Ubiquitin activation is essential for schizont maturation in Plasmodium falciparum blood-stage development

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites

Formation of the Food Vacuole in Plasmodium falciparum: A Potential Role for the 19 kDa Fragment of Merozoite Surface Protein 1 (MSP119)

Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP119), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP119 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP119, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP119 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP119 and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP119 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase

Bed of Roses

Photographic works evolved from the Women Sexual Imagery Project are introduced by a text defining the challenges encountered during the workshop in the making of "alternative erotica". Statements by the seven participants. Brief biographical notes

A series of diagrams summarising in greater detail the main conclusions relating to post-invasion MSP1₁₉ endocytosis and its progression through the asexual cycle.

<p>In each panel a portion of the parasite is shown in blue, with the adjacent RBC in red, and with the parasitophorous vacuole space between the two. The fragments of MSP1 are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003085#pone-0003085-g007" target="_blank">Figure 7</a>.</p

The distribution of MSP1 within late trophozoites and schizonts.

<p>Panels A–C show the appearance of the single food vacuole, prepared for EM morphology. Panel A shows a late trophozoite containing a typically dilated food vacuole, containing widely spaced hemozoin and several profiles of internal membranous structures. In panel B an early schizont stage vacuole contains some hemozoin crystals and internal membrane profiles, while the vacuole wall shows signs of inward folding (arrowheads). C shows another schizont where a lipid body has formed adjacent to the food vacuole. Panels D–G show specific immunogold labeling of early to mid-stage schizonts labeled with MSP1₁₉-specific antibodies, in all cases detecting the protein at the vacuole wall. In panel D a limited area of labeling is present along one side of the food vacuole (small arrows), and in E a similar distribution is seen on a more folded vacuole wall. Panels F and G show labeling along obliquely sectioned folds of the wall (arrows), where the label also lies close to hemozoin crystals. Panels H and I show food vacuoles of late-stage schizonts containing closely packed large hemozoin crystals and are almost completely surrounded by MSP1₁₉ labeled vacuole wall membrane. In Panel J (and the inset of a portion at higher magnification) an early-/mid-stage schizont has been immunostained with antibody reacting with N-terminal regions of MSP1 but not MSP1₁₉. This antibody labels the newly synthesized (full-length) MSP1 on the parasite's plasma membrane but fails to label the food vacuole, indicating that the MSP1₁₉-specific labeling, seen in panels D–I, is specific for MSP1₁₉ carried in on the parasite surface at invasion rather than new MSP1 recently synthesized by the schizont. <i>Abbreviations</i>: FV–food vacuole; Hz–hemozoin; Int mem–internal membranous structures; RBC–red blood cell.</p

The distribution of MSP1₁₉ in ring-stage parasites detected by IEM.

<p>Panels D and E show MSP1₁₉-specific labeling of the surface of early rings, and in panel E, a clear endocytic vacuole is also labeled. Panels F–H show that MSP1₁₉ has been endocytosed into the small dense food vacuoles, and is located mainly at the inner vacuolar membrane surface; in Panel G the food vacuoles have a tubular appearance. In Panel I, a late ring is labeled, showing the incorporation of MSP1₁₉ into larger vacuoles containing small hemozoin crystals (Hz), with the label again associated with the vacuole membrane. <i>Abbreviations</i>: Fvtu- tubular food vacuoles; Hz- hemozoin.</p

Immunofluorescence microscopy showing the localization of MSP1₁₉ and CRT in parasites that had invaded in the presence of mAb 1E1; showing that MSP1₁₉ moves to the food vacuole.

<p>Schizonts were allowed to release merozoites that invaded fresh erythrocytes in the presence of mAb 1E1, then ring-stage parasites were cultured and samples harvested every 3 hours over the asexual cycle. The mAb 1E1 antibody associates with MSP1₁₉ on the surface of the merozoite, and is detected within the ring-stage parasite; it was used to follow MSP1₁₉ in smears counterstained with rabbit anti-MSP1₁₉ (panel A) and rabbit anti-CRT (panel B), which is a marker of the food vacuole. Each row of four panels show an identical field from 1% formaldehyde-fixed thin smears of <i>P. falciparum</i> ring-stage parasites; columns A1 and B1 show mAb 1E1 associated with the parasite detected with Alexa Fluor 488 conjugated anti-mouse IgG (green); columns A2 rabbit anti-MSP1₁₉ and B2 rabbit anti-CRT, detected with Alexa Fluor 594 conjugated anti-rabbit IgG (red); columns A3 and B3 are composites of columns 1 and 2 with DAPI staining of the nucleus (blue) and any green and red fluorescence overlap is displayed in yellow. In columns A4 and B4, the parasitised erythrocyte is visualized by light microscopy, showing the location of the parasite within the infected erythrocyte. Only five time points are shown: 0, 6, 9, 12 and 18 hours post-invasion. The two anti-MSP1₁₉ antibodies, in panels A1 and A2, are seen to co-localize in panel A3, showing that 1E1 is still associated with MSP1₁₉ in the young parasite. CRT is clearly detectable from 6 to 9 hours onwards post-invasion (panel B2) and appears to be closely associated with MSP1₁₉ (panel B3). Pigment granules are clearly visible by light microscopy from about 18 hours (panels A4 and B4). Similar results were obtained in a separate experiment in which mAb 1E1 was used to detect MSP1₁₉ on formaldehyde fixed parasites at the same time points (data not shown) and at 24 hours (panel C). Panels C1–C4 show 1E1, (C1, green), rabbit anti-CRT (C2, red), composites of these antibodies with DAPI staining merged (C3) and light visualization (C4), as described above. CRT was clearly detectable (C2) and largely co-localized with MSP1₁₉ (C3). Both MSP1₁₉ and CRT were associated with the pigment detected by light microscopy in panel C4 and no longer around the surface of the parasite.</p