Malaria is today a disease of poverty and underdeveloped countries In Africa, mortality remains high because there is limited access to treatment in the villages We should follow in Pasteur's footsteps by using basic research to develop better tools for the control and cure of malaria Insight into the complexity of malaria pathogenesis is vital for understanding the disease and will provide a major step towards controlling it Those of us who work on pathogenesis must widen our approach and think in terms of new tools such as vaccines to reduce disease The inability of many countries to fund expensive campaigns and antimalarial treatment requires these tools to be highly effective and affordable

The pathogenic basis of malaria

The completion of the genome sequence for Plasmodium falciparum, the species responsible for most malaria human deaths, has the potential to reveal hundreds of new drug targets and proteins involved in pathogenesis. However, only approximately 35% of the genes code for proteins with an identifiable function. The absence of routine genetic tools for studying Plasmodium parasites suggests that this number is unlikely to change quickly if conventional serial methods are used to characterize encoded proteins. Here, we use a high-density oligonucleotide array to generate expression profiles of human and mosquito stages of the malaria parasite's life cycle. Genes with highly correlated levels and temporal patterns of expression were often involved in similar functions or cellular processes.

https://science.sciencemag.org/content/sci/301/5639/1503.full.pdf

Discovery of Gene Function by Expression Profiling of the Malaria Parasite Life Cycle

Ubiquitin regulates protein transport between membrane compartments by serving as a sorting signal on protein cargo and by controlling the activity of trafficking machinery. Monoubiquitin attached to integral plasma membrane proteins or to associated transport modifiers serves as a regulated signal for internalization into the endocytic pathway. Similarly, monoubiquitin attached to biosynthetic and endocytic membrane proteins is a signal for sorting of cargo into vesicles that bud into the late endosome lumen for delivery into the lysosome. Ubiquitination of trans-acting endocytic proteins is also required for transport, and key endocytic proteins are modified by monoubiquitin. Regulatory enzymes of the ubiquitination machinery, ubiquitin ligases, control the timing and specificity of plasma membrane protein downregulation in such diverse biological processes as cell fate specification and neurotransmission. Monoubiquitin signals appended by these ligases are recognized by endocytic proteins carrying ubiquitin-binding motifs, including UBA, UEV, UIM, and CUE domains. The UIM proteins epsins and Hrs are excellent candidates for adaptors that link ubiquitinated cargo to the clathrin-based sorting machinery at appropriate regions of the endosomal or plasma membranes. Other ubiquitin-binding proteins also play crucial roles in cargo transport, although in most cases the role of ubiquitin-binding is not defined. Ubiquitin-binding proteins such as epsins, Hrs, and Vps9 are monoubiquitinated, indicating the general nature of ubiquitin regulation in endocytosis and suggesting new models to explain how recognition of monoubiquitin signals may be regulated.

Regulation of Membrane Protein Transport by Ubiquitin and Ubiquitin-Binding Proteins

Cytoadhesion of infected red blood cells (iRBC) is mediated through parasite-encoded, clonally variant surface antigens (VSA) and is a central process in the pathogenesis of Plasmodium falciparum malaria. Pregnancy-associated malaria (PAM) has been linked to VSA-mediated adhesion of iRBC to the glycosaminoglycan chondroitin sulphate A (CSA) in the placental intervillous space. Several studies have pointed to members of the PfEMP1 VSA family as mediators of CSA-specific iRBC sequestration in the placenta. Here, we report marked upregulation of a single var gene in several P. falciparum parasite isolates after selection for adhesion to CSA in vitro. The gene belongs to a highly conserved and common var gene subfamily (var2csa). The var2csa genes are structurally distinct from all other var genes in the parasite genome in lacking both CIDR and DBL-gamma domains. These domains have previously been implicated in PfEMP1-mediated adhesion to CD36 and CSA. We also show that var2csa was transcribed at higher levels in three placental parasite isolates compared with transcription in parasites from peripheral blood of two children with P. falciparum malaria. This var gene thus has the properties expected of a gene encoding the parasite adhesion molecule that initiates the pathology associated with PAM.

Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria

Ubiquitin acts as a versatile cellular signal that controls a wide range of biological processes including protein degradation, DNA repair, endocytosis, autophagy, transcription, immunity, and inflammation. The specificity of ubiquitin signaling is achieved by alternative conjugation signals (monoubiquitin and ubiquitin chains) and interactions with ubiquitin-binding proteins (known as ubiquitin receptors) that decode ubiquitinated target signals into biochemical cascades in the cell. Herein, we review the current knowledge pertaining to the structural and functional features of ubiquitin-binding proteins and the mechanisms by which they recognize various types of ubiquitin topologies. The combinatorial use of diverse ubiquitin-binding domains (UBDs) in full-length proteins, selective recognition of chains with distinct linkages and length, and posttranslational modifications of ubiquitin receptors or multivalent interactions within protein complexes illustrate a few mechanisms by which a circuitry of signaling networks can be rewired by ubiquitin-binding proteins to control cellular functions in vivo.

Ubiquitin-Binding Proteins: Decoders of Ubiquitin-Mediated Cellular Functions

The protozoan Plasmodium falciparum causes lethal malaria1. Adhesion of erythrocytes infected with P. falciparum to vascular endothelium and to uninfected red blood cells (rosetting) may be involved in the pathogenesis of severe malaria2,3,4. The binding is mediated by the antigenically variant erythrocyte-membrane-protein-1 (PfEMP-1)5,6,7,8, which is encoded by members of the P. falciparum var gene family9,10. The control of expression and switching of var genes seems to lack resemblance to mechanisms operating in variant gene families of other microbial pathogens11,12. Here we show that multiple, distinct var gene transcripts (about 24 or more) can be detected by reverse transcription and polymerase chain reaction in bulk cultures of the rosetting parasite FCR3S1.2, despite the adhesive homogeneity of the cultures. We also detected several var transcripts in single erythrocytes infected with a ring-stage parasite of FCR3S1.2, and found that different var genes are transcribed simultaneously from several chromosomes in the same cell. In contrast, we detected only one var transcript, FCR3S1.2 var-1, which encodes the rosetting PfEMP-1 protein13, in individual rosette-adhesive trophozoite-infected cells, and we found only one PfEMP-1 type at the erythrocyte surface by labelling with 125iodine and immunoprecipitation. We conclude that a single P. falciparum parasite simultaneously transcribes multiple var genes but, through a developmentally regulated process, selects only one PfEMP-1 to reach the surface of the host cell.

Developmental selection of var gene expression in Plasmodium falciparum

Severe Plasmodium falciparum malaria is characterized by excessive sequestration of infected and uninfected erythrocytes in the microvasculature of the affected organ. Rosetting, the adhesion of P. falciparum-infected erythrocytes to uninfected erythrocytes is a virulent parasite phenotype associated with the occurrence of severe malaria. Here we report on the identification by single-cell reverse transcriptase PCR and cDNA cloning of the adhesive ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs. A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix. The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding. PfEMP1 is suggested to be the rosetting ligand and heparan sulfate, or a heparan sulfate-like molecule, the receptor both for PfEMP1 binding and naturally formed erythrocyte rosettes.

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Identification of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) as the Rosetting Ligand of the Malaria Parasite P. falciparum

Human infections with Plasmodium falciparum may result in severe forms of malaria. The widespread and rapid development of drug resistance in P. falciparum and the resistance of the disease-transmitting mosquitoes to insecticides make it urgent to understand the molecular background of the pathogenesis of malaria to enable the development of novel approaches to combat the disease. This review focuses on the molecular mechanisms of severe malaria caused by the P. falciparum parasite. The nature of severe malaria and the deleterious effects of parasite-derived toxins and host-induced cytokines are introduced. Sequestration, brought about by cytoadherence and rosetting, is linked to severe malaria and is mediated by multiple receptors on the endothelium and red blood cells. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is the ligand responsible for a majority of binding interactions, and the multiply adhesive features of this sticky molecule are presented. Antigenic variation is also a major feature of PfEMP1 and of the surface of the P. falciparum-infected erythrocyte. Possible mechanisms of P. falciparum antigenic variation in asexual stages are further discussed. We conclude this review with a perspective and suggestions of important aspects for future investigations.

https://cmr.asm.org/content/cmr/13/3/439.full.pdf

Martha Schlichtherle

Papers

Developmental selection of var gene expression in Plasmodium falciparum

Identification of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) as the Rosetting Ligand of the Malaria Parasite P. falciparum

Molecular Aspects of Severe Malaria