After invading human red blood cells (RBCs) the malaria parasite Plasmodium falciparum remodels the host cell by trafficking proteins to the RBC compartment. The virulence protein P falciparum ...erythrocyte membrane protein 1 (PfEMP1) is responsible for cytoadherence of infected cells to host endothelial receptors. This protein is exported across the parasite plasma membrane and parasitophorous vacuole membrane and inserted into the RBC membrane. We have used green fluorescent protein chimeras and fluorescence photobleaching experiments to follow PfEMP1 export through the infected RBC. Our data show that a knob-associated histidine-rich protein (KAHRP) N-terminal protein export element appended to the PfEMP1 transmembrane and C-terminal domains was sufficient for efficient trafficking of protein domains to the outside of the P falciparum–infected RBC. The physical state of the exported proteins suggests trafficking as a complex rather than in vesicles and supports the hypothesis that endogenous PfEMP1 is trafficked in a similar manner. This study identifies the sequences required for expression of proteins to the outside of the P falciparum–infected RBC membrane.
Plasmodium falciparum (Pf) has a family of 11 Rab GTPases to regulate its vesicular transport. However, PfRab5B is unique in lacking a C-terminal geranyl-geranylation motif, while having N-terminal ...palmitoylation and myristoylation motifs. We show that the N-terminal glycine is required for PfRab5B myristoylation in vitro and when an N-terminal PfRab5B fragment possessing both acylation motifs is fused to GFP and expressed in transgenic P. falciparum parasites, the chimeric PfRab5B protein localizes to the plasma membrane. Upon substitution of the modified glycine by alanine the staining becomes diffuse and GFP is found in soluble subcellular fractions. Immuno-electron microscopy shows endogenous PfRab5B decorating the parasite's plasma and food vacuole membranes. Using reverse genetics rab5b couldn't be deleted from the haploid genome of asexual blood stage P. berghei parasites. The failure of PbRab5A or PbRab5C to complement for loss of PbRab5B function indicates non-overlapping roles for the three Plasmodium Rab5s, with PfRab5B involved in trafficking MSP1 to the food vacuole membrane and CK1 to the plasma membrane. We discuss similarities between Plasmodium Rab5B and Arabidopsis thaliana ARA6, a similarly unusual Rab5-like GTPase of plants.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
During the maturation of intracellular asexual stages of Plasmodium falciparum parasite‐encoded proteins are exported into the erythrocyte cytosol. A number of these parasite proteins attach ...to the host cell cytoskeleton and facilitate transformation of a disk‐shaped erythrocyte into a rounded and more rigid infected erythrocyte able to cytoadhere to the vasculature. Knob formation on the surface of infected erythrocytes is critical for this cytoadherence to the host endothelium. P. falciparum proteins have been identified that localize to the parasite‐infected erythrocyte membrane: the variant cytoadherence ligand erythrocyte membrane protein 1 (PfEMP1), the knob‐associated histidine‐rich protein (KAHRP) and the erythrocyte membrane protein 3 (PfEMP3). In this study, we have generated parasites expressing PfEMP3‐green fluorescent protein chimeras and identified domains involved in entry to the secretory pathway, export across the parasitophorous vacuolar membrane and attachment to Maurer's clefts and the erythrocyte membrane. Solubility assays, fluorescence photobleaching experiments and immunogold electron microscopy suggest that the exported chimeric proteins are trafficked in a complex rather than in vesicles. This study characterizes elements involved in the tight but transient binding of PfEMP3 to Maurer's clefts and shows that the same elements are necessary for correct assembly under the erythrocyte membrane.
Plasmodium yoelii YM asexual blood stage parasites express multiple members of the py235 gene family, part of the super-family of genes including those coding for Plasmodium vivax reticulocyte ...binding proteins and Plasmodium falciparum RH proteins. We previously identified a Py235 erythrocyte binding protein (Py235EBP-1, encoded by the PY01365 gene) that is recognized by protective mAb 25.77. Proteins recognized by a second protective mAb 25.37 have been identified by mass spectrometry and are encoded by two genes, PY01185 and PY05995/PY03534. We deleted the PY01365 gene and examined the phenotype. The expression of the members of the py235 family in both the WT and gene deletion parasites was measured by quantitative RT-PCR and RNA-Seq. py235ebp-1 expression was undetectable in the knockout parasite, but transcription of other members of the family was essentially unaffected. The knockout parasites continued to react with mAb 25.77; and the 25.77-binding proteins in these parasites were the PY01185 and PY05995/PY03534 products. The PY01185 product was also identified as erythrocyte binding. There was no clear change in erythrocyte invasion profile suggesting that the PY01185 gene product (designated PY235EBP-2) is able to fulfill the role of EBP-1 by serving as an invasion ligand although the molecular details of its interaction with erythrocytes have not been examined. The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family. In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional redundancy at the protein level gives the parasite phenotypic plasticity in the absence of differences in gene expression.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The PfPCRCR complex
, containing PfRH5 (refs.
), PfCyRPA, PfRIPR, ...PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies
or nanobodies
against each of these conserved proteins, making them the leading blood-stage malaria vaccine candidates. However, little is known about how PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex
, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. We test the hypothesis that PfRH5 opens to insert into the membrane
, instead showing that a rigid, disulfide-locked PfRH5 can mediate efficient erythrocyte invasion. We show, through modelling and an erythrocyte-binding assay, that PfCyRPA-binding antibodies
neutralize invasion through a steric mechanism. We determine the structure of PfRIPR, showing that it consists of an ordered, multidomain core flexibly linked to an elongated tail. We also show that the elongated tail of PfRIPR, which is the target of growth-neutralizing antibodies
, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. A modular PfRIPR is therefore linked to the merozoite membrane through an elongated tail, and its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides fresh insight into the molecular mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.
The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites ...adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.
The Motor Complex of Plasmodium falciparum Green, Judith L.; Rees-Channer, Roxanne R.; Howell, Stephen A. ...
The Journal of biological chemistry,
11/2008, Letnik:
283, Številka:
45
Journal Article
Recenzirano
Odprti dostop
Calcium-dependent protein kinases (CDPKs) of Apicomplexan parasites are crucial for the survival of the parasite throughout its life cycle. CDPK1 is expressed in the asexual blood stages of the ...parasite, particularly late stage schizonts. We have identified two substrates of Plasmodium falciparum CDPK1: myosin A tail domain-interacting protein (MTIP) and glideosome-associated protein 45 (GAP45), both of which are components of the motor complex that generates the force required by the parasite to actively invade host cells. Indirect immunofluorescence shows that CDPK1 localizes to the periphery of P. falciparum merozoites and is therefore suitably located to act on MTIP and GAP45 at the inner membrane complex. A proportion of both GAP45 and MTIP is phosphorylated in schizonts, and we demonstrate that both proteins can be efficiently phosphorylated by CDPK1 in vitro. A primary phosphorylation of MTIP occurs at serine 47, whereas GAP45 is phosphorylated at two sites, one of which could also be detected in phosphopeptides purified from parasite lysates. Both CDPK1 activity and host cell invasion can be inhibited by the kinase inhibitor K252a, suggesting that CDPK1 is a suitable target for antimalarial drug development.
We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment ...during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of “pseudoschizonts,” which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition. Understanding the essential factors needed for malaria parasite development could help us find new therapeutic targets. This study reveals that N-myristoylation is a posttranslational modification of proteins essential for the parasites’ growth and their invasion of red blood cells.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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