The putative protein sorting receptor PfSortilin localizes to the <i>P. falciparum</i> Golgi apparatus.

<p>(A) Schematic diagram of the domain organization of PfSortilin with the number of amino acids in each domain indicated below. At bottom is the C-terminal sequence of PfSortilin with the site of incorporation of the HA tag indicated. TM, transmembrane. (B) Anti-HA immunoblot of the membrane fraction of parasites expressing PfSortilin-HA (clone C9) and of the parental 3D7 parasite line. PfSortilin-HA is indicated with an arrow. Two cross-reacting species are present in both lanes (asterisks). The membrane was reprobed with anti-plasmepsin V (PMV) antibodies for a loading control. The sizes of protein markers in kDa are indicated at left. (C) Co-localization of PfSortilin-HA and ERD2 in aldehyde-fixed clone C9 parasites. T, trophozoite; Sz, schizont. Scale bar, 2 µm.</p

Evidence for a Golgi-to-Endosome Protein Sorting Pathway in <i>Plasmodium falciparum</i>

<div><p>During the asexual intraerythrocytic stage, the malaria parasite <i>Plasmodium falciparum</i> must traffic newly-synthesized proteins to a broad array of destinations within and beyond the parasite's plasma membrane. In this study, we have localized two well-conserved protein components of eukaryotic endosomes, the retromer complex and the small GTPase Rab7, to define a previously-undescribed endosomal compartment in <i>P. falciparum</i>. Retromer and Rab7 co-localized to a small number of punctate structures within parasites. These structures, which we refer to as endosomes, lie in close proximity to the Golgi apparatus and, like the Golgi apparatus, are inherited by daughter merozoites. However, the endosome is clearly distinct from the Golgi apparatus as neither retromer nor Rab7 redistributed to the endoplasmic reticulum upon brefeldin A treatment. Nascent rhoptries (specialized secretory organelles required for invasion) developed adjacent to endosomes, an observation that suggests a role for the endosome in rhoptry biogenesis. A <i>P. falciparum</i> homolog of the sortilin family of protein sorting receptors (PfSortilin) was localized to the Golgi apparatus. Together, these results elaborate a putative Golgi-to-endosome protein sorting pathway in asexual blood stage parasites and suggest that one role of retromer is to mediate the retrograde transport of PfSortilin from the endosome to the Golgi apparatus.</p></div

PfRab6 but not PfRab7 or retromer rapidly redistributes to the ER upon brefeldin A treatment.

<p>(A) Distributions of DD-mCherry-PfRab6, DD-mCherry-PfRab7 and PfVps35-YFP in live parasites after one hour in the presence of 5 µg/mL brefeldin A. Redistribution of DD-mCherry-PfRab6 to the perinuclear ER is indicated with an arrow (top panel). FP, fluorescent protein. mCherry fluorescence is pseudocolored red, YFP fluorescence is pseudocolored green and Hoechst 33342 fluorescence is pseudocolored cyan. Scale bar, 2 µm. (B) Effect of brefeldin A (5 µg/mL, 1 hour) on the percentage of parasites exhibiting ER-associated DD-mCherry-PfRab6 fluorescence. Results are an average of three experiments, n = 75 to 85 parasites per condition. The <i>p</i>-value was determined using a two-tailed Student's t-test. (C) Effect of Brefeldin A (5 µg/mL, 1 hour) on the number of puncta labeled with DD-mCherry-PfRab6, DD-mCherry-PfRab7 (clones D9 and G9) or PfVps35-YFP. Results are averages of three experiments, n = 50 to 70 parasites per condition.</p

The retromer cargo-selective complex localizes to a novel, heritable subcellular compartment.

<p>(A) Wide-field epifluorescence images of live parasites expressing PfVps35-YFP. Parasites are shown at ring (R), trophozoite (T), schizont (Sz), segmenter (Sg), and extracellular merozoite (M) stages. Hoechst 33342 fluorescence (DNA) is pseudocolored red. (B) Images of live parasites expressing PfVps29-YFP. (C) Images of live parasites co-expressing PfVps35-YFP and PfVps29-mCherry in trophozoite (T) and schizont (Sz) stages. mCherry (mC) fluorescence is pseudocolored red. (D) Co-localization of PfVps35-YFP or PfVps35-HA and organellar markers in fixed parasites (except for MitoTracker, which was imaged live). PMV, plasmepsin V; ACP, acyl carrier protein; MT, MitoTracker Red CM-H2Xros; RAP1, rhoptry associated protein 1; AMA1, apical membrane antigen 1. The AMA1 panel shows free merozoites; all others are intraerythrocytic. Organelles labeled by the markers are indicated in parenthesis. Marker-derived fluorescence is pseudocolored red. (E) PfVps35-YFP is adjacent to developing rhoptries in a 2N parasite. Hoechst 33342 fluorescence (DNA) is pseudocolored cyan. In all panels, YFP fluorescence is pseudocolored green. Scale bars, 2 µm.</p

Antiplasmodial Sesquiterpenoid Lactones from <i>Trichospira verticillata</i>: Structure Elucidation by Spectroscopic Methods and Comparison of Experimental and Calculated ECD Data

A dichloromethane extract of <i>Trichospira verticillata</i> from the Natural Products Discovery Institute was discovered to have good antiplasmodial activity (IC₅₀ ∼5 μg/mL). After purification by liquid–liquid partition and C₁₈ reversed-phase HPLC, four new germacranolide-type sesquiterpenoid lactones named trichospirolides A–D (<b>1</b>–<b>4</b>) were isolated. The structures of the new compounds were elucidated by analysis of their 1D and 2D NMR and MS data. The relative and absolute configurations were assigned based on a comparison of calculated and experimental ECD and UV spectra, specific rotations, internuclear distances, and coupling constants for all possible diastereomers for each compound. Among these four compounds, the conjugated dienone <b>1</b> displayed the most potent antiplasmodial activity, with an IC₅₀ value of 1.5 μM

Biological Studies and Target Engagement of the 2‑<i>C</i>‑Methyl‑d‑Erythritol 4‑Phosphate Cytidylyltransferase (IspD)-Targeting Antimalarial Agent (1<i>R</i>,3<i>S</i>)‑MMV008138 and Analogs

Malaria continues to be one of the deadliest diseases worldwide, and the emergence of drug resistance parasites is a constant threat. <i>Plasmodium</i> parasites utilize the methylerythritol phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are essential for parasite growth. Previously, we and others identified that the Malaria Box compound MMV008138 targets the apicoplast and that parasite growth inhibition by this compound can be reversed by supplementation of IPP. Further work has revealed that MMV008138 targets the enzyme 2-<i>C</i>-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD) in the MEP pathway, which converts MEP and cytidine triphosphate (CTP) to cytidinediphosphate methylerythritol (CDP-ME) and pyrophosphate. In this work, we sought to gain insight into the structure–activity relationships by probing the ability of MMV008138 analogs to inhibit <i>Pf</i>IspD recombinant enzyme. Here, we report <i>Pf</i>IspD inhibition data for fosmidomycin (FOS) and 19 previously disclosed analogs and report parasite growth and <i>Pf</i>IspD inhibition data for 27 new analogs of MMV008138. In addition, we show that MMV008138 does not target the recently characterized human IspD, reinforcing MMV008138 as a prototype of a new class of species-selective IspD-targeting antimalarial agents

Neolignans and Other Metabolites from <i>Ocotea cymosa</i> from the Madagascar Rain Forest and Their Biological Activities1

Ten new neolignans including the 6′-oxo-8.1′-lignans cymosalignans A (<b>1a</b>), B (<b>2</b>), and C (<b>3</b>), an 8.O.6′-neolignan (<b>4a</b>), ococymosin (<b>5a</b>), didymochlaenone C (<b>6a</b>), and the bicyclo[3.2.1]­octanoids <b>7</b>–<b>10</b> were isolated along with the known compounds 3,4,5,3′,5′-pentamethoxy-1′-allyl-8.O.4′-neolignan, 3,4,5,3′-tetramethoxy-1′-allyl-8.O.4′-neolignan, didymochlaenone B, virologin B, ocobullenone, and the unusual 2′-oxo-8.1′-lignan sibyllenone from the stems or bark of the Madagascan plant <i>Ocotea cymosa</i>. The new 8.O.6′-neolignan <b>4a</b>, dihydrobenzofuranoid <b>5a</b>, and the bicyclo[3.2.1]­octanoid <b>7a</b> had in vitro activity against <i>Aedes aegypti</i>, while the new compounds <b>5a</b>, <b>7a</b>,<b> 8</b>, and <b>10a</b> and the known virolongin B (<b>4b</b>) and ocobullenone (<b>10b</b>) had antiplasmodial activity. We report herein the structure elucidation of the new compounds on the basis of spectroscopic evidence, including 1D and 2D NMR spectra, electronic circular dichroism, and mass spectrometry, and the biological activities of the new and known compounds