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European Biopharmaceutical Review

A Deadly Bite

Malaria has a suitably notorious reputation, affecting some of the most deprived regions of the world. The human death toll currently stands at approximately two million each year, with no available vaccine. Worryingly, in the tropics, the most common species of the human malaria pathogen – the parasite Plasmodium falciparum (P. falciparum) – is already developing resistance to tried and tested drugs, highlighting the urgent need for novel therapeutics.

With P. falciparum continuously adapting to whatever conditions and treatments it is exposed to, malaria is a hot topic in biomedical research, inspiring innovative strategies for tackling this global challenge. For example, a recent report describes a novel way to prevent host mosquitos from becoming vectors of P. falciparum by infecting mosquito embryos with the symbiotic bacterium Wolbachia (1). In order to develop such techniques, one must fi rst gain an in-depth understanding of malarial biology. Although the genome of P. falciparum has been fully mapped, many details of the parasite's biology are still not yet understood.

Life Stages

The lifecycle of P. falciparum is highly complex, comprising a whole series of stages and intricate tactics aimed at evading the defences of the host organism. Among the life stages of P. falciparum that occur in both mosquitoes and humans, it is the continuous asexual replication phase within human erythrocytes that is solely responsible for the symptoms of malaria.

Lasting approximately 48 hours, this ‘blood stage’ of development is extremely complex, with P. falciparum undergoing three successive morphological changes: the ring stage, the transition to trophozoite stage, and the trophozoite stage (see A in Figure 1). Hijacking the erythrocyte, this fi nal stage eventually leads to the generation of up to 32 daughter parasites, which rupture the host cell and escape into the blood to invade new erythrocytes.

For P. falciparum to thrive within this environment, the parasite develops within its own protective compartment, while making extensive modifi cations of the surrounding erythrocyte via the export of proteins out into the host cell. Appearing from the late ring stage, parasite-induced vesicular structures within the erythrocyte named ‘Maurer’s clefts’ (MCs) were initially believed to serve as platforms for the trafficking of these proteins to the erythrocyte surface (2-4). Since this is by far the most important stage to target via medication, it is vital to understand this process in greater detail.

Know Your Enemy

With many years of experience within malarial research, researchers at Bernhard Nocht Institute (BNI) in Hamburg are working towards identifying novel approaches for malarial drug targeting. Designated a National Reference Centre for the detection of tropical infections in 2002, the BNI has enjoyed much success. For example, in 2006, they discovered the merosomes, or the ‘transport media’, by which the malarial pathogen buds off from the host liver cell to enter the bloodstream (5).

It is suspected that being enclosed by the merosome assists in the parasite’s evasion of the host immune system. Focusing on the blood stage biology of P. falciparum, in vivo microscopy techniques are incredibly valuable for this research. While research at the molecular level provides insight into the fundamental complexities of malaria, in vivo observations are essential for understanding this dynamic parasite, and this, in turn, hinges on high-performance microscopy technology.

Under the Microscope

Although the destructive malarial blood stage was first described over a century ago, the knowledge of this stage is almost entirely based on static assays – mostly in fixed samples. This is because the survival of P. falciparum is incredibly fragile and dependent on physiological conditions; thus, live samples must be studied immediately after being removed from culture, or by very short-term analyses under the microscope. These approaches provide limited and, perhaps, misleading information on the lifecycle of such a dynamic organism, and its complex interaction with the host cell.

In contrast, time-lapse microscopy captures this elaborate behaviour, and a specialised microscopy system dedicated to the study of P. falciparum has been developed (6). This system employs the powerful technique of confocal laser scanning microscopy (CLSM), which has revolutionised live cell imaging, offering superior image quality over conventional fluorescence microscopy.

Furthermore, the ability to obtain three-dimensional optical sections at a defined focal plane enables unobstructed analysis of the malarial parasite internalised within the cell (see B in Figure 1). Time-lapse three-dimensional imaging – or four-dimensional imaging – has been instrumental in understanding cell biology in diverse systems. However, the continuous high-energy laser light emitted from many CLSM systems is lethal to the sensitive malaria pathogen residing within the erythrocyte.

CLSM Instruments

To overcome this hindrance, the most advanced CLSM instruments implement a highly sensitive detection system in order to increase the efficiency of light transmission through the microscope light path. Improving this optical efficiency reduces the laser intensity required for sample observation, minimising phototoxicity to living cells, which is especially important for extended periods of time-lapse imaging.

It was for this reason that the research group built their own unique system around the FluoView FV1000 microscope, as this allowed observation of the complete blood stage of the malarial parasite in precise detail. Owing to the minimal phototoxicity of the system, extended time-lapse observations of up to 80 hours could be undertaken, gaining unique insights into the biology of P. falciparum. In addition, the use of a laser intensity monitoring and feedback control system in the scan unit ensured stable excitation intensity during these long time-lapse observations.

Even overcoming the challenge posed by photo-toxicity, such long-term incubation of these highly sensitive pathogens demands precise control of environmental conditions, including temperature, humidity, CO2 and O2 levels. An incubator enclosure was therefore incorporated into the system, ensuring precise control of optimum conditions matching those in the host organism.

Replicating in vivo conditions also means P. falciparum behaves true to life, yielding observations that are far more valuable for drug discovery. As focus drift can be problematic for time-lapse observations, the third main component of the fourdimensional microscopy system was the compensation module. Emitting lowphototoxicity infrared light, this module automatically maintains a focused image over the duration of the time-lapse observation, and so is ideally suited to the aims of the research.

Revealing Secrets

This bespoke four-dimensional imaging system was a powerful tool for observing P. falciparum development (see B in Figure 1, page 35). Such detailed observations allowed the identification of visual indicators for referencing each stage of development, including the build-up and completion of host cell modifications, onset of feeding and active preparation for release of daughter parasites into the blood stream.

Interestingly, the parasite forms visualised using four-dimensional microscopy resemble early descriptions of malaria parasites observed in the blood from patients, but differ significantly from the current view influenced by the widely used fixed and stained parasites, or living parasites removed from culture (7).

A stage of profound changes representing the major turning point in the cycle was also discovered, and was named ‘transition to the trophozoite’, where the highly mobile but slowly growing ring stage parasite settles into the stationary, haemoglobin-ingesting and fast-growing trophozoite (see A in Figure 1, page 35). During this phase, MCs change from mobile to a fixed state, and a very mild echinocytosis – a morphological change – of the host cell occurs.

Once the parasite invades the erythrocyte, it must modify itself in order to develop undisturbed, so that it distracts the immune system from the invasion. The fact that it does so by exporting parasitic proteins into the host cell was wellknown, but just exactly when and how was unclear, with the leading theory relating this export path to MCs.

Analysing this in detail has disproved this theory. Observations yielded no indication of ongoing formation of new clefts throughout development, or even changes in the number of clefts after they first appeared at the early ring stage. In accordance with these observations, the export of malarial proteins to the membrane of the host cell does not require the generation of new clefts, and these proteins are instead trafficked to those already formed. Such insights into the mechanism of P. falciparum development are key to the development of new drugs.

New Possibilities

Long-term four-dimensional microscopy has proven to be incredibly valuable for investigating the subtle changes taking place during the blood stage of P. falciparum development. Made possible by advances in cutting-edge microscopy technology, research has not only provided an accurate reference for this stage of development, but has also revealed some unexpected discoveries. Research has identified a stage of profound changes representing the major turning point in the cycle – where the highly mobile ring-stage parasite settles into the fast-growing trophozoite. Furthermore, researchers have challenged the model that protein export in P. falciparum is linked to the host cell modifications.

The four-dimensional microscopy system employed for this research has opened up new possibilities for the evaluation of medical agents and alternative treatment strategies, such as the recent approach of rendering the male mosquito infertile via bacterial infection (1). Four-dimensional imaging is, therefore, providing novel insights for the study of this lethal and dynamic pathogen, while paving the way for innovative treatment strategies.

References
1. Bian G et al, Wolbachia invades anopheles stephensi populations and induces refractoriness to plasmodium infection, Science 340 (6133): pp748-751, 2013
2. Langreth SG et al, Fine structure of human malaria in vitro, J Protozool 25: pp443-452, 1978
3. Bannister LH et al, Three-dimensional ultrastructure of the ring stage of Plasmodium falciparum: evidence for export pathways, Microsc Microanal 10: pp551-562, 2004
4. Wickham ME et al, Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum infected human erythrocytes, EMBO J 20: pp5,636-5,649, 2001
5. Sturm A et al, Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids, Science 313 (5791): pp1,287-1,290, 2006
6. Grüring C et al, Development and host cell modifications of Plasmodium falciparum blood stages in four dimensions, Nat Commun 2: p165, 2011
7. Laveran A, Nature parasitaire des accidents de l'impaludism: description d'un nouveau parasite trouvé dans le sang des maladesatteints de fièvre palustre, J.B. Baillière, 1881

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About the author

Bülent Peker is Product Manager of Laser Scanning Microscopy, Olympus Europa, Germany. With a background in Chemistry and a focus on Materials Science and Physical Chemistry, Bülent received his PhD from the Technical University of Braunschweig in 2006, for which he built a two-photon laser scanning microscope for in vivo imaging. He moved into his current role in 2007.
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