Hijacking of the host cell Golgi by Plasmodium berghei liver stage parasites.

The intracellular lifestyle represents a challenge for the rapidly proliferating liver stage Plasmodium parasite. In order to scavenge host resources, Plasmodium has evolved the ability to target and manipulate host cell organelles. Using dynamic fluorescence-based imaging, we here show an interplay between the pre-erythrocytic stages of Plasmodium berghei and the host cell Golgi during liver stage development. Liver stage schizonts fragment the host cell Golgi into miniaturized stacks, which increases surface interactions with the parasitophorous vacuolar membrane of the parasite. Expression of specific dominant-negative Arf1 and Rab GTPases, which interfere with the host cell Golgi-linked vesicular machinery, results in developmental delay and diminished survival of liver stage parasites. Moreover, functional Rab11a is critical for the ability of the parasites to induce Golgi fragmentation. Altogether, we demonstrate that the structural integrity of the host cell Golgi and Golgi-associated vesicular traffic is important for optimal pre-erythrocytic development of P. berghei. The parasite hijacks the Golgi structure of the hepatocyte to optimize its own intracellular development. This article has an associated First Person interview with the first author of the paper.

Photosensitized INA-Labelled protein 1 (PhIL1) is novel component of the inner membrane complex and is required for Plasmodium parasite development.

Plasmodium parasites, the causative agents of malaria, possess a distinctive membranous structure of flattened alveolar vesicles supported by a proteinaceous network, and referred to as the inner membrane complex (IMC). The IMC has a role in actomyosin-mediated motility and host cell invasion. Here, we examine the location, protein interactome and function of PhIL1, an IMC-associated protein on the motile and invasive stages of both human and rodent parasites. We show that PhIL1 is located in the IMC in all three invasive (merozoite, ookinete-, and sporozoite) stages of development, as well as in the male gametocyte and locates both at the apical and basal ends of ookinete and sporozoite stages. Proteins interacting with PhIL1 were identified, showing that PhIL1 was bound to only some proteins present in the glideosome motor complex (GAP50, GAPM1-3) of both P. falciparum and P. berghei. Analysis of PhIL1 function using gene targeting approaches indicated that the protein is required for both asexual and sexual stages of development. In conclusion, we show that PhIL1 is required for development of all zoite stages of Plasmodium and it is part of a novel protein complex with an overall composition overlapping with but different to that of the glideosome.

Generation of transgenic rodent malaria parasites by transfection of cell culture-derived merozoites.

BACKGROUND: Malaria research is greatly dependent on and has drastically advanced with the possibility of genetically modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. Naturally, this transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. In this study, the potential of transfecting liver stage-derived merozoites was explored. In cell culture, upon merozoite development, infected cells detach from the neighbouring cells and can be easily harvested from the cell culture supernatant. This protocol offers robust experimental timing and temporal flexibility. METHODS: HeLa cells are infected with P. berghei sporozoites to obtain liver stage-derived merozoites, which are harvested from the cell culture supernatant and are transfected using the Amaxa Nucleofector® electroporation technology. RESULTS: Using this protocol, wild type P. berghei ANKA strain and marker-free PbmCherryHsp70-expressing P. berghei parasites were successfully transfected with DNA constructs designed for integration via single- or double-crossover homologous recombination. CONCLUSION: An alternative protocol for Plasmodium transfection is hereby provided, which uses liver stage-derived P. berghei merozoites for transfection. This protocol has the potential to substantially reduce the number of mice used per transfection, as well as to increase the temporal flexibility and robustness of performing transfections, if mosquitoes are routinely present in the laboratory. Transfection of liver stage-derived P. berghei parasites should enable generation of transgenic parasites within 8-18 days.

A Plasmodium plasma membrane reporter reveals membrane dynamics by live-cell microscopy.

During asexual replication within the Anopheles mosquito and their vertebrate host, Plasmodium parasites depend on the generation of a massive amount of new plasma membrane to produce thousands of daughter parasites. How the parasite plasma membrane (PPM) is formed has mostly been studied by electron microscopy, which does not allow an insight into the dynamics of this process. We generated a Plasmodium berghei reporter parasite line by GFP-tagging of a non-essential PPM-localized protein, and followed plasma membrane development in living parasites through the entire Plasmodium life cycle. By generating double-fluorescent parasites in which the PPM is visualized in combination with the parasite endoplasmic reticulum, we show that membrane contact sites are formed between both membrane systems during oocyst and liver stage development that might be used to deliver lipids to the dramatically expanding PPM. In conclusion, we have established a powerful tool to follow PPM development in living parasites, which promises to greatly expand our knowledge of membrane biology in the Plasmodium parasite.

Progress in imaging methods: insights gained into Plasmodium biology.

Over the past decade, major advances in imaging techniques have enhanced our understanding of Plasmodium spp. parasites and their interplay with mammalian hosts and mosquito vectors. Cryoelectron tomography, cryo-X-ray tomography and super-resolution microscopy have shifted paradigms of sporozoite and gametocyte structure, the process of erythrocyte invasion by merozoites, and the architecture of Maurer's clefts. Intravital time-lapse imaging has been revolutionary for our understanding of pre-erythrocytic stages of rodent Plasmodium parasites. Furthermore, high-speed imaging has revealed the link between sporozoite structure and motility, and improvements in time-lapse microscopy have enabled imaging of the entire Plasmodium falciparum erythrocytic cycle and the complete Plasmodium berghei pre-erythrocytic stages for the first time. In this Review, we discuss the contribution of key imaging tools to these and other discoveries in the malaria field over the past 10 years.

High resolution microscopy reveals an unusual architecture of the Plasmodium berghei endoplasmic reticulum.

To fuel the tremendously fast replication of Plasmodium liver stage parasites, the endoplasmic reticulum (ER) must play a critical role as a major site of protein and lipid biosynthesis. In this study, we analysed the parasite's ER morphology and function. Previous studies exploring the parasite ER have mainly focused on the blood stage. Visualizing the Plasmodium berghei ER during liver stage development, we found that the ER forms an interconnected network throughout the parasite with perinuclear and peripheral localizations. Surprisingly, we observed that the ER additionally generates huge accumulations. Using stimulated emission depletion microscopy and serial block-face scanning electron microscopy, we defined ER accumulations as intricate dense networks of ER tubules. We provide evidence that these accumulations are functional subdivisions of the parasite ER, presumably generated in response to elevated demands of the parasite, potentially consistent with ER stress. Compared to higher eukaryotes, Plasmodium parasites have a fundamentally reduced unfolded protein response machinery for reacting to ER stress. Accordingly, parasite development is greatly impaired when ER stress is applied. As parasites appear to be more sensitive to ER stress than are host cells, induction of ER stress could potentially be used for interference with parasite development.

The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites.

Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence.

Features of autophagic cell death in Plasmodium liver-stage parasites.

Analyzing molecular determinants of Plasmodium parasite cell death is a promising approach for exploring new avenues in the fight against malaria. Three major forms of cell death (apoptosis, necrosis and autophagic cell death) have been described in multicellular organisms but which cell death processes exist in protozoa is still a matter of debate. Here we suggest that all three types of cell death occur in Plasmodium liver-stage parasites. Whereas typical molecular markers for apoptosis and necrosis have not been found in the genome of Plasmodium parasites, we identified genes coding for putative autophagy-marker proteins and thus concentrated on autophagic cell death. We characterized the Plasmodium berghei homolog of the prominent autophagy marker protein Atg8/LC3 and found that it localized to the apicoplast. A relocalization of PbAtg8 to autophagosome-like vesicles or vacuoles that appear in dying parasites was not, however, observed. This strongly suggests that the function of this protein in liver-stage parasites is restricted to apicoplast biology.