Diseases can be caused by different agents called pathogens. You may have heard of bacteria and viruses, but infections can also be caused by fungi (e.g. yeast infections), and parasites (e.g. malaria, worms).
A parasite is an organism that gets its food from a host organism and requires the host for its survival. Parasitic diseases tend to cause huge problems in low income/rural areas due to the lack of sanitation and hygiene facilities, and hence are big killers in the developing world.
A particularly interesting parasite is the African trypanosome (Trypanosoma brucei), which causes African sleeping sickness. This disease is fatal without treatment and threatens almost 65 million people in sub-Saharan Africa, despite successful control efforts by the World Health Organisation (WHO) and other organisations. This parasite is transmitted by the tsetse fly via a similar manner to how malaria is transmitted by the female Anopheles mosquitoes.
T. brucei gambiense causes the chronic form of sleeping sickness (97% of cases), while T. brucei rhodesiense causes the acute form. T. brucei brucei causes a zoonotic (animal) form of the disease in livestock, known as nagana.
Trypanosoma brucei – Who is she?!
The African trypanosome is a kinetoplastid extracellular parasite. A kinetoplastid has a kinetoplast (see Figure 1), which contains mitochondrial DNA.
Figure 1: An overview of how Trypanosoma brucei looks in its bloodstream long slender form. The kinetoplast contains mitochondrial DNA, which in turn, encodes the proteins necessary for the normal mitochondrial functions.
It is important to note that the parasite has different forms depending on the stage of its life cycle. For instance, the trypanosome occurs in the bloodstream long-slender (B-LS) form when it is in the bloodstream of its host (i.e. humans). Each form is specifically designed to help the trypanosome survive in a particular environment and carry out special functions at that point in the life cycle.
An antigen is a molecule or group of molecules that triggers an immune response within the body. T. brucei contains antigens on its surface that can be recognised by the host immune system, which in turn, can trigger the formation of corresponding antibodies to help clear the parasite.
The main problem is that T. brucei has evolved an incredibly clever way of evading the immune system and thereby cause a persistent infection.
A brief recap of the immune system
Refers to the non-specific defence mechanisms that everyone is born with. Consists of defences that are activated almost immediately or within a few hours when the body encounters an unknown or new pathogen. Perfect examples of these would include physical barriers (skin), secretions (mucous, lysozyme), general immune response (complement, inflammation). In general, innate immune responses are constant and do not change throughout the organism’s lifetime.
The complement system, in particular, is very important for innate immunity and signals for more phagocytes to come to the infection area. They are also involved in creating the membrane attack complex (MAC), which helps to damage the cell membrane of the pathogen.
Refers to the processes that occur to create immunological memory after the body has encountered a pathogen. Adaptive immunity is specific to each pathogen, unlike innate immunity which attacks all pathogens in the same way. Adaptive immunity consists of humoral and cellular components.
Humoral immunity refers to antibodies, which are produced by B cells. Antibodies bind to antigens on the surface of the pathogen so that it can be easily recognised by phagocytes. On the other hand, cellular immunity refers to T cells (helper T cells and cytotoxic T cells), which directly kill infected cells by various mechanisms.
Trypanosomes are the masters of disguise
The trypanosome is an extracellular parasite, which means that it does not go inside cells, but instead floats around in the blood and within tissues. Since it cannot hide inside a cell, an extracellular parasite has to worry about a lot of things that are actively trying to kill it, such as the innate immune system (e.g. complement, phagocytes) and antibodies generated by the adaptive immune system. With that in mind, African trypanosomes have been an interesting subject of study for molecular biologists due to their unique gene expression and immune evasion tactics that allow them to persist in its host over long time periods.
The key to the trypanosome’s survival is the variant surface glycoprotein (VSG) surface coat. The VSG proteins are large and can shield the smaller antigens on the parasite’s surface from antibodies. In addition, the VSG can also protect the parasite from complement proteins (i.e. the innate immune system). Though ironically speaking, the VSG coat itself elicits a strong antibody response (that serves to clear out most of the parasite invasion). This sounds like the end of the story for the trypanosome, but it is just the beginning.
This parasite has the remarkable ability to switch the type of VSG that is expressed on its surface. Thus, it can become unrecognisable to the same antibodies when it changes the type of VSG. This process is known as antigenic variation (Check out Figure 2).
Figure 2: Antigenic variation in T. brucei. As the number of parasites in the blood increase, the antibody response builds up in order to initiate an attack, and most of the parasites are cleared. The interesting bit is – the surviving parasites would then express a different variant of VSG (shown by different colours), which allows them to escape immune detection and multiply until new antibodies are produced. Without intervention, the process will continue indefinitely.
The ability of T. brucei to switch its VSG coat is the main factor that makes it so deadly — as it is virtually impossible for the host immune system to effectively fight off the parasite on its own. Once the parasite enters the brain, treatment options become severely limited as there are next to no medicines that can cross the blood-brain barrier.
Not to mention, there are more than 1500 VSG genes in the trypanosome genome. So the parasite is essentially able to create an ever-changing camouflage that protects itself whilst it swims around in the host’s bloodstream.
More on VSG
VSG genes are unique in that they can only be expressed at specific sites of the T. brucei genome, which are called expression sites (ES). There are a total of 15 expression sites, but only one can be activated at any given time (See Figure 3). This is why the parasite will always have only one type of VSG being expressed on its surface, because only one gene can be transcribed at a time.
Figure 3: VSG expression sites. Expression sites are located in telomeric regions and contain expression site associated genes (ESAGs; not shown in figure) along with a VSG gene (shown as coloured boxes). Only one expression site is active at a time (transcription indicated by black arrow), while the rest are epigenetically silenced.
Telomeric regions are the ‘ends/tips’ of the chromosomes. Epigenetic silencing is the silencing of genes due to external factors (e.g. a protein binds to the promoter and blocks transcription, presence of histones means the DNA is tightly wrapped and inaccessible for transcription, etc.)
The characteristic of only expressing one VSG (or one ‘allele’) at a time is known as monoallelic exclusion. Monoallelic exclusion is governed by several factors, although the exact mechanism of activation/suppression is as yet unknown.
How does the wardrobe change work?
There are several ways in which T. brucei can change its VSG coat (see Figure 4):
The gene within the active expression site can be switched out for a new VSG gene
Existing VSG genes can be combined into new VSG genes. These new VSGs are termed as ‘mosaic’ VSGs
Homologous recombination (i.e. the crossover of DNA) between telomeres
The active ES can be silenced, whilst a new ES can become activated
Figure 4: Mechanisms of antigenic variation of VSG. Each mechanism has access to different sizes of the VSG pool. Duplicative and segmental gene conversion can access all VSGs (about 1500), while telomere exchange can only access the sub-telomeric VSGs (around 120 to 250 coats). On the other hand, expression site switching can only access approximately 20 VSG genes.
The specifics of these changes are quite complicated, but the important thing to know is that the trypanosome, effectively, has an infinite camouflage and thus can exist in the human body for years and years.
Most protein coding genes in eukaryotic cells are transcribed by RNA polymerase II. VSG, however, is transcribed by RNA polymerase I. RNA polymerase I is conveniently located very close to the expression site within the nucleus to facilitate the high demand of VSGs for the parasite. One possible reason for this anomaly is that RNA polymerase I works a lot faster than RNA polymerase II, and hence can provide the copious amounts of VSG more efficiently for the trypanosome.
In essence, these little quirks make trypanosomes a very hot topic for researchers.
Nuclear bodies in trypanosomes
The nucleus is a large organelle and is packed with chromatin, proteins, and RNA. This dense environment makes transport by simple diffusion very inefficient and inconvenient for many important cellular processes.
Non-membrane bound organelles termed nuclear bodies increase the local concentration of important proteins — a well known example is the nucleolus, where rRNA is transcribed and processed. These bodies can have various specialised functions depending on the proteins present. In order to understand how VSG expression is regulated, it is necessary to understand what is going on around the active expression site.
The expression site body (ESB) is a nuclear body which contains RNA polymerase I and other proteins necessary for VSG transcription and regulation. In its vicinity are a few other nuclear bodies which also contain regulatory proteins, some which help with splicing, and others which help with monoallelic exclusion (i.e. silencing the non-active expression sites). Hence, the latest trypanosome research aims to understand these nuclear bodies in more detail, and how they affect VSG expression.
So, why should we care?
African sleeping sickness is still prevalent in many countries, particularly the Congo, and treatment options are often complex and require technically trained staff which may not always be available – especially in rural areas. Researchers aim to understand trypanosome molecular biology so that they can find ways to perturb antigenic variation. If we can stop the parasite from switching its VSG, we can effectively stop it from camouflaging itself, and the antibodies can do the rest.
However, VSGs are regulated in a very complex and intricate manner in several different ways (as you saw in Figure 4, the parasite has several methods to replace its VSG), which makes it more difficult to halt antigenic variation.
In addition to curing the disease, trypanosomes have very interesting molecular biology quirks (besides VSG!) that make them an exciting topic of research. Trypanosomes also have the potential to be very good model organisms. More specifically, a good model organism is one that is easy to manage and manipulate, is well understood, and inspires researchers to ask interesting questions. Taking that into consideration, the trypanosome ticks every box.
Author: Devyani Saini, BSc Biochemistry