Melanie Rug

Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes

After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob‐associated histidine‐rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite‐derived structures, known as Maurers clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.

Cell-Cell Communication between Malaria-Infected Red Blood Cells via Exosome-like Vesicles

Cell-cell communication is an important mechanism for information exchange promoting cell survival for the control of features such as population density and differentiation. We determined that Plasmodium falciparum-infected red blood cells directly communicate between parasites within a population using exosome-like vesicles that are capable of delivering genes. Importantly, communication via exosome-like vesicles promotes differentiation to sexual forms at a rate that suggests that signaling is involved. Furthermore, we have identified a P. falciparum protein, PfPTP2, that plays a key role in efficient communication. This study reveals a previously unidentified pathway of P. falciparum biology critical for survival in the host and transmission to mosquitoes. This identifies a pathway for the development of agents to block parasite transmission from the human host to the mosquito.

Fluorescence photobleaching analysis for the study of cellular dynamics

Abstract The wide availability of the confocal microscope and the emergence of green fluorescent protein (GFP) transfection technology has led to the increasing use of photobleaching studies to examine aspects of cellular dynamics in living cells. In this review, we examine the theory and practice of performing photobleaching studies using a confocal microscope. We illustrate the application of photobleaching protocols using our own measurements of fluorescently labelled red blood cells and of malaria parasite-infected erythrocytes expressing GFP fusions and examine other examples from the literature.

Characterisation of a δ-COP homologue in the malaria parasite, Plasmodium falciparum ☆

The mature human erythrocyte is a simple haemoglobin-containing cell with no internal organelles and no protein synthesis machinery. The malaria parasite invades this cell and develops inside a parasitophorous vacuole (PV). The parasite exports proteins into the erythrocyte to bring about extensive remodelling of its adopted cellular home. Plasmodial homologues of two COPII proteins, PfSar1p and PfSec31p, are exported to the erythrocyte cytosol where they appear to play a role in the trafficking of proteins across the erythrocyte cytoplasm [Eur. J. Cell Biol. 78 (1999) 453; J. Cell Sci. 114 (2001) 3377]. We have now characterised a homologue of the COPI protein, delta-COP. A recombinant protein corresponding to 90% of the Pfdelta-COP sequence was used to raise antibodies. The affinity-purified antiserum recognised a protein with an apparent M(r) of 58 x 10(3) on Western blots of malaria parasite-infected erythrocytes but not on blots of uninfected erythrocytes. Pfdelta-COP was shown to be largely insoluble in non-ionic detergent, possibly suggesting cytoskeletal attachment. Confocal immunofluorescence microscopy of parasitised erythrocytes was used to show that, in contrast to the COPII proteins, Pfdelta-COP is located entirely within the parasite. The location of Pfdelta-COP partly overlaps that of the endoplasmic reticulum (ER)-located protein, PfERC, and partly that of the trans-Golgi-associated protein, PfRab6. Treatment of ring-stage plasmodium-infected erythrocytes with brefeldin A (BFA) inhibited development of the ER structure within the parasite cytosol and prevented the trafficking of the P. falciparum erythrocyte membrane protein-1, PfEMP1, to the erythrocyte cytosol. The Pfdelta-COP and PfSec31p populations each appear to be associated with the restricted ER structure in brefeldin-treated rings. When more mature stage parasites were treated with BFA, erythrocyte cytosol-located populations of parasite proteins were not reorganised, however, the overlap between Pfdelta-COP and PfERC in parasite cytosol was more complete suggesting a possible redistribution of the Golgi compartment into the ER. These data support the suggestion that both COPI and COPII proteins are involved in the trafficking of proteins within the parasite cytoplasm. However, only COPII proteins are exported to the erythrocyte cytosol to establish a vesicle-mediated protein trafficking pathway to the erythrocyte membrane.

Electron tomography of Plasmodium falciparum merozoites reveals core cellular events that underpin erythrocyte invasion

Erythrocyte invasion by merozoites forms of the malaria parasite is a key step in the establishment of human malaria disease. To date, efforts to understand cellular events underpinning entry have been limited to insights from non‐human parasites, with no studies at sub‐micrometer resolution undertaken using the most virulent human malaria parasite, Plasmodium falciparum. This leaves our understanding of the dynamics of merozoite sub‐cellular compartments during infectionincomplete, in particular that of the secretory organelles. Using advances in P.u2009falciparum merozoite isolation and new imaging techniques we present a three‐dimensional study of invasion using electron microscopy, cryo‐electron tomography and cryo‐X‐ray tomography. We describe the core architectural features of invasion and identify fusion between rhoptries at the commencement of invasion as a hitherto overlooked event that likely provides a critical step that initiates entry. Given the centrality of merozoite organelle proteins to vaccine development, these insights provide a mechanistic framework to understand therapeutic strategies targeted towards the cellular events of invasion.