Malaria: the master of disguise

The availability of faster and cheaper genome sequencing has provided a platform for delving further into the biological functions of lots of organisms. One of these organisms is the malaria parasite, Plasmodium falciparum. Since its genome was sequenced in 2002, scientists have identified a number of genes involved in how the parasite functions inside its host to cause malaria. The discovery of one family of genes called the var genes has provided some interesting clues about how the malaria parasite manages to survive and thrive inside the human body.

But first, some background…

Currently, the main focus for malaria research is on the species that causes the deadliest form of malaria, Plasmodium falciparum.

Once the parasite is inside the red blood cells it is hidden from the host’s immune system.

About a week after the P. falciparum parasite is injected by a mosquito into a human body, it attaches to and infects the red blood cells. Once it has done this, the parasite is hidden from the host’s immune system so it cannot be detected or attacked. This enables it to grow and divide uninterrupted.

Once the parasite has multiplied a number of times, the red blood cells bulge and eventually burst, releasing the parasite into the blood. This normally takes about 48 hours.

This growth and destruction of red blood cells causes the symptoms of malaria, such as fever, muscle aches and anaemia. Studying the blood stages of the parasite has therefore been a priority and scientists are trying to understand exactly how the parasite manages to avoid detection by the immune system over and over again.

So, what has been found out so far?

Scientists have investigated what the malaria parasite does when it is living inside human red blood cells. After making its way into a red blood cell, the parasite triggers proteins to appear on the surface of the red blood cell. These proteins are called P. falciparum Erythrocyte Membrane Protein-1s (PfEMP1s for short!) and are considered to be key in the malaria parasite’s ability to cause disease.

How does PfEMP1 work?

PfEMP1s are large, sticky proteins produced by the malaria parasite late on in the red blood cell stage of the life cycle.

PfEMP1s are large, sticky proteins that enable the malaria parasite to interact with its host from within the safety of the red blood cell.

PfEMP1s form ‘knobs’ on the surface of infected red blood cells and enable the parasite to interact with its host from within the safety of the red blood cell.

PfEMP1 then binds to other red blood cells to form bundles of cells called rosettes. These rosettes then stick to the walls of the blood vessels. This helps protect the parasites by stopping them from passing through the blood to the spleen, where they would be destroyed. Attached to the blood vessels, the malaria parasite can continue its life cycle uninterrupted. Unfortunately for the host, this also has the effect of blocking blood flow in small blood vessels. This causes tissue damage and inflammation which can in turn result in life threatening conditions, such as organ failure.

Why is PfEMP1 so interesting?

PfEMP1s are encoded by a family of 60 var genes that are constantly changing through a process called recombination.

PfEMP1s are encoded by a family of roughly 60 genes called var genes. Var genes are interesting because they are constantly changing through a process called recombination. This means that they are frequently shuffling their genetic information between each other to create lots of new variations of var gene and consequently, lots of variations of PfEMP1. If you consider 60 genes all shuffling genetic information over and over again you can imagine that is a lot of different variations.

Effectively, they were able to watch the parasites evolving in real-time.

In a study at the Wellcome Trust Sanger Institute, scientists grew malaria parasites in human red blood cells for over a year. Every few weeks or months they would then sequence the entire genome of the parasites to find out what genetic changes were occurring over that time. They were therefore able to get lots of snapshots of the malaria parasites’ genomes as they divided inside human red blood cells over the course of a year. They could see the results of recombination as well as other types of mutation, such as single base pair mutations. Effectively, they were able to watch the parasites evolving in real-time.

By doing this, the scientists found that var gene recombination was happening in around 0.2 per cent of parasite cells every 48 hours. This may not sound like a very significant fraction of the parasite population, but the average person with malaria might have a billion parasites inside them. So, 0.2 per cent of a billion is still two million newly-created var gene sequences being produced every 48-hour life cycle within a single person. This means that millions of new PfEMP1 variants can be produced inside one infected person every two days. And with 200 million new malaria infections happening around the world each year, that is a huge amount of genetic diversity that can spring up.

So what does this mean for the immune system?

In short, this is far too many changes for the immune system to be able to keep up with.

It’s a bit like the malaria parasite has about 60 different coloured coats that it can wear at any one time. So the parasite will start off wearing one coat, a blue one for example, and the immune system will produce antibodies to recognise blue coats. Then the parasite switches coats, to a green one maybe, but the body’s immune system is still looking for parasites with blue coats and not ones with green coats. Then the immune system realises it should be looking for green coats but by that point the parasite has changed coats again.

Through var gene recombination, the parasite can mix up the 60 colours of coat it starts with over the course of each infection, continuously producing different colour-combinations. This makes it incredibly difficult for the immune system to pin the parasite down.

The battle

To help illustrate the battle between parasites and the human immune system, imagine a person is infected with a particular malaria parasite. All of the parasites in their body are expressing the same var gene meaning their red blood cells all have the same PfEMP1 on their surface. The immune system then starts to produce antibodies that recognise that specific PfEMP1 and direct the immune system to destroy the infected red blood cells, along with the malaria parasite inside. 1-0 to the immune system.

One pioneer parasite will express a brand new var gene that encodes a PfEMP1 protein that the immune system has not seen before.

But (and here’s the clever bit), while this is happening, recombination is taking place between var genes in the genomes of dividing malaria parasites. One pioneer parasite will express a brand new var gene that encodes a PfEMP1 protein that the immune system has not seen before and therefore has no specific antibodies against. This means that the parasite has an advantage over the others as it is not recognised by the immune system and can multiply rapidly, out-competing other malaria parasites with the old var gene. The malaria parasite population increases again. So now it’s 1-1!

However, the immune system’s not been beaten yet! As the numbers of this new parasite increase the immune system kicks into action again, producing new antibodies that recognise the new PfEMP1. The immune system therefore takes the upper hand again reducing the numbers of this new malaria parasite. However, var gene recombination is continually taking place within the dividing parasites and eventually the parasite will gain an advantage once more.

Now, here, you see, it takes all the running you can do, to keep in the same place.

_{The Red Queen in Through the Looking-Glass}

This somewhat exhausting process is sometimes referred to as a Red Queen battle. The Red Queen theory is an evolutionary hypothesis based on the Red Queen’s race in Lewis Carols’ book Through the Looking-Glass. In the book, the Red Queen and Alice are running but appearing to remain in the same spot. The Red Queen then comments, ‘Now, here, you see, it takes all the running you can do, to keep in the same place’. In biology this is often used to illustrate that organisms must constantly adapt and evolve to survive in an ever-changing environment. In the case of malaria, the immune system is constantly trying to destroy the parasite to get rid of the infection, but the parasite is consistently managing to evade it by generating diversity through processes such as genetic recombination.

Add in the fact that PfEMP1 proteins are only one out of many hundreds of antigens expressed by the malaria parasite, and you can get an idea of how complicated the interaction between host and parasite becomes.

Var gene recombination was originally thought to happen in malaria parasites when they were in the mosquito. Discovering that this recombination occurs continuously when the parasites are in the human host may help to explain how the parasite can survive for many months inside infected people during the dry season, when there aren’t many mosquitoes biting. It means the parasites don’t have to rely on being inside mosquitoes to recombine their var genes and produce new PfEMP1 variants – they can do it all from within infected people.

What now?

This isn’t the end of the story. Although scientists know that this recombination of var genes is happening, they still don’t know how the DNA is cut and stuck back together again during recombination. More work is therefore needed to find this mechanism, which may open up new avenues for malaria treatments, for example by interfering with whatever is cutting and splicing the DNA in the parasite.

The first malaria genome took over a decade to sequence.

The ability to sequence entire genomes has made it easier to track genetic changes in the parasite. This has helped us to learn more about how the parasite evolves and adapts in response to the human immune system and to the efforts of modern medicine to control the disease. In the var gene study, hundreds of malaria genomes were sequenced in just a couple of years. Yet only 10 years ago, the first malaria genome took over a decade to sequence, so huge progress has already been made.

This new-found knowledge is bringing scientists closer to finding new weapons to add to their arsenal against malaria, and will hopefully give them the upper hand to defeat this deadly disease in the future.