Biting back at malaria
Max Planck researchers are searching for vulnerabilities in the life cycle of the malaria parasite
Many parasites have complex life cycles. They change hosts over the course of their lives and completely alter their way of life in the process. The malaria pathogen Plasmodium falciparum also lives in different organisms: humans and mosquitoes. Scientist Kai Matuschewski from the Berlin-based Max Planck Institute for Infection Biology is therefore searching for a weakness in the life cycle of the pathogen that could be used to prevent it from spreading.
Text: Catarina Pietschmann
The humidity is tropical, the temperature is a constant 28 degrees, and there’s an abundant supply of fresh water, as well as Brekkies cat food to eat - and there are droves of mosquitoes of the same age. Even the sunrise and sunset are simulated - perfect conditions for taking things easy or swimming a few leisurely laps. The lab is nothing short of a luxury spa for mosquito larvae! Scientist Kai Matuschewski laughs: "Yes, we really pamper them."
Matuschewski’s colleagues, who constantly position, feed up and remove the offspring from the surface of the water before they fledge, find that their work has a meditative calm about it. It’s easy to see why this is so, as the environment is not particularly exciting - white walls, against which white units rest, each containing numerous shelves filled with flat white containers, in which the "next scientific generation" swim: around 200 larvae of the species Anopheles stephensi. Despite originating in India, the mosquito colony is already used to life in the laboratory. Initially not much more than black dots around the size of vanilla seeds, the larvae develop into insects that are (almost) ready for take off, but are still shrouded in their pupae. Clearly labelled so that each researcher can find "his" or "her" mosquitoes, they are still "clean", that is not infected with plasmodium, the malaria pathogen.
A breeding station for Plasmodium
The next room contains airy gauze containers around the size of shoeboxes. The larvae hatch in the boxes which, again, are stored on shelves. There must be several thousand mosquitoes here! Thanks to the air conditioning, the dreadful buzzing cannot be heard. In nature, they would suck on sweet plant juices. Here, they are given sugar water on cotton wool and when they are old enough, they receive their first meal of blood from mice infected with plasmodia. The mosquitoes are kept mainly here as "factories" - plasmodia factories.
Kai Matuschewski has been head of the Department of Parasitology at the Max Planck Institute for Infection Biology on the Charité Campus in Berlin for two years. He completed his undergraduate and post-graduate studies in Tübingen and Heidelberg, and did his doctorate on the genetics of yeast cells. He has long been fascinated by unicellular organisms with a nucleus, as they have a genome with a manageable size.
Plasmodia only have 5,600 genes. Matuschewski, a native of Berlin, has been working intensively on this scourge since 1998 when he did his post-doctoral work at the School of Medicine in New York University. He is looking for answers to a series of questions: How does the pathogen get into the host cell? How does it develop in there? And how does it get out again? The answers are located in the genes. For this reason, he switches off one gene after another and observes whether and how such knock-outs interfere with the behaviour of the parasite.
The codes on the storage boxes refer to the various mutants that reside inside the mosquitoes. A doctoral student will come again soon, pull out a box and examine how a plasmodium, in which the formation of a certain protein is increased or suppressed, has developed in the mosquito collective. Many mutants are stained with fluorescent dyes and are luminous red or green. They can be identified in the live mosquito under the microscope. The scientist will photograph individual insects and isolate pathogens to examine them in greater detail.
But...there’s one flying around the room! "It won’t do any harm," says Matuschewski smiling and echoing a statement often uttered by dog owners. But it’s not intent on playing either. The scientist follows it quickly with his eye and - clap! - the beast meets its end between the palms of his hands. Was that one already infected? That’s irrelevant, as the unicellular spore organisms, of which there are far in excess of 100 species, are host-specific. They live in the blood of reptiles, birds, rodents and primates; and, unfortunately, Plasmodium falciparum lives in ours. However, the mosquitoes kept here homed in on mice many millions of generations ago. For safety reasons alone, the research group works mainly with the mouse pathogen Plasmodium berghei. It is native to the highlands of the Congo, which is why the temperature in the incubators containing the plasmodium-infected mosquitoes is only 20 degrees Celsius.
With the exception of the polar regions, plasmodia are found all over the world. However, the species that pose a threat to humans are limited to tropical and subtropical countries. This means that three billion people throughout the world are exposed to the risk of malaria infection. According to WHO, between 300 and 500 million are infected annually as a result of the nocturnal bite of an Anopheles female. One million of those infected die, the majority of who are children. Somewhere in the world, a child dies of malaria every minute.
Vaccination protects longer than drugs
Kai Matuschewski is looking for plasmodium’s Achilles heel, the most vulnerable phase in the insect’s eventful life cycle, so that lasting immunity against the treacherous pathogen, which was discovered back in 1880, can be developed. And, to come straight to the point: he has already found a weakness! In the mouse variant, at least.
But why is vaccination so important? Malaria is curable. "Yes, of course. We have drugs that work effectively to counteract the pathogenic blood stages and kill plasmodia. For example, artemisinin, which is derived from the Chinese mugwort." Similarly, the drugs mefloquine, ato-vaquone/proguanil and doxycycline can also be taken as a prophylactic treatment during short visits to malarial regions.
However, it is not practical to take a drug for life. And a substance that provides reliable and long-term protection against malaria is still not available. For this reason, the next mosquito bite can mean the next infection. "You see this in Mali, for example, where almost every child becomes infected during the rainy season," explains Matuschewski. Having been treated successfully, the children are back in hospital the following year, at the latest. And not that many mosquitoes are actually infected - just one or, at most, five percent. However, being bitten numerous times over a period of several days increases the hit rate. "The repeated treatment generates enormous costs, which are met by the global community. But can we sustain this over decades without offering a better alternative?"
The word malaria originates from Italian and translates literally as "bad air". The "bad air" in question rises from marshes and swamps, in which millions of mosquitoes breed. The disease is also referred to as swamp fever and intermittent fever as patients experience cyclical spikes in temperatures over a two to three day cycle. However, these temperature spikes are sometimes completely absent from the most serious form of the disease, Malaria tropica, which is triggered by Plasmodium falciparum.
Yet whatever might be said about the mosquitoes and their bites, they are not really the ones at fault here. They are just the disease vector, they merely transmit the pathogen without noticing anything themselves, and they do not develop the disease. In ancient times, the bearers of bad tidings were beheaded. Today, specialists in infectious diseases refer to this approach as "vector control": the mosquitoes are destroyed over a large area using insecticides. Attempts are also made to keep them away from their victims at night with the help of barriers (mosquito nets). These tried-and-trusted anti-malaria strategies have made a significant contribution to eradicating the infection from the Mediterranean region and parts of Southeast Asia. However, less success has been achieved in Africa using such measures.
It is simply not very easy to bring insects under control. This circumstance is demonstrated, for example, by the German control programme on the Upper Rhine, which merely concerns the nuisance caused by swarms of mosquitoes. Every spring, solutions containing Bacillus thuringiensis israelensis, which is highly toxic to the native mosquito species Aedes und Culex, but are harmless to humans, are sprayed between Lörrach and Koblenz. "If the wrong mixture was to be used for just one week, the population would increase sharply," says Matuschewski. "And if that should happen, all of the summer festivals would have to be cancelled."
Dozen of pathogens transmitted with every bite
Human - mosquito - human: the chain of malaria infection. Female mosquitoes need blood after fertilisation because it contains proteins that play an important role in oviposition. Thus, attracted by the smell of carbon-dioxide from human breath, they bite people they come across while flying blind at night. If the victim is already infected, they consume the pathogen with the five microlitres of blood they suck. If the mosquito is infected, like a Trojan horse, it immediately and unknowingly unloads its disastrous freight with its saliva. Several dozen plasmodia gain access to the victim’s body in the form of sporozoites. What happens then, however, is far more adventurous than anything found in Greek mythology.
The beginning of the plasmodia’s long journey is marked by their unexpected arrival in the human skin: an unaccustomed environment and 35 degrees Celsius! "A regular heat shock for sporozoites, which activates a new programme," explains Matuschewski. The view through a microscope reveals that they move faster. Like sniffing dogs, the oblong cells search around until they meet a blood capillary. They then prick the capillaries and gain access to the bloodstream in no time.
In a matter of between 10 and 30 minutes they reach the liver, clamp on to endothelial cells and bore through to the interior of the organ. Once there, they’ve made it to a safe harbour where they remain invisible to the immune system’s guards. Therefore, this phase of the infection does not attract any clinical attention. The sporozoites then get down to business. They become spherical, feed, grow and divide - again and again, over twelve to 15 cell cycles. "One sporozoite soon produces 10,000 daughter cells," explains Matuschewski. This army leaves the liver and floods the bloodstream where it boards 100,000 red blood corpuscles. The entire process unfolds very rapidly and the immune system notices nothing.
As tiny, sealed swimming capsules, that are constantly on the move and reach every corner of the body, red blood corpuscles are ideal host cells. While they circulate in the blood, the blind passenger inside them reaches maturity and begins to divide again. One becomes two, then four, then eight, 16, 32. And that’s as much as the tiny capsule can hold. This process takes two days and then the cell bursts. A vast armada then enters the water. The invaders, which are known as merozoites, boldly present their antigens, proteins that are foreign to the body, on the surface of the cell. But there are so many of them! What can the immune system do? It raises the alarm, lights a fire and heats up the body, and the first temperature spike occurs. Twelve days have now passed since the mosquito bite. However, the pathogens quickly retreat under cover again. They hijack more blood corpuscles and throw themselves into the next round of cell division. Meanwhile, the body temperature goes down again. However, the number of pathogens increases by a factor of ten with each cycle - every two days.
Malaria is usually treated at this stage, which is known as the blood phase. "Typically, one percent of the patient’s blood cells are found to be parasitised, giving a total of 100 billion cells," explains Matuschewski. The treacherous plasmodium causes the infected blood corpuscles to discard molecular anchors from their surface and to adhere to the blood vessel wall. A plaque forms that can block the blood vessels locally. If this process arises in brain capillaries, the patient may go into a coma (cerebral malaria). Other organs, like the lungs, may also suffer from an undersupply, which can be fatal if left untreated. Some patients, in particular children, simply die of anaemia because too many red blood corpuscles are destroyed.
"Whereas the blood phase is crucial from the perspective of tropical medicine specialists, as this is when the symptoms arise, for biologists, it’s just one window of many," says Matuschewski. So why do they not choose an earlier one? His team specifically looked for genes that are only active in the liver phase, and their search was fruitful. If a particular gene is missing, the liver cell acts as a prison for plasmodium: that is, it can get in but it can never get out again.
Some of these genes are transcribed into messenger RNA in the sporozoites and stored in the cell’s cytoplasm. These mRNAs are not read and translated into proteins until a sporozoite has penetrated a liver cell. The proteins manipulate the host cell from the surface of the parasite organelle, known as the parasitophorous vacuole, and form long tube-like structures. If one of these proteins on the border between the parasite and host cell is missing, the parasite can round itself off but it cannot grow any more.
Presented to the immune system on a plate
This hiatus in the life cycle of the parasite is not planned, and stimulates the immune system. Antigens, which only make a brief appearance in an infection with natural pathogens and unhampered growth in the liver, are presented in detail here to the immune system. If immunisations with these genetically outwitted parasites are repeated, the host remains completely immune to new infections for many months.
Similar - or even better - results are obtained when a well-tolerated antibiotic is administered following infection with the natural (non-mutated) pathogen. Surprisingly, the immune system can then build up long-lasting protection against new infections. Mice, in which sporozoites were injected into the blood and were then given azithromycin or clindamycin for three days, did not develop the disease - even when they were re-infected 40 days, three and six months later. Merozoites still form, but they are no longer capable of penetrating the red blood corpuscles. Their key for moving to another host cell, a small cell organelle called apicoplast, is blocked. "The fact that this approach functions in the clinically inconspicuous liver phase and goes wrong in the blood phase is probably due to the different immunological micro-climate that prevails in the liver," says Matuschewski. Drugs like clindamycin and azithromycin are authorised for use in children and are reasonably priced.
Both vaccination strategies have only been tested on the mouse model up to now. Whether safe and lasting immunoprotection can also be developed in humans, particularly in young African children, still must be investigated in clinical tests. Meanwhile, the team has found a master gene that is responsible for the entire development programme of the sporozoites in the liver, and controls over 100 other genes. The researchers assume that this kind of central switch also provides the further plan of action when the parasite changes host.
The deliberate switching on and off of these master genes makes it possible to interrupt the lifecycle completely at will. This enables the researchers to understand the parasite’s development programme and to undertake a systematic search for a tailor-made, particularly powerful genetically-modified vaccine strain.
One possible approach would involve the large-scale infection of people with a genetically modified plasmodia strain or the natural pathogen, and the simultaneous, controlled administration of antibiotics, therefore immunising them in that way. That might work in Europe - but in Africa? "Could we really drive from village to village on a moped with a live pathogen?" Matuschewski is sceptical. Periodic and temporary administration of antibiotics could even be sufficient in malaria areas where constant infection must be expected. The decisive experiments with the human pathogen Plasmodium falciparum still have to be carried out; therefore there is still time to find the optimal strategy.
From free-living unicelluar organism to parasite
Let’s get back to plasmodium, which we abandoned at the merozoite stage. Its journey continues as reproduction through asexual division alone is not sufficient. Its DNA must also be re-mixed - and that can only be achieved through sexual reproduction. This requires a change of host - so, it’s back to the mosquito. With the next bite, it takes in infected blood. Sexual pathogen stages are formed in the mosquito’s intestine that exchange their genetic information, and a fertilised pathogen stage arises. "Hundreds of them sit like thick bobbles on the mosquito’s midgut, but, surprisingly, this does not harm it whatsoever."
So how did the plasmodium actually get into the mosquito? "Without doubt, it was originally a free-living unicellular organism that later opted for life inside the insect - just as all animals are inhabited by microorganisms," says Matuschewski. A complex parasite-host relationship then formed over the course of evolution.
The parasite has already been accompanying Homo sapiens for between 30,000 and 50,000 years. This contact has left certain life-threatening traces in the genomes of many people who live in former malaria regions. The genome adapted - for example, through the haemoglobin S-allele, which protects against the serious complications of malaria when present as a single copy, but causes death in childhood when two copies are present. These incredibly high genetic costs can only be explained by the enormous selection pressure of the pathogen. Gene defects like this one, which is also known as sickle cell anaemia, mainly arise in Africa. Others, like thalassemia, arise more frequently in Sardinia, Sicily and Cyprus, Greece and the Middle East.
To get back to the fertilised pathogen stages in the mosquito: at this stage, they move off. They begin by crossing the mosquito’s midgut wall and then take a break for two weeks. Meanwhile, the new generation develops: up to 1,000 sporozoites form from each fertilised ovum. When it bursts, they head off in the direction of the salivary glands where they assemble for take-off - they leap from the mosquito to the human. The circle closes here, and the odyssey of the next plasmodium generation begins. From the perspective of a ten micrometre-long parasite, it must look like a journey into space.
Many individual molecular steps in this process are still unexplained. How does the pathogen suddenly manage to move through the gut wall? How does it make its way to the salivary gland? And how do they hold on there? These are all questions being investigated by Kai Matuschewski’s team of 15 biologists, biochemists and medics. It would appear that certain recognition molecules that are transmitted on the parasite give the marching orders: "Off you go! Crawl through the gut wall!"
It is also clear that a system consisting of actin and myosin proteins is involved. The tiny organisms also have something remotely resembling muscles that can expand and contract, and enable active movement.
The team has succeeded in demonstrating that actin-binding proteins play a crucial part in the rapid sliding movements. If a heat shock protein is missing, which stabilises actin filaments, the sporozoites are unable to penetrate the salivary glands. Without the protein, the sporozoites are too slow and remain stuck in the skin.
Insects are also part of the ecosystem
Despite everything he knows about parasite-host relationships, in Kai Matuschewski’s opinion neither mosquitoes nor pathogens are monsters. He examines them under the microscope with his children, who are three, ten and eleven years of age, and conveys the fascination of nature’s incredible variety. "Irrespective of the threat it poses, every insect has its place in the ecosystem," he stresses. "It’s amazing to see how well our immune system works! Despite our constant contact with pathogens, we rarely contract diseases."
Matuschewski is repeatedly drawn to Africa, and not only for professional reasons. "At first, it was just for the nature. There is simply nowhere better to observe it than the Ethiopian highlands, the Ugandan rainforest or the savannah in Mali." Now, however, he is almost more fascinated by the cultural diversity and the enthusiasm of the people. "Often the best experiences arise when you find yourself stranded in a village because the bus has broken down and you become part of everyday life there for an evening." He knows all about the problems and injustices here. "That’s why it is my fervent wish as a scientist to contribute to finding innovative ways of controlling malaria."
Germany also has around 500 to 1000 cases of malaria each year, mostly infections brought back by holidaymakers who were a bit careless "with the prophylaxis". Too many cases, but not nearly enough to facilitate research on the tropical disease. "Ultimately, it’s about finding practical approaches to malaria. For that reason, equal partnerships with local research institutes are so important for us." Kai Matuschewski is in the process of establishing such a partnership with the Kemri-Wellcome-Trust in Kilifi in Kenya, a renowned and excellently resourced institute where staff of the MPI are now testing patient blood as part of their doctoral studies. Whether or not the human immune system can be fired up to provide long-term protection against malaria like that of the mouse will emerge in the years to come. The prospects are not at all bad.
One final question: Why does plasmodium lead such a complicated life? Kai Matuschewski laughs. "Compared to bacteria and viruses, parasites have very long generation times. So they have to be smart." But even the smartest of them have a weak spot. And we need to know what will bring about their downfall.
An organelle of bacterial origin that probably arose like the chloroplasts in plants through the endosymbiosis of a bacterium. Thererfore, it is sensitive to antibiotics and herbicides. Many single-cell parasites (Apicomplexa) need the apicoplast to infect host cells, for example the toxoplasmosis and malaria pathogens.
Alternation of generations
Lifecycle with sexual and asexual reproduction. The advantages of both types of reproduction are combined: that is, the mixing of the gene pool and production of large numbers of offspring. Organisms that exhibit alternation of generations include, for example, corals, aphids, water fleas, mosses, ferns and seed plants. In many parasites, the alternation of generations is accompanied by a change of host.