APOL-1 Variants, Susceptibility and Resistance to Trypanosomiasis
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African trypanosomes are flagellate protozoans that are transmitted to mammals after inoculation by flies of the genus Glossina, also known as tsetse flies. Chronic infection with these parasites is fatal if untreated. In humans, the disease is also called “sleeping sickness” and affects thousands of people per year in sub-Saharan Africa (Pays et al. 2014).
Apolipoprotein L1 (APOL-1), an ionic channel-forming protein, is the lytic component of the innate immune system that protects humans against infection from the majority of the African trypanosomes, except for Trypanosoma brucei rhodesiense in East Africa and Trypanosoma brucei gambiense in West Africa. These two species have evolved mechanisms to become resistant to lysis mediated by APOL-1 (Cooper et al. 2016).
In recent years, variants of APOL-1 have been identified that either knock down the trypanolytic activity of the protein and thus predispose the carrier to infections by atypical trypanosomes (Cuypers et al. 2016), or are able to lyse and kill the East African Trypanosoma brucei rhodesiense by hindering the resistance mechanism of the parasite. The latter variants were found in a subset of Old World monkeys (Cooper et al. 2016) but now also in some humans with African ancestry (Genovese et al. 2010). Part of the importance of knowing about these variants is the potential for developing alternative therapeutic options for infected patients, who currently receive medications that are mostly unsatisfactory (Cooper et al. 2016).
Trypanosomes are protozoan parasites that are able to infect a wide variety of mammals and cause major public health and agricultural development problems in Africa. Only two of the subspecies are able to infect humans: Trypanosoma brucei gambiense in West Africa and Trypanosoma brucei rhodesiense in East and Southern Africa. These cause a debilitating and many times fatal human disease that is often referred to as sleeping sickness. The western parasite (>97% of all cases of trypanosomiasis) typically causes a chronic disease, whereas the eastern one results in an acute and rapidly progressing disease (Cooper et al. 2016).
Trypanosomiasis caused by T.b. gambiense is characterized by an early hemolymphatic phase associated with nonspecific symptoms such as intermittent fever and headache, followed by a meningoencephalitic phase where the parasite invades the central nervous system leading to the characteristic neurological disorder and death if left untreated. In T.b. rhodesiense infection, the symptoms are very similar, yet the progression is more rapid. Also, an inoculation “chancre” is often observed at the site of the bite by the tsetse fly, in contrast with T. b. gambiense infection in which the occurrence of the chancre is rare (Bucheton et al. 2011).
Hundreds of thousands of trypanosomiasis cases occurred in the first part of the twentieth century due to exploitation of tsetse-infested areas. Systematic screening and treatment of millions of people lead to almost a halt in transmission by the 1960s. However, the disease progressively flared up again since the 1970s and reached almost half a million cases at the end of the last century (Bucheton et al. 2011).
Despite efforts to improve diagnosis and treatment, trypanosomiasis continues to be an important public health issue in Africa. Current anti-trypanosomal drugs are mostly unsatisfactory due to toxicity, difficulty to follow regimens, and the emergence of resistance (Cooper et al. 2016).
Human Immunity Against Trypanosomes
The ability to control parasitemia in trypanosomiasis in humans involves at least four mechanisms: (1) antibody/complement lysis, (2) antibody-mediated phagocytosis, (3) innate immunity in terms of trypanolytic complexes in human serum (i.e. APOL-1), and (4) release of trypanotoxic molecules such as reactive nitrogen intermediates or reactive oxygen radicals by macrophages, cells that provide the first line of defense (Bucheton et al. 2011).
With regards to the innate immunity mechanism against trypanosomes, it is now known that these parasites are lysed by APOL-1, a component of human high-density lipoprotein (HDL) particles that are also characterized by the presence of haptoglobin-related protein (Vanhollebeke et al. 2008).
APOL-1 is one of the six members of the APOL gene family. The function of these proteins is mainly unknown and only humans, gorillas, and baboons express the APOL1 gene. APOL-1 was first discovered within the high-density lipoprotein 3 (HDL-3) particle (Limou et al. 2015).
APOL-1 is the trypanolytic protein that provides resistance against Trypanosoma brucei infection (Vanhamme et al. 2003). The parasite internalizes the protein through endocytosis and is then transported to the lysosomes. APOL-1 is bound to an HDL particle but is released after progressive acidification of the environment within the lysosome. APOL-1 then forms an ionic channel in the lysosomal membrane that causes an influx of ions, which provokes osmotic swelling, and death of the parasite (Pérez-Morga et al. 2005; Limou et al. 2015). Haptoglobin-related protein (HPR), another primate-specific protein, is involved in targeting the complex that contains APOL-1 to the parasite after association with hemoglobin (Pays et al. 2014). Thus, in humans, the presence of haptoglobin-related protein has diverted the function of the trypanosome haptoglobin-hemoglobin receptor to elicit innate host immunity against the parasite (Vanhollebeke et al. 2008).
The APOL-1 protein has a pore-forming domain, a pH-sensitive membrane-addressing domain, and an SRA-interacting domain (Vanhollebeke and Pays 2006) and shares some structural and functional similarities with bacterial colicins (from different strains of E. coli), diphtheria toxin, and mammalian Bcl-2 family members, which suggest a similar activity for all these proteins. Interestingly, the diphtheria pathway through the infected cell is highly comparable to APOL-1’s pathway in the trypanosome (Limou et al. 2015).
Furthermore, other studies suggest that APOL-1 may have other protective roles in innate immunity in general and not just for protection against trypanosomal infection. It is known that APOL genes are upregulated by pro-inflammatory cytokines such as IFNγ and TNF, and also that APOL-1 can ameliorate other parasitic infections such as leishmaniasis (Limou et al. 2015; Samanovic et al. 2009) and even restrict in vitro replication of HIV-1 in macrophages (Taylor et al. 2014).
Evasion of Human Immune Responses by Trypanosomes
Being extracellular parasites that are continually exposed to the host’s immune system, African Trypanosomes have evolved sophisticated evasion mechanisms to survive in the chronically infected host (Bucheton et al. 2011).
One of the first mechanisms that prevents the immediate elimination of trypanosomes is the parasite’s ability to limit early on the production of tumor necrosis factor by myeloid cells. This is caused by the stress-induced activation of adenylyl cyclases of the trypanosome plasma membrane when the parasite is phagocytosed, leading to the release of cyclic AMP by the parasite into the myeloid cells, the consecutive activation of protein kinase A, and inhibition of TNF synthesis (Salmon 2012).
After that, the main strategy is by antigenic variation in which the variant surface glycoproteins (VSG), the main surface antigens of the parasite, change continuously to avoid antibody-mediated clearance (Hall et al. 2013).
Most importantly, and as part of an arms race against human immunity, trypanosomes have evolved different mechanisms to resist the killing by APOL-1. T.b. rhodesiense evolved a serum resistance associated (SRA) glycoprotein that binds to APOL-1 within the lysosome to prevent its toxicity (Vanhamme et al. 2003), whereas T.b. gambiense has a specific glycoprotein (TgsGP) that forms hydrophobic β-sheets that stiffen the endolysosomal membrane to prevent the insertion and thus toxicity of APOL-1. Also, the uptake of this protein by the parasite is limited and the degradation is enhanced (Uzureau et al. 2013).
SRA in T.b. rhodesiense is derived from a mutated VSG. Instead of being targeted to the plasma membrane, as would normally occur for other VSGs, SRA is targeted to the endolysosomal system where it encounters APOL-1 and binds strongly to it (Pays et al. 2014).
The mechanism of resistance is different for T.b. gambiense as TgsGP does not interact directly with APOL-1 but rather inhibits its toxicity by inducing endosomal membrane stiffening. This process prevents or slows down insertion of APOL-1 into the membrane and results in its degradation by endosomal proteases (Pays et al. 2014). The lysosomal cathepsin I is known to be the main cysteine protease involved in APOL-1 degradation, and it seems that, in Trypanosoma brucei, APOL-1 is prone to efficient degradation by cathepsin I, unless it is inserted into endosomal membranes, where it will generate ionic pores.
As mentioned above, there are two additional processes which are necessary for full resistance of T.b. gambiense to human serum, both responsible for reducing the intracellular levels of APOL-1: the limitation of its uptake and increased degradation. The reduced uptake results from the inactivation by a single point mutation of the receptor that allows entry of the APOL-1-containing complex into the parasite, known as T. brucei surface receptor for haptoglobin-hemoglobin or TbHpHbR (Kieft et al. 2010). The increased degradation comes from lowering endosomal pH, causing earlier acidification of endosomes which enables faster cysteine protease-mediated digestion of APOL-1 (Uzureau et al. 2013).
APOL-1 Variants and Their Consequences
APOL-1 variants that confer either susceptibility or resistance to African trypanosomiasis have been found in humans and other primates.
Recently, a patient in Ghana infected with an atypical T.b. gambiense lacking the TgsGP defense mechanism against APOL-1 was found to have homozygous missense substitution in the membrane-addressing domain of APOL-1, which knocked down the trypanolytic activity, allowing the trypanosome to avoid APOL-1 mediated immunity. It is thought that populations with this variant may be at increased risk of contracting trypanosomiasis (Cuypers et al. 2016).
On the other hand, APOL-1 variants that confer resistance to trypanosomiasis have been found in African-American and western African populations. The two genotypes known as G1 and G2 have been found to confer resistance to T.b. rhodesiense but, unfortunately, they do so at the expense of high probability of developing end-stage kidney disease in homozygotes. The resistance is owed to reduced interaction of these APOL-1 variants with SRA (Genovese et al. 2010).
The pathophysiology behind the development of kidney disease in those expressing these variants is an area of active research. APOL proteins share structural and functional similarities with proteins of the BCL2 family, as mentioned above, which control apoptosis and autophagy. APOL-1 could then be contributing to glomerulosclerosis by apoptosis, autophagy and/or endocytosis and lysosomal stimulation (Limou et al. 2015). Autophagy is a recognized major pathway in kidney function and disease, and some propose that APOL-1 may be interfering with autophagy in podocytes, leading to progressive kidney sclerosis (Pays et al. 2014). Furthermore, the fact that APOL-1 circulates with HDL particles is suggestive of a role in lipid transport and metabolism, which could be very important to maintain the plasma membranes of podocytes (Limou et al. 2015).
There is currently no evidence for natural APOL-1 variants that confer resistance to T.b. gambiense in humans. However, an APOL-1 variant was recently found in a West African baboon species that is able to kill both T.b. rhodesiense and T.b. gambiense. This variant could be a potential candidate for anti-trypanosomal therapies targeted at all pathogenic trypanosome species. Indeed, this knowledge has led to the experimental design of APOL-1 variants that are able to kill these parasites and could eventually be used as therapeutic tools (Cooper et al. 2016).
Trypanosomiasis is a potentially fatal parasitosis that affects thousands of people in sub-Saharan Africa. The two species that cause the disease in humans are T.b. rhodesiense and T.b. gambiense. An evolutionary arms race between these protozoa and humans has led to the development of mechanisms of resistance to each other’s killing. One such important factor in humans is APOL-1, a protein that ultimately lyses the lysosomal membrane of the parasite, leading to its death. Thus, APOL-1 should be considered as part of the innate immunity’s armament in humans.
As it would be expected, genetic variations of the APOL-1 protein can lead to either increased susceptibility or resistance to infection by these and other Trypanosoma species. Finding and understanding these variants could lead to therapeutic alternatives against this public health concern.
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