MAHRP2, an exported protein of Plasmodium falciparum,is an essential component of Maurer's cleft tethers

Upon invasion into erythrocytes, the malaria parasite Plasmodium falciparum must refurbish the host cell. The objective of this study was to elucidate the location and function of MAHRP2 in these processes. Using immunofluorescence and immunoelectron microscopy we showed that the membrane‐associated histidine‐rich protein‐2 (MAHRP2) is exported during this process to novel cylindrical structures in the erythrocyte cytoplasm. We hypothesize that these structures tether organelles known as Maurer's clefts to the erythrocyte skeleton. Live cell imaging of parasite transfectants expressing MAHRP2–GFP revealed both mobile and fixed populations of the tether‐like structures. Differential centrifugation allowed the enrichment of these novel structures. MAHRP2 possesses neither a signal peptide nor a PEXEL motif, and sequences required for export were determined using transfectants expressing truncated MAHRP2 fragments. The first 15 amino acids and the histidine‐rich N‐terminal region are necessary for correct trafficking of MAHRP2 together with a predicted hydrophobic region. Solubilization studies showed that MAHRP2 is membrane associated but not membrane spanning. Several attempts to delete the mahrp2 gene failed, indicating that the protein is essential for parasite survival.     

Targeted mutagenesis of the ring-exported protein-1 of Plasmodium falciparum disrupts the architecture of Maurer's cleft organelles

Mature red blood cells have no internal trafficking machinery, so the intraerythrocytic malaria parasite, Plasmodium falciparum , establishes its own transport system to export virulence factors to the red blood cell surface. Maurer's clefts are parasite-derived membranous structures that form an important component of this exported secretory system. A protein with sequence similarity to a Golgi tethering protein, referred to as ring-exported protein-1 (REX1), is associated with Maurer's clefts. A REX1–GFP chimera is trafficked to the Maurer's clefts and preferentially associates with the edges of these structures, as well as with vesicle-like structures and with stalk-like extensions that are involved in tethering the Maurer's clefts to other membranes. We have generated transfected P. falciparum expressing REX1 truncations or deletion. Electron microscopy reveals that the Maurer's clefts of REX1 truncation mutants have stacked cisternae, while the 3D7 parent line has unstacked Maurer's clefts. D10 parasites, which have lost the right end of chromosome 9, including the rex1 gene, also display Maurer's clefts with stacked cisternae. Expression of full-length REX1–GFP in D10 parasites restores the 3D7-type unstacked Maurer's cleft phenotype. These studies reveal the importance of the REX1 protein in determining the ultrastructure of the Maurer's cleft system.     

Sequence requirements for the export of the Plasmodium falciparum Maurer's clefts protein REX2

A short motif termed Plasmodium export element (PEXEL) or vacuolar targeting signal (VTS) characterizes Plasmodium proteins exported into the host cell. These proteins mediate host cell modifications essential for parasite survival and virulence. However, several PEXEL-negative exported proteins indicate that the currently predicted malaria exportome is not complete and it is unknown whether and how these proteins relate to PEXEL-positive export. Here we show that the N-terminal 10 amino acids of the PEXEL-negative exported protein REX2 (ring-exported protein 2) are necessary for its targeting and that a single-point mutation in this region abolishes export. Furthermore we show that the REX2 transmembrane domain is also essential for export and that together with the N-terminal region it is sufficient to promote export of another protein. An N-terminal region and the transmembrane domain of the unrelated PEXEL-negative exported protein SBP1 (skeleton-binding protein 1) can functionally replace the corresponding regions in REX2, suggesting that these sequence features are also present in other PEXEL-negative exported proteins. Similar to PEXEL proteins we find that REX2 is processed, but in contrast, detect no evidence for N-terminal acetylation.     

Export of PfSBP1 to the Plasmodium falciparum Maurer's Clefts

The human malaria parasite Plasmodium falciparum exports determinants of virulence and pathology to destinations within the host erythrocyte, including the erythrocyte cytoplasm, plasma membrane and membrane profiles of parasite origin termed Maurer's clefts. Most of the exported proteins contain a conserved pentameric motif termed plasmodial export element (PEXEL)/vacuolar transfer signal (VTS) that functions as a cleavable sorting signal permitting export to the host erythrocyte. However, there are some exported proteins, such as the skeleton-binding protein 1 (PfSBP1) that lack the PEXEL/VTS motif and that are not N-terminally processed, suggesting the presence of alternative sorting signals and/or mechanisms. In this study, we have investigated trafficking of PfSBP1 to the Maurer's clefts. Our data show that the transmembrane domain of PfSBP1 functions as an internal signal sequence for entry into the parasite's secretory pathway and for transport to the parasite plasma membrane. Trafficking beyond the parasite's plasma membrane required additional N-terminal domains, which are characterized by a high negative net charge. Biochemical data indicate that these domains affect the solubility and extraction profile, the orientation of the protein within the membrane and the subcellular localization. Our findings suggest new principles of protein export in P.   falciparum -infected erythrocytes.     

Maurer's clefts-restricted localization, orientation and export of a Plasmodium falciparum RIFIN

RIFINs are clonally variant antigens expressed in Plasmodium falciparum. Transfection and the green fluorescence protein (GFP) tagged either internally or C-terminally to the 3D7 PFI0050c RIFIN gene product were used to investigate protein localization, orientation and trafficking. Green fluorescence pattern emerging from live transfectant parasites expressing each of the RIFIN-GFP chimera was different. The internally GFP-tagged protein was exported to Maurer's clefts (MC) in the erythrocyte cytosol, whereas the C-terminally GFP-tagged full-length RIFIN chimera was not trafficked out of the parasite. Interestingly, when some RIFIN-specific C-terminal amino acid sequences were removed, the resulting truncated molecule reached the MC. Using anti-RIFIN and anti-GFP antibodies to probe both live and fixed transfectants, staining was confined to MC and was not detected on the erythrocyte surface, a location previously suggested for this protein family. From selective permeabilization experiments, the highly variable portion of the RIFIN-GFP-insertion chimera appeared to be exposed to the erythrocyte cytosol, presumably anchored in the MC membrane via the two transmembrane domains. Trafficking of both chimeras in young ring stages was sensitive to Brefeldin A (BFA), although older rings showed differential sensitivity to BFA.     

MAHRP-1, a novel Plasmodium falciparum histidine-rich protein,binds ferriprotoporphyrin IX and localizes to the Maurer's clefts

Using a stage-specific cDNA library from Plasmodium falciparum we have identified a gene coding for a novel histidine-rich protein (MAHRP-1). The gene is exclusively transcribed during early erythrocyte stages and codes for a small transmembrane protein. The C-terminal region contains a polymorphic stretch of histidine-rich repeats. Fluorescence microscopy studies using polyclonal mouse sera revealed that MAHRP-1 is located at the Maurer's clefts, which represent parasite-induced structures within the cytosol of infected erythrocytes. Biochemical studies showed that recombinant MAHRP-1 binds the toxic hemoglobin degradation product, ferriprotoporphyrin (FP) with a submicromolar dissociation constant and a stoichiometry determined by the number of DHGH motifs. The bound FP has increased peroxidase-like activity and is 10-fold more susceptible to H2O2-induced degradation compared with unbound FP. These properties of MAHRP-1 suggest it may play a protective role against oxidative stress, and its location at the Maurer's clefts suggests a function in promoting the correct trafficking of exported proteins, such as P. falciparum erythrocyte membrane protein-1.     

3-D analysis of the Plasmodium falciparum Maurer's clefts using different electron tomographic approaches

The human malaria parasite Plasmodium falciparum exports a large number of proteins into its host erythrocyte to install functions necessary for parasite survival. Important structural components of the export machinery are membrane profiles of parasite origin, termed Maurer's clefts. These profiles span much of the distance between the parasite and the host cell periphery and are believed to deliver P. falciparum-encoded proteins to the erythrocyte plasma membrane. Although discovered more than a century ago, Maurer's clefts remain a mysterious organelle with little information available regarding their origin, their morphology or their precise role in protein trafficking. Here, we evaluated different techniques to prepare samples for electron tomography, including whole cell cryo-preparations, vitreous sections, freeze-substitution and chemical fixation. Our data show that the different approaches tested all have their merits, revealing different aspects of the complex structure of the Maurer's clefts.     

The Maurer's clefts of Plasmodium falciparum: parasite-induced islands within an intracellular ocean

It is suggested that Maurer's clefts, membranous structures observed within the cytoplasm of Plasmodium-falciparum-infected human erythrocytes, play an important role in trafficking virulence proteins from the parasite to the surface of the host cell. How they fulfil this role, however, still is unclear. A recent study by Bhattacharjee et al. now suggests that the clefts function as the major conduit through which parasite-encoded proteins pass before entering the host cell. In this article we comment on the significance of this information in our understanding of the novel 'extracellular' secretory pathway of this important human pathogen.     

Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum-infected erythrocytes reveals novel structural features

During intraerythrocytic development, the human malaria parasite, Plasmodium falciparum, establishes membrane-bound compartments, known as Maurer's clefts, outside the confines of its own plasma membrane. The Maurer's compartments are thought to be a crucial component of the machinery for protein sorting and trafficking; however, their ultrastructure is only partly defined. We have used electron tomography to image Maurer's clefts of 3D7 strain parasites. The compartments are revealed as flattened structures with a translucent lumen and a more electron-dense coat. They display a complex and convoluted morphology, and some regions are modified with surface nodules, each with a circular cross-section of approximately 25 nm. Individual 25 nm vesicle-like structures are also seen in the erythrocyte cytoplasm and associated with the red blood cell membrane. The Maurer's clefts are connected to the red blood cell membrane by regions with extended stalk-like profiles. Immunogold labelling with specific antibodies confirms differential labelling of the Maurer's clefts and the parasitophorous vacuole and erythrocyte membranes. Spot fluorescence photobleaching was used to demonstrate the absence of a lipid continuum between the Maurer's clefts and parasite membranes and the host plasma membrane.     

The Maurer's cleft protein MAHRP1 is essential for trafficking of PfEMP1 to the surface of Plasmodium falciparum-infected erythrocytes

During the intra-erythrocytic development of Plasmodium falciparum, the parasite modifies the host cell surface by exporting proteins that interact with or insert into the erythrocyte membrane. These proteins include the principal mediator of cytoadherence, P. falciparum erythrocyte membrane protein 1 (PfEMP1). To implement these changes, the parasite establishes a protein-trafficking system beyond its confines. Membrane-bound structures called Maurer's clefts are intermediate trafficking compartments for proteins destined for the host cell membrane. We disrupted the gene for the membrane-associated histidine-rich protein 1 (MAHRP1). MAHRP1 is not essential for parasite viability or Maurer's cleft formation; however, in its absence, these organelles become disorganized in permeabilized cells. Maurer's cleft-resident proteins and transit cargo are exported normally in the absence of MAHRP1; however, the virulence determinant, PfEMP1, accumulates within the parasite, is depleted from the Maurer's clefts and is not presented at the red blood cell surface. Complementation of the mutant parasites with mahrp1 led to the reappearance of PfEMP1 on the infected red blood cell surface, and binding studies show that PfEMP1-mediated binding to CD36 is restored. These data suggest an important role of MAHRP1 in the translocation of PfEMP1 from the parasite to the host cell membrane.     

The motor complex of Plasmodium falciparum: phosphorylation by a calcium-dependent protein kinase

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.     

Integral membrane protein located in the apical complex of Plasmodium falciparum.

We describe the cloning of a novel antigen of Plasmodium falciparum which contains a hydrophobic domain typical of an integral membrane protein. This antigen is designated apical membrane antigen 1 because it appears to be located in the apical complex. Apical membrane antigen 1 appears to be transported to the merozoite surface near the time of schizont rupture.     

PEXEL-independent trafficking of Plasmodium falciparum SURFIN4.2 to the parasite-infected red blood cell and Maurer's clefts

SURFIN_4.2 is a parasite-infected red blood cell (iRBC) surface associated protein of Plasmodium falciparum. To analyze the region responsible for the intracellular trafficking of SURFIN_4.2 to the iRBC and Maurer's clefts, a panel of transgenic parasite lines expressing recombinant SURFIN_4.2 fused with green fluorescent protein was generated and evaluated for their localization. We found that the cytoplasmic region containing a tryptophan rich (WR) domain is not necessary for trafficking, whereas the transmembrane (TM) region was. Two PEXEL-like sequences were shown not to be responsible for the trafficking of SURFIN_4.2, demonstrating that the protein is trafficked in a PEXEL-independent manner. N-terminal replacement, deletion of the cysteine-rich domain or the variable region also did not prevent the protein from localizing at the iRBC or Maurer's clefts. A recombinant SURFIN_4.2 protein possessing 50 amino acids upstream of the TM region, TM region itself and a part of the cytoplasmic region was shown to be trafficked into the iRBC and Maurer's clefts, suggesting that there are no essential trafficking motifs in the SURFIN_4.2 extracellular region. A mini-SURFIN_4.2 protein containing WR domain was shown by Western blotting to be more abundantly detected in a Triton X-100-insoluble fraction, compared to the one without WR domain. We suggest that the cytoplasmic region containing the WR may be responsible for their difference in solubility.     

Plasmodium falciparum expresses a multidrug resistance-associated protein

Plasmodium falciparum proteins that efflux toxic metabolic products such as oxidised glutathione (GSSG) are possible targets for anti-malarial drug development. Proteins capable of transporting GSSG and glutathione conjugates include the multidrug resistance-associated transporters (MRPs). A gene, PFA0590w, encoding a MRP homologue, has been identified in P. falciparum. Here we show the presence of full-length mRNA (5.5 kb) of this PfMRP in trophozoites by RT-PCR and Northern blotting. A polyclonal anti-PfMRP antibody generated against two unique, hydrophilic peptides in the predicted sequence produced a strong immunoreactive protein band of 210-215 kDa on Western blots of schizonts of chloroquine-sensitive and chloroquine-resistant strains, confirming expression of PfMRP protein. Using confocal microscopy the protein was seen to be localised at the edge of the schizonts with no obvious staining of the food vacuole. We suggest that PfMRP may act as the GSSG transporter in the parasite plasma membrane.     

Rab7 of Plasmodium falciparum is involved in its retromer complex assembly near the digestive vacuole

Background:Intracellular protein trafficking is crucial for survival of cell and proper functioning of the organelles; however, these pathways are not well studied in the malaria parasite. Its unique cellular architecture and organellar composition raise an interesting question to investigate.Methods:The interaction of Plasmodium falciparum Rab7 (PfRab7) with vacuolar protein sorting-associated protein 26 (PfVPS26) of retromer complex was shown by coimmunoprecipitation (co-IP). Confocal microscopy was used to show the localization of the complex in the parasite with respect to different organelles. Further chemical tools were employed to explore the role of digestive vacuole (DV) in retromer trafficking in parasite and GTPase activity of PfRab7 was examined.Results:PfRab7 was found to be interacting with retromer complex that assembled mostly near DV and the Golgi in trophozoites. Chemical disruption of DV by chloroquine (CQ) led to its disassembly that was further validated by using compound 5f, a heme polymerization inhibitor in the DV. PfRab7 exhibited Mg²⁺ dependent weak GTPase activity that was inhibited by a specific Rab7 GTPase inhibitor, CID 1067700, which prevented the assembly of retromer complex in P. falciparum and inhibited its growth suggesting the role of GTPase activity of PfRab7 in retromer assembly.Conclusion:Retromer complex was found to be interacting with PfRab7 and the functional integrity of the DV was found to be important for retromer assembly in P. falciparum.General significance:This study explores the retromer trafficking in P. falciparum and describes amechanism to validate DV targeting antiplasmodial molecules.     

Plasmodium falciparum: glycosylation status of Plasmodium falciparum circumsporozoite protein expressed in the baculovirus system

We expressed the main surface antigen of Plasmodium falciparum sporozoites, the circumsporozoite protein (CSP), in High Five (Trichoplusia ni) insect cells using the baculovirus system. Significant amounts of the recombinant protein could be obtained, as judged by SDS-PAGE, Western blot, and immunofluorescence analysis. The cellular localization for recombinant CSP was determined by immunofluorescence. The high fluorescence signal of the permeabilized cells, relative to that of fixed nonpermeabilized cells, revealed a clear intracellular localization of this surface antigen. Analysis of possible posttranslational modifications of CSP showed that this recombinant protein is only N-glycosylated in the baculovirus system. Although DNA-sequence analysis revealed a GPI-cleavage/attachment site, no GPI anchor could be demonstrated. These analyses show that the glycosylation status of this recombinant protein may not reflect its native form in P. falciparum. The impact of these findings on vaccine development will be discussed.     

Characterization of a membrane-associated rhoptry protein of Plasmodium falciparum

Invasive forms of apicomplexan parasites contain secretory organelles called rhoptries that are essential for entry into host cells. We present a detailed characterization of an unusual rhoptry protein of the human malaria parasite Plasmodium falciparum, the rhoptry-associated membrane antigen (RAMA) that appears to have roles in both rhoptry biogenesis and host cell invasion. RAMA is synthesized as a 170-kDa protein in early trophozoites, several hours before rhoptry formation and is transiently localized within the endoplasmic reticulum and Golgi within lipid-rich microdomains. Regions of the Golgi membrane containing RAMA bud to form vesicles that later mature into rhoptries in a process that is inhibitable by brefeldin A. Other rhoptry proteins such as RhopH3 and RAP1 are found in close apposition with RAMA suggesting direct protein-protein interactions. We suggest that RAMA is involved in trafficking of these proteins into rhoptries. In rhoptries, RAMA is proteolytically processed to give a 60-kDa form that is anchored in the inner face of the rhoptry membrane by means of the glycosylphosphatidylinositol anchor. The p60 RAMA form is discharged from the rhoptries of free merozoites and binds to the red blood cell membrane by its most C-terminal region. In early ring stages RAMA is found in association with the parasitophorous vacuole.     

Dissecting the mechanism and assembly of a complex virulence mycobacterial lipid

Mycobacterium tuberculosis cell envelope is a treasure house of biologically active lipids of fascinating molecular architecture. Although genetic studies have alluded to an array of genes in biosynthesis of complex lipids, their mechanistic, structural, and biochemical principles have not been investigated. Here, we have dissected the molecular logic underlying the biosynthesis of a virulence lipid phthiocerol dimycocerosate (PDIM). Cell-free reconstitution studies demonstrate that polyketide synthases, which are usually involved in the biosynthesis of secondary metabolites, are responsible for generating complex lipids in mycobacteria. We show that PapA5 protein directly transfers the protein bound mycocerosic acid analogs on phthiocerol to catalyze the final esterification step. Based on precise identification of biological functions of proteins from Pps cluster, we have rationally produced a nonmethylated variant of mycocerosate esters. Apart from elucidating mechanisms that generate chemical heterogeneity with PDIMs, this study also presents an attractive approach to explore host-pathogen interactions by altering mycobacterial surface coat.