The rapid intraerythrocytic replication of Plasmodium falciparum, a deadly species of malaria parasite, requires a quick but constant supply of phospholipids to support marked cell membrane expansion. In the malarial parasite, many enzymes functioning in phospholipid synthesis pathway have not been identified or characterized. Here, we identify P. falciparum lysophospholipid acyltransferase 1 (PfLPLAT1) and show that PfLPLAT1 is vital for asexual parasite cell cycle progression and cytostome internalization. Deficiency in PfLPLAT1 results in decreased parasitemia and prevents transition to the schizont stage. Parasites lacking PfLPLAT1 also exhibit distinctive omega-shaped vacuoles, indicating disrupted cytostome function. Transcriptomic analyses suggest that this deficiency impacts DNA replication and cell cycle regulation. Mass spectrometry-based enzyme assay and lipidomic analysis demonstrate that recombinant PfLPLAT1 exhibits lysophospholipid acyltransferase activity with a preference for unsaturated fatty acids as its acyl donors and lysophosphatidic acids as an acceptor, with its conditional knockout leading to abnormal lipid composition and marked morphological and developmental changes including stage arrest. These findings highlight PfLPLAT1 as a potential target for antimalarial therapy, particularly due to its unique role and divergence from human orthologs. PfLPLAT1 has important roles in the phospholipid synthesis pathway of Plasmodium falciparum, particularly phosphatidic acid as an essential substance for cytostome internalization. Its dysfunction leads to cell cycle arrest and death of parasites.
The structure of Escherichia coli FoF1-ATPase (ATP synthase) and its Fo sector reconstituted in lipid membranes1 was analyzed using atomic force microscopy (AFM), a technique that does not require protein crystals and reveals the spatial organization of membrane proteins. Most FoF1-ATPases (Fig. 1A and C) were visualized as spheres with a calculated diameter of ∼90 Å and a height of ∼100 Å from the membrane surface.2 These dimensions are consistent with the information about the F1 sector obtained by electron microscopy3 and X-ray crystallography.4,5 In contrast to the height of the F1 sectors, the height of the Fo sectors out of the membrane was very small (<10 Å) (Table 1). Two ring-like structures of Fo with a calculated outer diameter of ∼130 Å were consistently observed, one with a central hollow of ≥ 18 Å in depth and a similar one with a central mass (Fig. 1B and D).2 These images possibly represent two surface views of Fo oriented differently in the reconstituted membrane. Further analysis of these two ring-like structures at higher resolution revealed that in both types of rings, half the ring was about twice as thick as the other half; however, both halves (ridges) of the ring-like structures exhibited similar heights (<10 Å) (Table 1). Such asymmetry of the Fo sector was also found in a recent electron microscopic study.6 It is noteworthy that regardless of the presence of a central mass, a similar ring structure was observed. The bacterial Fo sector is composed of a, b, and c subunits with a stoichiometry of 1:2:10–12, and all three subunits are necessary to reconstitute a functional sector.7 Each subunit of Fo traverses the membrane; subunit a is very hydrophobic and crosses the membrane several times (probably 6 times), whereas subunit b crosses the membrane once. Subunit c has a hairpin structure with two transmembrane helical domains with a polar cytoplasmic loop in between as supported by nuclear magnetic-resonance studies.8.9 The two asymmetric AFM images suggest a possible structure of Fo comprising a, b, and c subunits having 6, 1, and 2 transmembrane helices. respectively. The central mass of the ring-like image may not represent the transmembrane regions of these subunits because other images showed a hollow. Thus, a model with central a and b subunits surrounded by 10–12 copies of the c subunit is unlikely (Fig. 2B). In our model, the a and b subunits are attached to the ring-like structure formed by the c subunits (Fig. 2A). This allows at least 12 c subunits to form a symmetrical ring, taking into account the fact that each c subunit contains two transmembrane α helices (total 24 α helices for 12 c subunits). An asymmetric Fo structure could be formed through the association with a and b subunits to one side of the symmetric ring formed by the c subunits. About 15 residues at the cytoplasmic amino terminus of the c subunit may extend beyond one side of the membrane and form the central mass observed in a ring-like structure. Subunit b crosses the membrane with a single transmembrane helix, leaving most of the polar domain exposed to the cytoplasm,7 allowing the formation of the stalk region, along with parts of F1 subunits, that connects F1 and Fo. However, such a structure could not be observed on AFM, although the Fo preparation contained a stoichiometric amount of subunit b. Therefore, it is possible that the b subunit could not be maintained as part of the stalk and thus formed the central mass observed on AFM.
Malaria parasites cannot multiply in host erythrocytes without cholesterol because they lack complete sterol biosynthesis systems. This suggests parasitized red blood cells (pRBCs) need to capture host sterols, but its mechanism remains unknown. Here we identified a novel high-density lipoprotein (HDL)-delivery pathway operating in blood-stage Plasmodium. In parasitized mouse plasma, exosomes positive for scavenger receptor CD36 and platelet-specific CD41 increased. These CDs were detected in pRBCs and internal parasites. A low molecular antagonist for scavenger receptors, BLT-1, blocked HDL uptake to pRBCs and suppressed Plasmodium growth in vitro. Furthermore, platelet-derived exosomes were internalized in pRBCs. Thus, we presume CD36 is delivered to malaria parasites from platelets by exosomes, which enables parasites to steal HDL for cholesterol supply. Cholesterol needs to cross three membranes (RBC, parasitophorous vacuole and parasite's plasma membranes) to reach parasite, but our findings can explain the first step of sterol uptake by intracellular parasites.
The rapid intraerythrocytic replication of Plasmodium falciparum, a deadly species of malaria parasite, requires a quick but constant supply of phospholipids to support marked cell membrane expansion. In the malarial parasite, many enzymes functioning in phospholipid synthesis pathway have not been identified or characterized. Here, we identified P. falciparum lysophospholipid acyltransferase 1 (PfLPLAT1; PF3D7_1444300) and showed that PfLPLAT1 is vital for asexual parasite cell cycle progression and cytostome internalization. Deficiency in PfLPLAT1 resulted in decreased parasitemia and prevented transition to the schizont stage. Parasites lacking PfLPLAT1 also exhibited distinctive omega-shaped vacuoles, indicating disrupted cytostome function. Transcriptomic analyses suggested that this deficiency impacted DNA replication and cell cycle regulation. Mass spectrometry-based enzyme assay and lipidomic analysis demonstrated that recombinant PfLPLAT1 exhibited lysophospholipid acyltransferase activity with a preference for unsaturated fatty acids as its acyl donors and lysophosphatidic acids as an acceptor, with its conditional knockout leading to abnormal lipid composition and marked morphological and developmental changes including stage arrest. These findings highlight PfLPLAT1 as a potential target for antimalarial therapy, particularly due to its unique role and divergence from human orthologs.
SignificanceDevelopments of anti-gametocyte drugs have been delayed due to insufficient understanding of gametocyte biology. We report a systematic workflow of data processing algorithms to quantify changes in the absorption spectrum and cell morphology of single malaria-infected erythrocytes. These changes may serve as biomarkers instrumental for the future development of antimalarial strategies, especially for anti-gametocyte drug design and testing. Image-based biomarkers may also be useful for nondestructive, label-free malaria detection and drug efficacy evaluation in resource-limited communities.AimWe extend the application of hyperspectral microscopy to provide detailed insights into gametocyte stage progression through the quantitative analysis of absorbance spectra and cell morphology in malaria-infected erythrocytes.ApproachMalaria-infected erythrocytes at asexual and different gametocytogenesis stages were imaged through hyperspectral confocal microscopy. The preprocessing of the hyperspectral data cubes to transform them to color images and spectral angle mapper (SAM) analysis were first used to segment hemoglobin (Hb)- and hemozoin (Hz)-abundant areas within the host erythrocytes. Correlations between changes in cell morphology and increasing Hz-abundant areas of the infected erythrocytes were then examined to test their potential as optical biomarkers to determine the progression of infection, involving transitions from asexual to various gametocytogenesis stages.ResultsFollowing successful segmentation of Hb- and Hz-abundant areas in malaria-infected erythrocytes through SAM analysis, a modest correlation between the segmented Hz-abundant area and cell shape changes over time was observed. A significant increase in both the areal fraction of Hz and the ellipticity of the cell confirms that the Hz fraction change correlates with the progression of gametocytogenesis.ConclusionsOur workflow enables the quantification of changes in host cell morphology and the relative contents of Hb and Hz at various parasite growth stages. The quantified results exhibit a trend that both the segmented areal fraction of intracellular Hz and the ellipticity of the host cell increase as gametocytogenesis progresses, suggesting that these two metrics may serve as useful biomarkers to determine the stage of gametocytogenesis.
We report a well controlled method to make carbon nanotube tips for a scanning probe microscope (SPM). A multiwalled carbon nanotube, which is purified by the electrophoresis, is transferred onto a conventional Si tip for a SPM using a scanning electron microscope (SEM) equipped with two independent specimen stages. The nanotube is fixed on the Si tip by electron beam deposition of carbon. A force curve measurement of nanotubes using the nanotube tips in the SEM reveals that Young's modulus of a nanotube of 20 nm diameter is 1.1 TPa and the fixing of nanotubes by the carbon deposit is effective. The nanotube tips are used to image plasmid deoxyribonucleic acids on mica by tapping mode. The average resolution by using the nanotube tips is about two times higher than that by the best Si tips.
