Molecular Basis of Disease State

History and Metabolic Context

Molecular Basis of Disease State

Treatment and Disease Management

Conclusions and Proposals for Future Work

Annotated Bibliography

The trypanosomes that cause human African trypanosomiasis (HAT) owe much of their success to their ability to disable and evade the human immune system.

Inhibition of Production of Tumor Necrosis Factor
One of their first lines of attack is an assault on the production of tumor necrosis factor (TNF). As stated on the “History and Metabolic Context” page, TNF is a cytokine primarily produced by immune cells such as macrophages, but it can be expressed in various cell types. In the case of HAT, the TNF that is affected is that produced by myeloid cells (Salmon et al, 2012). It is involved in several signaling pathways that activate transcription factors for cell death/cell proliferation, as well as being responsible for cell death itself (Chen & Goeddel, 2002). In 2012, Salmon et al. demonstrated that Trypanosoma brucei hinders the production of TNF through the activation of adenylyl cyclases on the plasma membrane of the trypanosome. These adenylyl cyclases are activated by stress when the body is attempting to destroy the trypanosome via phagocytosis. This sets off a cascade in which the trypanosome releases cyclic AMP into myeloid cells, which activates protein kinase A; this inhibits the synthesis of TNF (Salmon et al, 2012). Perhaps trypanosomes make destroying TNF a priority because of its role in recognizing and destroying abnormal cells.

TNF pathway
The TNF signaling pathway, from Chen & Goeddel, 2002.

 

 

Natural Resistance to Trypanosomes Due to TrypanosomalMethod of Heme Acquisition
There are two trypanosomes responsible for African sleeping sickness: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. T. b. gambiense causes the much more common, chronic form of trypanosomiasis, so it can be assumed that, from now on, if T.b. rhodesiense is not specifically mentioned, the text is referring to T.b. gambiense  (Stich, Abel, & Krishna, 2002).

 

The disease occurs in two stages. The first is known as the hemolymphatic stage. During this stage, trypanosomes are living in circulatory system, multiplying and scavenging heme (Pays et al, 2014). Trypanosomes need heme because they have several heme proteins (proteins that require heme as a cofactor), yet completely lack a biosynthetic pathway for heme. Heme proteins in the parasite are used for the biosynthesis of essential molecules like sterols and polyunsaturated fatty acids (Tripodi, Menendez, & Cricco, 2011).
As was briefly explained on the title page about HAT, the human immune system has learned to exploit a trypanosome’s need for heme as a way to destroy most unwanted trypanosome guests. Trypanosomes capture heme in the form of hemoglobin complexed with haptoglobin (Vanhollebeke et al, 2008). Haptoglobin is a protein that binds hemoglobin in order to prevent it from circulating and causing oxidative damage  .The trypanosome T. brucei brucei (a close relative of pathogenic trypanosomes) possesses a receptor called the TbHpHbR, which binds the haptoglobin-hemoglobin complex so that the trypanosome can obtain heme from hemoglobin (Vanhollebeke et al, 2008).

 

The structure of the TbHpHbR from T. brucei (A), T. congolese (B), and a close up of the kink in the receptor due to several hydrophobic residues, as well as a alignment of helix sequences. From Lane-Serff et al, 2014.
The structure of the TbHpHbR from T. brucei (A), T. congolese (B), and a close up of the kink in the receptor due to several hydrophobic residues, as well as a alignment of helix sequences. From Lane-Serff et al, 2014.

 

 

The immune system has worked out a defense system in which a serum factor called Trypanosome Lytic Factor 1 (TLF1), which is comprised of high-density lipoprotein particles (the most important component of TLF1 is an apolipoprotein called APOL1, but more on that later) can bind to a protein called HPR (Pays et al, 2006). HPR is haptoglobin-derived, and can therefore bind hemoglobin in a similar manner. The TLF1/HPR/hemoglobin complex can then bind to the TbHpHbR, allowing APOL1 to infiltrate the parasite and kill it from within (Vanhollebeke et al, 2008). For some time it was thought that HRP also functioned trypanocidally, but it is now thought to function as a type of GPS to locate trypanosomes (Vanhollebeke et al, 2007).
APOL1 kills trypanosomes by forming chloride pores in their endosomal membranes, which eventually causes an imbalance of chloride ions, osmotic pressure, and, eventually, lysis (Perez-Morga et al, 2005). The exact mechanism of how APOL1 does this is still, however, unknown.

 

What determines whether trypanosomes take up APOL1? The answer lies in the relative concentrations of TLF1/HPR/hemoglobin and haptoglobin/hemoglobin, as they are competing for the TbHpHbR on the trypanosome surface. Under normal physiological conditions, there is an excess of haptoglobin to HRP present in the blood, and it has been shown that haptoglobin levels can inhibit trypanosomal lysis (Vanhollebeke et al, 2008), (Smith & Hadjuk, 1995). In fact, due to the high concentration of haptoglobin in the blood, many think it plausible that another trypanosome lytic factor (TLF2, whose mechanism is not as well studied) may play the primary trypanocidal role (Smith & Hadjuk, 1995). That being said, TLF1 is also at work in lymph fluid, where haptoglobin concentration is not as high, so it could be lysing trypanosomes there. In addition, populations in which HAT is prevalent also have a high incidence of malaria. One of the byproducts of malarial infection is a low haptoglobin concentration in the bloodstream, which could potentially allow TLF1 to play a large role in trypanosomal lysis (Pays et al, 2014). Either way, there seems to be a consensus that, with either TLF1 or TLF2, APOL1 is the trypanosomal toxin.

 

Counter-resistance of T.b. gambiense
The question then, is why T. b. gambiense causes disease in humans, if the immune system has developed such natural strategies of resistance. The answer is three pronged, and lies in altered pathways for the acquisition, control, and degradation of APOL1.
The plasma membrane surface of T.b. gambiense is covered with molecules called variant surface glycoproteins (VSGs). One certain type of VSG is expressed at a time all over the plasma membrane, forming a kind of glycoprotein coat (Hall, Wang, & Barry, 2013). There are over 1000 genes that code for these different VSGs, and the trypanosomes can switch them on and off (Koumando et al, 2013), changing the composition of its coat and evading the immune system- this evasion is one of the main reasons that T.b. gambiense is so effective when causing disease (Pays et al, 2006).
These VSGs don’t just have a function in evasion. One VSG, called TgsGP (for T.b. gambiense specific glycoprotein) is partially responsible for conferring resistance to APOL1 in T.b. gambiense (Capewell et al, 2012). It does this by introducing a hydrophobic beta sheet in between helix A and B of the protein. This induces an antiparalllel peptide organization, which causes the membrane to stiffen and become more resistant to rupture by APOL1 (Uzureau et al, 2013). This VSG was proven to be important in 2013, when Capewell et al. showed that the deletion of TgsGP causes trypanosomes to be sensitive to human serum.

 

From Capewell et al, 2012. This shows trypanosomal survival under various conditions, noted at the top of the figure. This figure indicates that the TgsGP gene is necessary for T.b. gambiense resistance to human serum.
From Capewell et al, 2012. This shows trypanosomal survival under various conditions, noted at the top of the figure. This figure indicates that the TgsGP gene is necessary for T.b. gambiense resistance to human serum.

This mechanism of resistance to APOL1 is coupled with two other forms of resistance. First, the TbHpHbR in T.b.gambiense has a single point mutation that effectively prevents the TLF1/HRP/hemoglobin complex from binding, severely reducing APOL1 entry into the parasite (DeJesus et al, 2013). Still, if any APOL1 gets in, the trypanosome has one more form of resistance, in the form of increased APOL1 degradation. It was previously mentioned that APOL1 works by forming chloride pores in endosomal membranes. Endosomes in T.b. gambiense have a lower pH than those of T. b. brucei, which is thought to aid in cysteine protease-mediated digestion of APOL1 (Uzureau et al, 2013). The reason for T.b. gambiense’s resistance to innate human immunity is therefore a three-pronged assault on APOL1: prevention of its entry, improved resistance to its pore-forming abilities, and its increased degradation within the trypanosome.
When the trypanosomes have finished living in (and taking resources from) the circulatory system, it manages to cross over into the central nervous system and cause neurological symptoms that are characteristic of HAT (Cnops, Magez, & De Trez, 2015). The exact molecular mechanism for this infiltration is not known, although there are theories. While many still believe that trypanosomes still directly cross the blood-brain-barrier (BBB) into the brain parenchyma, there are other theories of infection that are more complex, and seem more likely given previous work (Mogk et al, 2013). One of these theories is that the trypanosomes actually do not cross the BBB, but rather cross the BCB (the blood-CSF-barrier), which is the barrier between blood and cerebrospinal fluid. It is hypothesized they cross the barrier cyclically, whenever trypanosome populations are highest in the blood, travel in the CSF, hiding in cells called pial cells where the CSF cannot destroy them, and then infect the brain parenchyma (Mogk et al, 2013).

 

Stage Two HAT
Once they are in the brain, little is known about what they do, as trypanosomes can only be found in the brain post-mortem. There was a study done in 1979 by Cornford et al., however, that suggests that the sleep disturbances characteristic of HAT are caused by rapid distribution of tryptophol (3-indole ethanol) in the brain. Tryptophol is an aromatic compound known to induce sleep in humans that is produced by trypanosomes from tryptophan, obtained from their human hosts. This paper demonstrated that tryptophol can easily cross the BBB due to its lipophilic nature, and then binds to something in the brain so that it cannot be easily discarded by the CNS. Their theory is that it is this easy distribution of tryptophol that causes the daytime sleep and nighttime insomnia associated with HAT (Cornford et al, 1979). There are other neurological symptoms that have not yet been molecularly explained, such as psychosis and and an inability to cope with surroundings (Stich, Abel, & Krishna, 2002), but the inevitable outcome (without treatment) is death.

 

4 Replies to “Molecular Basis of Disease State”

  1. Hi Gabbie,

    Great job! As I was reading through your summary, though, I had a few questions. I noticed that one of the proposed mechanisms for the disease involves the movement of the parasite through the blood-CSF barrier. I know from my histology class that the BCB is much less effective barrier than the BBB because the BCB is composed of fenestrated capillaries, which are capillaries that have tiny holes in them; the BCB is more solid and lacks these small holes, which prevents uncontrolled entry of molecules. The pores of the BCB are extremely small, and generally only allow for the transfer of extremely small particles, usually ions, through the barrier. Although you didn’t mention anything about the size of the parasite (perhaps this might be useful?), I imagine that it is a relatively large size that wouldn’t be able to normally cross through the BCB. Do you know, then, how it actually gets through the BCB, and then eventually into the brain? Does the parasite perhaps secrete something to weaken the BCB, and allow it to squeeze through the pores? Or is it somehow transported into this space inside another cell?

    If the parasite does in fact travel through the BCB, then this region may serve as a potential target for future treatment methods. This seems extremely useful because I noticed that a lot of the treatments seem especially cumbersome, and may have many side effects. For example, one of the current treatments, Pentamidine, targets mitochondrial DNA synthesis. While this may be useful to target the parasite, wouldn’t this also have an effect on human mitochondrial DNA? Does that mean that this type of treatment causes major side effects in patients, perhaps causing problems with metabolism and energy production? While side effects are obviously undesirable, you also mentioned that the lack of treatment causes death. Is the mechanism through which the parasite actually causes death known?

    1. Hi Mike- thank you for your feedback! You ask good questions.

      In terms of crossing the BCB (as opposed to the BBB), a mechanism is not known, but one has been hypothesized. Mogk et al believe that the trypanosomes may used a membrane-localized protease in order to enter the stroma of the choroid plexus. In order to do this, the authors suggest that parasites must become “stumpy:” i.e., they must stop expressing a variant surface glyocprotein coat and instead express the necessary membrane-localized protease on their plasma membranes. They support this idea by referencing the fact that trypanosomes lacking a VSG coat have been previously found in the stroma. The authors do make it clear, however, that it is still unknown how EXACTLY the parasite overcome the epithelial cells of the BCB, yet electron microscopy studies have apparently supported the hypothesis that the BCB is the barrier that is either crossed first, or only barrier that is crossed by the parasite. I suppose the best summary of this is that it is proposed that trypanosomes shed their VSG coat in order to express a protease that can essentially hack holes in the barrier, allowing the trypanosomes to cross (Mogk et al, 2013).

      That’s an interesting observation about pentamidine. It is usually reported as being well-tolerated by patients. I believe that is because of the manner by which the trypanosomes take up pentamidine: they use a nucleoside transporter that is specific to the trypanosomes (Bacchi, 2009). Human cells do not have this transporter, so they cannot take up pentamidine at the prodigious rates that trypanosomes do. Perhaps this accounts for its lack of toxicity.

      As for your last question, no, the exact mechanism by which the parasite causes death in the brain is unknown. Researching this has been extremely difficult due to the lack of a good animal model for the disease, as well as the drawbacks of only being to examine an infected brain post-mortem.

  2. Hi Gabbie,

    Great paper!

    I was particularly curious about the lack of current research options presented for mitigating HAT2, given the reach of non-intrusive scanning tools and alternative mammalian models. Do you feel there is any solvency in utilizing radiation tagged parasites, coupled with penetrative scanning, to explore the mechanism by which the parasite brings about stage 2 HAT? I feel as though a more competent understanding of the parasite’s activity in the brain might at the very least allow for better palliative care options within the affected population.

    1. Hi Anthony! You have a good point. From my understanding, according to Mogk et al, radio-tagged parasites have been used in animal models to try to elucidate the mechanism by which trypanosomes cause disease in the brain. While these studies have suggested a different mechanism by which the trypanosomes invade the CNS (crossing the BCB instead of the BBB, they have yet to elucidate how exactly the trypanosomes cause disease once they have reached the brain. I believe that you’re right, and this needs to be a focus for future research.

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