The Plasmodium falciparum parasitophorous vacuole protein P113 interacts with the parasite protein export machinery and maintains normal vacuole architecture.

Infection with Plasmodium falciparum parasites results in approximately 627,000 deaths from malaria annually. Key to the parasite's success is their ability to invade and subsequently grow within human erythrocytes. Parasite proteins involved in parasite invasion and proliferation are therefore intrinsically of great interest, as targeting these proteins could provide novel means of therapeutic intervention.

Characterisation of complexes formed by parasite proteins exported into the host cell compartment of Plasmodium falciparum infected red blood cells.

During its intraerythrocytic life cycle, the human malaria parasite Plasmodium falciparum supplements its nutritional requirements by scavenging substrates from the plasma through the new permeability pathways (NPPs) installed in the red blood cell (RBC) membrane. Parasite proteins of the RhopH complex: CLAG3, RhopH2, RhopH3, have been implicated in NPP activity. Here, we studied 13 exported proteins previously hypothesised to interact with RhopH2, to study their potential contribution to the function of NPPs.

Proteomic Analysis of Antigen 60 Complex of Bacillus Calmette-Guérin Reveals Presence of Extracellular Vesicle Proteins and Predicted Functional Interactions.

Tuberculosis (TB) is ranked among the top 10 causes of death worldwide. New biomarker-based serodiagnostics and vaccines are unmet needs stalling disease control. Antigen 60 (A60) is a thermostable mycobacterial complex typically purified from Bacillus Calmette-Guérin (BCG) vaccine.

The N-terminus of EXP2 forms the membrane-associated pore of the protein exporting translocon PTEX in Plasmodium falciparum.

In order to facilitate a number of processes including nutrient acquisition and immune evasion, malaria parasites extensively remodel their host erythrocyte. This remodelling is to a large extent accomplished through protein export, a crucial process mediated by the Plasmodium translocon for exported proteins (PTEX) translocon which is comprised of three core components, HSP101, PTEX150 and EXP2. EXP2 has been structurally and electrophysiologically shown to form the pore that spans the vacuole membrane enveloping the parasite.

Spatial organization of protein export in malaria parasite blood stages.

Plasmodium falciparum, which causes malaria, extensively remodels its human host cells, particularly erythrocytes. Remodelling is essential for parasite survival by helping to avoid host immunity and assisting in the uptake of plasma nutrients to fuel rapid growth. Host cell renovation is carried out by hundreds of parasite effector proteins that are exported into the erythrocyte across an enveloping parasitophorous vacuole membrane (PVM).

The malaria PTEX component PTEX88 interacts most closely with HSP101 at the host-parasite interface.

The pathogenic nature of malaria infections is due in part to the export of hundreds of effector proteins that actively remodel the host erythrocyte. The Plasmodium translocon of exported proteins (PTEX) has been shown to facilitate the trafficking of proteins into the host cell, a process that is essential for the survival of the parasite. The role of the auxiliary PTEX component PTEX88 remains unclear, as previous attempts to elucidate its function through reverse genetic approaches showed that in contrast to the core components PTEX150 and HSP101, knockdown of PTEX88 did not give rise to an export phenotype.

Functional Conservation of the AMA1 Host-Cell Invasion Ligand Between P. falciparum and P. vivax: A Novel Platform to Accelerate Vaccine and Drug Development.

Plasmodium vivax and P. falciparum malaria species have diverged significantly in receptor-ligand interactions and host-cell invasion. One protein common to both is the merozoite invasion ligand AMA1.

The exported chaperone Hsp70-x supports virulence functions for Plasmodium falciparum blood stage parasites.

Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P.

parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate.

parasites, the causative agents of malaria, modify their host erythrocyte to render them permeable to supplementary nutrient uptake from the plasma and for removal of toxic waste. Here we investigate the contribution of the rhoptry protein RhopH2, in the formation of new permeability pathways (NPPs) in -infected erythrocytes. We show RhopH2 interacts with RhopH1, RhopH3, the erythrocyte cytoskeleton and exported proteins involved in host cell remodeling.

Differing rates of antibody acquisition to merozoite antigens in malaria: implications for immunity and surveillance.

Antibodies play a key role in acquired human immunity to (Pf) malaria and target merozoites to reduce or prevent blood-stage replication and the development of disease. Merozoites present a complex array of antigens to the immune system, and currently, there is only a partial understanding of the targets of protective antibodies and how responses to different antigens are acquired and boosted. We hypothesized that there would be differences in the rate of acquisition of antibodies to different antigens and how well they are boosted by infection, which impacts the acquisition of immunity.

Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component, PTEX150.

The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality.

Identification of potent phosphodiesterase inhibitors that demonstrate cyclic nucleotide-dependent functions in apicomplexan parasites.

Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, the causative agents of severe malaria and toxoplasmosis, respectively, undergo several critical developmental transitions during their lifecycle. Most important for human pathogenesis is the asexual cycle, in which parasites undergo rounds of host cell invasion, replication, and egress (exit), destroying host cell tissue in the process. Previous work has identified important roles for Protein Kinase G (PKG) and Protein Kinase A (PKA) in parasite egress and invasion, yet little is understood about the regulation of cyclic nucleotides, cGMP and cAMP, that activate these enzymes.

Plasmodium falciparum transfected with ultra bright NanoLuc luciferase offers high sensitivity detection for the screening of growth and cellular trafficking inhibitors.

Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase.

PTEX is an essential nexus for protein export in malaria parasites.

During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol. These proteins have a variety of roles that are essential to virulence or parasite growth. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins.

The Plasmodium translocon of exported proteins (PTEX) component thioredoxin-2 is important for maintaining normal blood-stage growth.

Plasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX. Previously PTEX150, HSP101 and EXP2 have been shown to be bona fide members of PTEX. Here we validate that PTEX88 and TRX2 are also genuine members of PTEX and provide evidence that expression of PTEX components are also expressed in early gametocytes, mosquito and liver stages, consistent with observations that protein export is not restricted to asexual stages.

Inhibition of Plasmodium falciparum CDPK1 by conditional expression of its J-domain demonstrates a key role in schizont development.

PfCDPK1 [Plasmodium falciparum CDPK1 (calcium-dependent protein kinase 1)] is highly expressed in parasite asexual blood and mosquito stages. Its role is still poorly understood, but unsuccessful gene knockout attempts suggest that it is essential for parasite replication and/or RBC (red blood cell) invasion. In the present study, by tagging endogenous CDPK1 with GFP (green fluorescent protein), we demonstrate that CDPK1 localizes to the parasite plasma membrane of replicating and invasive forms as well as very young intracellular parasites and does not appear to be exported into RBCs.

Biochemical and functional analysis of two Plasmodium falciparum blood-stage 6-cys proteins: P12 and P41.

The genomes of Plasmodium parasites that cause malaria in humans, other primates, birds, and rodents all encode multiple 6-cys proteins. Distinct 6-cys protein family members reside on the surface at each extracellular life cycle stage and those on the surface of liver infective and sexual stages have been shown to play important roles in hepatocyte growth and fertilization respectively. However, 6-cys proteins associated with the blood-stage forms of the parasite have no known function.

A newly discovered protein export machine in malaria parasites.

Several hundred malaria parasite proteins are exported beyond an encasing vacuole and into the cytosol of the host erythrocyte, a process that is central to the virulence and viability of the causative Plasmodium species. The trafficking machinery responsible for this export is unknown. Here we identify in Plasmodium falciparum a translocon of exported proteins (PTEX), which is located in the vacuole membrane.

MSP1(19) miniproteins can serve as targets for invasion inhibitory antibodies in Plasmodium falciparum provided they contain the correct domains for cell surface trafficking.

Antibodies from malaria-exposed individuals can agglutinate merozoites released from Plasmodium schizonts, thereby preventing them from invading new erythrocytes. Merozoite coat proteins attached to the plasma membrane are major targets for host antibodies and are therefore considered important malaria vaccine candidates. Prominent among these is the abundant glycosylphosphatidylinositol (GPI)-anchored merozoite surface protein 1 (MSP1) and particularly its C-terminal fragment (MSP1(19)) comprised of two epidermal growth factor (EGF)-like modules.

Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum schizonts.

Merozoite surface proteins of the human malaria parasite Plasmodium falciparum are involved in initial contact with target erythrocytes, a process that begins a cascade of events required for successful invasion of these cells. In order to identify complexes that may play a role in invasion we purified detergent-resistant membranes (DRMs), known to be enriched in merozoite surface proteins, and used blue native-polyacrylamide gel electrophoresis (BN-PAGE) to isolate high molecular weight complexes for identification by mass spectrometry. Sixty-two proteins were detected and these mostly belonged to expected DRM proteins classes including GPI-anchored, multi-membrane spanning and rhoptry proteins.

A set of glycosylphosphatidyl inositol-anchored membrane proteins of Plasmodium falciparum is refractory to genetic deletion.

Targeted gene disruption has proved to be a powerful approach for studying the function of important ligands involved in erythrocyte invasion by the extracellular merozoite form of the human malaria parasite, Plasmodium falciparum. Merozoite invasion proceeds via a number of seemingly independent alternate pathways, such that entry can proceed with parasites lacking particular ligand-receptor interactions. To date, most focus in this regard has been on single-pass (type 1) membrane proteins that reside in the secretory organelles.

Distinct protein classes including novel merozoite surface antigens in Raft-like membranes of Plasmodium falciparum.

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol.

Plasmodium falciparum merozoite surface protein 8 is a ring-stage membrane protein that localizes to the parasitophorous vacuole of infected erythrocytes.

To date, the following seven glycosylphosphatidylinositol (GPI)-anchored merozoite antigens have been described in Plasmodium falciparum: merozoite-associated surface protein 1 (MSP-1), MSP-2, MSP-4, MSP-5, MSP-8, MSP-10, and the rhoptry-associated membrane antigen. Of these, MSP-1, MSP-8, and MSP-10 possess a double epidermal growth factor (EGF)-like domain at the C terminus, and these modules are considered potential targets of protective immunity. In this study, we found that surprisingly, P.

A subset of Plasmodium falciparum SERA genes are expressed and appear to play an important role in the erythrocytic cycle.

The Plasmodium falciparum serine repeat antigen (SERA) has shown considerable promise as a blood stage vaccine for the control of malaria. A related protein, SERPH, has also been described in P. falciparum.