An exported protein-interacting complex involved in the trafficking of virulence determinants in Plasmodium-infected erythrocytes

The malaria parasite, Plasmodium falciparum, displays the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells (RBCs). We here examine the physical organization of PfEMP1 trafficking intermediates in infected RBCs and determine interacting partners using an epitope-tagged minimal construct (PfEMP1B). We show that parasitophorous vacuole (PV)-located PfEMP1B interacts with components of the PTEX (Plasmodium Translocon of EXported proteins) as well as a novel protein complex, EPIC (Exported Protein-Interacting Complex). Within the RBC cytoplasm PfEMP1B interacts with components of the Maurer\u27s clefts and the RBC chaperonin complex. We define the EPIC interactome and, using an inducible knockdown approach, show that depletion of one of its components, the parasitophorous vacuolar protein-1 (PV1), results in altered knob morphology, reduced cell rigidity and decreased binding to CD36. Accordingly, we show that deletion of the Plasmodium berghei homologue of PV1 is associated with attenuation of parasite virulence in vivo

Fenoldopam use in a burn intensive care unit: a retrospective study

<p>Abstract</p> <p>Background</p> <p>Fenoldopam mesylate is a highly selective dopamine-1 receptor agonist approved for the treatment of hypertensive emergencies that may have a role at low doses in preserving renal function in those at high risk for or with acute kidney injury (AKI). There is no data on low-dose fenoldopam in the burn population. The purpose of our study was to describe our use of low-dose fenoldopam (0.03-0.09 μg/kg/min) infusion in critically ill burn patients with AKI.</p> <p>Methods</p> <p>We performed a retrospective analysis of consecutive patients admitted to our burn intensive care unit (BICU) with severe burns from November 2005 through September 2008 who received low-dose fenoldopam. Data obtained included systolic blood pressure, serum creatinine, vasoactive medication use, urine output, and intravenous fluid. Patients on concomitant continuous renal replacement therapy were excluded. Modified inotrope score and vasopressor dependency index were calculated. One-way analysis of variance with repeated measures, Wilcoxson signed rank, and chi-square tests were used. Differences were deemed significant at p < 0.05.</p> <p>Results</p> <p>Seventy-seven patients were treated with low-dose fenoldopam out of 758 BICU admissions (10%). Twenty (26%) were AKI network (AKIN) stage 1, 14 (18%) were AKIN stage 2, 42 (55%) were AKIN stage 3, and 1 (1%) was AKIN stage 0. Serum creatinine improved over the first 24 hours and continued to improve through 48 hours (<it>p </it>< 0.05). There was an increase in systolic blood pressure in the first 24 hours that was sustained through 48 hours after initiation of fenoldopam (<it>p </it>< 0.05). Urine output increased after initiation of fenoldopam without an increase in intravenous fluid requirement (<it>p </it>< 0.05; <it>p </it>= NS). Modified inotrope score and vasopressor dependency index both decreased over 48 hours (<it>p </it>< 0.0001; <it>p </it>= 0.0012).</p> <p>Conclusions</p> <p>These findings suggest that renal function was preserved and that urine output improved without a decrease in systolic blood pressure, increase in vasoactive medication use, or an increase in resuscitation requirement in patients treated with low-dose fenoldopam. A randomized controlled trial is required to establish the efficacy of low-dose fenoldopam in critically ill burn patients with AKI.</p

Disrupting assembly of the inner membrane complex blocks <i>Plasmodium falciparum</i> sexual stage development

<div><p>Transmission of malaria parasites relies on the formation of a specialized blood form called the gametocyte. Gametocytes of the human pathogen, <i>Plasmodium falciparum</i>, adopt a crescent shape. Their dramatic morphogenesis is driven by the assembly of a network of microtubules and an underpinning inner membrane complex (IMC). Using super-resolution optical and electron microscopies we define the ultrastructure of the IMC at different stages of gametocyte development. We characterize two new proteins of the gametocyte IMC, called PhIL1 and PIP1. Genetic disruption of PhIL1 or PIP1 ablates elongation and prevents formation of transmission-ready mature gametocytes. The maturation defect is accompanied by failure to form an enveloping IMC and a marked swelling of the digestive vacuole, suggesting PhIL1 and PIP1 are required for correct membrane trafficking. Using immunoprecipitation and mass spectrometry we reveal that PhIL1 interacts with known and new components of the gametocyte IMC.</p></div

PhIL1 is located at the IMC in gametocytes.

<p>(A) Immunofluorescence microscopy showing anti-PhIL1 (green) at the periphery of a 3D7 stage IV gametocyte, showing fluorescence close to the anti-β-tubulin (red) labeling. (B) Immunofluorescence microscopy of a stage IV gametocyte showing overlap of GAP50-GFP (red) and PhIL1 (green) at the periphery of the cell. (C) Western blot analysis of saponin-treated pellets of 3D7 and GAP50-GFP stage IV gametocytes. Gametocytes were probed with PhIL1 pre-immune serum, anti-PhIL1 antiserum, anti-GAP45, anti-GFP and anti-ERC. (D, E) Immunofluorescence microscopy of stage IV PhIL1-GFP (D) and PhIL1-HA (E) gametocyte transfectants. Parasites were labeled with anti-PhIL1 (green) and anti-GFP or anti-HA (red). Nuclei were labeled with DAPI. Scale bars: 5 μm. (F) Purified, saponin-lysed PhIL1-HA gametocytes were solubilized with different extraction agents for 30 min. Pellet and supernatant fractions were separated and loaded in equivalent amounts on 4–12% acrylamide gels. PhIL1 and GAP45 bands were visualized using mouse anti-HA or rabbit anti-GAP45 primary and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, respectively. P = pellet; S = supernatant. Western analysis and immunofluorescence of PhIL1 is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006659#ppat.1006659.s003" target="_blank">S3 Fig</a>.</p

Model and schematic of the gametocyte IMC development.

<p>(A) Schematic showing the proposed positioning of proteins in the gametocyte IMC. Red blood cell membrane (RBCM); Parasitophorous vacuole membrane (PVM); parasite plasma membrane (PPM); inner membrane complex (IMC); microtubules (MT); Photosensitized 5-[125I] iodonaphthalene-1-azide labeled protein-1 (PhIL1); PhIL1-interacting protein 1 (PIP); Glideosome-associated protein 45 and 50 (GAP45 and 50); Glideosome-associated protein with multiple membrane spans (GAPM); myosin-A (MyoA); myosin-A tail domain interacting protein (MTIP). (B) Proposed model of plate formation and expansion during development and elongation of wild type gametocytes. IMC plates are deposited as 13 disk-like structures on the cytoplasmic side of the parasite plasma membrane. These plates act as a scaffold for microtubule formation. As the parasite develops, the plates expand through the addition of new membrane to the leading edges of the plates. The microtubule network aligns on these plates and drives parasite elongation. These plates continue to develop through to stage IV of development. At stage V, the microtubule network is disassembled but the IMC remains at the parasite periphery. (C) Gametocyte maturation in the absence of PhIL1 or PIP1. IMC plate formation is initiated as in wild type parasites, but new membrane is not added to the IMC membranes resulting in failure to organize the microtubule network and elongate the parasite. The membrane is miss-trafficked to the digestive vacuole leading to its swelling and enlargement. Disruption of PhIL1 and PIP1 leads to a significant reduction in parasite numbers and arrested morphological development.</p

Knockdown of PhIL1 arrests gametocyte development.

<p>(A) PhIL1-HA-<i>glmS</i> (green) is located at the parasite periphery, closely associated with β-tubulin (red). (B) Stage IV PhIL1-HA-<i>glmS</i> gametocytes treated with a range of glucosamine concentrations and analyzed by Western blotting. Probing with anti-HA shows efficient knockdown of the PhIL1-HA protein when compared to the anti-<i>Pf</i>ERC loading control. (C) Glucosamine-treated (+GLCN, 5 mM) and untreated (-GLCN, 0 mM) cultures were induced to form gametocytes and the parasitemia estimated at day 6. Data represent mean ± SEM; n = 3 experiments performed in triplicate. *** P <0.001, unpaired t-test. (D) Representative Giemsa-stained smears highlighting the altered morphology following PhIL1 knockdown. (E) Stage progression counts for PhIL1-HA-<i>glmS</i> parasites plus or minus glucosamine, highlighting the arrest in development following knockdown. Days 3, 5 and 7 only are shown. The complete data set, including wild type parasites plus or minus glucosamine, are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006659#ppat.1006659.s006" target="_blank">S6C and S6D Fig</a>. Data from 2 separate experiments performed in triplicate. Mean values are shown. (F) SBF-SEM images of PhIL1-HA-<i>glmS</i> parasites, plus or minus glucosamine. Rendered models are shown on the left, highlighting the IMC in purple and the digestive vacuole in green. A swollen digestive vacuole (DV; yellow arrow) is observed in glucosamine-treated parasites. Scale bars: 5 μm. (G) Quantification of the SBF-SEM images. Mean volumes for the parasite and the digestive vacuole are shown. Data represent mean ± SEM. n = 5. ** P <0.01, unpaired t-test. (H) PhIL1-HA-<i>glmS</i> parasites plus or minus glucosamine were labeled with LysoSensor to highlight the acidic digestive compartments. Swollen compartments are seen in the treated group, but not in the untreated control group (blue arrows).</p

Whole cell reconstructions reveal organelle organization during gametocyte development.

<p>(A-H) Individual SBF-SEM images of different stages (top rows), and rendered images of serial SBF-SEM sections (bottom rows). The following structures are labeled in the sections and color-coded in the rendered images. Parasite plasma membrane/parasitophorous vacuole membrane (PM, blue), mitochondrion (M, red), nucleus (N, yellow), hemozoin (Hz), the apicoplast (A, orange), osmophillic bodies (OB, light blue), Dimples (Di) and RBC. Scale bars: 5 μm. The dotted line marks the plane of sectioning. Rotations of these models and translations through the SBF-SEM images can be seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006659#ppat.1006659.s011" target="_blank">S1</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006659#ppat.1006659.s016" target="_blank">S6</a> Videos. Quantitative measurements and relative organelle positions are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006659#ppat.1006659.s001" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006659#ppat.1006659.s002" target="_blank">S2</a> Figs and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006659#ppat.1006659.s019" target="_blank">S1 Table</a>.</p

Genesis and development of the gametocyte IMC.

<p>(A-G) 3D-SIM immunofluorescence microscopy reveals the location of PhIL1-HA (red) relative to β-tubulin (green) for PhIL1-HA gametocytes from stage I-V of development. (A) A row of PhIL1-HA labeled puncta (yellow arrow) is observed along one edge of the parasite, interleaved with β-tubulin staining (blue arrow). (B) In stage II gametocytes, PhIL1-HA is observed as 13 ring-like structures (yellow arrows) aligned at the periphery of the parasites. Accumulations of β-tubulin staining can be seen in the centers of these disks (blue arrow). (C) The nascent IMC plates are more homogenously labeled with PhIL1-HA (yellow arrow), forming a ribbon-like structure at the periphery of the cell. Microtubules align with the IMC plates (blue arrow) or cross the parasite cytoplasm (white arrow). (D-E) In stage III, the IMC plates are clearly defined with homogenous PhIL1-HA labeling (yellow arrow). Bundles of microtubules underlie the IMC (blue arrow) or cross the parasite cytoplasm (white arrow). (F) In stage IV gametocytes, the IMC largely encases the parasite. The PhIL1-HA plates (yellow arrow) are still clearly visible and the microtubule network is tightly associated with the IMC (blue arrow). (G) By stage V, the microtubule network is disassembled but the IMC (labeled with PhIL1-HA) remains at the parasite periphery (yellow arrows; sutures are indicated with white arrows). 5x and 2.5X zoom images are shown on the right hand side. Scale bars: 5 μm. Scale bars: zoom images: 1 μm. The represented area is marked on the images with a white box. Rotations of these 3D-SIM images are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006659#ppat.1006659.s017" target="_blank">S7</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006659#ppat.1006659.s018" target="_blank">S8</a> Videos.</p

Identification of new IMC proteins in gametocytes.

<p>(A) 3D7 parent and PhIL1-GFP transfectant gametocytes, purified at stage IV, were detergent-solubilized and proteins were immunoprecipitated using GFP-Trap. The input and precipitated eluates (IP) were prepared for SDS-PAGE and Western blot and probed with anti-GFP, anti-GAP50 and anti-GAP45 antibodies. Molecular masses of markers are shown in kDa. (B) List of the PhIL1-interacting proteins. Two independent experiments were performed. Proteins that returned ≥2 significant MS/MS peptides in each experiment are included. A complete list of significant and non-significant proteins identified are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006659#ppat.1006659.s020" target="_blank">S2 Table</a>.</p