Variant Surface Glycoproteins (VSGs) of Trypanosomes

Trypanosomes

Electron micrograph of the Ebola Zaire virus. This was the first photo ever taken of the virus, on 10/13/1976. By Dr. F.A. Murphy, now at U.C. Davis, then at the CDC.

By Kelly Wahl


Trypranasomes are single celled flagellated eukaryotic parasites that infect humans and livestock causing trypanosomiasis, more commonly as sleeping sickness in Africa and Chagas disease in the Americas. These parasites are usually introduced into the body via an arthropod vector. The tsetse fly (subgenus Glossina morsitans), transmits the strains Trypanosoma brucei gambiense (95% of reported cases and found exclusively in West Africa) and Trypanosoma brucei rhodesiense (5% of reported cases, only seen in East Africa) responsible for sleeping sickness through its bite. Oddly the geographic range of these two strains do not overlap. The kissing bug (subfamily : Triatominae) spreads Trypanosoma cruzi when its feces enter the body through the mucosal membranes, or broken skin (bite marks or scratches). Besides T. brucei, and T. cruzi, there are many other strains of trypanosomes that affect mainly domesticated animals like; cows, camels, deer, horses, antelope, and donkey.

These parasites fall under the order Kinetoplastidia and are related to another pathogenic parasite, Leishmania. Kinetoplasts are called such because of the kinetoplast at the base of their flagellum. A kinetoplast is a mitochondrion with coiled mitochondrial DNA and 10% of the total genomic DNA of the parasite. The kinetoplast changes size and shape throughout the parasites life cycle and is the target site of some anti-parasitic drugs (like?). Kinetoplasts broke off from the common ancestor fairly early on and consequently transcription of their genome is unlike most other organism and is under intensive study as a potential vaccine or drug target. (Epigenetics and…)(kinetoplast wiki)(that cool site)

Trypanosome Infection; Sleeping Sickness, and Chagas Disease

Sleeping Sickness

Stage one of T. brucei infection is the haemolymphatic stage. Parasites circulate and multiply by binary fission in the blood and lymph systems. infection with T. b. rhodesiense often begins as a sore called a chancre and is followed a week or two of headaches, fevers, and muscle and joint pain. Enlarged lymph nodes are common and some patients also present with a rash. Large inflamed lymph nodes is a characteristic sign of infection called Winterbottom’s sign. T. b. gambiense infection shows the same symptoms but they are not as severe and last for a longer period of time.

The second stage of infection is neurological. The parasites have crossed the blood-brain barrier and are now multiplying in the central nervous system. classic symptoms include rapid weight loss, impaired motor skills, seizures, confusion, and changes in personality. A disrupted circadian rhythm where a patient lies awake at night but sleeps during the day is where this disease gets its namesake. Without treatment T. b. rhodesiense infection progress to coma and death within a few months. Often times T. b. rhodesiense infection moves so quickly that malnutrition, heart failure, and pneumonia cause death before the neurological problems ever manifest themselves. The progression of T. b. gambiense infection takes much longer and a person can take years to show neurological symptoms. The different clinical manifestations of the two strains stems from differences in the two strains stimulation of the immune system. T. b. gambiense infection stimulates a huge response from the immune system that results in death of host cells and observed anemia, weakness, pain, and inflammation.

Chagas Disease

Chagas disease is similar to sleeping sickness in many ways. There is an acute stage when the parasite is circulating in the bloodstream and the initial site of infection swells and a patient may present with a fever or show no signs of infection. One of the classic signs of acute Chagas infection is Romana’s sign, the swelling of the area around the eye where the conjunctiva (mucosal tissue around the eye) or a site near the eye was point of infection. Serious symptoms are rare in this stage of infection but are possible. They include heart failure, chest pain, seizures, or paralysis.

Unlike sleeping sickness, Chagas disease only progresses to the chronic form of the disease in 20 to 30% or people infected. The remaining 70 to 80% of people infected remain asymptomatic for life and may never know they are infected. As the parasite replicates in the target tissues; cardiac muscle and digestive smooth muscle those infected show a variety of cardiac problems such as irregular heartbeat, blood flow disorders, issues with heart muscle function, and blood clots. As well as problems with the digestive tracts such as ulcers.

Treatment

Symptomatic trypanasome infection is usually fatal if left untreated. While there is no vaccine or prophylactic drugs to prevent T. brucei infection there are drugs available for treatment a recent infection as well as cases that have already become symptomatic. The earlier the diagnosis is made the easier it is to manage symptoms with less toxic drugs that target the parasite outside of the central nervous system. once the infection has become symptomatic strong drugs with much higher toxicity must be used to cross the blood-brain barrier and attack the parasite in the CNS. If the infection has progressed to this point it is likely that a lengthy hospital stay is required to clear up infection. There is a need for new drugs to combat this disease because the toxicity associated with some of the drugs used to treat the infection are almost as bad as the infection itself.

Why Are Trypanosomes of Public health and Economic Interest?

African trypanosomiasis is of public health and economic importance for a number of reasons. Besides affecting humans, trypanosomes also infect domesticated cattle making cattle sick with disease called Nagana and hindering economic development in affected areas. The inability to raise cattle in sub-Saharan African further contributes to malnutrition in the area. Animals also serve as reservoirs to the parasites, most importantly the T. b. rhodesiense strain making it difficult to eradicate the parasite in a given area. These parasites are only found within the tsetse flys’ geographic range, sub-Saharan Africa and have devastating economic effects on the people infected because of the length of infection and the nature of those infected. Those who rely on farming, fishing, and hunting are most at risk to be bitten by an infected tsetse fly and are also commonly the breadwinners in poor, rural populations. While the WHO only reports about 10,000 new cases a year this number is most likely a gross underestimate of the true number of cases and other estimates put the number around 100,000 although others still caution that these numbers are underestimated when close to 50,000,000 are at risk.

Both African trypanosomiasis and Chagas disease considered neglected tropical diseases under which a projected 534,000 deaths a year and account for millions of year of functional life lost due to disability or years of life lost due to ill health. Those affected are primarily low and middle income countries in areas where the disease is most prevalent. Under international public health trypanosome diseases are important for the lives lost and emerging rural economies stunted by this disease. Domestically, Chagas presents a threat to blood and organ transplant recipients. It is estimated that there are more than 300,000 people living with T. cruzi infections in the U.S.. Although natural transmission through triatomines found in North America is possible it is very rare and iatrogenic (through transplants) exposure represents the greatest risk of route of domestic exposure. In this increasingly globalized world diseases that once hid in the jungles of South America and the plains of sub-Saharan Africa have ended up on the doorstep of industrialized nations. Although trypanosomiasis is still considered a third-world disease infections are continually diagnosed in the first world and represent a real risk to anyone that can get on a plane and travel to an affected area.

Transmission and Life Cycle

Life cycle of T. brucei

Transmission

Although the most common route of infection is for T. brucei, and T. cruzi are run-ins with the arthropods Glossina morsitans and : Triatominae these parasites can be passed from mother to child during pregnancy. Blood transfusion and sex can also spread these parasites but this is very rare.

Life cycle

Over the course of its lifetime a trypanosome cycles through five different morphological forms between arthropod vector and mammalian host. The different forms allow these parasites to live in very different host environments, from the gut of an insect to the central nervous system in a human.

The forms found within the mammalian body are distinctly different from the forms found in insects because of the presence of variant surface glycoproteins (VSGs). These sugar-proteins are the secret weapon of trypanosomes that allow them to remain undetected by the immune system almost indefinitely. Ten million of them form homodimers with each other and stick out of the cell membrane like trees in a dense forest. Although VSGs amino acid sequences and attached sugars vary wildly from variant to variant their secondary and tertiary structures are highly conserved. In a way, VSGs are similar to the T cell receptors found on the surface of T cells in the immune system. Allelic exclusion ensures that only one VSG is expressed at a time. This monotypic surface coat is recognizable to B cells independent of signals from helper T cells that usually signal activation and differentiation of B cells but before the body can clear all of the parasites they have already silenced expression of that particular VSG and begun expressing a new VSG that has yet to be seen by the immune system.

(From early detection article)

In the midgut of the tsetse fly procyclic trypomastigotes proliferate and move to the salivary gland where they become morphologically distinct epimastigotes. After three weeks from the initial introduction of the trypanosomes into the midgut of the tsetse fly there are mature non-dividing metacyclic epimastigotes with VSGs ready for another round of mammalian infection. When the metacyclic epimastigotes enter the mammalian body through the bite of the fly they differentiate into “slender” form and travel in the lymph and blood circulatory systems reproducing by binary fission in almost all organs of the body. Some of these dividing slender forms become “stumpy” and stop dividing. Although the stumpy form is no longer dividing within the host this form can survive in the midgut where it differentiates into the procyclic form and complete the cycle of infection (her book, website).

While T. brucei multiplys in the extracelluar space between cells T. cruzi actually hides intracellularly (VSGs?????????????????)

Variant Surface Glycoproteins (VSGs)

VSGs

Variant surface glycoproteins (VSGs) are sugar-proteins that coat the surface of trypomastigotes. There are at least 200 active VSG alleles and more than 1000 silent alleles and psuedogenes. Active genes can recombine with silent genes to form new unique active genes. This process is called antigentic switching and allows the blood-stream, slender form of the parasite to continually change its “appearance” to the immune system and remain undetected through the course of infection. While the immune system eventually detects and makes antibodies to a particular VSG it can make antibodies to every type of VSG present on the surface of all the circulating trypomastigotes. The immune response continually selects for the expression of new VSG variants. It is a case of the Red Queen’s race only the parasite eventually wins because it has more possible VSG variants than the immune system could ever catch given the amount of time the immune system has to act before parasite proliferation in vital organs causes death. VSGs can also actively modulate various components of the immune system to further multiply throughout the body undetected (her book horn). The mechanism that regulate this entire process is fairly unique but has been studied in yeast.

VSG expression

Only one of 10-20 VSG telomeric sites expression sites is available for transcription at a time when trypanosomes are in the bloodstream. this strategy allows only one VSG coat with a given set of epitopes to be expressed at a time. The adaptive immune system eventually forms antibodies that bind to these epitopes and elicit an immune response but by the time this happens the parasite has silenced that expression site and begins expressing antigenetically different VSGs from another telomeric site (horn). Oddly enough these ESs are transcribed by RNA polymerase I. In eukaryotes RNA pol I is responsible only for the transcription of ribosomal RNA while RNA pol II is normally used for the transcription of other types of RNA (horn).

There are two ways that VSGs are mono-telomerically expressed (only one site/one VSG is transcribed). Transcription switching is where an active telomeric site that was being actively transcribed is repressed and another active telomeric site that was silenced begins active transcription. Recombination switching is the other way switch VSG expression. One of the thousand or so pseudogenes that cannot be transcribed on its own recombines with an active telomeric site so it can be expressed. Active expression sites are flexible in their recombination. Psuedogenes are found in silent expression sites, the telomeres of chromosomes, and in subtelomeric VSG arrays. When recombing with VSG arrays either whole genes or segments of genes can be moved to active expression sites greatly increasing the possible number of VSGs that can be expressed. The exact proteins involved and the mechanism by which they are able to recombine is still under study.

The mechanism that allows one telomeric expression site (ES) to be actively transcribed while the rest are silenced generally know some of the details need to be worked out. It seems to involve the coiling and uncoiling of DNA from nucleosomes and other chromatin proteins. Nucleosomes are the spooling proteins that help organize DNA and play a role in gene expression. Expression of certain genes is repressed by these proteins because they twist up the DNA in such a way that RNA polymerase cannot physically bind the promoter region of the gene and begin transcription.

ES silencing requires at least six protein factors and operates by two different mechanism; short-range, and long-range telomeric silencing. In short-range silencing, the modifications by the histone deacetylase SIR2rp1 is within 5 kbp of the expression site silence expression. HAT1 is the histone acetylase that reverses silencing and allows the histones to relax enough so active transcription can take place. Long-range silencing is controlled farther away from the expression site (about 50 kbp upstream) most likely through chromatin remodeling with a variety of histone deacetylases, histone acetylases, histone methylases, chromatin remodelers (assembly and disassembly of histones). The default for both of these mechanism is silencing and almost all are required for growth. One of the factors being considered as a drug target is the deacetylase DAC3.

While the mechanism of silencing expression itself is well understood the factors that control which genes are silenced or activated remains to be studied. Indeed it is amazing that with all of the potential VSG alleles available for transcription only one allele is transcribed at a time (monoallelic expression). (all of this is the horn review)

Current Research

Expression of a Mosaic VSG Coat to Escape Early Detection by B-cells

Although only one VSG gene is expressed at a time the surface coat isn’t always made up of one VSG. Even though a new gene is being expressed the existing VSG coat doesn’t just disappear overnight. It takes about 48 hours for the VSG coat to completely switch from one variant to another. This lag period is due to the time is takes to transcribe, translate, and translate the new VSGs to the surface and get rid of residual mRNA and protein from the previous coat.

Recently Dubois et al. (2005) found that this 48 period of a mosaic VSG coat during antigenetic variation actually helps prevent B cells from recognizing the new VSGs that are appearing on the surface of the cell amongst VSGs that have already been recognized by B cells independent of T cells. It is not until the surface coat again becomes monotypic with a single VSG present are B cells able to recognize the parasite.

Athymic mice (lacking T-cells) were infected with a strain of T. b. rhodesiense that had been transformed to express two VSGs at the same time. Figure X shows dual VSG expression of transformed trypanosomes. Wild-type trypanosomes could not be used because very few parasites in a given population are switching coats at any one time and dual expression of VSGs on the surface of cells is brief. An ELISPOT assay was done on B-cells from the spleen. They found that fewer B-cells were active and producing antibodies to either expressed VSG for the five says of infection. Figure X shows that the number of B-cells activated by VSG-coexpressing trypanosomes was near the level of B-cells activated in uninfected mice. It seems that B-cells are fooled by the insertion of variant VSGs in a motypic coat during switching because the new VSGs breakup the pattern of identical epitopes that are required for B-cell activation. (escape early detection of immune system)

Trypanosomes Effects on Immune Function

Besides hiding from the immune system with an ever-changing VSG surface coat trypanosomes also manage to down-regulate the function of various components of the immune system. one example of down-regulated function is the decreased ability of white blood cells to process and present other exogenous antigens after trypanosome infection.

In 2009 Dagenais et al. showed that white blood cells taken from infected mice displayed a decreased ability to respond to newly encountered hen egg lysozyme (HEL) antigen ex vivo. This assay had to be done ex vivo in order to measure this type of activity in a controlled way. Extracted T-cells were irradiated and hybridized with hybridomas so the cells could be grown in cell culture. The ability to process and present antigen was measured by the amount of interlukin-2 (IL-2) that was produced. IL-2 is an important cytokine that helps stimulate cytoxic T-cells and affects the process of T-cell immunologic memory, among other functions.