David J. Sullivan

Chemical genetics of Plasmodium falciparum

Malaria caused by Plasmodium falciparum is a disease that is responsible for 880,000 deaths per year worldwide. Vaccine development has proved difficult and resistance has emerged for most antimalarial drugs. To discover new antimalarial chemotypes, we have used a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library—many of which showed potent in vitro activity against drug-resistant P. falciparum strains—and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in several organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Our findings provide the scientific community with new starting points for malaria drug discovery.

Plasmodium Hemozoin Formation Mediated by Histidine-Rich Proteins

The digestive vacuole of Plasmodium falciparum is the site of hemoglobin degradation, heme polymerization into crystalline hemozoin, and antimalarial drug accumulation. Antibodies identified histidine-rich protein II (HRP II) in purified digestive vacuoles. Recombinant or native HRP II promoted the formation of hemozoin, and chloroquine inhibited the reaction. The related HRP III also polymerized heme, and an additional HRP was identified in vacuoles. It is proposed that after secretion by the parasite into the host erythrocyte cytosol, HRPs are brought into the acidic digestive vacuole along with hemoglobin. After hemoglobin proteolysis, HRPs bind the liberated heme and mediate hemozoin formation.

Host-mediated regulation of superinfection in malaria

In regions of high rates of malaria transmission, mosquitoes repeatedly transmit liver-tropic Plasmodium sporozoites to individuals who already have blood-stage parasitemia. This manifests itself in semi-immune children (who have been exposed since birth to Plasmodium infection and as such show low levels of peripheral parasitemia but can still be infected) older than 5 years of age by concurrent carriage of different parasite genotypes at low asymptomatic parasitemias. Superinfection presents an increased risk of hyperparasitemia and death in less immune individuals but counterintuitively is not frequently observed in the young. Here we show in a mouse model that ongoing blood-stage infections, above a minimum threshold, impair the growth of subsequently inoculated sporozoites such that they become growth arrested in liver hepatocytes and fail to develop into blood-stage parasites. Inhibition of the liver-stage infection is mediated by the host iron regulatory hormone hepcidin, whose synthesis we found to be stimulated by blood-stage parasites in a density-dependent manner. We mathematically modeled this phenomenon and show how density-dependent protection against liver-stage malaria can shape the epidemiological patterns of age-related risk and the complexity of malaria infections seen in young children. The interaction between these two Plasmodium stages and host iron metabolism has relevance for the global efforts to reduce malaria transmission and for evaluation of iron supplementation programs in malaria-endemic regions.

The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.

Human immunodeficiency virus type 1 long-term non-progressors: the viral, genetic and immunological basis for disease non-progression.

A small subset of human immunodeficiency virus type 1 (HIV-1)-infected, therapy-naive individuals--referred to as long-term non-progressors (LTNPs)--maintain a favourable course of infection, often being asymptomatic for many years with high CD4(+) and CD8(+) T-cell counts (>500 cells  μl(-1)) and low plasma HIV-RNA levels (<10 ,000 copies ml(-1)). Research in the field has undergone considerable development in recent years and LTNPs offer a piece of the puzzle in understanding the ways that persons can naturally control HIV-1 infection. Their method of control is based on viral, genetic and immunological components. With respect to virological features, genomic sequencing has shown that some LTNPs are infected with attenuated strains of HIV-1 and harbour mutant nef, vpr, vif or rev genes that contain single nuclear polymorphisms, or less frequently, large deletions, in conserved domains. Studies have also shown that some LTNPs have unique genetic advantages, including heterozygosity for the CCR5-Δ32 polymorphism, and have been found with excitatory mutations that upregulate the production of the chemokines that competitively inhibit HIV-1 binding to CCR5 or CXCR4. Lastly, immunological factors are crucial for providing LTNPs with a natural form of control, the most important being robust HIV-specific CD4(+) and CD8(+) T-cell responses that correlate with lower viral loads. Many LTNPs carry the HLA class I B57 allele that enhances presentation of antigenic peptides on the surface of infected CD4(+) cells to cytotoxic CD8(+) T cells. For these reasons, LTNPs serve as an ideal model for HIV-1 vaccine development due to their natural control of HIV-1 infection.

A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells

Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection, but specific interactions involved in cerebral homing of infected erythrocytes (IEs) are poorly understood. In this study, P. falciparum-IEs were characterized for binding to primary human brain microvascular endothelial cells (HBMECs). Before selection, CD36 or ICAM-1–binding parasites exhibited punctate binding to a subpopulation of HBMECs and binding was CD36 dependent. Panning of IEs on HBMECs led to a more dispersed binding phenotype and the selection of three var genes, including two that encode the tandem domain cassette 8 (DC8) and were non-CD36 binders. Multiple domains in the DC8 cassette bound to brain endothelium and the cysteine-rich interdomain region 1 inhibited binding of P. falciparum-IEs by 50%, highlighting a key role for the DC8 cassette in cerebral binding. It is mysterious how deadly binding variants are maintained in the parasite population. Clonal parasite lines expressing the two brain-adherent DC8-var genes did not bind to any of the known microvascular receptors, indicating unique receptors are involved in cerebral binding. They could also adhere to brain, lung, dermis, and heart endothelial cells, suggesting cerebral binding variants may have alternative sequestration sites. Furthermore, young African children with CM or nonsevere control cases had antibodies to HBMEC-selected parasites, indicating they had been exposed to related variants during childhood infections. This analysis shows that specific P. falciparum erythrocyte membrane protein 1 types are linked to cerebral binding and suggests a potential mechanism by which individuals may build up immunity to severe disease, in the absence of CM.

A Common Mechanism for Blockade of Heme Polymerization by Antimalarial Quinolines

The antimalarial quinolines are believed to work by blocking the polymerization of toxic heme released during hemoglobin proteolysis in intraerythrocytic Plasmodium falciparum. In the presence of free heme, chloroquine and quinidine associate with the heme polymer. We have proposed that this association of the quinoline-heme complex with polymer caps the growing heme polymer, preventing further sequestration of additional heme that then accumulates to levels that kill the parasite. In this work results of binding assays demonstrate that the association of quinoline-heme complex with heme polymer is specific, saturable, and high affinity and that diverse quinoline analogs can compete for binding. The relative quinoline binding affinity for heme polymer rather than free heme correlates with disruption of heme polymerization. Mefloquine, another important antimalarial quinoline, associated with polymer in a similar fashion, both in cultured parasites and in the test tube. In parasite culture, blocking heme release with protease inhibitor was antagonistic to mefloquine action, as it is to chloroquine action. These data suggest a common mechanism for quinoline antimalarial action dependent on drug interaction with both heme and heme polymer.

Highly penetrative, drug-loaded nanocarriers improve treatment of glioblastoma

Current therapy for glioblastoma multiforme is insufficient, with nearly universal recurrence. Available drug therapies are unsuccessful because they fail to penetrate through the region of the brain containing tumor cells and they fail to kill the cells most responsible for tumor development and therapy resistance, brain cancer stem cells (BCSCs). To address these challenges, we combined two major advances in technology: (i) brain-penetrating polymeric nanoparticles that can be loaded with drugs and are optimized for intracranial convection-enhanced delivery and (ii) repurposed compounds, previously used in Food and Drug Administration-approved products, which were identified through library screening to target BCSCs. Using fluorescence imaging and positron emission tomography, we demonstrate that brain-penetrating nanoparticles can be delivered to large intracranial volumes in both rats and pigs. We identified several agents (from Food and Drug Administration-approved products) that potently inhibit proliferation and self-renewal of BCSCs. When loaded into brain-penetrating nanoparticles and administered by convection-enhanced delivery, one of these agents, dithiazanine iodide, significantly increased survival in rats bearing BCSC-derived xenografts. This unique approach to controlled delivery in the brain should have a significant impact on treatment of glioblastoma multiforme and suggests previously undescribed routes for drug and gene delivery to treat other diseases of the central nervous system.

Platelet factor 4 mediates inflammation in experimental cerebral malaria.

Cerebral malaria (CM) is a major complication of Plasmodium falciparum infection in children. The pathogenesis of CM involves vascular inflammation, immune stimulation, and obstruction of cerebral capillaries. Platelets have a prominent role in both immune responses and vascular obstruction. We now demonstrate that the platelet-derived chemokine, platelet factor 4 (PF4)/CXCL4, promotes the development of experimental cerebral malaria (ECM). Plasmodium-infected red blood cells (RBCs) activated platelets independently of vascular effects, resulting in increased plasma PF4. PF4 or chemokine receptor CXCR3 null mice had less severe ECM, including decreased T cell recruitment to the brain, and platelet depletion or aspirin treatment reduced the development of ECM. We conclude that Plasmodium-infected RBCs can directly activate platelets, and platelet-derived PF4 then contributes to immune activation and T cell trafficking as part of the pathogenesis of ECM.

The effects of placental malaria on mother-to-child HIV transmission in Rakai, Uganda.

We examined the association of placental malaria and mother-to-child transmission (MTCT) of HIV in a prospective community-randomized trial in Rakai District, Uganda. In the 746 HIV-positive mother-infant pairs, the MTCT rate was 20.4%. Placental malaria was more common in HIV-positive than HIV-negative women. After multivariate adjustment for HIV viral load, the risk of MTCT associated with placental malaria was 2.89 and with HIV viral load the risk was 2.85. Interventions to prevent malaria during pregnancy could potentially reduce MTCT.