Giel G. van Dooren

Tropical infectious diseases: metabolic maps and functions of the Plasmodium falciparum apicoplast.

Discovery of a relict chloroplast (the apicoplast) in malarial parasites presented new opportunities for drug development. The apicoplast – although no longer photosynthetic – is essential to parasites. Combining bioinformatics approaches with experimental validation in the laboratory, we have identified more than 500 proteins predicted to function in the apicoplast. By comparison with plant chloroplasts, we have reconstructed several anabolic pathways for the parasite plastid that are fundamentally different to the analogous pathways in the human host and are potentially good targets for drug development. Products of these pathways seem to be exported from the apicoplast and might be involved in host-cell invasion.

Dynamics of neutrophil migration in lymph nodes during infection.

Although the signals that control neutrophil migration from the blood to sites of infection have been well characterized, little is known about their migration patterns within lymph nodes or the strategies that neutrophils use to find their local sites of action. To address these questions, we used two-photon scanning-laser microscopy to examine neutrophil migration in intact lymph nodes during infection with an intracellular parasite, Toxoplasma gondii. We found that neutrophils formed both small, transient and large, persistent swarms via a coordinated migration pattern. We provided evidence that cooperative action of neutrophils and parasite egress from host cells could trigger swarm formation. Neutrophil swarm formation coincided in space and time with the removal of macrophages that line the subcapsular sinus of the lymph node. Our data provide insights into the cellular mechanisms underlying neutrophil swarming and suggest new roles for neutrophils in shaping immune responses.

Development of the endoplasmic reticulum, mitochondrion and apicoplast during the asexual life cycle of Plasmodium falciparum

Plasmodium parasites are unicellular eukaryotes that undergo a series of remarkable morphological transformations during the course of a multistage life cycle spanning two hosts (mosquito and human). Relatively little is known about the dynamics of cellular organelles throughout the course of these transformations. Here we describe the morphology of three organelles (endoplasmic reticulum, apicoplast and mitochondrion) through the human blood stages of the parasite life cycle using fluorescent reporter proteins fused to organelle targeting sequences. The endoplasmic reticulum begins as a simple crescent‐shaped organelle that develops into a perinuclear ring with two small protrusions, followed by transformation into an extensive reticulated network as the parasite enlarges. Similarly, the apicoplast and the mitochondrion grow from single, small, discrete organelles into highly branched structures in later‐stage parasites. These branched structures undergo an ordered fission – apicoplast followed by mitochondrion – to create multiple daughter organelles that are apparently linked as pairs for packaging into daughter cells. This is the first in‐depth examination of intracellular organelles in live parasites during the asexual life cycle of this important human pathogen.

Evolution: red algal genome affirms a common origin of all plastids.

Photosynthetic organelles (plastids) come in many forms and were originally thought to have multiple origins. The complete genome of the thermophilic red alga Cyanidioschizon merolae provides further evidence that all plastids derive from a single endosymbiotic event more than 600 million years ago.

Processing of an apicoplast leader sequence in Plasmodium falciparum, and the identification of a putative leader cleavage enzyme

The plastid (apicoplast) of the malaria-causing parasite Plasmodium falciparum was derived via a secondary endosymbiotic process. As in other secondary endosymbionts, numerous genes for apicoplast proteins are located in the nucleus, and the encoded proteins are targeted to the organelle courtesy of a bipartite N-terminal extension. The first part of this leader sequence is a signal peptide that targets proteins to the secretory pathway. The second, so-called transit peptide region is required to direct proteins from the secretory pathway across the multiple membranes surrounding the apicoplast. In this paper we perform a pulse-chase experiment and N-terminal sequencing to show that the transit peptide of an apicoplast-targeted protein is cleaved, presumably upon import of the protein into the apicoplast. We identify a gene whose product likely performs this cleavage reaction, namely a stromal-processing peptidase (SPP) homologue. In plants SPP cleaves the transit peptides of plastid-targeted proteins. The P. falciparum SPP homologue contains a bipartite N-terminal apicoplast-targeting leader. Interestingly, it shares this leader sequence with a Δ-aminolevulinic acid dehydratase homologue via an alternative splicing event.

Toxoplasma gondii Tic20 is essential for apicoplast protein import

Apicomplexan parasites harbor a secondary plastid that has lost the ability to photosynthesize yet is essential for the parasite to multiply and cause disease. Bioinformatic analyses predict that 5–10% of all proteins encoded in the parasite genome function within this organelle. However, the mechanisms and molecules that mediate import of such large numbers of cargo proteins across the four membranes surrounding the plastid remain elusive. In this work, we identify a highly diverged member of the Tic20 protein family in Apicomplexa. We demonstrate that Tic20 of Toxoplasma gondii is an integral protein of the innermost plastid membrane. We engineer a conditional null-mutant and show that TgTic20 is essential for parasite growth. To characterize this mutant functionally, we develop several independent biochemical import assays to reveal that loss of TgTic20 leads to severe impairment of apicoplast protein import followed by organelle loss and parasite death. TgTic20 is the first experimentally validated protein import factor identified in apicoplasts. Our studies provide experimental evidence for a common evolutionary origin of import mechanisms across the innermost membranes of primary and secondary plastids.

Translocation of proteins across the multiple membranes of complex plastids

Secondary endosymbiosis describes the origin of plastids in several major algal groups such as dinoflagellates, euglenoids, heterokonts, haptophytes, cryptomonads, chlorarachniophytes and parasites such as apicomplexa. An integral part of secondary endosymbiosis has been the transfer of genes for plastid proteins from the endosymbiont to the host nucleus. Targeting of the encoded proteins back to the plastid from their new site of synthesis in the host involves targeting across the multiple membranes surrounding these complex plastids. Although this process shows many overall similarities in the different algal groups, it is emerging that differences exist in the mechanisms adopted.

Dynamics of T cell, antigen-presenting cell, and pathogen interactions during recall responses in the lymph node.

Memory T cells circulate through lymph nodes where they are poised to respond rapidly upon re-exposure to a pathogen; however, the dynamics of memory T cell, antigen-presenting cell, and pathogen interactions during recall responses are largely unknown. We used a mouse model of infection with the intracellular protozoan parasite, Toxoplasma gondii, in conjunction with two-photon microscopy, to address this question. After challenge, memory T cells migrated more rapidly than naive T cells, relocalized toward the subcapsular sinus (SCS) near invaded macrophages, and engaged in prolonged interactions with infected cells. Parasite invasion of T cells occurred by direct transfer of the parasite from the target cell into the T cell and corresponded to an antigen-specific increase in the rate of T cell invasion. Our results provide insight into cellular interactions during recall responses and suggest a mechanism of pathogen subversion of the immune response.

Genetic Evidence that an Endosymbiont-derived Endoplasmic Reticulum-associated Protein Degradation (ERAD) System Functions in Import of Apicoplast Proteins

Most apicomplexan parasites harbor a relict chloroplast, the apicoplast, that is critical for their survival. Whereas the apicoplast maintains a small genome, the bulk of its proteins are nuclear encoded and imported into the organelle. Several models have been proposed to explain how proteins might cross the four membranes that surround the apicoplast; however, experimental data discriminating these models are largely missing. Here we present genetic evidence that apicoplast protein import depends on elements derived from the ER-associated protein degradation (ERAD) system of the endosymbiont. We identified two sets of ERAD components in Toxoplasma gondii, one associated with the ER and cytoplasm and one localized to the membranes of the apicoplast. We engineered a conditional null mutant in apicoplast Der1, the putative pore of the apicoplast ERAD complex, and found that loss of Der1Ap results in loss of apicoplast protein import and subsequent death of the parasite.

Building the perfect parasite: cell division in apicomplexa.

Apicomplexans are pathogens responsible for malaria, toxoplasmosis, and crytposporidiosis in humans, and a wide range of livestock diseases. These unicellular eukaryotes are stealthy invaders, sheltering from the immune response in the cells of their hosts, while at the same time tapping into these cells as source of nutrients. The complexity and beauty of the structures formed during their intracellular development have made apicomplexans the darling of electron microscopists. Dramatic technological progress over the last decade has transformed apicomplexans into respectable genetic model organisms. Extensive genomic resources are now available for many apicomplexan species. At the same time, parasite transfection has enabled researchers to test the function of specific genes through reverse and forward genetic approaches with increasing sophistication. Transfection also introduced the use of fluorescent reporters, opening the field to dynamic real time microscopic observation. Parasite cell biologists have used these tools to take a fresh look at a classic problem: how do apicomplexans build the perfect invasion machine, the zoite, and how is this process fine-tuned to fit the specific niche of each pathogen in this ancient and very diverse group? This work has unearthed a treasure trove of novel structures and mechanisms that are the focus of this review.