malaria and placental malaria when sequestration occurs in the brain or placenta. Hemolysis of infected and bystander (uninfected) RBCs causes anemia that may be exacerbated by impaired erythropoiesis. Hemolysis also contributes to endothelial injury and dysfunction as free hemoglobin (Hb) catalyzes oxidative damage and consumes nitric oxide (NO), a regulator of endothelial cells. Merozoites develop in the sequestered RBCs, and the rupture of infected erythrocytes causes fever and rigors. Most merozoites invade uninfected RBCs and circulate as ring-stage parasites, but a small fraction of merozoites develop into male and female gametocytes that infect mosquitoes when taken up during a blood meal. Gametocytes continue to circulate after treatment at the asexual blood stages; therefore, safe drugs to kill circulating gametocytes would help in P. falciparum elimination. ROS, reactive oxygen species; BBB, blood-brain barrier. (b) Progression of malaria in a susceptible population and opportunities for treatment. Approximately 2 billion people live in areas where malaria is transmitted. In regions where malaria is endemic, asymptomatic parasitemia is common and contributes to transmission. Intermittent presumptive treatment given to a population helps to eliminate parasites from asymptomatic carriers. Of the ∼500 million symptomatic cases of malaria globally each year, only about 1% progress to severe malaria. The major severe malaria syndromes are cerebral malaria, acidosis (respiratory distress) and severe anemia. Effective antidisease therapies that can be combined with parasite-killing drugs are needed to improve survival from severe malaria.

Figure 2

Quinine and artemisinin discoveries led to the development of many synthetic antimalarial drugs. In the digestive vacuole (DV) of the intraerythrocytic forms of the parasite, hemoglobin (Hb) is digested, and hematin is released, which is detrimental to the parasite. The parasite can reduce the harmful effects of hematin by converting it into hemozoin; however, this reaction is inhibited by chloroquine (CQ). Heme activates artemisinin activity, resulting in parasite killing. RBC, red blood cell; Mut, mutant; PV, parasitophorous vacuole.

Figure 3

Merozoite invasion of the erythrocyte involves five steps, including the movement of erythrocyte membrane past the junction to form the parasitophorous vacuole (PV). RBC, red blood cell; RH, reticulocyte homology ligand; RIPR, RH5 interacting protein.

Figure 4

Malaria infection disrupts nitric oxide metabolism and causes harmful endothelial activation. Intravascular hemolysis limits nitric oxide (NO) bioavailability: destruction of the erythrocyte releases hemoglobin, arginase and ADMA into plasma. Hemoglobin (Hb) reacts with endothelial nitric oxide and converts it to biologically inactive nitrate (NO₃^–), diminishing nitric oxide signaling. Hemoglobin can also catalyze the production of reactive oxygen species (ROS). Arginase is released from erythrocytes and metabolizes arginine to ornithine (Orn), limiting the arginine that is available to NOS. Erythrocytes have high concentrations of ADMA incorporated in proteins; hemolysis and proteolysis releases free ADMA into plasma. Impaired nitric oxide synthesis: NOS catalyzes the generation of nitric oxide from the substrates oxygen (O₂), Arg and NADPH. Tetrahydrobiopterin (BH₄) is an essential NOS cofactor. In the absence of either arginine or BH₄, NOS functions as an oxidase, generating superoxide from NADPH and molecular oxygen. The product citrulline (Cit) can be recycled to arginine by argininosuccinate synthase (ASS1) and argininosuccinate lyase (ASL). ADMA is a NOS inhibitor that is generated by the methylation of arginine in polypeptides by protein arginine methyltransferase (PRMT) followed by proteolysis to release free ADMA. DDAH regulates nitric oxide synthesis by metabolizing ADMA. Arginine, ADMA and symmetrical dimethylarginine (SDMA) cross the endothelial cell membrane by a cationic amino acid transporter (CAT). Inflammation: glycosylphosphatidylinositol (GPI) from malaria parasites can trigger inflammation through Toll-like receptor 2 (TLR2) signaling, leading to NF-κB activation and transcription of inflammatory cytokines (such as TNF), adhesion molecules (such as ICAM1) and procoagulant molecules (such as tissue factor, TF). Nitric oxide can downregulate NF-κB to exert anti-inflammatory effects. Adhesion: infected erythrocytes (iRBCs) bind to endothelial cells through parasite-encoded PfEMP1 and endothelial receptors such as ICAM1 and heparan sulfate (HS). Binding to ICAM1 triggers RhoA and Rho kinase, leading to cytoskeletal rearrangements that cause cell retraction and disrupt cell-cell junctions. Rho kinase also activates NF-κB to exert proinflammatory, proadhesive and procoagulant effects. Rho kinase downregulates NOS activity, but nitric oxide can suppress RhoA activation by nitrosylation of protein kinase C (PKC). Loss of barrier integrity: tight junctions between endothelial cells are maintained by signaling through the Ang1–endothelial-specific receptor tyrosine kinase (Tie2) axis. Ang2 binds to Tie2 but does not transduce a signal, thereby interrupting constitutive Ang1-Tie2 signaling. In the setting of proinflammatory cytokines such as TNF, this leads to loss of integrity of the endothelial cell layer. Weibel-Palade (WP) body exocytosis: Weibel-Palade bodies contain vWF multimers that can bind to circulating platelets and trigger thrombosis. vWF multimers are cleaved by the vWF protease ADAMTS13 to limit the extent of thrombosis. Patients with severe malaria have reduced ADAMTS13 activity and an overabundance of vWF multimers. Tethered vWF multimers can bind to platelets and form a bridge to infected erythrocytes. Thrombosis and coagulation: adherent infected erythrocytes trigger a display of tissue factor on the endothelial cell apical surface and recruit circulating coagulation factors to activate thrombin. Thrombin generates fibrin to form a blood clot that can block the lumen of small vessels and stop blood flow. Thrombin also activates protease-activated receptors (PARs) that couple with multiple G proteins to cause cytoskeletal retraction and expression of inflammatory cytokines and adhesion molecules.