Monday, January 29, 2018

The Death Defying Stunts of Trypanosomes

by EB

How much do you hate being sick, feeling better for a while, and then getting sick again? In the two deadly human illnesses caused by Trypanosoma brucei and Trypanosoma cruzi—African Sleeping Sickness and Chagas’ Disease, respectively—this cycle becomes a reality. These diseases are known for their unique symptoms: for example, African Sleeping Sickness causes the host to go through cycles of feeling ill and then feeling better every few weeks, while Chagas Disease there can be no symptoms after the initial infection for 20+ years [1, 2]!
Figure 1: Trypanosome. The arrow points to a Trypanosome.
African Sleeping Sickness, which is caused by T. brucei, had large outbreaks during the 1800s in rural sub-Saharan Africa and was almost eradicated in the 1960s [1]. However, there are still significant numbers of new cases of this disease, about 10,000, that occur each year [2]. In comparison, Chagas’ Disease, caused by T. cruzi, is endemic in almost all Latin American countries and is only present in the western hemisphere [3]. These diseases are very hard to treat because Trypanosomes are eukaryotes, meaning that many of the mechanisms that antibiotics use to treat other diseases are ineffective against them. Additionally, as they are eukaryotes, like us, stronger drugs that could treat them cause very bad side-effects, as the drugs can’t differentiate between a human cell and a Trypanosoma cell [4].

Additionally, Trypanosoma has unique mechanisms for evading the immune system, and therefore death. The best characterized mechanism is that of variable surface glycoprotein coats (VSG), which are proteins on the surface of cell [5]. The VSG that the Trypanosoma expresses changes throughout the course of the infection due to gene conversion and telomere exchanges during reproduction and replication of the parasite [5], please see Figure 2. Gene conversion is where a piece of ‘donor’ DNA gets placed into other chromosomes, which causes a change in the expression of the genes [6]. Telomeric exchanges are similar in that a piece of ‘donor’ DNA is inserted into another chromosome, however, in this case, the piece of DNA that is replaced is on the end of the chromosome—the telomere—instead of anywhere on the chromosome as in gene conversion.
Figure 2: VSG segments change by two major mechanisms as shown above. Gene conversion: First, a double stranded break in the DNA occurs, and DNA around the broken area is deleted, so that the cell can more easily repair it. Then, a ‘donor’ piece of DNA from a different chromosome comes into the broken area, where it is replicated as a template for the cell. This ‘donor’ piece of DNA is now present in the broken area, creating non-broken DNA once again. Telomeric VSG Conversion: During replication of the chromosomes, one end of the DNA on one chromosome ‘invades’ the telomere of another chromosome while it is being replicated. Due to this ‘invasion’, the DNA from the telomere of the first chromosome will become the DNA sequence at the telomere of the chromosome that was ‘invaded’. From here.
The cycle of feeling sick and then better that characterizes African Sleeping Sickness is caused by the immune system and the parasite being in opposition. When the patient feels ill, the parasite load in the blood is high and the immune system is attacking T. brucei. However, when the patient feels better, the parasites have simply changed their VSG thus evading the immune system for a few days until the immune system can make more ‘soldier’ cells to defend the body. Due to the chronic infections seen in these diseases and the difficulty in treatment, it is important to understand how Trypanosoma can evade the immune system. As not much is known about how they do this, research is being conducted upon two types of proteins: expressed and secreted. This research has determined that these types of protein play a large role in the ability of Trypanosoma in escaping death.

Secreted proteins are important to living organisms in regulation of protein activities and cell-cell communication. Due to this variation of function, there are several groups of proteins that include secreted proteins; however, we’ll be focusing on the proteins involved in folding and degradation, and enzymes of nucleotide metabolism [7]. Now for some more definitions. Folding is necessary for protein function—think of it like origami, if you’re trying to make a crane but instead make a crumpled ball it obviously can’t be a crane. Folding of proteins is exactly like that, except that if they aren’t folded in the correct ways they can’t perform their functions. Degradation of proteins is also necessary, as if too many proteins are floating around but not being used, energy is being wasted and potential harmful effects could occur. Think of this like garbage floating in a delicate ecosystem. If the garbage isn’t gotten rid of, the animals living there start dying or moving away. Next, Nucleotide metabolism is the degradation of extra nucleotides [7]. Finally, let’s define what a secretome is. Secretomes are defined as all the secreted proteins of an organism. Therefore, the investigation of secretome proteins was of high importance, especially because the VSG proteins are in the secretome. As stated above, VSG proteins are highly variable and switch every couple of weeks during the course of an infection. This variation of the parasite allows it to better evade the immune system because the immune system trigger that infected cells present changes, and so the B and T cells—think of as the generals of the immune system—for the new trigger must be activated and the ‘old’ B and T cells must become memory cells, so that is the trigger goes back to the ‘old’ trigger, the generals can be ready to marshal the troops to fight once again.
Figure 3: B and T Cells Broken Down.
The figure above breaks down the components of B and T cells and what they do for the immune system. B cells are divided into two types of cells—memory, which hang around and are ready to defend the body if the immune reactant re-emerges, and effector B cells which secrete antibodies as an immune response. T cells are initially divided into two types—CD4 and CD8 cells. CD4 T cells secrete compounds to activate other immune cells for help in clearing out the immune reactant, while CD8 T cells secrete cytokines to kill infected cells.
This research identified two important proteins that were involved in the folding and degradation of proteins. They were called cyclophilin A and heat shock protein. Cyclophilin A and heat shock proteins are known to regulate the immune system of mammals and to act as mediators for intercellular signaling [7]. Intercellular signaling allows nearby cells to communicate with each other, so that if one is sick of dying the other cells around it know. These functions are important for the escape of Trypanosoma from the host’s immune system, as effectively monitoring intercellular signaling and modulation of the immune system would aid the parasites in evasion of the killer immune cells. The proteins in the enzymatic nucleotide metabolism group are thought to be important to the parasites to degrade nucleotides around them to decrease inflammatory reactions of signals carried by them to the immune system and other cells [7]. These inflammatory reactions are one of the most basic defenses of our immune systems, and so if this reaction isn’t carried out precisely, it won’t be effective, and could even kill healthy cells. Based upon this evidence, it is clear that secreted proteins may have an effect upon the Trypanosoma’s escape from the host’s immune system, as the proteins found modulate the immune system, the inflammatory response, and intercellular signaling.

Before we go on talking about Trypanosoma’s death defying stunts, let’s define a few more terms. First, what are surface proteins? Surface proteins are proteins that are located on the cell membrane of a cell and are exposed to the environment around the cell. They also allow transport in and out of the cell and engage in communication between cells; they are important to look at for further applications for the creation of drugs. CD4 T cells are part of the immune system, and are a type of ‘general’. These ‘generals’ release cytokines and interleukins, which are the ‘soldiers’ of the immune system to combat the enemy, in this case Trypanosoma, through inflammatory reactions that cause death, and the activation of more ‘generals’ and ‘soldiers’.

Now that we know what these terms are, let’s continue our discussion about how Trypanosoma evade the immune system. Further research was conducted upon a group of surface proteins called Sialoglycoproteins of Trypanosoma cruzi. These It was found that the Sialoglycoproteins, specifically one called O-glycoslyated Thr/Ser/Pro-rich mucin molecules, also known as TcMuc, can cause inhibitory effects on CD4 T cells and a decrease in the production of cytokines [8]. In fact, very small TcMuc concentrations resulted in inhibition of CD4 T cell proliferation. This inhibition of one of the ‘generals’ of the immune system was not overcome with the inclusion of Interleukin-2 (IL-2) [8]. This interleukin is necessary for the activation of the CD4 general cell, which demonstrates how bad this protein is for the human immune system. As CD4 T cells are a major component of the human adaptive immune system, therefore, without CD4 T cells, an immunocompromised individual can occur. IL-2 meanwhile, is an important signal to both CD4 T cells and CD8 T cells; it is a cytokine that helps T cells to proliferate. Therefore, this one molecule on the surface of T. cruzei effectively cripples the adaptive immune system. If T cells can’t proliferate, a large response to the parasite would not be able to be formed and the immune response compromised.

Furthermore, TcMuc downregulates expression of certain cytokines, the ‘soldiers’, see Figure 3 [8]. These cytokines that are expressed at lower dosages are highly important in the immune response as they activate various immune cells, such as different varieties of CD4 T cells like TH1, TH2, and Treg, and cause systemic effects that tell the host that they are sick. These other CD4 T cells are like ‘captains’ of units that have specific functions; for example, Treg ‘captains’ molecules that regulate autoimmunity—without these cells, everyone would have at least one autoimmune disease—while TH1, and TH2 CD4 T cells participate in the immune response towards the parasite. With downregulated expression of these cytokines, the immune system has a harder job to complete in order to become activated (IL-2) and prepared to fight the parasitic infection (cytokines). Furthermore, TcMuc sialylation decreases dendritic cell function [8]. Dendritic cells are a major part of one’s innate immune system. They process immune triggers and bring them into the lymph nodes, where B and T cells mature, in an attempt to activate the appropriate adaptive cells. Therefore, if their ability to present antigen and bring it to the lymph nodes is compromised, the adaptive immune system is unable to complete their job and clear the infection.

Trypanosomes engage various mechanisms to evade the immune system of their hosts. Commonly, these mechanisms are associated with secreted proteins on the surface of the cell. Treatment for Trypanosoma sickness is complex because the drugs used also target human cells. As these organisms are eukaryotes, drug targets that do not have homologs in humans are challenging to find. The evasion mechanisms using the proteins discussed above all have homologs in humans; therefore, more research is needed on secreted and surface protein functions of Trypanosomes. This research may uncover potential drug targets that do not have homologs in humans and open new paths for treatment of African Sleeping Sickness and Chagas’ Disease.


References:
[1] Brun, R., Blum, J., Chappuis, F. and Burri, C. (2010). Human African trypanosomiasis. The Lancet, 375(9709), pp.148-159.

[2] Cdc.gov. (2017). CDC - African Trypanosomiasis. [online] Available at: https://www.cdc.gov/parasites/sleepingsickness/index.html [Accessed 27 Nov. 2017].

[3]Desforges, J. and Kirchhoff, L. (1993). American Trypanosomiasis (Chagas' Disease) -- A Tropical Disease Now in the United States. New England Journal of Medicine, 329(9), pp.639-644.

[4] Cdc.gov. (2017). CDC - African Trypanosomiasis - Resources for Health Professionals. [online] Available at: https://www.cdc.gov/parasites/sleepingsickness/health_professionals/index.html#tx [Accessed 27 Nov. 2017].

[5] Marcello, L. and Barry, J. (2007). Analysis of the VSG gene silent archive in Trypanosoma brucei reveals that mosaic gene expression is prominent in antigenic variation and is favored by archive substructure. Genome Research, 17(9), pp.1344-1352.

[6] Chen, J., Cooper, D., Chuzhanova, N., Férec, C. and Patrinos, G. (2007). Gene conversion: mechanisms, evolution and human disease. Nature Reviews Genetics, 8(10), pp.762-775.

[7] Geiger, A., Hirtz, C., Bécue, T., Bellard, E., Centeno, D., Gargani, D., … Peltier, J.-B. (2010). Exocytosis and protein secretion in TrypanosomaBMC Microbiology10, 20. http://doi.org/10.1186/1471-2180-10-20

[8] Nunes, M., Fortes, B., Silva-Filho, J., Terra-Granado, E., Santos, L., Conde, L., de Araújo Oliveira, I., Freire-de-Lima, L., Martins, M., Pinheiro, A., Takyia, C., Freire-de-Lima, C., Todeschini, A., DosReis, G. and Morrot, A. (2013). Inhibitory Effects of Trypanosoma cruzi Sialoglycoproteins on CD4+ T Cells Are Associated with Increased Susceptibility to Infection. PLoS ONE, 8(10), p.e77568.

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