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Host Immune System Evasion by the Protozoan Parasites

Trypanosoma - Copy
Biological Sciences (Bournemouth University)

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Host Immune System Evasion and Manipulation by the
Protozoan Parasite Trypanosoma brucei (Protozoa:
Kinetoplastida)
Abstract

The single celled protozoan Trypanosoma brucei is responsible for human African
trypanosomiasis in sub-Saharan Africa, affecting some 65 million people in 36 countries.
Carried by the tsetse fly (Glossina sp.) mammals are the definitive host. The pathology
associated with T. brucei is notoriously difficult to treat, and this is due to the fact that the
parasite uses a number of molecules and proteins to not only evade detection and
elimination of the parasite by the host immune system, but is also capable of manipulating
the host’s own biological molecules in order to promote growth of the parasite. Here we
review the molecular mechanisms employed by T. brucei in order to evade detection and
destruction by immune cells of the host organism, and how this parasite can use the host’s
immune system to its own advantage in order to proliferate in the host both the
mammalian host and the tsetse fly vector).

Key words: Trypanosoma brucei, African sleeping sickness, human African trypanosomiasis,
immune evasion, growth promoting factors, mammalian host, tsetse fly, kinetoplastid
Introduction

The protozoan parasite Trypanosoma brucei is the causative agent of African

trypanosomiasis, otherwise known as human African sleeping sickness. The disease has two
forms, depending on the subspecies of Trypanosoma involved; Trypanosoma brucei
gambiense (West African sleeping sickness) accounts for around 97% of cases, whereas
Trypanosoma brucei rhodesiense (East African sleeping sickness) is responsible for less than
3% of reported cases (Koffi et al. 2009). Other species of Trypanosoma can be pathogenic to
wild animals such as T. vivax, which causes nagana in antelope and other ungulate
mammals (Rodrigues et al. 2017). The lifecycle of T. brucei is complex and involves an insect
vector in the form of the tsetse fly (Glossina spp.). The definitive hosts of T. brucei includes
ungulate mammals and humans, and between 300,000 and 500,000 cases of human African
sleeping sickness are reported per year, with the majority of cases being fatal if left
untreated (Njiokou et al. 2010). Trypanosoma brucei is an extracellular parasite and as such
has evolved several mechanisms in order to modulate the host’s immune response and also
to activate certain pathways of the host’s immune system to stimulate growth of the
parasite (Zimmer 2001).

Biology of Trypanosoma brucei

Trypanosoma is a kinetoplastid haemoflagellate, closely related to Leishmania, another

parasitic protozoan whose vector is the sand fly (Bates 2007). Kinetoplastids are so named
after the kinetoplast, an organelle found only in this group of flagellates which consists of a
mass of mitochondrial DNA which codes for mitochondrial proteins (Gluenz et al. 2011).
However not all kinetoplastids are parasitic; indeed the group also includes the
mixotrophic free-living flagellate Euglena (Ahmadinejad et al 2007). Trypanosomes have
several distinct morphological forms depending on the stage of development (Ooi et al.
2016).

Two subspecies of T. brucei, T. b. rhodesiense and T. b. gambiense, are the causative

agents of East African and West African sleeping sickness (respectively) in humans, which
can be lethal if left untreated (Koffi et al. 2009), whilst other subspecies such as
Trypanosoma brucei brucei are only pathogenic in wild animals such as mice (Wang et al.
2008). Infection can lead to muscle aches, headaches, fever and swollen lymph glands, and
as the parasites reach the central nervous system confusion, slurred speech and
alterations to sleeping patterns are noticed as the parasites affect the circadian rhythm of
the host (Kristensson et al. 2010).
Lifecycle and Transmission

The lifecycle of Trypanosoma brucei is indirect and involves two hosts – the tsetse fly (genus
Glossina) which acts as the vector, and large mammals including humans. In T. b.
gambiense and T. b. rhodesiense, humans are the definitive host for the parasite, with
mammals such as game animals and cattle acting as reservoir hosts (Njiokou et al. 2010).

When an infected tsetse fly takes a blood meal from a mammal, metacyclic trypomastigotes
residing in the salivary glands of the fly are injected into the bloodstream of the mammal
(Poinsignon et al. 2007), where they develop into bloodstream trypomastigotes, and spread
through the hosts lymphatic and circulatory systems, dividing by binary fission (Vertommen
et al. 2008). The bloodstream trypomastigotes are then taken up a feeding tsetse fly. Upon
uptake, the bloodstream trypomastigotes develop into procyclic trypomastigotes in the
midgut of the fly, and replicate further by binary fission. After replication, the procyclic
trypomastigotes then leave the midgut and develop into epimastigotes, which multiply
within the salivary glands by binary fission (Peacock et al. 2011). The epimastigotes then
develop into metacyclic trypomastigotes which will be injected into the bloodstream of a
mammal when the fly takes its next meal.

Epidemiology of Human African Trypanosomiasis (HAT)

Human African trypanosomiasis is restricted to sub-Saharan Africa with a risk population of

approximately 65 million people in 36 countries. East African and West African
trypanosomiasis are geographically isolated from each other by the African Rift Valley, and
Uganda is the only country which has cases of both T. b. rhodesiense and T. b. gambiense
(Selby et al. 2013). West African trypanosomiasis, caused by T. b. gambiense, is prevalent in
West and Central African nations, such as the Democratic Republic of Congo and Angola,
and is responsible for the majority of cases of HAT (Koffi et al. 2007). East African
trypanosomiasis, caused by T. b. rhodesiense, is much rarer and found in East and Southern
Africa, in countries such as Uganda, and is responsible for less than 3% of HAT cases (Selby
et al. 2013).

Immune System Evasion by Trypanosoma brucei

Unlike some species of protozoon parasites, such as Plasmodium, the causative agent of
malaria which inhabits the erythrocytes of the host, Trypanosoma brucei is an extracellular
parasite, spending the some of its lifecycle in the bloodstream of the host. As such, the
parasite should be vulnerable to the innate immune defences of the host including
phagocytes and lymphocytes (Gebreselassie et al. 2012). In order to evade detection by the
immune system of the host, Trypanosoma has evolved several mechanisms which are able
to manipulate the host’s immune system to both modulate host defences ensuring the
parasite is not destroyed, and also to activate certain processes to stimulate growth and
development of the parasite.

Once the trypanosomes have developed into metacyclic trypomastigotes in the salivary
glands of the tsetse fly, they must enter the bloodstream of the mammalian host. The skin
of the mammal represents a significant anatomical barrier to T. brucei, and in order to
penetrate the skin’s defences Trypanosoma uses a combination of saliva components and
trypanosome-derived factors to create a trypanosome-receptive microenvironment in the
skin, allowing the parasite to enter the bloodstream undetected. When feeding, infected fly
injects saliva and along with it the metacyclic trypomastigotes intradermally, and the saliva
constituents TTI and Adenosine-Deaminase (ADA) related proteins prevent coagulation of
blood and the aggregation of platelet cells to the site of penetration (Van Den Abbeele et al.
2010). Also, the allergen TAg5 stimulates activation of the host’s mast cells, which causes
degranulation of the mast cells. As a result the mast cells release histamine and TNF, and
this causes vasodilation of the blood vessels and also increases membrane permeability of
blood vessels, allowing Trypanosoma to enter the blood stream (Caljon et al. 2009).
Simultaneously, the immunoregulatory peptide Gloss2 downregulates the mammalian
inflammatory response which is triggered upon breaching of the skin by the proboscis of the
tsetse fly and in response to metacyclic trypomastigotes (Bai et al. 2015).

In addition to tsetse salivary components trypanosome factors are also involved in the
inoculation of Trypanosoma into the mammalian bloodstream. Before entering the
bloodstream, the metacyclic trypomastigotes develop into bloodstream forms, however the
pathogen associated molecular patterns (PAMP) of this form, particularly variant surface
glycoproteins (VSG) and CpG oligodeoxynucleotides activate host T cells and keratinocytes,
leading to an increased immune response (Paulnock et al. 2010).

Despite this, T. brucei is able to evade the host’s immune system using a variety of different
biological molecules. The T. brucei-derived kinesin heavy chain (TbKHC1) is one molecule
deployed by T. brucei which is able to dampen the inflammatory response of host
macrophages. When TbKHC1 binds to the SIGN-R1 molecule, arginase activity is favoured
which leads to an increased production of L-ornithine and by extension polyamines by the
host, which are required for the growth of trypanosomes within the host. This also
compromises the ability of arginine-producing immune cells to destroy the trypanosomes,
allowing the parasite to grow and establish itself in the bloodstream of the host (De
Muldyer et al. 2013).

Trypanosoma brucei is also able to utilise adenylate cyclases (AdCs), namely T. brucei
adenylate cyclase (TbAdC), an enzyme which catalyses the conversion of ATP to cyclic
adenosine monophosphate (cAMP). During situation of immunological stress, for example
when phagocytosis is taking place, cAMP levels are elevated within phagocytes, and this
activates protein kinase A, leading to the inhibition of TNF synthesis, enabling the
parasites to establish whilst avoiding destruction by host organism phagocytes (Salmon et
al. 2012).

Considering that Trypanosoma brucei is an extracellular parasite, they are directly exposed
to the humoral immune response of the host. Once the metacyclic form of the
trypanosome is inoculated by the infected tsetse fly, it rapidly develops into a LS
bloodstream form. This change involves the remodelling of the trypanosome cell surface,
with a change in the structure of the VSG (variant surface glycoprotein) coat (Hutchinson et
al. 2007). The VSG coat has two main functions, which are to protect bloodstream parasites
from complementary-mediated lysis by the host’s immune cells, and to prevent recognition
of cell surface proteins on the trypanosome by the innate immune system of the host. This
way the immune cells of the host are unable to attach to the antigens and other
extramembranous proteins on the cell surface of the trypanosomes, and thus the innate
immune defences of the host are compromised (Kim and Cross 2010).

However, as mentioned previously, VSGs are susceptible to detection and activation of T

cells which can initiate antibody-mediated lysis of the trypanosome cell (trypanolysis). In
order to prevent this from occurring, T. brucei has evolved to frequently change the gene
expression and by extension structure, of VSGs, meaning that the cell-surface antigens of
the trypanosome are frequently mutating (Marcello and Barry 2007), much like the surface
proteins of a virus. Again, this causes complications for the host immune system, as host
antibodies are unable to bind to the cell surface antigens of the trypanosome (Kim and
Cross 2010). In addition, premature host B-cell expansion triggered by VSGs and non-
mammalian CpG DNA causing the B-cells to differentiate into short-lived plasmablasts
results in the production of non-specific IgM antibodies, which eventually leads to a decline
in the population of host B-cells as cell death (apoptosis) occurs (Radwanska et al. 2008).

Another trypanosome-derived factor which is linked to promotion of parasite growth is the

trypanosome-derived lymphocyte-triggering factor (TLTF). This secreted glycoprotein plays
an important role in host-parasite interactions by stimulating the production of interferon
gamma (IFN-γ), a type of cytokine produced by T-cells. Although IFN-γ is associated with a
reduction in TLTF in the presence of anti-TLTF antibodies (De Alencar et al. 2009), in vitro
studies have shown that IFN-γ is actually able to trigger TLTF secretion, promoting growth
of the parasite (Hamadien et al. 2000, Holzmuller et al. 2008). This shows that both TLTF
and IFN-γ are critical molecules for bidirectional cellular communication between T. brucei
trypomastigotes and host T-lymphocytes, and highlights the regulatory function of these
molecules in the host-parasite interactions in T. brucei.

The T. brucei-derived trypanosome suppression immunological factor (TbTSIF) is another key

molecule produced by Trypanosoma brucei which is known to initiate NO-dependent
suppression of T-cell populations by stimulating macrophage activity (Magez et al. 2006).
TbTSIF has two main routes of action against the immune response of the host. First, the
molecule is able to inhibit proliferation of host T-lymphocytes by utilising IFN-γ dependent
pathways, and secondly TbTSIF is able to down regulate the secretion of interleukin 10 (IL-
10), an anti-inflammatory cytokine which plays a key role in immunological defence
against pathogens. This is done by activating M2 macrophages, which reduce the effects of
M1 macrophages. The overall effect of this is the suppression of the action of both M1
macrophages and T-lymphocytes, resulting in establishment of T. brucei and suppression
of the host immune response (Stijlemans et al. 2014). By this effect, it can be considered
that TbTSIF is an essential molecule for parasite proliferation in the mammalian host.

Alongside host immune system evasion, trypanosome-derived factors are also capable of
actively impairing healthy functioning and development of B-lymphocytes. The high
antigenic variability and constant mutation of VSG proteins mean result in a loss of humoral
immune function against the parasite, until a new set of antigen-specific antibodies are
produced, a process which can take up to 10 days post-immunisation (Magez and
Radwanska 2009). In addition, VSGs have two direct effects on B-lymphocyte growth and
development. Firstly, VSGs stimulate the production of non-specific polyclonal B-
lymphocytes which leads to polyclonal exhaustion, leading to a failed immune response
(Radwanska et al. 2008). Secondly, the VSGs are able to destroy the splenic B-lymphocyte
compartment, resulting in massive depletion of B-cell proliferation and development
(Cnops et al. 2015). This results in a complete compromise of B-cell mediated immune
response by the host, alleviating antibody-related pressures from the parasite and allowing
T. brucei to successfully establish itself within the host, further resulting in trypanosome-
related pathogenicity.

Conclusion

To conclude, over the course of evolution, Trypanosoma brucei has evolved many
mechanisms for not only evading detection by the immune system of the host, for example
by using tsetse salivary components to establish a trypanosome-tolerant
microenvironment and escape detection by mast cells, but also to avoid elimination by
host immune cells such as B-lymphocytes, achieved by manipulating immune cells and
using the host’s own immunological molecules, such as INF-γ, to not only supress B- and T-
lymphocytes, and to stimulate production of growth-promoting molecules such as TNF and
TLTF. In addition, the constant mutation and structural changes of VSGs due to
morphological changes in the lifecycle of T. brucei mean that there is a constant ‘arms race’
between parasite and host, as each time the surface antigens of the parasite change, the
host’s immune system produces complimentary antibodies exerting an antibody-mediated
pressure on the parasite.

Trypanosoma brucei is a perfect example of a parasite which, although simple in body

structure, being a microbial eukaryote, has incredibly complex molecular mechanism
involved in interactions with the hosts, demonstrating a specialisation to mammalian
definitive hosts.
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