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.
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 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.
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.