The complexity of the apicomplexans

The phylum of Apicomplexa comprises a large group of highly successful parasites. It includes many human and animal pathogens such as the famous malaria species, Toxoplasma and the dinosaur of the parasite world, Cryptosporidia.

Numerous apicomplexan parasites are also of significant economic importance, particularly in the agricultural and veterinary fields in relation to livestock pathogenesis.

For the biologist, these organisms also provide extremely good models for studying the basics of parasitic mechanisms, and one of the fundamental questions in the field is how a eukaryotic cell can penetrate, survive and replicate within another eukaryotic cell.

Addressing such questions can also provide wider insights into biological diversity and the mechanistic origins of mammalian systems.

Working at the University of Geneva in Switzerland as a Howard Hughes Medical Institute international scholar, Swiss-born Dr Dominique Soldati-Favre is a world-expert on the apicomplexan parasites and the cell biology underlying how these clever little creatures literally glide into mammalian cells to set up camp.

In fact, over the last decade Soldati-Favre’s group has contributed much to our current understanding of host-parasite interactions in general, from host-cell recognition to infection.

The parasite of choice for Soldati-Favre over many years has been Toxoplasma gondii, the human pathogen that causes Toxoplasmosis.

“It is gentle in terms of pathogenesis compared to related parasites like Plasmodium falciparum (malaria), and is far easier to manipulate genetically in the laboratory,” Soldati-Favre says.

“So, we use this organism as a model to study parasite mechanisms for the Apicomplexa group.”

Her primary focus is the mechanism of host invasion – understanding how these parasites actually contact and then enter the host cell.

Obligate intracellular organisms like the apicomplexans have to do so to gain nutrients, replicate and potentially evade the host immune system.

This dependence on the host cell makes their entry stage quite vulnerable, with any major component of the machinery that orchestrates cell invasion a potentially good target for therapeutic intervention.

“Understanding how it all goes together is thus imperative not only to improve our fundamental knowledge of parasite biology, but also to improve the chances of preventing parasitic disease,” she says.

---PB--- Self-contained and single-minded

Unlike viruses and pathogenic bacteria that are phagocytosed by their host cells, or most other parasites that use host-mediated processes, apicomplexans employ a unique mechanism of adhesion-based host-cell entry – a highly dynamic and energy-dependent process known as gliding motility.

The mechanism is facilitated via parasitic machinery called the glideosome, a macromolecular complex consisting of adhesive proteins that are released from the apical pole of the parasite and translocated to the posterior pole.

This well orchestrated and tightly controlled mechanism relies heavily on the parasite cytoskeleton anchored in the inner membrane of the parasite – particularly actin polymerisation and myosin motor protein activity – as well as numerous interconnecting molecules such as proteases that are highly conserved throughout the apicomplexans.

The gliding motility of apicomplexan parasites is crucial for migrating across host biological barriers and thus establishing infection.

This active mode of penetration presents numerous advantages for the pathogenic parasite: i) the parasite can enter virtually any cell type including red blood cells as in the case of malarial species; ii) the smooth mode of entry prevents the stimulation of host cell defences; and iii) it leads to the formation of a unique parasitophorous vacuole in the host cell, a membrane-bound compartment formed by an invagination of the host cell membrane on invasion and segregated from host cellular trafficking pathways, enabling the parasite to survive for a long time even in unfriendly intracellular environments.

Everything orchestrating the host-parasite interaction for apicomplexans takes place within the very confined environment of the parasite plasma membrane.

In this space, the parasite is turning on the myosin motors and actin polymerisation needed to enable the motility in a process that is very tightly regulated because of the short need-to-use basis of this step. The entire invasion takes only 15 to 20 seconds and once inside, the parasite immediately turns off the motility mechanisms and initiates a completely different program for survival and replication.

The gliding motility used by Toxoplasma and its relatives to invade mammalian cells is also very interesting from an evolutionary standpoint. Although unique to this phylum of parasites, is it virtually completely conserved across the group.

Analysis of the genome sequences now available for a number of closely related free-living organisms has now revealed that the apicomplexans have simply adapted machinery that existed in some of these organisms already for functions such as predation, and they have evolved and refined it to successfully and efficiently access their host cell.

---PB--- Big questions in the field

There are many ‘big’ questions to ask about the cell biology of host-parasite interactions in general, and this has been a very active area in recent years.

Some of the main conundrums for anyone studying parasites in general is how they gain access to a host mammalian cell, what controls the mechanism of both host recognition and invasion and what are the virulence factors leading to pathogenesis?

Soldati-Favre says that over the last 10 years or so, her group and others have described much about the invasion process for the apicomplexan parasites, both morphologically and biochemically.

“We have compiled the basic building blocks and identified the major players in the process and we now need to unravel how these components work in a concerted action that is finely controlled both temporally and spatially within the context of the parasite/host membranes,” she says.

“It becomes clear that sophisticated signalling cascades are governing and triggering the invasion process and that complex patterns of post-translational modifications regulate the activity of each individual invasion factors to turn the whole process on and off.”

Soldati-Favre’s group has focused much of its research on the motility machinery, such as the myosin motor proteins and the whole regulation scheme for the necessary actin polymerisation.

They are now looking into how each of the identified protein components is controlled during invasion, particularly at the post-translational level of regulating protein activity.

“The formins and profilin proteins we have identified are now recognised as key players in the process. They orchestrate the actin polymerisation, hence building an essential roadway (actin filament) for the myosin motor to power the gliding motion.

This motility allows the parasite to literally propel itself into the host cell. It is extraordinary to watch down a microscope.”

---PB--- Playing with the players

Recently developed techniques in parasite biology have allowed gene expression to be specifically turned down or otherwise disrupted. Soldati-Favre’s group is exploiting this novel methodology in their parasites to further address exactly what protein players such as the formins are doing during invasion.

“All of the relevant factors we are looking at are critical for the parasite viability, and finding ways to look at these essential genes is not a trivial thing … we cannot just knock out essential genes, so technology such as this is a major advance.”

They have also developed an inducible system in collaboration with Dr Brendan Crabb in Melbourne. Originally developed for the malarial parasites, it also works well for T. gondii.

Additionally, a novel strategy controlling protein stability has been adapted to T. gondii by Dr Markus Meissner in Heidelberg. This approach offers an alternative and complementary opportunity to examine gene function, and will prove invaluable in the next, discovery stage being undertaken by Soldati-Favre and others in the field.

---PB--- Role of the host cell

For a long time, it was believed that apicomplexan parasites were driving invasion and that the host cell was a rather passive partner in the interaction.

The fact that these parasites can access red blood cells, which do not have much of their own machinery to contribute anyway, seemed to confirm this prevailing hypothesis. However, research about to be published now suggests that the host cell does indeed participate in the invasion process, at least to some extent.

The general impression in the field according to Soldati-Favre is that this contribution of the host cell will still be fairly minor, but that this finding is still quite significant from a fundamental biology angle and for drug target development.

“What is becoming very clear now, however, and what is really a booming area for parasite biology, is that these parasites do indeed subvert and reprogram the host cell processes for their own benefit.

“Effector molecules modulating host cell functions have been extensively characterised in bacteria and viruses and recent studies uncover similar strategies adopted by the apicomplexans.”

Soldati-Favre thinks that such revelations are just the tip of the iceberg in terms of what will be revealed about parasite-host interactions in the coming years.

Some of the effector molecules running this parasite-driven renovation of the host machinery have been identified. These discoveries came about largely through genome comparison, although it was not at all a simple process, according to Soldati-Favre.

“The equivalent molecules in viruses and other organisms used for comparison have quite different sequence information,” she says.

In fact, the major breakthrough in this area came about with new technologies that allowed the identification of virulence factors using a genetic cross between parasites. “These techniques aimed to reveal how the virulence factors in a strain that is attenuated compare to those in a highly virulent strain.”

Protein kinases feature as an important group of these parasite effector molecules. It seems that these important cellular enzymes hit at some point in a general pathway of kinase signalling in the host cell to substantially reprogram host gene expression.

“For example and very interestingly, sets of genes involved in innate immunity are remodelled, which of course disarms the host defences against the pathogen. This pathogen arrives, invades and then changes the whole workings of its immune defences.”

As an example, collaboration between Dr Alan Sher, an immunologist at the NIH/NIAID in the US, and Soldati-Favre recently showed that profilin is both an actin regulator and a modulator of the host immune response in their model organism, T. gondii.

In addition to its key contribution to actin polymerisation, Toxoplasma profilin binds to Toll-like receptors on the host cell membrane and induces production of the protective host cytokine, interleukin-12, by dendritic cells.

In contrast, whilst still acting as an actin regulator, malarial profilin does not fulfil an immunomodulatory function despite being highly homologous to T. gondii profilin.

“This can be explained,” says Soldati-Favre, “by the fact that two parasites have completely different goals in life.”

Toxoplasma infection is transmitted mainly via carnivorism. These parasites need to get into the host cells and remain there until another predator eats the host…activating the host immune system to destroy the infected cell before this happens would be counter-productive and just plain dumb.

In contrast, Plasmodium induces a high parasitemia to assure transmission by a mosquito biting the host, so the parasite action on the immune system is of a very different nature – such a different strategy favouring dissemination has been adopted at any cost including death of the host.

---PB--- From basics to bedside (or pasture)

Understanding how the machinery of invasion works is obviously a good way to identify novel approaches for intervention in the case of obligate intracellular parasites because without invasion there is no establishment of infection.

“However, finding the right target and the right strategy is not always so obvious,” Soldati-Favre says.

Although her work has always been focused more towards understanding the fundamental processes of parasite cell biology and not directly towards drug development or finding an intervention, she remains quietly optimistic about that task. She knows that by understanding the fundamentals more carefully and then keeping the potential clinical benefits always at the back of her mind, researchers should be able to find promising and ultimately, effective drug targets.

As part of this, companies are currently developing and testing attenuated Toxoplasma vaccines for use in the veterinary arena. However, according to Soldati-Favre, these vaccine prototypes are not all that safe, as the attenuated forms are poorly defined and could revert.

“I think there is a great potential today to generate much safer live vaccines for animals based on recently accumulated knowledge, and this is clearly where fundamental cell biology of the causative organism holds the key. Using Toxoplasma as an organism model, we can ask and address the key questions more easily and in a way that applies to the whole group of parasites.”

At the moment, Soldati-Favre and collaborators are surprised and delighted about some quite basic findings they have made into protein lipidation and lipid-modifying enzymes in the lytic cycle of these parasites “What we are now studying about the post-translational modifications of proteins, which is quite fundamental, is unexpectedly revealing a promising avenue for intervention.

“We have identified a series of compounds that interfere with protein acylation and have very dramatic effects on the invasion process and on the intracellular replication of the parasites.

“Importantly, this happens without deleterious effects on the host cells. This definitely encourages us to pursue our central and basic aim of identifying the parasite targets. There can always be a surprise in science by looking at the detail.”