Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems

Degradation of host hemoglobin by the human malaria parasite Plasmodium falciparum is a massive metabolic process. What role this degradation plays and whether it is essential for parasite survival have not been established, nor have the roles of the various degradative enzymes been clearly defined. We report that P. falciparum can grow in medium containing a single amino acid (isoleucine, the only amino acid missing from human hemoglobin). In this medium, growth of hemoglobin-degrading enzyme gene knockout lines (missing falcipain-2 and plasmepsins alone or in combination) is impaired. Blockade of plasmepsins with the potent inhibitor pepstatin A has a minimal effect on WT parasite growth but kills falcipain-2 knockout parasites at low concentrations and is even more potent on falcipain-2, plasmepsin I and IV triple knockout parasites. We conclude that: (i) hemoglobin degradation is necessary for parasite survival; (ii) hemoglobin degradation is sufficient to supply most of the parasite's amino acid requirements; (iii) external amino acid acquisition and hemoglobin digestion are partially redundant nutrient pathways; (iv) hemoglobin degradation uses dual protease families with overlapping function; and (v) hemoglobin-degrading plasmepsins are not promising drug targets.     

The hydration state of human red blood cells and their susceptibility to invasion by Plasmodium falciparum

In most inherited red blood cell (RBC) disorders with high gene frequencies in malaria-endemic regions, the distribution of RBC hydration states is much wider than normal. The relationship between the hydration state of circulating RBCs and protection against severe falciparum malaria remains unexplored. The present investigation was prompted by a casual observation suggesting that falciparum merozoites were unable to invade isotonically dehydrated normal RBCs. We designed an experimental model to induce uniform and stable isotonic volume changes in RBC populations from healthy donors by increasing or decreasing their KCl contents through a reversible K(+) permeabilization pulse. Swollen and mildly dehydrated RBCs were able to sustain Plasmodium falciparum cultures with similar efficiency to untreated RBCs. However, parasite invasion and growth were progressively reduced in dehydrated RBCs. In a parallel study, P falciparum invasion was investigated in density-fractionated RBCs from healthy subjects and from individuals with inherited RBC abnormalities affecting primarily hemoglobin (Hb) or the RBC membrane (thalassemias, hereditary ovalocytosis, xerocytosis, Hb CC, and Hb CS). Invasion was invariably reduced in the dense cell fractions in all conditions. These results suggest that the presence of dense RBCs is a protective factor, additional to any other protection mechanism prevailing in each of the different pathologies.     

Fatty acid synthase is preferentially degraded by autophagy upon nitrogen starvation in yeast

Autophagy, an evolutionarily conserved intracellular catabolic process, leads to the degradation of cytosolic proteins and organelles in the vacuole/lysosome. Different forms of selective autophagy have recently been described. Starvation-induced protein degradation, however, is considered to be nonselective. Here we describe a novel interaction between autophagy-related protein 8 (Atg8) and fatty acid synthase (FAS), a pivotal enzymatic complex responsible for the entire synthesis of C16- and C18-fatty acids in yeast. We show that although FAS possesses housekeeping functions, under starvation conditions it is delivered to the vacuole for degradation by autophagy in a Vac8- and Atg24-dependent manner. We also provide evidence that FAS degradation is essential for survival under nitrogen deprivation. Our results imply that during nitrogen starvation specific proteins are preferentially recruited into autophagosomesAutophagy is a major cellular catabolic pathway in eukaryotes, responsible for the degradation of organelles and large protein aggregates. This process is induced under various stress conditions, such as amino acid starvation, hypoxia, and oxidative stress (1, 2).In yeast (Saccharomyces cerevisiae), nitrogen starvation is known to induce macroautophagy (hereafter termed autophagy), which is essential mainly for supplementing amino acids needed for protein synthesis (3). Autophagy has long been considered to be a nonselective process except for the recruitment of ribosomes into autophagosomes, which was suggested to be selective under conditions of nitrogen starvation (4). Selective autophagy is thought to play a role mainly in cell homeostasis, and it uses the core autophagy genes and varying sets of proteins for different cargos (5). Autophagy-related protein 8 (Atg8), a key autophagic factor essential for autophagosome biogenesis, also plays a major role in selective autophagy by recruiting cargo proteins into the autophagosome (6).Fatty acid synthase (FAS) is a large (2.6 MDa), essential enzymatic complex responsible for the entire synthesis of C16- and C18-fatty acids (7). FAS is composed of two subunits, Fas1 (Fasβ) and Fas2 (Fasα), which are arranged in an α₆β₆ macromolecular complex. Deletion of one of the FAS subunits has been shown to result in degradation of the other (8). Whereas the unassembled Fas2 subunit is short-lived and degrades in the proteasome, the unassembled Fas1, once induced, is degraded by autophagy. Moreover, the FAS complex is known to be delivered to the vacuole under potassium acetate starvation (8).Here we investigated the role of autophagy in the degradation of the FAS complex. We report a physical interaction between Atg8 and FAS, and show that autophagy preferentially degrades FAS and decreases its activity during nitrogen starvation. We suggest that the selective degradation of FAS under nitrogen starvation requires the participation of Atg24 and Vac8, two factors that each play a role in selective autophagy (9–12). Finally, we show that FAS degradation is important for cell viability. We propose that selective degradation of proteins under nitrogen starvation occurs to maintain cell homeostasis.     

A calpain unique to alveolates is essential in Plasmodium falciparum and its knockdown reveals an involvement in pre-S-phase development

Plasmodium falciparum encodes a single calpain that has a distinct domain composition restricted to alveolates. To evaluate the potential of this protein as a drug target, we assessed its essentiality. Both gene disruption by double cross-over and gene truncation by single cross-over recombination failed. We were also unable to achieve allelic replacement by using a missense mutation at the catalytic cysteine codon, although we could obtain synonymous allelic replacement parasites. These results suggested that the calpain gene and its proteolytic activity are important for optimal parasite growth. To gain further insight into its biological role, we used the FKBP degradation domain system to generate a fusion protein whose stability in transfected parasites could be modulated by a small FKBP ligand, Shield1 (Shld1). We made a calpain-GFP-FKBP fusion through single cross-over integration at the endogenous calpain locus. Calpain levels were knocked down and parasite growth was greatly impaired in the absence of Shld1. Parasites were delayed in their ability to transition out of the ring stage and in their ability to progress to the S phase. Calpain is required for cell cycle progression in Plasmodium parasites and appears to be an attractive drug target. We have shown that regulated knockdowns are possible in P. falciparum and can be useful for evaluating essentiality and function.     

经输血感染恶性疟原虫1例

本文报道了1例因急性淋巴细胞白血病输血后感染恶性疟原虫的病例。通过对该患者及相关供血者进行流行病学调查和实验室检测，发现传染源为1名非洲籍留学生。该供血者血样经快速诊断试纸条检测为阳性，其外周血涂片镜检及PCR检测结果均提示恶性疟原虫阳性。     

Inactivation of Plasmodium falciparum by photodynamic excitation of heme-cycle intermediates derived from delta-aminolevulinic acid

Transfusion-transmitted malaria (TTM), especially that caused by Plasmodium falciparum, is of great concern in malaria-endemic areas. As a result of increased international travel, migration, and the spread of drug-resistant parasites, TTM is also a growing problem in industrialized nations. An effective and inexpensive means of inactivating malaria parasites in blood products would represent an important advance. In this report, we demonstrate that photoactivation of plasmodial heme-cycle intermediates, derived from supplemental delta -aminolevulinic acid (ALA), by exposure to simple white light in the presence of ALA, reduces P. falciparum in culture to levels that are undetectable by light microscopy or lactate dehydrogenase assay. Photodynamic excitation of presumed heme-cycle intermediates, which was revealed by fluorescence microscopy, did not appear to adversely affect the viability of erythrocytes. These data suggest that this pathogen-inactivation strategy, which uses inexpensive reagents and white light, may represent an appropriate means of inactivating malaria parasites in blood products in resource-poor settings.     

Malaria transmitted to humans by mosquitoes infected from cultured Plasmodium falciparum

Malaria was transmitted to six normal human volunteers by mosquitoes infected from cultured gametocytes of Plasmodium falciparum. This method, which offers advantages over other methods of infecting volunteers, will be useful for evaluating the efficacy of human malaria vaccines.     

Interspersed blocks of repetitive and charged amino acids in a dominant immunogen of Plasmodium falciparum.

We describe an antigen of Plasmodium falciparum that is a dominant immunogen in man. The corresponding cDNA clone, Ag231, expressing this antigen in Escherichia coli reacted in an in situ colony assay with sera from up to approximately equal to 93% of 65 people living in an area in which P. falciparum is endemic. Human antibodies affinity purified on immobilized Ag231 lysates identified the corresponding parasite antigen as a polypeptide of Mr approximately equal to 300,000. It was present in schizonts and also in ring-stage trophozoites, where a speckled immunofluorescence pattern suggested an association with the erythrocyte. Its mRNA was enriched in merozoites relative to other blood stages, a distinctive property shared by a recently described antigen located on the surface of ring-infected erythrocytes, and it is encoded by a single gene having a number of allelic variants. The complete nucleotide sequence of Ag231 revealed a structural unit composed of 13 hexapeptide repeats flanked by a highly charged region containing both acidic and basic amino acids. This structural unit is itself repeated, so that blocks of repeats and charged units are interspersed along the molecule. The sequences within the repeats vary much more extensively than those in the charged units.     

From the Cover: Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH₂]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.     

Measuring allelic heterogeneity in Plasmodium falciparum by a heteroduplex tracking assay

We developed a novel Plasmodium falciparum genotyping strategy based on the heteroduplex tracking assay (HTA) method commonly used to genotype viruses. Because it can detect both sequence and size polymorphisms, we hypothesized that HTA is more sensitive than current methods. To test this hypothesis, we compared the ability of HTA and a nested polymerase chain reaction (PCR) to detect genetic diversity in 17 Thai samples. The HTA detected more MSP1 sequence variants in eight isolates (47%), less sequence variants in three isolates (18%), and an equal number of sequence variants in six isolates (35%), suggesting that HTA is equal to or more sensitive than the nested PCR. This study is a proof of concept that HTA is a sensitive allelic discrimination method able to determine genetic diversity in P. falciparum and warrants its use in studies of antimalarial drug efficacy.     

Characterization of a Plasmodium falciparum erythrocyte-binding protein paralogous to EBA-175

A member of a Plasmodium receptor family for erythrocyte invasion was identified on chromosome 13 from the Plasmodium falciparum genome sequence of the Sanger Centre (Cambridge, U.K.). The protein (named BAEBL) has homology to EBA-175, a P. falciparum receptor that binds specifically to sialic acid and the peptide backbone of glycophorin A on erythrocytes. Both EBA-175 and BAEBL localize to the micronemes, organelles at the invasive ends of the parasites that contain other members of the family. Like EBA-175, the erythrocyte receptor for BAEBL is destroyed by neuraminidase and trypsin, indicating that the erythrocyte receptor is a sialoglycoprotein. Its specificity, however, differs from that of EBA-175 in that BAEBL can bind to erythrocytes that lack glycophorin A, the receptor for EBA-175. It has reduced binding to erythrocytes with the Gerbich mutation found in another erythrocyte, sialoglycoprotein (glycophorin C/D). The interest in BAEBL's reduced binding to Gerbich erythrocytes derives from the high frequency of the Gerbich phenotype in some regions of Papua New Guinea where P. falciparum is hyperendemic.     

Rabbit and human antibodies to a repeated amino acid sequence of a Plasmodium falciparum antigen, Pf 155, react with the native protein and inhibit merozoite invasion.

The Plasmodium falciparum-derived antigen of Mr 155,000 designated Pf 155, deposited in the membrane of infected erythrocytes, contains at least two blocks of tandemly repeated amino acid sequences. The peptide Glu-Glu-Asn-Val-Glu-His-Asp-Ala, which corresponds to a subunit of a C-terminally located repeat, was synthesized. Rabbits immunized with the octapeptide conjugated with either keyhole limpet hemocyanine or tetanus toxoid formed antibodies against the octapeptide. These antibodies reacted with Pf 155 as detected by immunoblotting or a modified immunofluorescence assay. Sera from humans exposed to P. falciparum also contained antibodies binding to the octapeptide in a dot-blot immunoassay. Their anti-octapeptide titers were correlated with their immunofluorescence titers in the assay detecting Pf 155 and other parasite antigens in the membrane of infected erythrocytes. Human octapeptide-reactive antibodies were isolated on an affinity column with the octapeptide conjugated to bovine serum albumin as ligand. These human antibodies reacted with Pf 155 in immunoblotting and strongly stained the surface of infected erythrocytes in the modified immunofluorescence assay. Approximately 20% of this immunofluorescence activity in a high-titered human serum could be recovered from the octapeptide column, indicating that a significant fraction of these anti-parasite antibodies react with epitopes associated with the octapeptide. Furthermore, the human octapeptide-reactive antibodies very efficiently inhibited merozoite reinvasion into erythrocytes in vitro. Similarly purified rabbit antibodies also significantly inhibited reinvasion. Our results suggest that the C-terminal segment of repeated peptides in Pf 155 is a major antigenic region of the molecule and may contain target sites for protective immunity in P. falciparum malaria.     

复式PCR检测间日疟原虫和恶性疟原虫的现场应用

目的探讨复式PCR方法在疟疾诊断中的现场应用价值。方法根据疟原虫18 S rRNA基因序列,合成疟原虫属特异性上游引物和间日疟原虫、恶性疟原虫种特异性下游引物,优化PCR反应体系,建立在同一PCR反应体系中同时检测间日疟原虫、恶性疟原虫基因组特异性DNA片段的疟疾诊断方法,并评价其现场应用价值。结果间日疟原虫和恶性疟原虫基因组DNA经过复式PCR后,分别扩增出1 451 bp和833 bp的特异性条带,而伯氏疟原虫、食蟹猴疟原虫及健康人血样均无扩增带出现,用该反应体系可检出原虫血症为1.1×10-6和5.6×10-7的间日疟原虫和恶性疟原虫感染。与镜检法相比,复式PCR检测119份现场样本,112份与镜检结果相同,阳性率为54%,漏诊率为0.8%,误诊率为0,而镜检法依次分别为53%、1.7%和3.4%。结论复式PCR方法检测疟原虫具有简便、快速、特异、敏感等优点,在疑似病例的鉴别诊断和分子流行病学调查中具有良好的应用价值。     

Plasmodium falciparum genotyping by microsatellites as a method to distinguish between recrudescent and new infections

In vivo tests for susceptibility to antimalarial drugs require molecular methods to distinguish recrudescence from new infection. The most commonly used DNA markers (merozoite surface proteins [MSPs]) are under immune selective pressure, which might lead to misclassification. We evaluated immunologically neutral microsatellite markers in blood samples collected during a drug efficacy trial in Rwanda. Fifty percent of the infections classified as recrudescent by MSP were classified as new by microsatellite markers. Reciprocally, 23.3% of infections classified as recrudescent by microsatellite markers were identified as new by MSP. In drug efficacy studies, microsatellite markers should complement MSP genotyping to distinguish a recrudescence from a new infection.     

维和人员恶性疟原虫感染合并支气管肺炎1例

疟疾是由疟原虫感染引起的一种急性发热疾病.在非洲疟疾高负担地区,呼吸道感染等引起的发热症状会影响疟疾诊断,诊断不明且已服用抗疟药物的患者尤其应引起注意.本文报道1例恶性疟合并支气管肺炎病例.     

Differential antibody recognition of FC27-like Plasmodium falciparum merozoite surface protein MSP2 antigens which lack 12 amino acid repeats

Three alleles of the FC27-type allelic family of the MSP2 gene of the malaria parasite Plasmodium falciparum have been sequenced from parasites from the field (The Gambia and Tanzania). These alleles lack the 12 amino acid repeat units which are usual in this family of MSP2 alleles. We have investigated the recognition by sera from an endemic area (The Gambia) of three recombinant MSP2 proteins that have 5, 1 and no copies of this repeat region. Antibody recognition of these recombinant proteins varied according to the number of repeats present. High titre antibody levels were seen with most sera using the recombinant protein with 5 × 12-mer repeats, whereas only low responses were measured using proteins containing 1 or no 12-mer repeats. Several sera entirely failed to recognise the protein which lacked 12-mer repeats. The data suggest that variation in the number of tandem repeat sequences could allow the parasite to avoid high avidity antibody binding and this may allow escape from immune recognition.