Lawrence W. Bergman

A combined transcriptome and proteome survey of malaria parasite liver stages

For 50 years since their discovery, the malaria parasite liver stages (LS) have been difficult to analyze, impeding their utilization as a critical target for antiinfection vaccines and drugs. We have undertaken a comprehensive transcriptome analysis in combination with a proteomic survey of LS. Green fluorescent protein-tagged Plasmodium yoelii (PyGFP) was used to efficiently isolate LS-infected hepatocytes from the rodent host. Genome-wide LS gene expression was profiled and compared with other parasite life cycle stages. The analysis revealed ≈2,000 genes active during LS development, and proteomic analysis identified 816 proteins. A subset of proteins appeared to be expressed in LS only. The data revealed exported parasite proteins and LS metabolic pathways including expression of FASII pathway enzymes. The FASII inhibitor hexachlorophene and the antibiotics, tetracycline and rifampicin, that target the apicoplast inhibited LS development, identifying FASII and other pathways localized in the apicoplast as potential drug targets to prevent malaria infection.

Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites.

Apicomplexan host cell invasion and gliding motility depend on the parasites actomyosin system located beneath the plasma membrane of invasive stages. Myosin A (MyoA), a class XIV unconventional myosin, is the motor protein. A model has been proposed to explain how the actomyosin motor operates but little is known about the components, topology and connectivity of the motor complex. Using the MyoA neck and tail domain as bait in a yeast two-hybrid screen we identified MTIP, a novel 24 kDa protein that interacts with MyoA. Deletion analysis shows that the 15 amino-acid C-terminal tail domain of MyoA, rather than the neck domain, specifically interacts with MTIP. In Plasmodium sporozoites MTIP localizes to the inner membrane complex (IMC), where it is found clustered with MyoA. The data support a model for apicomplexan motility and invasion in which the MyoA motor protein is associated via its tail domain with MTIP, immobilizing it at the outer IMC membrane. The head domain of the immobilized MyoA moves actin filaments that, directly or via a bridging protein, connect to the cytoplasmic domain of a transmembrane protein of the TRAP family. The actin/TRAP complex is then redistributed by the stationary MyoA from the anterior to the posterior end of the zoite, leading to its forward movement on a substrate or to penetration of a host cell.

A co-ligand complex anchors Plasmodium falciparum merozoites to the erythrocyte invasion receptor band 3.

In Plasmodium falciparum malaria, erythrocyte invasion by circulating merozoites may occur via two distinct pathways involving either a sialic acid-dependent or -independent mechanism. Earlier, we identified two nonglycosylated exofacial regions of erythrocyte band 3 termed 5ABC and 6A as an important host receptor in the sialic acid-independent invasion pathway. 5ABC, a major segment of this receptor, interacts with the 42-kDa processing product of merozoite surface protein 1 (MSP142) through its 19-kDa C-terminal domain. Here, we show that two regions of merozoite surface protein 9 (MSP9), also known as acidic basic repeat antigen, interact directly with 5ABC during erythrocyte invasion by P. falciparum. Native MSP9 as well as recombinant polypeptides derived from two regions of MSP9 (MSP9/Δ1 and MSP9/Δ2) interacted with both 5ABC and intact erythrocytes. Soluble 5ABC added to the assay mixture drastically diminished the binding of MSP9 to erythrocytes. Recombinant MSP9/Δ1 and MSP9/Δ2 present in the culture medium blocked P. falciparum reinvasion into erythrocytes in vitro. Native MSP9 and MSP142, the two ligands binding to the 5ABC receptor, existed as a stable complex. Our results establish a novel concept wherein the merozoite exploits a specific complex of co-ligands on its surface to target a single erythrocyte receptor during invasion. This new paradigm poses a new challenge in the development of a vaccine for blood stage malaria.

Pyrazoleamide compounds are potent antimalarials that target Na+ homeostasis in intraerythrocytic Plasmodium falciparum

The quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na+ regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na+ homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na+ homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes.

Functional Characterization of a Redundant Plasmodium TRAP Family Invasin, TRAP-Like Protein, by Aldolase Binding and a Genetic Complementation Test

ABSTRACT Efficient and specific host cell entry is of exquisite importance for intracellular pathogens. Parasites of the phylum Apicomplexa are highly motile and actively enter host cells. These functions are mediated by type I transmembrane invasins of the TRAP family that link an extracellular recognition event to the parasite actin-myosin motor machinery. We systematically tested potential parasite invasins for binding to the actin bridging molecule aldolase and complementation of the vital cytoplasmic domain of the sporozoite invasin TRAP. We show that the ookinete invasin CTRP and a novel, structurally related protein, termed TRAP-like protein (TLP), are functional members of the TRAP family. Although TLP is expressed in invasive stages, targeted gene disruption revealed a nonvital role during life cycle progression. This is the first genetic analysis of TLP, encoding a redundant TRAP family invasin, in the malaria parasite.

Structure-based Design of Novel Small-Molecule Inhibitors of Plasmodium falciparum

Malaria is endemic in most developing countries, with nearly 500 million cases estimated to occur each year. The need to design a new generation of antimalarial drugs that can combat the most drug-resistant forms of the malarial parasite is well recognized. In this study, we wanted to develop inhibitors of key proteins that form the invasion machinery of the malarial parasite. A critical feature of host-cell invasion by apicomplexan parasites is the interaction between the carboxy terminal tail of myosin A (MyoA) and the myosin tail interacting protein (MTIP). Using the cocrystal structure of the Plasmodium knowlesi MTIP and the MyoA tail peptide as input to the hybrid structure-based virtual screening approach, we identified a series of small molecules as having the potential to inhibit MTIP-MyoA interactions. Of the initial 15 compounds tested, a pyrazole-urea compound inhibited P. falciparum growth with an EC(50) value of 145 nM. We screened an additional 51 compounds belonging to the same chemical class and identified 8 compounds with EC(50) values less than 400 nM. Interestingly, the compounds appeared to act at several stages of the parasites life cycle to block growth and development. The pyrazole-urea compounds identified in this study could be effective antimalarial agents because they competitively inhibit a key protein-protein interaction between MTIP and MyoA responsible for the gliding motility and the invasive features of the malarial parasite.

A Multigene Family That Interacts with the Amino Terminus of Plasmodium MSP-1 Identified Using the Yeast Two-Hybrid System

ABSTRACT Merozoite surface protein 1 (MSP-1) is a high-molecular-weight protein expressed on the surface of the malaria merozoite in a noncovalent complex with other protein molecules. MSP-1 undergoes a series of proteolytic processing events, but no precise biological role for the various proteolytic fragments of MSP-1 or for the additional proteins present in the complex is known. Through the use of the yeast two-hybrid system, we have isolated genes encoding proteins that interact with a region of the amino-terminal proteolytic fragment of MSP-1 from the mouse parasite Plasmodium yoelii. This analysis has led to the isolation of two sequence-related molecules, one of which is the P. yoelii homologue of MSP-7 originally described in Plasmodium falciparum. BLAST analysis of the P. falciparum database has revealed that there are six related protein molecules present in this species encoded near each other on chromosome 13. In P. falciparum, we designated these molecules MSRP-1 to -5. Analysis of the P. yoelii database indicates a similar chromosomal organization for the two genes in the mouse parasite species. The three P. falciparum sequences with the highest degree of homology to the P. yoelii sequences isolated in the two-hybrid screen have been characterized at the molecular level (MSRP-1 to -3). Expression analysis indicated that the mRNAs are expressed at various levels in the different asexual stages. Immunofluorescence studies colocalized the expression of the MSRP molecules and the amino-terminal portion of MSP-1 to the surfaces of trophozoites. In vitro binding experiments confirmed the interaction between MSRP-1, MSRP-2, and the amino-terminal region of P. falciparum MSP-1.

Structure-function relationships of the yeast cyclin-dependent kinase Pho85.

The PHO85 gene of Saccharomyces cerevisiae encodes a cyclin-dependent kinase involved in both transcriptional regulation and cell cycle progression. Although a great deal is known concerning the structure, function, and regulation of the highly homologous Cdc28 protein kinase, little is known concerning these relationships in regard to Pho85. In this study, we constructed a series of Pho85-Cdc28 chimeras to map the region(s) of the Pho85 molecule that is critical for function of Pho85 in repression of acid phosphatase (PHO5) expression. Using a combination of site-directed and ethyl methanesulfonate-induced mutagenesis, we have identified numerous residues critical for either activation of the Pho85 kinase, interaction of Pho85 with the cyclin-like molecule Pho80, or substrate recognition. Finally, analysis of mutations analogous to those previously identified in either Cdc28 or cdc2 of Schizosaccharomyces pombe suggested that the inhibition of Pho85-Pho80 activity in mechanistically different from that seen in the other cyclin-dependent kinases.

High Mobility Group Protein HMGB2 Is a Critical Regulator of Plasmodium Oocyst Development

The sexual cycle of Plasmodium is required for transmission of malaria from mosquitoes to mammals, but how parasites induce the expression of genes required for the sexual stages is not known. We disrupted the Plasmodium yoelii gene encoding high mobility group nuclear factor hmgb2, which encodes a DNA-binding protein potentially implicated in transcriptional regulation of malaria gene expression. We investigated its function in vivo in the vertebrate and invertebrate hosts. Δpyhmgb2 parasites develop into gametocytes but have drastic impairment of oocyst formation. A global transcriptome analysis of the Δpyhmgb2 parasites identified ∼30 genes whose expression is down-regulated in the Δpyhmgb2 parasites. These genes are conserved in all malaria species, and more than 90% of these genes show a peak of mRNA expression at the gametocyte stage. Surprisingly, the transcripts coding for the Plasmodium berghei orthologues of those genes are stored and translated in the ookinete stage. Therefore, sexual stage protein expression appears to be both transcriptionally and translationally regulated with Plasmodium HMGB2 acting as an important regulator of malaria sexual stage gene expression.

Interaction ofSaccharomyces cerevisiae Pho2 with Pho4 increases the accessibility of the activation domain of Pho4

InSaccharomyces cerevisiae, expression of acid phosphatase, encoded by thePHO5 gene, requires two positive regulatory factors, Pho4 and Pho2 (also called Bas2 or Grf10). UsingGAL4-PHO4 fusions, we demonstrate that a functional interaction between these two proteins is necessary for transcriptional activation to occur. This functional interaction between Pho4 and Pho2 is independent of the presence of the negative regulatory factor, Pho80, which also interacts with Pho4. Interestingly, truncations of Pho4 missing amino acids 252–265, which encompass the basic region of the basic helix-loop-helix (bHLH) DNA binding motif, exhibit high transcriptional activation that is independent of the Pho2 molecule. Single amino acid mutations of highly conserved residues within this area all display this Pho2-independent phenotype. A region near the C-terminus of Pho2 appears to be critical for this interaction with Pho4. A model to account for the requirement for Pho2 in Pho4-dependent transcriptional activation is proposed.