Comparative spatial proteomics of Plasmodium-infected erythrocytes.

Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized subcellular compartments to extensively remodel the host cell for its survival. The information to fully understand these compartments is likely hidden in the so far poorly characterized Plasmodium species spatial proteome. To address this question, we determined the steady-state subcellular location of more than 12,000 parasite proteins across five different species by extensive subcellular fractionation of erythrocytes infected by Plasmodium falciparum, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. This comparison of the pan-species spatial proteomes and their expression patterns indicates increasing species-specific proteins associated with the more external compartments, supporting host adaptations and post-transcriptional regulation. The spatial proteome offers comprehensive insight into the different human, simian, and rodent Plasmodium species, establishing a powerful resource for understanding species-specific host adaptation processes in the parasite.

Comparative Heterochromatin Profiling Reveals Conserved and Unique Epigenome Signatures Linked to Adaptation and Development of Malaria Parasites.

Heterochromatin-dependent gene silencing is central to the adaptation and survival of Plasmodium falciparum malaria parasites, allowing clonally variant gene expression during blood infection in humans. By assessing genome-wide heterochromatin protein 1 (HP1) occupancy, we present a comprehensive analysis of heterochromatin landscapes across different Plasmodium species, strains, and life cycle stages. Common targets of epigenetic silencing include fast-evolving multi-gene families encoding surface antigens and a small set of conserved HP1-associated genes with regulatory potential. Many P. falciparum heterochromatic genes are marked in a strain-specific manner, increasing the parasite's adaptive capacity. Whereas heterochromatin is strictly maintained during mitotic proliferation of asexual blood stage parasites, substantial heterochromatin reorganization occurs in differentiating gametocytes and appears crucial for the activation of key gametocyte-specific genes and adaptation of erythrocyte remodeling machinery. Collectively, these findings provide a catalog of heterochromatic genes and reveal conserved and specialized features of epigenetic control across the genus Plasmodium.

Host immune evasion strategies of malaria blood stage parasite.

Host immune evasion is a key strategy for the continual survival of many microbial pathogens including Apicomplexan protozoan: Plasmodium spp., the causative agent of Malaria. The malaria parasite has evolved a variety of mechanisms to evade the host immune responses within its two hosts: the female Anopheles mosquito vector and vertebrate host. In this review, we will focus on the molecular mechanisms of the immune evasion strategies used by the Plasmodium parasite at the blood stage which is responsible for the clinical manifestations of human malaria. We also aim to provide some insights on the potential targets for malaria interventions through the recent advancement in understanding the molecular biology of the parasite.

An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum.

Variegated surface antigen expression is key to chronic infection and pathogenesis of the human malaria parasite Plasmodium falciparum. This protozoan parasite expresses distinct surface molecules that are encoded by clonally variant gene families such as var, rif and stevor. The molecular mechanisms governing activation of individual members remain ill-defined. To investigate the molecular events of the initial transcriptional activation process we focused on a member of the apicomplexan ApiAP2 transcription factor family predicted to bind to the 5' upstream regions of the var gene family, AP2-exp (PF3D7_1466400). Viable AP2-exp mutant parasites rely on expressing no less than a short truncated protein including the N-terminal AP2 DNA-binding domain. RNA-seq analysis in mutant parasites revealed transcriptional changes in a subset of exported proteins encoded by clonally variant gene families. Upregulation of RIFINs and STEVORs was validated at the protein levels. In addition, morphological alterations were observed on the surface of the host cells infected by the mutants. This work points to a complex regulatory network of clonally variant gene families in which transcription of a subset of members is regulated by the same transcription factor. In addition, we highlight the importance of the non-DNA binding AP2 domain in functional gene regulation.

Three Is a Crowd - New Insights into Rosetting in Plasmodium falciparum.

The intracellular malaria parasites extensively modify host erythrocytes to allow nutrient uptake, ensure homeostasis, and evade the host's immune response. To achieve this, the parasite exports several proteins to the erythrocyte surface. In Plasmodium falciparum, the parasite responsible for the most severe form of human malaria, three major variant surface antigen families - PfEMP1, STEVOR, and RIFIN - have been implicated in contributing to immune evasion, parasite sequestration, and parasite-mediated rosetting of uninfected erythrocytes. Sequestration and rosetting have been linked to parasite-mediated pathology, making the variant surface antigens of P. falciparum major virulence factors. Here we review our current understanding of rosetting mechanism, recent findings of STEVOR, RIFIN-mediated rosetting, and their implication on the severity and pathology of the disease.

Characterization of the Plasmodium Interspersed Repeats (PIR) proteins of Plasmodium chabaudi indicates functional diversity.

Plasmodium multigene families play a central role in the pathogenesis of malaria. The Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium spp. However their function(s) remains unknown. Using the rodent model of malaria, Plasmodium chabaudi, we show that individual CIR proteins have differential localizations within infected red cell (iRBC), suggesting different functional roles in a blood-stage infection. Some CIRs appear to be located on the surface of iRBC and merozoites and are therefore well placed to interact with host molecules. In line with this hypothesis, we show for the first time that a subset of recombinant CIRs bind mouse RBCs suggesting a role for CIR in rosette formation and/or invasion. Together, our results unravel differences in subcellular localization and ability to bind mouse erythrocytes between the members of the cir family, which strongly suggest different functional roles in a blood-stage infection.

Identification of a new export signal in Plasmodium yoelii: identification of a new exportome.

Development of the erythrocytic malaria parasite requires targeting of parasite proteins into multiple compartments located within and beyond the parasite confine. Beyond the PEXEL/VTS pathway and its characterized players, increasing amount of evidence has highlighted the existence of proteins exported using alternative export-signal(s)/pathway(s); hence, the exportomes currently predicted are incomplete. The nature of these exported proteins which could have a prominent role in most of the Plasmodium species remains elusive. Using P. yoelii variant proteins, we identified a signal associated to lipophilic region that mediates export of P. yoelii proteins. This non-PEXEL signal termed PLASMED is defined by semi-conserved residues and possibly a secondary structure. In vivo characterization of exported-proteins indicated that PLASMED is a bona fide export-signal that allowed us to identify an unseen P. yoelii exportome. The repertoire of the newly predicted exported proteins opens up perspectives for unravelling the remodelling of the host-cell by the parasite, against which new therapies could be elaborated.

Proteomic analysis of detergent-resistant membrane microdomains in trophozoite blood stage of the human malaria parasite Plasmodium falciparum.

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum.

Plasmodium falciparum STEVOR proteins are highly expressed in patient isolates and located in the surface membranes of infected red blood cells and the apical tips of merozoites.

The human parasite Plasmodium falciparum has the potential to express a vast repertoire of variant proteins on the surface of the infected red blood cell (iRBC). Variation in the expression pattern of these proteins is linked to antigenic variation and thereby evasion of host antibody-mediated immunity. The genes in the stevor multigene family code for small variant antigens that are expressed in blood-stage parasites where they can be detected in membranous structures called Maurer's clefts (MC). Some studies have indicated that STEVOR protein may also be trafficked to the iRBC membrane. To address the location of STEVOR protein in more detail, we have analyzed expression in several cultured parasite lines and in parasites obtained directly from patients. We detected STEVOR expression in a higher proportion of parasites recently isolated from patients than in cultured parasite lines and show that STEVOR is trafficked in schizont-stage parasites from the MC to the RBC cytosol and the iRBC membrane. Furthermore, STEVOR protein is also detected at the apical end of merozoites. Importantly, we show that culture-adapted parasites do not require STEVOR for survival. These findings provide new insights into the role of the stevor multigene family during both the schizont and merozoite stages of the parasite and highlight the importance of studying freshly isolated parasites, rather than parasite lines maintained in culture, when investigating potential mediators of host-parasite interactions.