Effects on growth, hemoglobin metabolism and paralogous gene expression resulting from disruption of genes encoding the digestive vacuole plasmepsins of Plasmodium falciparum

Four of the plasmepsins of Plasmodium falciparum are localised in the digestive vacuole (DV) of the asexual blood stage parasite (PfPM1, PfPM2, PfPM4 and PfHAP), and each of these aspartic proteinases has been successfully targeted by gene disruption. This study describes further characterisation of the single-plasmepsin knockout mutants, and the creation and characterisation of double-plasmepsin knockout mutants lacking complete copies of pfpm2 and pfpm1 or pfhap and pfpm2. Double-plasmepsin knockout mutants were created by transfecting pre-existing knockout mutants with a second plasmid knockout construct. PCR and Southern blot analysis demonstrate the integration of a large concatamer of each plasmid construct into the targeted gene. All mutants have been characterised to assess the involvement of the DV plasmepsins in sustaining growth during the asexual blood stage. Analyses reaffirmed that knockout mutants Deltapfpm1 and Deltapfpm4 had lower replication rates in the asexual erythrocytic stage than the parental line (Dd2), but double-plasmepsin knockout mutants lacking intact copies of either pfpm2 and pfpm1, or pfpm2 and pfhap, had normal growth rates compared with Dd2. The amount of crystalline hemozoin produced per parasite during the asexual cycle was measured in each single-plasmepsin knockout to estimate the effect of each DV plasmepsin on hemoglobin digestion. Only Deltapfpm4 had a statistically significant reduction in hemozoin accumulation, indicating that hemoglobin digestion was impaired in this mutant. In the single-plasmepsin knockouts, no statistically significant differences were found in the steady state levels of mRNA from the remaining intact DV plasmepsin genes. Disruption of a DV plasmepsin gene does not affect the accumulation of mRNA encoding the remaining paralogous plasmepsins, and Western blot analysis confirmed that the accumulation of the paralogous plasmepsins in each knockout mutant was similar among all clones examined.     

The digestive food vacuole of the malaria parasite is a dynamic intracellular Ca2+ store

The acidic food vacuole of Plasmodium falciparum has been the subject of intense scientific investigation in the 40 years since its role in the digestion of host hemoglobin was first suggested. This proposed role has important implications for the complex host-parasite inter-relationship and also for the mode of action of several of the most effective antimalarial drugs. In addition, adaptive changes in the physiology of this organelle are implicated in drug resistance. Here we show that in addition to these functions, the digestive food vacuole of the malaria parasite is a dynamic internal store for free Ca2+, a role hitherto unsuspected. With the aid of live-cell laser scanning confocal imaging, spatiotemporal studies revealed that maintenance of elevated free Ca2+ in the digestive food vacuole (relative to cytosolic levels) is achieved by a thapsigargin (and cyclopiazonic acid)-sensitive Ca2+-pump in cooperation with a H+-dependent Ca2+ transporter. Redistribution of free cytosolic and vacuolar Ca2+ during parasite growth also suggests that vacuolar Ca2+ plays an essential role in parasite morphogenesis. These data imply that the digestive food vacuole of the malaria parasite is functionally akin to the vacuole of plants (tonoplast) and the small electron-dense granules of some parasites (acidocalcisomes) whereby H+-coupled Ca2+ transport is involved in ion transport, Ca2+ homeostasis, and signal transduction. These findings have significant implications for parasite development, antimalarial drug action, and mechanisms of drug resistance.     

Critical roles for the digestive vacuole plasmepsins of Plasmodium falciparum in vacuolar function

Knockout mutants of Plasmodium falciparum lacking pfpm1, pfpm2 and pfhap (triple-PM KO), and mutants lacking all four digestive vacuole (DV) plasmepsins (pfpm4, pfpm1, pfpm2 and pfhap; quadruple-PM KO), were prepared by double cross-over integration effecting chromosomal deletions of up to 14.6 kb. The triple-PM KO was similar to the parental line (3D7) in growth rate, morphology and sensitivity to proteinase inhibitors. The quadruple-PM KO showed a significantly slower rate of growth in standard medium, which manifested as delayed schizont maturation accompanied by reduced formation of haemozoin. In amino acid-limited medium, the reduction in growth rate of the quadruple-PM KO was pronounced. The sensitivity of both the triple- and quadruple-PM KOs to six different HIV aspartic proteinase inhibitors was comparable to that of 3D7, thus establishing that the DV plasmepsins were not the primary targets of the antimalarial activity of these clinically important compounds. Electron microscopic analysis revealed the presence of multilamellar bodies resembling ceroid in the DV of the quadruple-PM KO, and intermediates of the autophagic pathway accumulated as determined by Western blot analysis. Thus, the DV plasmepsins, although not essential, contribute significantly to the fitness of the parasite and are required for efficient degradation of endosomal vesicles delivered to the DV.     

Trafficking of the Phosphoprotein PfCRT to the Digestive Vacuolar Membrane in Plasmodium falciparum

The digestive vacuole plays an important role in the pathophysiology of the human malaria parasite Plasmodium falciparum. It is a terminal degradation organelle involved in the proteolysis of the host erythrocyte's haemoglobin; it is the site of action of several antimalarial drugs and its membrane harbours transporters implicated in drug resistance. How the digestive vacuole recruits residential proteins is largely unknown. Here, we have investigated the mechanism underpinning trafficking of the chloroquine resistance transporter, PfCRT, to the digestive vacuolar membrane. Nested deletion analysis and site‐directed mutagenesis identified threonine 416 as a functional residue for sorting PfCRT to its site of residence. Mass spectroscopy demonstrated that threonine 416 can be phosphorylated. Further phosphorylation was detected at serine 411. Our data establish PfCRT as a phosphoprotein and suggest that phosphorylation of threonine 416 is a possible deciding signal for the sorting of PfCRT to the digestive vacuolar membrane.     

Quantitative pH measurements in Plasmodium falciparum-infected erythrocytes using pHluorin

The digestive vacuole of the malaria parasite Plasmodium falciparum is the site of action of several antimalarial drugs, such as chloroquine, which accumulate in this organelle due to their properties as amphiphilic weak bases that inhibit haem detoxification. It has been suggested that changes in the pH of the digestive vacuole, affecting either drug partitioning or haem solubility and/or biomineralization rates, would correlate with reduced intracellular chloroquine accumulation and, hence, would determine the chloroquine-resistance phenotype. The techniques previously used to quantify digestive vacuolar pH mainly relied on lysed or isolated parasites, with unpredictable consequences on internal pH homeostasis. In this study, we have investigated the baseline steady-state pH of the cytoplasm and digestive vacuole of a chloroquine-sensitive (HB3) and a chloroquine-resistant (Dd2) parasite using a pH-sensitive green fluorescent protein, termed pHluorin. This non-invasive technique allows for in vivo pH measurements in intact P. falciparum-infected erythrocytes under physiological conditions. The data suggest that the pH of the cytoplasm is approximately 7.15 +/- 0.07 and that of the digestive vacuole approximately 5.18 +/- 0.05. No significant differences in baseline pH values were recorded for the chloroquine-sensitive and chloroquine-resistant parasites.     

Luciferase, when fused to an N-terminal signal peptide, is secreted from transfected Plasmodium falciparum and transported to the cytosol of infected erythrocytes

Plasmodium falciparum, a unicellular parasite that causes human malaria, infects erythrocytes where it develops within a vacuole. The vacuolar membrane separates the parasite from the erythrocyte cytosol. Some secreted parasite proteins remain inside the vacuole, and others are transported across the vacuolar membrane. To identify the protein sequences responsible for this distribution we investigated the suitability of the green fluorescent protein and luciferase as reporters in transiently transfected parasites. Because of the higher sensitivity of the enzymatic assay, luciferase was quantified 3 days after transfection, whereas reliable detection of green fluorescent protein required prolonged drug selection. Luciferase was confined to the parasite cytosol in subcellular fractions of infected erythrocytes. When parasites were transfected with a hybrid gene coding for the cleavable N-terminal signal peptide of a secreted parasite protein fused to luciferase, the reporter protein was secreted. It was recovered with the vacuolar content and the erythrocyte cytosol. The results suggest that no specific protein sequences are required for translocation across the vacuolar membrane. The high local concentration of luciferase within the vacuole argues against free diffusion, and thus transport into the erythrocyte cytosol must involve a rate-limiting step.     

Disruption of Toxoplasma gondii parasitophorous vacuoles by the mouse p47-resistance GTPases

The p47 GTPases are essential for interferon-gamma-induced cell-autonomous immunity against the protozoan parasite, Toxoplasma gondii, in mice, but the mechanism of resistance is poorly understood. We show that the p47 GTPases, including IIGP1, accumulate at vacuoles containing T. gondii. The accumulation is GTP-dependent and requires live parasites. Vacuolar IIGP1 accumulations undergo a maturation-like process accompanied by vesiculation of the parasitophorous vacuole membrane. This culminates in disruption of the parasitophorous vacuole and finally of the parasite itself. Over-expression of IIGP1 leads to accelerated vacuolar disruption whereas a dominant negative form of IIGP1 interferes with interferon-gamma-mediated killing of intracellular parasites. Targeted deletion of the IIGP1 gene results in partial loss of the IFN-gamma-mediated T. gondii growth restriction in mouse astrocytes.     

The role of Plasmodium falciparum food vacuole plasmepsins

Plasmepsins (PMs) are thought to have an important function in hemoglobin degradation in the malarial parasite Plasmodium falciparum and have generated interest as antimalarial drug targets. Four paralogous plasmepsins reside in the food vacuole of P. falciparum. Targeted gene disruption by double crossover homologous recombination has been employed to study food vacuole plasmepsin function in cultured parasites. Parasite clones with deletions in each of the individual PM I, PM II, and HAP genes as well as clones with a double PM IV/PM I disruption have been generated. All of these clones lack the corresponding PMs, are viable, and appear morphologically normal. PM II and PM IV/I disruptions have longer doubling times than the 3D7 parental line in rich RPMI medium. This appears to be because of a decreased level of productive progeny rather than an increased cell cycle time. In amino acid-limited medium, all four knockouts exhibit slower growth than the parental strain. Compared with 3D7, knock-out clone sensitivity to aspartic and cysteine protease inhibitors is changed minimally. These results suggest substantial functional redundancy and have important implications for the design of antimalarial drugs. The slow growth phenotype may explain why P. falciparum has maintained four plasmepsin genes with overlapping functions.     

Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum

The P-glycoprotein homolog of the human malaria parasite Plasmodium falciparum (Pgh-1) has been implicated in decreased susceptibility to several antimalarial drugs, including quinine, mefloquine and artemisinin. Pgh-1 mainly resides within the parasite's food vacuolar membrane. Here, we describe a surrogate assay for Pgh-1 function based on the subcellular distribution of Fluo-4 acetoxymethylester and its free fluorochrome. We identified two distinct Fluo-4 staining phenotypes: preferential staining of the food vacuole versus a more diffuse staining of the entire parasite. Genetic, positional cloning and pharmacological data causatively link the food vacuolar Fluo-4 phenotype to those Pgh-1 variants that are associated with altered drug responses. On the basis of our data, we propose that Pgh-1 imports solutes, including certain antimalarial drugs, into the parasite's food vacuole. The implications of our findings for drug resistance mechanisms and testing are discussed.     

A nonpermeant biotin derivative gains access to the parasitophorous vacuole in Plasmodium falciparum-infected erythrocytes permeabilized with streptolysin O

In its host erythrocyte, the malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole, the membrane of which forms a barrier between the host cell cytosol and the parasite surface. The vacuole is a unique compartment because it contains specific proteins that are believed to be involved in cell biological functions essential for parasite survival. As a prerequisite for the characterization of the vacuolar proteome, we have developed an experimental approach that allows the selective biotinylation of soluble vacuolar proteins. This approach utilizes nonpermeant biotin derivatives that can be introduced into infected erythrocytes after selective permeabilization of the erythrocyte membrane with the pore-forming protein streptolysin O. The derivatives gain access to the vacuolar lumen but not to the parasite cytosol, thus providing supportive evidence for the existence of nonselective pores within the vacuolar membrane that have been postulated based on electrophysiological studies. Soluble vacuolar proteins that are biotin-labeled can be isolated by affinity chromatography using streptavidin-agarose.     

Characterization of the Chloroquine Resistance Transporter Homologue in Toxoplasma gondii

Mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein confer resistance to the antimalarial drug chloroquine. PfCRT localizes to the parasite digestive vacuole, the site of chloroquine action, where it mediates resistance by transporting chloroquine out of the digestive vacuole. PfCRT belongs to a family of transporter proteins called the chloroquine resistance transporter family. CRT family proteins are found throughout the Apicomplexa, in some protists, and in plants. Despite the importance of PfCRT in drug resistance, little is known about the evolution or native function of CRT proteins. The apicomplexan parasite Toxoplasma gondii contains one CRT family protein. We demonstrate that T. gondii CRT (TgCRT) colocalizes with markers for the vacuolar (VAC) compartment in these parasites. The TgCRT-containing VAC is a highly dynamic organelle, changing its morphology and protein composition between intracellular and extracellular forms of the parasite. Regulated knockdown of TgCRT expression resulted in modest reduction in parasite fitness and swelling of the VAC, indicating that TgCRT contributes to parasite growth and VAC physiology. Together, our findings provide new information on the role of CRT family proteins in apicomplexan parasites.     

Toxoplasma gondii PPM3C,a secreted protein phosphatase,affects parasitophorous vacuole effector export

The intracellular parasite Toxoplasma gondii infects a large proportion of humans worldwide and can cause adverse complications in the settings of immune-compromise and pregnancy. T. gondii thrives within many different cell types due in part to its residence within a specialized and heavily modified compartment in which the parasite divides, termed the parasitophorous vacuole. Within this vacuole, numerous proteins optimize intracellular survival following their secretion by the parasite. We investigated the contribution of one of these proteins, TgPPM3C, predicted to contain a PP2C-class serine/threonine phosphatase domain and previously shown to interact with the protein MYR1, an essential component of a putative vacuolar translocon that mediates effector export into the host cell. Parasites lacking the TgPPM3C gene exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. Phosphoproteomic assessment of TgPPM3C deleted parasite cultures demonstrated alterations in the phosphorylation status of many secreted vacuolar proteins including two exported effector proteins, GRA16 and GRA28, as well as MYR1. Parasites lacking TgPPM3C are defective in GRA16 and GRA28 export, but not in the export of other MYR1-dependant effectors. Phosphomimetic mutation of two GRA16 serine residues results in export defects, suggesting that de-phosphorylation is a critical step in the process of GRA16 export. These findings provide another example of the emerging role of phosphatases in regulating the complex environment of the T. gondii parasitophorous vacuole and influencing the export of specific effector proteins from the vacuolar lumen into the host cell.     

The plasmodium digestive vacuole: metabolic headquarters and choice drug target

The Plasmodium digestive (food) vacuole is an acidic proteolytic compartment central to the metabolism of the parasite. Here haemoglobin is degraded, haem is polymerized, amino acid are transported, oxygen radicals are detoxified, drugs are accumulated, acidification is maintained and free iron may be generated. Despite these crucial roles in parasite development, a number of questions about the digestive vacuole and the haemoglobin ingestion pathway remain unanswered; in consequence, a number of attractive drug targets remain to be exploited. Piero Olliaro and Daniel Goldberg here review the morphology, metabolism and pharmacological disruption of this specialized organelle.     

Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae. II. VAM7, a gene for regulating morphogenic assembly of the vacuoles.

VAM7 gene function has shown to be required for proper morphogenesis of the vacuole in yeast. The DNA fragments that complemented the defective vacuolar morphology of the vam7-1 mutation were isolated from a yeast genomic library. An overlapping 2.5-kilobase BglII-HindIII region was found to be sufficient for complementation of the vam7-1 phenotype. This fragment was integrated at the chromosomal VAM7 locus, indicating that it contained an authentic VAM7 gene. On nucleotide sequencing of the VAM7 gene, an open reading frame of 948 base pairs, coding for a hydrophilic polypeptide of 316 amino acid residues, was identified. The deduced amino acid sequence of the carboxyl-terminal region of the VAM7 gene product has heptad repeats and shows potential ability to form a coiled-coil structure. Disruption of VAM7 was not lethal; cells with a disrupted VAM7 gene did not, however, have a prominent large vacuoles but rather numerous small compartments that accumulated the histochemical marker molecule of the vacuolar compartment. They contained mature forms of the vacuolar marker proteins carboxypeptidase Y and vacuolar glycoprotein vgp72. A mutant with both vam7 and vam5 null mutations was constructed and shown to have neither vacuolar structures stained with ade-related fluorochrome nor mature forms of vacuolar marker proteins. These findings suggested that the VAM7 gene product functions in the process of morphogenic assembly of the vacuolar compartment but is not involved in the protein sorting and delivery to the vacuole.     

Structural studies of vacuolar plasmepsins

Plasmepsins (PMs) are pepsin-like aspartic proteases present in different species of parasite Plasmodium. Four Plasmodium spp. (P. vivax, P. ovale, P. malariae, and the most lethal P. falciparum) are mainly responsible for causing human malaria that affects millions worldwide. Due to the complexity and rate of parasite mutation coupled with regional variations, and the emergence of P. falciparum strains which are resistant to antimalarial agents such as chloroquine and sulfadoxine/pyrimethamine, there is constant pressure to find new and lasting chemotherapeutic drug therapies. Since many proteases represent therapeutic targets and PMs have been shown to play an important role in the survival of parasite, these enzymes have recently been identified as promising targets for the development of novel antimalarial drugs. The genome of P. falciparum encodes 10 PMs (PMI, PMII, PMIV-X and histo-aspartic protease (HAP)), 4 of which (PMI, PMII, PMIV and HAP) reside within the food vacuole, are directly involved in degradation of human hemoglobin, and share 50-79% amino acid sequence identity. This review focuses on structural studies of only these four enzymes, including their orthologs in other Plasmodium spp.. Almost all original crystallographic studies were performed with PMII, but more recent work on PMIV, PMI, and HAP resulted in a more complete picture of the structure-function relationship of vacuolar PMs. Many structures of inhibitor complexes of vacuolar plasmepsins, as well as their zymogens, have been reported in the last 15 years. Information gained by such studies will be helpful for the development of better inhibitors that could become a new class of potent antimalarial drugs. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Protein targeting to the yeast vacuole

Mutational and gene fusion studies have identified localization signals that target proteins to the yeast lysosome-like vacuole. Genetic analyses have also identified groups of genes (VPS and PEP) whose products are required for recognition of these signals, and sorting and transport of proteins to the vacuole. One of the components involved in protein sorting has been shown to be the vacuolar H+-ATPase, presumably via its role in vacuolar acidification.     

Vac14 controls PtdIns(3,5)P(2) synthesis and Fab1-dependent protein trafficking to the multivesicular body

BACKGROUND: The PtdIns3P 5-kinase Fab1 makes PtdIns(3,5)P(2), a phosphoinositide essential for retrograde trafficking between the vacuole/lysosome and the late endosome and also for trafficking of some proteins into the vacuole via multivesicular bodies (MVB). No regulators of Fab1 were identified until recently. RESULTS: Visual screening of the Eurofan II panel of S. cerevisiae deletion mutants identified YLR386w as a novel regulator of vacuolar function. Others recently identified this ORF as encoding the vacuolar inheritance gene VAC14. Like fab1 mutants, yeast lacking Vac14 have enlarged vacuoles that do not acidify correctly. FAB1 overexpression corrects these defects. vac14Delta cells make very little PtdIns(3,5)P(2), and hyperosmotic shock does not stimulate PtdIns(3,5)P(2) synthesis in the normal manner, implicating Vac14 in Fab1 regulation. We also show that, like fab1Delta mutants, vac14Delta cells fail to sort GFP-Phm5 to the MVB and thence to the vacuole: irreversible ubiquitination of GFP-Phm5 overcomes this defect. In the BY4742 genetic background, loss of Vac14 causes much more penetrant effects on phosphoinositide metabolism and vacuolar trafficking than does loss of Vac7, another regulator of Fab1. Vac14 contains motifs suggestive of a role in protein trafficking and interacts with several proteins involved in clathrin-mediated membrane sorting and phosphoinositide metabolism. CONCLUSIONS: Vac14 and Vac7 are both upstream activators of Fab1-catalysed PtdIns(3,5)P(2) synthesis, with Vac14 the dominant contributor to the hierarchy of control. Vac14 is essential for the regulated synthesis of PtdIns(3,5)P(2), for control of trafficking of some proteins to the vacuole lumen via the MVB, and for maintenance of vacuole size and acidity.     

Loss of pH Control in Plasmodium falciparum Parasites Subjected to Oxidative Stress

The intraerythrocytic malaria parasite is susceptible to oxidative stress and this may play a role in the mechanism of action of some antimalarial agents. Here we show that exposure of the intraerythrocytic malaria parasite to the oxidising agent hydrogen peroxide results in a fall in the intracellular ATP level and inhibition of the parasite''s V-type H⁺-ATPase, causing a loss of pH control in both the parasite cytosol and the internal digestive vacuole. In contrast to the V-type H⁺-ATPase, the parasite''s digestive vacuole H⁺-pyrophosphatase is insensitive to hydrogen peroxide-induced oxidative stress. This work provides insights into the effects of oxidative stress on the intraerythrocytic parasite, as well as providing an alternative possible explanation for a previous report that light-induced oxidative stress causes selective lysis of the parasite''s digestive vacuole.     

The pH of the Plasmodium falciparum digestive vacuole: holy grail or dead-end trail?

The maintenance of acidic pH in the digestive vacuole of the malaria parasite is thought to be crucial to the digestion of host cell haemoglobin and the subsequent process of heme detoxification. It may also be important in the mode of action of chloroquine and in the mechanism of resistance to the drug. Obtaining a definitive measurement of digestive vacuole pH has been surprisingly difficult. Some of the techniques for the measurement of pH in acid vesicles are outlined here along with some key aspects that are specific to malaria parasites. The use of acridine orange and dextran-tagged dyes as probes for the measurement of digestive vacuole pH has proved problematic, yet some surprising findings have emerged from work with these compounds.     

Acidification of the young phagosomes ofParamecium is mediated by proton pumps derived from the acidosomes

Summary Although it is generally accepted that phagosome acidification is induced through the activity of a vacuolar proton pump (V-ATPase) present on the phagosome membrane, exactly how these pumps are delivered to the phagosomes is not well understood. To study this question inParamecium, it was necessary to first show that an authentic V-ATPase was present on their phagosomal membranes. Three antibodies raised against V-ATPases or their subunits were each found to label one or two large digestive vacuoles (DVs) inParamecium multimicronucleatum when immunofluorescence microscopy was used. Using horseradish peroxidase immunocytochemistry to increase sensitivity, about 10 DVs were shown to contain a V-ATPase. In high magnification images and cryoultramicrotomy these proton pumps were found to be located on the acidosomes, suggesting the vacuolar proton pumps on the DVs originate from the acidosomes. The authenticity of the V-ATPase was further confirmed by its sensitivity to cold temperature and to the V-ATPase specific inhibitor, concanamycin B, which at 10 nM doubled the t_1/2 for vacuole acidification. Thus, we conclude that (1) acidosomes and some DVs ofParamecium have a bona-fide concanamycin B-sensitive and cold-sensitive V-ATPase, (2) the V-ATPase is delivered to the young DVs during acidosome fusion, and (3) the V-ATPase is involved in vacuole acidification. Finally, we have now determined thatParamecium has two immunologically related V-ATPases that are involved in two very different functions, (1) the acidification of phagosomes and (2) fluid segregation in the contractile vacuole complexes.Abbreviations BS-FITC bovine serum albumin-fluorescein isothiocyanate - CVC contractile vacuole complex - DV-I to DV-IV digestive vacuole stages 1 to 4 - HRP horseradish peroxidase - V-ATPase vacuolar proton pump