Luciferase, When Fused to an N-terminal Signal Peptide, Is Secreted from Transfected Plasmodium falciparum and Transported to the Cytosol of Infected Erythrocytes

Abstract

Plasmodium falciparum, a unicellular parasite that causes human malaria, infects erythrocytes where it develops within a vacuole. The vacuolar membrane separates the parasite from the erythrocyte cytosol. Some secreted parasite proteins remain inside the vacuole, and others are transported across the vacuolar membrane. To identify the protein sequences responsible for this distribution we investigated the suitability of the green fluorescent protein and luciferase as reporters in transiently transfected parasites. Because of the higher sensitivity of the enzymatic assay, luciferase was quantified 3 days after transfection, whereas reliable detection of green fluorescent protein required prolonged drug selection. Luciferase was confined to the parasite cytosol in subcellular fractions of infected erythrocytes. When parasites were transfected with a hybrid gene coding for the cleavable N-terminal signal peptide of a secreted parasite protein fused to luciferase, the reporter protein was secreted. It was recovered with the vacuolar content and the erythrocyte cytosol. The results suggest that no specific protein sequences are required for translocation across the vacuolar membrane. The high local concentration of luciferase within the vacuole argues against free diffusion, and thus transport into the erythrocyte cytosol must involve a ratelimiting step.Plasmodium falciparum, the parasite that causes the most severe form of malaria, spends part of its life cycle in human erythrocytes. Here it resides within the so-called parasitophorous vacuole, which is bound by the parasitophorous vacuolar membrane (PVM).1 The vacuole constitutes a separate compartment in the infected red blood cell (iRBC) that is distinct from the cytosol of the parasite and from the cytosol of the erythrocyte, respectively (1). Most proteins secreted from P. falciparum are not released into an extracellular space but are transported to various destinations within the iRBC: the parasitophorous vacuole or the erythrocyte cytosol. Membranebound proteins are found in the PVM and erythrocyte plasma membrane (2, 3). It is completely unknown which information within a polypeptide chain determines whether a protein is transported across the PVM. Recently we described experimental evidence for a transport pathway that involves the release of secreted parasite proteins into the vacuolar space and translocation across the PVM in a subsequent step (4). This model infers that a sorting mechanism must operate within the vacuole that discriminates between vacuolar resident proteins and proteins destined for a location beyond the PVM (5). Sorting could involve two different principles: the retention of vacuolar proteins or, alternatively, the recognition of protein signals that mediate translocation across the PVM.A widely used experimental approach for the identification of protein targeting and sorting signals in eukaryotic cells is the fusion of putative signal sequences to reporter proteins and their subsequent localization in the transfected cell. Although...

An enhancer-like region regulates hrp3 promoter stage-specific gene expression in the human malaria parasite Plasmodium falciparum

“…The absence of recognizable sequence homology amongst Plasmodium promoters suggests that stage specific regulation may require complex mechanisms that could include transcriptional initiation and/or nuclear position amongst others. A wide variety of eukaryotic genes, including those of Plasmodium, are regulated by DNA structure such as formation of kinks by poly T tracts flanking the core promoter [37][38][39] and poly(dA:dT) sequences [40], both of which are very abundant in the Plasmodium genome. Other DNA-dependent structures that could play a role in the promoter function such as poly purine-pyrimidine sequences have also been described [41].…”

Trafic protéique dans le globule rouge infecté parPlasmodium

“…Cette étape dépendante de l'ATP serait donc critique pour l'exportation et conduirait à l'accumulation provisoire de ces protéines dans l'espace vacuolaire jouant le rôle de compartiment de transit [13]. Certains auteurs suggèrent qu'une séquence signal serait indispensable pour retenir les protéines dans la VP, alors que l'exportation des protéines vers le cytosol érythrocytaire ne serait en définitive qu'une voie par défaut [14]. À l'inverse, selon d'autres travaux, la voie de sécrétion par défaut concernerait le transit jusqu'à la VP, et une séquence signal serait indispensable à l'exportation au-delà de la VP [15,16].…”

A Nonpermeant Biotin Derivative Gains Access to the Parasitophorous Vacuole in Plasmodium falciparum-infected Erythrocytes Permeabilized with Streptolysin O

“…Streptolysin O (SLO) was kindly provided by S. Bhakdi and prepared as described elsewhere (10). Polyclonal rabbit antibodies directed against GBP, SERP, parasite aldolase, and PfBiP have been described previously (12,19). The mouse monoclonal antibody to band 3, clone III-136, was obtained from Sigma.…”

“…Estimates based on morphological data suggest a 1:10,000 ratio of vacuolar volume to erythrocyte cytosol in IRBCs infected with stages of the parasite that have completed ϳ30 h of the 48-h intraerythrocytic development (12). Cell fractionation experiments on IRBCs infected with the same developmental stages show that more than 70% of the total protein are erythrocyte cytosolic proteins, predominantly hemoglobin (18).…”

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ Recipient of a scholarship from the German Academic Exchange Service.ʈ (12,19). The mouse monoclonal antibody to band 3, clone III-136, was obtained from Sigma.…”