VIRULENCE

2023, VOL. 14, NO. 1, 2150445

https://doi.org/10.1080/21505594.2022.2150445

REVIEW ARTICLE

Pathogenicity and virulence of African trypanosomes: From laboratory models

to clinically relevant hosts
a a b b
Liam J. Morrison , Pieter C. Steketee , Mabel D. Tettey , and Keith R. Matthews
a
Roslin Institute, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, UK; bInstitute for Immunology and Infection
Research, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

ABSTRACT ARTICLE HISTORY

African trypanosomes are vector-borne protozoa, which cause significant human and animal Received 26 August 2022
disease across sub-Saharan Africa, and animal disease across Asia and South America. In humans, Revised 14 November 2022
infection is caused by variants of Trypanosoma brucei, and is characterized by varying rate of Accepted 17 November
progression to neurological disease, caused by parasites exiting the vasculature and entering the 2022
brain. Animal disease is caused by multiple species of trypanosome, primarily T. congolense, KEYWORDS
T. vivax, and T. brucei. These trypanosomes also infect multiple species of mammalian host, and Trypanosome; human
this complexity of trypanosome and host diversity is reflected in the spectrum of severity of African trypanosomiasis;
disease in animal trypanosomiasis, ranging from hyperacute infections associated with mortality animal African
to long-term chronic infections, and is also a main reason why designing interventions for animal trypanosomiasis;
trypanosomiasis is so challenging. In this review, we will provide an overview of the current pathogenicity; virulence
understanding of trypanosome determinants of infection progression and severity, covering
laboratory models of disease, as well as human and livestock disease. We will also highlight
gaps in knowledge and capabilities, which represent opportunities to both further our funda­
mental understanding of how trypanosomes cause disease, as well as facilitating the development
of the novel interventions that are so badly needed to reduce the burden of disease caused by
these important pathogens.

Introduction trypanosomiasis (HAT) in recent decades, in particular

for T. b. gambiense, with an elimination program in
African trypanosomes are protozoan parasites, trans­
mitted either cyclically by tsetse flies or mechanically place that aims to remove T. b. gambiense HAT as
by other biting flies. Several species infect a range of a disease of public health importance by 2030, an objec­
mammals and cause disease, impacting upon both ani­ tive that seems achievable from recent progress [4].
mal and human health. Animal disease is caused by However, the methods used to control T. b. gambiense
multiple species, with T. congolense, T. vivax and HAT (active case detection) will not eliminate
T. brucei the main pathogens of cattle, sheep, goats, T. b. rhodesiense HAT, due to the truly zoonotic nature
equids, and wild animals in sub-Saharan Africa, and large animal reservoir of the latter pathogen [5].
T. simiae and T. suis infecting pigs in the same region, This outline serves to illustrate the point that trypano­
and T. brucei evansi and T. vivax infecting cattle, equids, somiasis is caused by a wide diversity of species or
camels, and Asian buffalo across North Africa, Asia variants – and indeed AT can be caused by concurrent
(T. b. evansi) and South America (T. b. evansi and infections of multiple species. The genetic diversity
T. vivax). T. brucei equiperdum causes a venereally trans­ within this complex of organisms has begun to be
mitted form of trypanosomiasis in horses and donkeys, much better understood in the post-genomic era,
mostly in sub-Saharan Africa. Variants of T. brucei, which has underlined that the species are not only
T. b. gambiense and T. b. rhodesiense, also cause genetically divergent, but that there is also substantial
human infections and disease in sub-Saharan Africa. genetic diversity within species (e.g. T. congolense
The economic and health impact of these pathogens is Savannah, Forest and Kilifi subtypes). This inevitably
collectively enormous, with Animal Trypanosomiasis means that genetic diversity translates to phenotypic
(AT) remaining widespread and causing millions of diversity, and this includes virulence.
infections and deaths per year [1–3]. There has been What do we mean by virulence in trypanosome
substantial progress in combating human African infections? Virulence can be a very loosely used term

CONTACT Liam J. Morrison Liam.Morrison@roslin.ed.ac.uk

© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
2 L. J. MORRISON ET AL.

in trypanosome literature, often applied to simple phe­ multiple ways. In this article, we define virulence as
notypes such as parasite growth rate (including the ability to cause disease in the recipient host, i.e. the
in vitro), but it is also used to refer to more complex more virulent a trypanosome is, the more severe the
traits such as host-specific infectivity or vector trans­ disease is in the mammalian host. In this context, we
missibility. In reality, many of these phenotypes inter­ aim to describe what is currently known about the
act to determine the virulence of a trypanosome. spectrum of virulence diversity in trypanosomes, the
However, virulence is also clearly an outcome of the variety of mechanisms that underpin virulence in try­
interaction(s) of the trypanosome with the host, and panosomes, and the virulence factors that have thus far
host factors (for example, host species) can also shape been identified in trypanosomes (see Figure 1 for over­
and influence the virulence of trypanosomes in view). Additionally, we will outline the important

Figure 1. Overview of virulence in African trypanosomes. (from left to right); Human infectivity: ApoL1 is the main component of
human trypanosome lytic factor (TLF), a high-density lipoprotein subclass that confers protection against animal-infective trypano­
somes through parasite lysis. The human-infective trypanosome species, T. b. rhodesiense and T. b. gambiense, have evolved
mechanisms to evade ApoL1-mediated lysis, strongly influencing virulence in human hosts. For example, T. b. rhodesiense can
express SRA, a protein that neutralises ApoL1 through direct interaction. Another mechanism is reduced ApoL1 uptake via an L210S
mutation in the haptoglobin-haemoglobin receptor (HpHbr) that inactivates it. Coinfection: Infection with multiple species and/or
strains can lead to multiple virulence phenotypes as described. For example, the presence of a less virulent strain can suppress the
pathology associated with a more virulent strain of the same species in a coinfection setting. In addition, coinfection of multiple
trypanosome species can impact differentiation dynamics. Immune response: The interaction of trypanosomes and the host
immune response can greatly impact virulence phenotypes. Antigenic variation is undoubtedly a paradigm of trypanosome biology.
Hydrodynamic flow of VSGs across the cell surface sweep bound antibodies to the cell posterior, where they are degraded following
endocytosis. Furthermore, trypanosomes regularly switch the identity of the expressed VSG, leading to waves of parasitaemia with
host antibodies eventually raised to the dominant VSG in the parasite population. A further parasite virulence phenotype associated
with the host immune response is the ablation of B cell memory via killing of host B cells. Extravasation/sequestration: A key
symptom of HAT is an ability of T. brucei to extravasate and enter extravascular tissues, in particular the brain, adipose tissue and the
skin. A related virulence phenotype has also been described in animal-infective trypanosomes, albeit caused by intravascular
sequestration rather than extravasation (e.g. strain and tissue specific sequestration in T. congolense). Secreted factors/EVs:
Trypanosomes release a significant amount of metabolites, proteins and vesicles into the host environment, several of which
have been characterised. In particular, virulence associated with secreted peptidases has been established, with oligopeptidase
B (targeting atrial natriuretic factor), type 1 proglutamyl peptidase (targeting gonadotropin-releasing hormone and thyrotropin-
releasing hormone) and prolyl oligpeptidase (type I and native collagen) all targeting host effectors. As-of-yet unidentified parasite-
derived secretome components also target the maturation of host LPS-induced dendritic cells. Abbreviations: ApoL1: apolipoprotein
L1; HpHbr: haptoglobin-haemoglobin receptor; SRA: serum resistance-associated protein; VSG: variant surface glycoprotein; OPB:
oligopeptidase B; ANF: atrial natriuretic factor; PGP: proglutamyl peptidase; GnRH: gonadotropin-releasing hormone; TRH: thyro­
tropin-releasing hormone; POP: prolyl oligpeptidase; COL I: type I collagen; COL-N: native collagen; LPS: lipopolysaccharide; EVs:
extracellular vesicles. Inset graph in immune response panel adapted from [6].
 VIRULENCE 3

current gaps in knowledge, and consider the opportu­ evolution and accumulation of mutations in the
nities that recent research presents for advancing homologous chromosomes, which can only occur
understanding in this area. Finally, we will propose with long-term absence of sexual recombination
priorities for research on trypanosome virulence going [8]. In contrast, evidence from both experimental
forward. and population genetics data indicates that
T. b. rhodesiense and T. b. gambiense Group 2
are capable of frequent sexual recombination
Evidence of virulence diversity of field isolates [8,11–15].
● T. b. gambiense Group 1 and T. b. rhodesiense
Human trypanosomiasis
require treatment by different drugs, particularly
Disease caused by trypanosomiasis in humans is char­ in the meningoencephalitic stage 2 of infection
acterized by two stages. The first (the hemolymphatic when parasites have entered the brain [16].
stage or stage 1) is initiated by the deposition of trypa­ ● The nature of the disease caused is very different -
nosomes into the skin by tsetse bite, from where para­ T. b. gambiense Group 1 is classically described as
sites disseminate, initially via the lymphatics, to spread leading to a chronic infection that takes many
throughout the vascular system. This stage is typified months to years to culminate in the neurological
by fever and lymphadenopathy, alongside malaise, end stage of infection [17], whereas
weakness, and headaches. The second stage occurs T. b. rhodesiense is usually described as causing
when the parasites invade the brain (meningoencepha­ infections that tend to be much more acute
litic stage or stage 2) which is associated with motor [18,19], in some instances reaching the critical
and sensory dysfunction (including abnormal sleep neurological complications in a matter of days
patterns, giving rise to the colloquial name of sleeping [20].
sickness), and if untreated, eventually leads to seizures,
coma, and death. The rate of progression from stage 1 However, virulence is complex in human-infective try­
to 2 disease is a classical metric of virulence in human panosomes, as there is a clear contribution of host
infections, and here we will discuss the parasite-driven genetic variation to disease progression and outcome –
aspects of this. this has mostly been defined in T. b. gambiense Group
As described above, animal and human disease is 1, where there is evidently a spectrum of disease pre­
caused by an assemblage of trypanosome species. The sentation, including long-term asymptomatic indivi­
diversity within species can contribute to phenotypic duals and even apparent self-cure [21]. This difference
divergence, and in particular can be critically linked to in outcome has been linked to differential immune
parasite virulence and disease outcome. This is exem­ response signatures, with high IL10 and low TNFα
plified by the variants (often termed subspecies) of being associated with an increased risk of developing
T. brucei that are able to infect humans and cause HAT, whereas increased IL8 was associated with indi­
disease; T. b. rhodesiense, T. b gambiense Group 1 and viduals becoming seronegative [22] – in contrast
T. b. gambiense Group 2. In fact, the current status of macrophage inhibitory factor (MIF) was shown to be
T. b. gambiense Group 2 in the area of West Africa elevated in infected people (HAT patients and latent
from where it was originally described is uncertain infections) but to not contribute to pathology [23].
[7] – and how these parasites evade lysis by human Differential disease outcome has also been shown to
serum is detailed below. These human infective variants be associated with different Apolipoprotein-1 geno­
are genetically distinct from each other, and reflect types (see section below on human serum resistance),
multiple independent emergences of human infectivity with patients having G2 alleles of this gene showing
as a trait [8]. The genetic divergence also manifests as improved disease outcome [24], and genome-wide
differences across multiple phenotypes: association studies have corroborated this observation
[25]. The analysis of host genetic factors is a continuing
● T. b. gambiense Group 1 is primarily anthropono­ area of work, and how host genetic diversity may inter­
tic while T. b. rhodesiense and T. b. gambiense act with parasite genetic diversity is a remaining ques­
Group 2 are zoonotic. tion. Indeed, there have been attempts to link
● T. b. gambiense Group 1 does not undergo sexual T. b. gambiense Group 1 genotype to disease presenta­
recombination and all data suggest this organism tion in HAT patients [26], but given the remarkable
reproduces strictly clonally [8–10] – indeed, geno­ clonality and high levels of homozygosity across multi­
mic analysis demonstrated the Meselson effect in ple isolates [8] it may be that this will prove difficult,
this organism whereby there is independent and this homogeneity across T. b. gambiense Group 1
4 L. J. MORRISON ET AL.

exemplifies why it may be a good model to explore the isolated from different disease foci with differing sever­
human genetic contribution to disease. Having said ity of disease presentation [33,34], with circulating
that, inoculation of T. b. gambiense Group 1 isolates IFNγ levels correlating with progression to the neuro­
into mice deriving from human patients that differed in logical stage of disease [33]. In a separate study, geno­
clinical severity demonstrated that pathology in mice mic analysis was carried out on Ugandan
also broadly separated into three categories (“highly T. b. rhodesiense isolates deriving from human patients
pathogenic,” “intermediate pathogenic,” and “low with differing clinical presentation. This analysis sug­
pathogenicity”) that mirrored the pattern of disease gested that the genetic divergence of these pathogeni­
severity in the patients they were isolated from [27]. cally distinct isolates to some extent derived from
Therefore, there may be a pathogen genetic factor introgression from West African T. b. brucei, with
underpinning at least some of the variation in disease a region on chromosome 8 originating from West
outcome observed in T. b. gambiense Group 1 infec­ African T. b. brucei containing a gene(s) whose alleles
tions, perhaps sited in the highly variable subtelomeric underpin the virulence differences observed between
regions of the trypanosome genome that are very diffi­ the isolates [35]. Interestingly, the virulence differences
cult to assemble and which would not be covered or between these T. b. rhodesiense isolates variants also
captured by low-resolution approaches, such as micro- was recapitulated to some extent in murine infection
or minisatellites, or by most genome approaches/ models, with isolates of the strain that presented with
assemblies. The substantial challenge of incorporating more severe human disease (Z310) resulting in infec­
these regions in analysis is evidenced by one study tions with substantially higher parasitemia and more
currently being the only genome assembly that has severe symptoms than isolates from the strain derived
managed to provide a complete picture of T. brucei from milder human infections (B17) [35].
subtelomeric regions [28], despite the organism being There have been relatively few attempts to directly
the focus of multiple genome sequencing efforts for examine the genetic determinants of parasite-driven
many years. differential pathology, exemplified by T. b. rhodesiense
The population genetics of T. b. rhodesiense is much and T. b gambiense, in T. brucei. One approach has
more complex than that of T. b. gambiense, deriving been to utilize a classical genetics approach, exploiting
from the fact that T. b. rhodesiense is defined by the cloned progeny derived from a genetic cross between
carrying of a single gene, the serum resistance asso­ two strains of T. brucei (TREU927 and STIB247) that
ciated (SRA) gene [29] that confers resistance to the cause very different severity of infection in mice [36].
trypanolytic factors in human serum. T. b. rhodesiense Analysis of the inheritance pattern of induced pathol­
are therefore essentially variants of T. brucei that carry ogy during infections with progeny in mice identified
the SRA gene, and the genetic diversity of a locus on chromosome 3 that was linked to differential
T. b. rhodesiense reflects that of the underlying organomegaly (enlarged liver and spleen) [37]. While
T. brucei population that it is a member of, with clonal the causative gene(s) in this locus remain to be identi­
expansions occurring when outbreaks occur fied, the phenotype observed involved differential argi­
[8,11,14,30]. As a result of T. b. rhodesiense being nase expression and alternative macrophage activation
genetically diverse, there is unsurprisingly also evidence [36], reminiscent of the parasite-induced arginase-
for variation in virulence within T. b. rhodesiense in mediated reduction of NO synthesis and consequent
human cases [31]. This is perhaps best characterized by liver damage by T. brucei kinesin heavy chain 1
studies in Uganda and Malawi, where it was demon­ (TbKHC1 – see section below for further details) [38].
strated that there were differing population dynamics of With improved sequencing technologies enabling
T. b. rhodesiense, with semi-stable clonal lineages in assembly and annotation of the previously difficult to
Uganda and frequent mating in Malawi [14]. These assemble and highly repetitive subtelomeric regions
differences likely correlate with their varying transmis­ [28], in which many trypanosome virulence factors
sion intensity and population dynamics resulting in are located, it may be timely to revisit the utility of
different levels of interactions and mating with the genetic crosses and exploit these resources as routes to
underlying non-human infective T. b. brucei popula­ identifying virulence factors that may be important for
tion. This also correlated with differing clinical presen­ disease.
tations, there being a more chronic form of disease in
Malawi and acute disease in Uganda [32]. Within
Animal trypanosomiasis
Uganda, with the increased power to test associations
afforded by the expansion of clonal lineages, it was While HAT is caused by different variants of T. brucei,
possible to link genetically distinct T. b. rhodesiense Animal Trypanosomiasis (AT) is caused by multiple
 VIRULENCE 5

species of trypanosome [39], which are very genetically T. simiae Tsavo) on livestock has been poorly docu­
divergent indeed. These differences are such that traits mented, partially due to lack of molecular tools to
defined in T. brucei as paradigms of African trypano­ enable detection, but also due to a relative paucity of
some biology, such as reliance upon high-rate glucose research focus. Similarly, the diversity of trypanosomes
metabolism in the mammalian host, or the mechanistic identified through molecular screening of tsetse is
utilization of variant surface glycoproteins (VSGs) in clearly greater than that of the traditionally described
antigenic variation, have been shown to either not hold livestock pathogens T. brucei, T. congolense, and
(reliance upon glucose metabolism is much reduced in T. vivax, (for example the recently described “T. vivax-
T. congolense [40]) or be achieved by very distinct like” species [66,67], but the role that such trypano­
mechanisms (the VSG content and family structure somes play in causing disease in livestock is currently
indicate that antigenic variation is achieved in unclear.
T. congolense and T. vivax by different mechanisms to This brief outline of pathology in animal trypanoso­
T. brucei [41–45]). This means that animal disease is miasis [covered in more detail elsewhere - [46,47,68]
caused by a much greater spectrum of trypanosome serves to illustrate the enormous complexity of the
genetic diversity, making it a very broad disease in disease, and this can make identifying the contribution
terms of clinical severity, duration and presentation of parasite virulence to clinical severity difficult to
[46]. This complexity is one reason why developing ascertain – even before considering the added compli­
interventions such as drugs and vaccines against AT cation and contribution of coinfections (see section
has been and remains such a challenge, as any product below) and host susceptibility/tolerance. However, it is
needs to be effective against multiple divergent patho­ clear that some of the variation in clinical presentation
gen species. does derive from the parasite genotype. T. congolense
Typically in ruminants (cattle, sheep and goats) try­ groups broadly into three genetically distinct subtypes,
panosome infection manifests as a chronic wasting dis­ Forest, Kilifi, and Savannah [39,44,69], and evidence
ease, with intermittent pyrexia, lymphadenopathy, indicates that there is frequent genetic exchange in
weight loss and reduction in activity [47]. Anemia is the field resulting in substantial genetic diversity, at
a consistent clinical sign in ruminants, and can be used least within T. congolense Savannah [70–72], and
as a diagnostic indicator of infection [48] - albeit with furthermore suggestion that T. congolense Savannah
the caveat that several co-endemic pathogens also cause and Forest may hybridize [72]. It has been demon­
anemia. As well as reduced production in terms of strated that field isolates representative of the three
weight loss, trypanosome infections also result in subtypes differed in virulence in cattle infections – a
reduced milk yield and fertility [49–51], with abortions Savannah isolate (Sam 28.1) causing more severe dis­
in some cases [52,53]. In contrast to the human disease, ease (higher parasitemia, lower packed cell volume and
neurological symptoms are not a common feature of eventually death) than Forest (Dind.3.1) or Kilifi
ruminant infections, although seem to be reported (K60.1A) subtypes, which either showed minimal clin­
more frequently in T. vivax infections [54,55]. ical signs compared to control animals (Kilifi) or even
Donkeys and horses are also commonly infected with apparent self-cure (or inability to detect parasites in the
trypanosomes [56–58]; in these hosts neurological dis­ blood) in all five animals infected with Forest after 95
ease is much more common, with data indicating that days of infection (with animals followed for 295 days
T. brucei (including T. b. evansi and T. b. equiperdum) post-infection) [73]. This strain-specific pattern of viru­
may be implicated in being the primary causative agent lence was mirrored in mice infections, with Sam 28.1
of neurological complications [59,60]. Asian or water giving rise to lethal infections that lasted less than
buffalo (Bubalus bubalis) are an important livestock a week, whereas Dind.3.1 and K60.1A produced
species across Asia, as well as increasingly in South chronic infections with low parasitemia and low mor­
America, and the impact of T. b. evansi (as well as tality rate (one death in each group of seven mice over
T. vivax in S. America) upon buffalo is important and 130 days of infection) [74]. While the caveat of these
currently underappreciated, where it causes a similar studies is that there was a single isolate per subtype, the
chronic production disease to that seen in cattle [46], observations would fit with the increased representa­
with occasional outbreaks of high mortality [61]. tion of T. congolense Savannah isolates in AT field
T. suis, T. simiae and T. godfreyi all either only or studies in the literature [39] – the expansion of loca­
preferentially infect pigs, causing either acute or tions across the African subcontinent where
chronic presentation, which is seemingly dependent T. congolense Kilifi (and to a lesser extent Forest) has
upon age of infection [62–65]. The extent of the impact been detected has coincided with the advent of more
of the latter species (as well as other species such as sensitive molecular diagnostics, and this suggests that
6 L. J. MORRISON ET AL.

these subtypes may be reasonably widespread but either to potentially relate to the smaller VSG repertoire of this
cause limited severe disease or present with very low species [42,45,50] and therefore “exhaustion” of avail­
parasitemia in livestock, and are therefore picked up able VSGs during infections, and/or a reduced amount
less frequently in surveillance efforts. However, there of VSG protein on the cell surface that may result in
are really very significant knowledge gaps around greater exposure of other invariant antigens to the host
T. congolense Kilifi and Forest, including the extent of immune response. However, while there are reduced
the role they play in livestock disease, and these sub­ levels of cellular VSG gene transcripts compared to
types certainly warrant further investigation. T. brucei and T. congolense [88], evidence that this
Virulence variation within T. congolense Savannah translates to reduced levels of VSG protein at the cell
has also been demonstrated, with 31 field isolates surface is less clear [50]. There is reported strain-specific
derived from cattle in Zambia being tested by inocula­ virulence in T. vivax, perhaps the most notable being
tion into two mice each (with all strains used at their a reported hemorrhagic form of T. vivax infection that
fifth or sixth passage from cattle isolation). The isolates seems be more commonly reported in isolates deriving
grouped into three categories – termed “extremely viru­ from East Africa [50,89–92] – this is associated with
lent,” “moderately virulent” or “low virulence,” as a hyperacute infection profile with very high and sus­
determined by parasitemia profile, survival time, pre­ tained parasitemia, fever, profound anemia and multiple
patent period, and degree of anemia induced [75]. How hemorrhages of visceral and mucosal surfaces. The
these virulence categories translate to clinical presenta­ hemorrhagic stage is correlated with thrombocytopenia
tion in cattle is unclear and requires further work, but it and dysregulation of the clotting cascade, as well as
is worth noting that highly virulent field strains of generation of autoantibodies that bind to and cause
T. congolense Savannah have been isolated, which lysis of erythrocytes and platelets [93]. Additionally,
reproducibly give rise to very acute infections in cattle, other highly virulent field strains (without the hemor­
resulting in death in 9–10 days unless treatment is rhagic presentation) have been isolated that give rise to
provided [76–78]. experimental infections with very acute profile and short
T.vivax broadly splits into two genetic groupings, East duration (9–10 days before rescue treatment is required)
and West African [45,79,80]. Several studies using dif­ [76,77]. However, while we have good evidence for there
ferent (low resolution) genetic markers indicate that being parasite-driven differences in virulence in T. vivax,
South American strains are derived from West African a barrier to understanding the parasite factors that con­
T. vivax [81–83]. While the population genomic analysis tribute to these differences is that very few T. vivax
of Silva Pereira et al. [45] robustly demonstrated group­ strains grow in mice and only one strain has been repro­
ing of South American T. vivax with Ugandan strains, ducibly cultured in vitro (Y486) [94,95] - although it
Uganda is closely linked with West Africa (including via should be noted that the culture of bloodstream form
trade routes and transfer of livestock) by being on the Y486 has only been successful to a limited extent. This
edge of the Congo basin, and therefore it is possible that limitation to ruminant in vivo experimental work for all
Ugandan T. vivax may be more representative of West but very few strains means that there has not been the
African strains than those from elsewhere in East and ability to either assess translation of variable virulence in
Southern Africa (which were unable to be included in the murine model, or functionally assess potential
the analysis). This is backed up by a study that assessed mechanisms in vitro.
cross-reactivity of sera from cattle inoculated with
strains from West and East Africa, which showed that
Trypanosome interactions with the host
sera from cattle infected with Ugandan strains cross-
immune response
reacted with that from cattle infected with West
African strains, but not those infected with East The host immune system and its interaction with the
African strains [84]. Such data indicate that further pathogen evidently is a major component of how virulence
genomic analysis is therefore required to fully resolve presents in the infected mammalian host. The details of
the continental picture of diversity for T. vivax. With immunology in trypanosome infections are well covered
respect to sexual recombination influencing genetic elsewhere [68,96–98], and in this section, we will aim to
diversity, all evidence suggests that, like focus on aspects of the host immune response that are
T. b. gambiense, T. vivax is clonal and does not undergo driven by the parasite (i.e. how parasite virulence influences
sexual recombination [45,85]. A feature of T. vivax elements of the host immune system).
infections of cattle (as opposed to T. congolense and The paradigmal trypanosome interaction with the
T. brucei) is that self-cure is reasonably frequently host immune system is antigenic variation.
reported (e.g. [50,86,87]), and this has been suggested Trypanosomes have developed an incredibly elaborate
 VIRULENCE 7

and extensive system of antigenic variation, which is free movement of VSGs across the cell surface, results in
driven by a large gene family of variant surface glyco­ hydrodynamic pressures at the cell surface effectively
proteins (VSGs), one of which is expressed in each cell sweeping bound antibodies to the cell posterior, where
through a monoallelic expression system that results in they are engulfed and removed by endocytosis in the
the parasite coat being covered in homodimers of the flagellar pocket [102]. This provides an extended time
expressed VSG protein. The VSG N-terminal domains window for trypanosomes to switch VSG identity before
are the primary point of contact for the host adaptive antigen-specific antibodies reach a concentration thresh­
immune response, and VSG epitope-specific antibodies old that can overcome the hydrodynamic flow effect.
are generated that clear parasites expressing the rele­ Initially described in T. brucei, this has since been
vant VSG. However, the parasites regularly change the shown to also occur in T. congolense and T. vivax
identity of the expressed VSG, meaning that within [103] – with species-specific differences in motility char­
a population cells emerge that are not susceptible to acteristics postulated to link to the differential infection
the VSG-specific antibodies raised against epitopes on biology of the parasites, such as extravascular tissue
the previously expressed VSG. Through a combination invasion for T. brucei, cellular adherence for
of a very large VSG gene repertoire (2,000 in T. brucei – T. congolense and intravascular circulation for T. vivax.
approximately 20% of the coding genome) and elabo­ The VSG coat structure has long been posited to
rate recombinatorial processes that massively amplify provide a near insurmountable barrier in terms of
the potential encoded genetic VSG variation, antigenic targeting the host immune response to underlying and
variation in trypanosomes is a powerful tool that conserved antigen epitopes, via vaccination for exam­
matches the host ability to generate antibodies, and is ple. How this barrier functions as such, given there are
key to their ability to establish and maintain long- invariant proteins whose structure suggested they
lasting chronic infections. The intricacies of antigenic should protrude above the protective VSG monolayer,
variation, particularly in T. brucei, have been the sub­ has long been debated [104]. However, the generation
ject of much research over many decades, and the of the first model of a full T. brucei VSG structure
mechanistic understanding is highly developed provided insight that the C-terminal domain of the
[43,99,100]. While it is very evident that T. congolense VSG is likely to be remarkably conformationally flex­
and T. vivax also undergo antigenic variation, the struc­ ible, sufficiently so to enable VSGs to possibly shield
ture and content of the VSG repertoire in these species invariant surface proteins [105]. This is supported by
is very different to that of T. brucei [42,101], and the efforts that have targeted invariant antigens in vaccina­
degree of recombination-driven amplification of diver­ tion efforts providing at best partial protection
sity also appears quite distinct. For example, while [106,107]. Strategies have been implemented to try
T. brucei massively multiplies antigenic diversity and bypass this structural barrier, such as using single-
through recombination between VSGs that belong to chain camel-derived nanobodies against invariant anti­
one of two subfamilies (a-VSG and b-VSG), evidence gens [108]. Overall, this strategy has also met with
suggests that T. vivax does not employ recombinational limited success, although some protective effect was
VSG switching [45], with genes in four subfamilies demonstrated. However, recent data has demonstrated
corresponding to 174 phylotypes (where a phylotype that vulnerabilities can be identified by targeting invar­
is a clade of highly related VSGs based on amino acid iant antigens. Through a process of expressing recom­
alignment). T. congolense lies somewhere between, with binant versions of proteins predicted to be expressed on
a repertoire indicating recombination largely occurring the cell surface of T. vivax, and immunization and
within 15 phylotypes split between two subfamilies challenge experiments in mice, the extracellular domain
[44,101]. Currently, it is unclear if these repertoire of one protein (“invariant flagellum from T. vivax,”
differences translate to mechanistic differences in IFX) resulted in reproducibly sterile protection against
terms of how antigenic variation is expressed in rechallenge [109]. These remarkable data provide proof
T. congolense and T. vivax [43]. Additionally, the impli­ of principle that vaccination using surface-expressed
cations of the VSG repertoire differences with respect proteins may be achievable, after decades of skepticism.
to host-parasite interactions, such as the putative dif­ The localization of IFX, between the flagellum and cell
ferent effective repertoire sizes, remain to be elucidated. body, suggests it may play a role in flagellum structure
The structure of the VSG coat and limited presenta­ or function, and this may provide a reason as to why it
tion of epitopes to the host response are one mechanism represents a vulnerability for the parasite, as due to its
of immune evasion, but trypanosomes also elegantly location it may not be subject to hydrodynamic clear­
exploit their motility as an immune evasion technique – ance of bound antibodies. Whether this vulnerability
the motility driven by the flagellum, combined with the also applies to T. congolense and T. brucei awaits
8 L. J. MORRISON ET AL.

further study. Additionally, the translation of successful formally described in cattle infections, memory loss was
immunization against IFX from the mouse model to observed in cattle immunized with irradiated T. brucei,
a clinically relevant host species (goats) has been tried infected with T. congolense and then re-challenged with
in pilot vaccination and challenge experiments, but did homologous irradiated T. brucei, with the memory
not result in protection [109]. Therefore, significant response against T. brucei being impaired in three of
hurdles clearly remain to be overcome in order to the five cattle [120], suggesting that this process also
replicate the promising protection observed in mice in occurs in cattle. The extent of any parasite-driven B cell
disease-relevant hosts such as cattle. destruction in human trypanosome infections is also
The host antibody response is clearly important in not clear, although one study has demonstrated
clearance of trypanosomes during infection [110]. reduced anti-measles antibody levels in HAT patients
Debate continues about the role and efficacy of parti­ [121]. Both cattle and human data, although scanty,
cular antibody isotypes; for example, the key isotype indicate that the phenomenon indeed may occur, but
that conferred protection against T. vivax in IFX vacci­ the extent of B cell memory loss may not be as exten­
nations studies was shown to be IgG2a [109], but recent sive as in infected mice. The B cell destruction is known
data demonstrated that activation-induced cytidine to be mediated by host natural killer (NK) cells and is
deaminase (AID)-deficient mice, which are incapable perforin-mediated [122], but the identity of any para­
of somatic hypermutation and therefore cannot gener­ site ligand that may stimulate and drive this interaction
ate IgG antibodies, were more efficient at clearing chal­ is yet to be identified. While the impact of this parasite-
lenge with T. b. evansi than their wild-type controls driven phenomenon clearly benefits immune escape
through IgM [111] – consistent with previous studies and survival of trypanosomes within infections, it also
showing the importance of IgM in controlling T. brucei has potentially serious implications for the epidemiol­
infections in mice [112]. Nguyen et al. [111] interest­ ogy of other infectious diseases in endemic areas. The
ingly hypothesized that the rapid onset of B cell follicle impairment of immune memory in trypanosome
activation and isotype switching to IgG may in fact be infected animals or humans may mean hosts become
driven by the trypanosome, as switching to the lower more permissive for particular coinfections, and as
efficacy IgG would benefit parasite survival. These suggested by other authors [96], the trypanosome-
aspects of antibody response remain to be fully eluci­ mediated destruction of immune memory would in
dated in the mouse model of T. brucei, let alone host theory also disrupt vaccination-mediated protection,
species such as cattle, in which the mechanisms of with consequent implications for disease control
antibody generation are very different and for which efforts. This latter suggestion is backed up by several
the antibodies can have some unique features that may observations of diminished antigen-specific antibody
impact upon antigen binding [113,114], and for responses in trypanosome-infected Asian buffalo,
T. congolense and T. vivax. If vaccine prospects for goats, and cattle to vaccinations ranging from
AT are to be achieved from candidates such as IFX, Pasteurella multocida, Bacillus anthracis, contagious
clarity on what constitutes an effective antibody bovine pleuropneumonia to foot and mouth disease
response, and how this would be optimally induced, virus [96,123–127]. This aspect of trypanosome infec­
in the eventual host species and against the AT-relevant tion biology deserves further attention, and in particu­
trypanosome species, will be needed. lar fuller understanding of the extent of immune
Given the key role of antigen-specific antibodies, memory loss in clinically relevant hosts, as this could
a notable parasite-driven phenotype is the destruction be an important factor in both general disease suscept­
of host immune memory, with trypanosome infection ibility and epidemiology, and, for example, if efforts to
of mice resulting in ablation of B cell memory via generate a vaccine against AT bear fruit.
killing of B cells. This included the loss of memory to The symptomology of human trypanosomiasis is
previously exposed non-trypanosome antigens [115]; defined by the ability of T. brucei to extravasate and
this was recently shown to specifically involve the loss enter extravascular tissues, leading to encephalitis-
of memory B cells from infected animals [116]. This related clinical signs. The description of adipose-
effect has also been shown to occur in mouse infections and skin-resident trypanosomes in mouse and
with T. congolense [117], and observed disruptions to human infections with T. brucei sensu lato [128–
splenic architecture including lymphocyte-depleted 130] has focused attention on this aspect of
germinal centers and depletion of splenic B cells in T. brucei infection biology, with its obvious relevance
mice infected with T. vivax are consistent with the for disease progression, transmission, diagnosis,
phenotype also occurring in infections with this species parasite metabolism, and interactions with the host
[118,119]. While the destruction of B cells has not been immune response. Indeed, the extravasation has been
 VIRULENCE 9

shown in a mouse model to be active (i.e. occurs prior Human infectivity

to any vascular compromise induced by inflamma­
Another defense mechanism elicited by the mammalian
tion) and if the process is blocked by introducing
host is the presence of apolipoprotein L1 (ApoL1) in
antibodies against host molecules involved in cellular
normal human serum. ApoL1 is a component of the
adhesion (E-selectin, P-selectin, ICAM2, CD36, and
trypanosome lytic factor-1 and −2 (TLF1 and TLF2)
PECAM1) mouse survival was improved, indicating
which is a subclass of high-density lipoprotein (HDL)
that extravasation is a key virulence phenotype in
[138–140]. Human ApoL1 lyses exclusively animal
T. brucei infections [131]. Notably, CD36 was
infective trypanosomes through the formation of pH-
shown to preferentially inhibit extravasation into adi­
dependent ionic pores in the lysosomal membrane.
pose depots, indicating potential tissue-specific inter­
This causes the inflow of chloride ions from the cyto­
action in extravasation. Brain involvement in the
plasm leading to lysosomal swelling [141–144], and
mouse model has also been well defined in infections
ultimately, parasite death. Permeabilization of the mito­
with T. vivax and T. congolense [132,133]. In the case
chondrial membrane has also been reported [145].
of T. congolense, which binds to endothelial cells and
However, the human infective forms of the parasite,
is considered an intravascular parasite [134], brain
T. b. rhodesiense and T. b. gambiense, are resistant to
pathology was associated with trypanosome seques­
these TLFs. T. b rhodesiense evades ApoL1 lysis by the
tration in brain vasculature and the consequent
possession of serum resistance-associated (SRA) pro­
immune response; interestingly this effect was strain-
tein [146], which neutralizes the ApoL1 toxin by direct
specific (T. congolense 1/148 caused sequestration
interaction. However, some variants of ApoL1, variants
and pathology, while IL3000 did not), suggestive of
G1 and G2, are able to avoid this deactivation resulting
a differentially expressed parasite virulence factor(s).
While similar sequestration in cerebral capillaries in the killing of T. b. rhodesiense [142]. These variants
and sequelae have been observed in cattle experimen­ are primarily found in African Americans and West
tally infected with T. congolense [135,136], neurolo­ Africans [147], potentially contributing to the absence
gical clinical signs associated with T. congolense of T. b. rhodesiense infections in west Africa, and factor
infections are not frequently reported in the field in in the spectrum of disease severity in T. b. rhodesiense
livestock [18,46]. It is not completely clear whether patients (see “Evidence of virulence diversity of field
T. vivax readily extravasates or sequesters, and mouse isolates” section), with the G2 allele being associated
data has either described mainly vascular lesions with less severe disease in a genetic analysis of
[119] or used non-invasive bioluminescence imaging T. b. rhodesiense patients in Malawi [148].
techniques that would not discriminate between T. b. gambiense, consisting of two groups, Group 1
intravascular and extravascular parasites [132]. and Group 2, are both resistant to ApoL1 lysis. While
While neurological clinical signs have been reported T. b. gambiense Group 1 stably avoids TLF lysis,
from T. vivax livestock infections in the field T. b. gambiense Group 2 shows variable TLF resistance
[54,137], as with T. congolense it is also not in a way seemingly similar to T. b. rhodesiense but
a frequent clinical presentation. However, clearly which does not involve SRA, and thus, remains to be
a fuller exploration of tissue distribution in all three fully elucidated [149,150]. T. b. gambiense Group 1 on
parasites, and in clinically relevant hosts as well as the other hand, uses the specific glycoprotein (TgsGP)
mice, is needed before the potentially important to inhibit ApoL1-mediated lysosomal damage by mem­
implications of tissue specificity and adaptation are brane stiffening when it interacts with lipids [151].
understood. Other mechanisms employed by T. b. gambiense
The interaction of trypanosomes with the immune Group 1 to escape ApoL1 killing include reduced sen­
response is clearly multifaceted, and we have chosen sitivity to ApoL1 by cysteine proteases [152], reduced
here to focus on key parasite-driven aspects. The fol­ uptake of ApoL1 due to an L210S substitution in the
lowing sections also contain multiple examples of para­ haptoglobin-hemoglobin receptor, resulting in inactiva­
site biology whose interaction with the hosts, including tion [151,153], and increased digestion of ApoL1 [142].
with the immune response, also determine virulence There have been reports of atypical infections of
and infection outcome. The examples outlined above humans with species of trypanosome not normally
particularly serve to illustrate gaps in our knowledge – infective to humans, including T. lewisi and
many of these derive from the need to translate find­ T. b. evansi, and very rarely T. b. brucei, T. vivax and
ings from either T. brucei to T. congolense and T. vivax, T. congolense – while sporadic and clearly rare,
or from in vitro or mouse models to clinically relevant instances of such infections either are increasing or
hosts. are being detected more often [154]. While often the
10 L. J. MORRISON ET AL.

basis for human infectivity in such infections has not TbKHC1 secretion is not the only form of host NO
been able to be fully investigated, T. b. evansi infections modulation. Earlier work highlighted that soluble VSG
have been identified to occur both in an individual (sVSG), a form of VSG released by trypanosomes,
lacking APoL1 due to null mutations [155], but also modulates host gene expression in macrophages [165].
recently in a patient with no observable ApoL1 defi­ Importantly, the timing of sVSG exposure in relation to
ciency [156], suggesting there remain aspects yet to be that of IFN-γ is crucial. Whereas IFN- γ exposure
explained in this intensively studied and important followed by sVSG exposure leads to the expression of
host-parasite interaction. TNF-α, IL-6, and IL12p40, treatment of macrophages
with sVSG prior to IFN-γ led to a reduction in IFN-γ-
induced responses, including reduced NO synthase
expression and NO secretion [165]. Further work
Parasite metabolism and virulence
showed that the glycosylinositolphosphate moiety of
Parasite metabolism is crucial to enable generation of the sVSG is crucial for these host modulatory effects
sufficient ATP to persist in the host bloodstream. It is [165,166].
well established that African trypanosomes rely on host Metabolism of fatty acids also impacts virulence. In
carbohydrates in the form of glucose for ATP production. particular, phospholipase A1 (PLA1) activity is thought
However, metabolic enzymes and their products can also to correlate with pathogenesis [167] and indeed, PLA1
impact host gene expression and metabolism in ways that activity in plasma and tissue fluid from experimentally
maximize parasite survival, modulate host immunity, and infected rabbits correlates with parasitemia [168]. It is
directly contribute to virulence phenotypes. The role of thought PLA1 (and potentially PLA2) activity is
parasite metabolism in mediating host immune responses responsible for the severe changes seen in plasma lipids
has been studied in several pathogenic protozoan parasite in infected animals, in particular a reduction in phos­
species, including Trypanosoma cruzi and Leishmania phocholines (phosphatidylcholine) accompanied by
spp., in addition to African trypanosomes [157–159]. increased levels of choline, indicative of phospholipase
These parasites all release a significant number of proteins action [169,170]. Interestingly, the phospholipase activ­
and metabolites into their host environment (the former ity from non-pathogenic trypanosome species such as
detailed by studies of the secretome [160–162]), although Trypanosoma lewisi is relatively low compared to that
relatively few studies have detailed the impacts of meta­ of pathogenic species, suggesting a correlation between
bolism on host-pathogen dynamics, and thus, virulence, PLA1 action and virulence/pathogenesis [167].
during infection. Nonetheless, there is clear evidence that Trypanosomiasis is also associated with significant
parasite-derived metabolites and proteins impact host perturbations in serum/plasma levels of amino acids
immune responses with implications for parasite viru­ [170]. In particular, there is depletion of the aromatic
lence [161,163]. amino acids L-tryptophan, L-tyrosine and
Nitric oxide (NO) is a key host effector molecule in L-phenylalanine [171–174]. Concomitantly, T. brucei
the defense against trypanosome infection and NO excretes biologically relevant levels of aromatic ketoa­
exhibits cytostatic and cytolytic properties. To counter cids, specifically indolepyruvate (IP), hydroxyphenyl­
the effects of NO, Kinesin Heavy Chain (TbKHC)-1 is pyruvate (HPP) and phenylpyruvate (PP)
a protein secreted by T. brucei under both in vitro and [171,172,175,176]. These aromatic ketoacids are gener­
in vivo conditions, and has been shown to induce ated through degradation of aromatic amino acids by
arginase-1 activity in host myeloid cells, even those the cytosolic aspartate aminotransferase (cASAT)
from uninfected mice [38]. Arginase-1 converts [163,177]. This protein, as well as the reactions it cat­
L-arginine to L-ornithine and urea, and its activity alyzes, are essential to the parasite [163,178], but the
leads to reduction in the synthesis of NO. products of these reactions possess several important
Presumably, increased competition for L-arginine (an immunomodulatory properties.
important substrate for NO synthesis) leads to this The most studied excretory aromatic keto acid,
reduction. Indeed, it has previously been shown that indolepyruvate (IP), has been implicated in several
L-arginine bioavailability is an important determinant virulence roles [163,179]. IP is a product of
of NO production and parasite killing [164]. L-tryptophan metabolism through cASAT action
Recombinant TbKHC1 was shown to trigger IL-10 [163]. Firstly, IP treatment of host cells leads to reduced
and arginase-1 expression mediated by a C-type lectin glycolytic capacity by interfering with the transcription
(SIGN-R1; CD209b) receptor. Importantly, host survi­ factor hypoxia-inducible factor-1α (HIF-1α) [163].
val time is significantly prolonged in TbKHC1 KO- Furthermore, this study showed that IP inhibits the
infected mice, compared to wild-type controls [38]. induction of pro-IL-1β, a potent pro-inflammatory
 VIRULENCE 11

cytokine. More recent work on IP has highlighted the energy [170]. This is partially due to competition for
modulation of host eicosanoid production associated the main energy source, glucose [188–190]. Ultimately,
with this trypanosome-derived metabolite [179], speci­ hypoglycemia can play a role in host survival [and has
fically the downregulation of a class of eicosanoids been noted in cattle infections, e.g. [54]], and, therefore,
called prostaglandins (PGs). In this study, Diskin and parasite glycolytic rates have the potential to impact
colleagues further showed that IP acts as a direct inhi­ upon parasite virulence.
bitor of cyclooxygenase (COX) activity, an upstream Several other important metabolic processes have
mediator of PG production, and this effect is replicated been shown to impact upon virulence, including pro­
in human macrophages [179]. Thus, IP is a powerful teases such as serine peptidase 2 (ISP2) [191] and the
modulator of host activity, in particular that of the pro- cysteine proteases Cathepsin L and Cathepsin B [192].
inflammatory and innate immune response to In addition, increased levels of O- and N-acetylated
infection. glycoproteins have been detected in T. brucei-infected
Whilst the aforementioned studies were in large part plasma, which are likely T. brucei derived [193]. Whilst
carried out in murine trypanosomiasis models, recent the underlying mechanisms remain to be elucidated, it
evidence shows that the immunomodulatory properties is clear that these proteins are involved in trypano­
of IP (in addition to HPP) are replicated in primary some-mediated attenuation of the immune response
human dendritic cells [180], with HO-1 induction [191–193]. Finally, T. brucei, like other pathogens, exhi­
through Nrf2 activation, suppressed production of pro- bits an ability for metabolic mimicry, where T. brucei
inflammatory cytokines, reduced CD4+ T cell activa­ derived inositol phosphate glycans released from GPI
tion and modulation of host cell metabolism, including anchors are able to affect the host in the same way as
downregulation of glycolytic capacity [180]. To our insulin, an important hormone for glucose regula­
knowledge, there are no reports on any immunomodu­ tion [194].
latory effects of PP, another aromatic ketoacid excreted Recent evidence has revealed that T. brucei is capable
at high levels by African trypanosomes. Unlike IP, PP of invading adipose tissue [130], a site abundant in
has no effect on the ability of LPS to induce IL-1β glycerol. Indeed, T. brucei is capable of proliferation
[163], but other roles cannot be ruled out. in glycerol-based medium [195], and these findings
In murine models, trypanosome infection is asso­ may also contribute to our understanding of trypano­
ciated with global host metabolic disturbances, includ­ some virulence in vivo. Furthermore, imbalances in
ing in the bloodstream, but also in other anatomical plasma lipid bioavailability have also been detected in
locations such as the gut and brain [170,181,182]. These plasma samples derived from experimental model and
changes are the result of both host and parasite meta­ human infections [196–198].
bolism. The main glycolytic end-product from trypano­ Whilst the majority of studies on trypanosome meta­
somes is pyruvate, which accumulates to high levels in bolism and its impact on virulence have focused on the
the host plasma [170]. There are also increased plasma relevant model for human infection, T. brucei, com­
concentrations of lactate and these are, to an extent, paratively few studies have investigated the relevant
indicative of upregulated glycolysis in host cells [170]. species for Animal Trypanosomiasis - T. congolense
T. brucei does not encode lactate dehydrogenase (LDH) and T. vivax. Recent studies, however, have shown
[183], and, therefore, cannot generate lactate via fer­ that the former differs from T. brucei in key metabolic
mentation of glucose [184]. However, glucose-derived areas, such as glycolysis and lipid metabolism, and this
L-lactate is excreted from T. brucei at low levels, likely may impact metabolic phenotypes associated with viru­
via methylglyoxal detoxification [183,185]. It should be lence [40]. For example, it was hypothesized some time
noted that procyclic form T. brucei, as well as blood­ ago that free fatty acids released from autolyzing trypa­
stream form T. lewisi, also excrete L-lactate, the latter nosomes can significantly impact pathogenesis and
able to do so via lactate dehydrogenase [186,187]. It is virulence [199]. The differences in fatty acid metabo­
currently unknown whether the livestock trypanosomes lism between T. congolense and T. brucei that have been
T. congolense and T. vivax generate L-lactate via fer­ observed recently could underpin differences in viru­
mentation, although LDH is not annotated in their lence between the species [40]. Furthermore, differ­
respective genomes. It is plausible that both host and ences in metabolite uptake and excretion may also
parasite-derived lactate likely contribute to metabolic play an important role in differing virulence between
acidosis, a significant contributor to pathology. As the African trypanosome species, but these are as yet
disease progresses, the host can enter a ketotic state, unstudied.
characterized by increased levels of Whilst the genetic basis of differential virulence in
D-3-hydroxybutyrate, where lipids are metabolized for livestock trypanosomes has not been elucidated, there is
12 L. J. MORRISON ET AL.

clear evidence that strain-dependent variation in viru­ reduced levels of glucose and substantially increased
lence exists in T. congolense [73–75], and it is likely that levels of small volatile fatty acids (e.g. propionic acid
differential metabolism underlies at least some aspects and butyric acid) [210–212] compared to human or
of this variation. T. vivax is unique amongst African mouse blood. Thus, given that, as an example, glycoly­
trypanosomes in that it encodes a proline racemase not sis is a cornerstone of trypanosome metabolism, host
found in T. brucei or T. congolense [200]. This enzyme metabolic differences may play an important role in
was subsequently shown to be a potent B-cell mitogen influencing host–pathogen interactions and virulence
and thus, can be considered a virulence factor under­ (Figure 2 summarizes our current understanding of
pinning hypergammaglobulinemia, a symptom how trypanosome metabolism influences infection
observed during acute stages of T. vivax infection in severity and outcome).
mice [200]. Furthermore, unlike T. brucei, adhesion to
host cells is an important aspect of T. congolense and
Quorum sensing
T. vivax bloodstream form biology, as well as patho­
genesis [134,201,202]. In both T. vivax and Although trypanosomes are single-celled parasites,
T. congolense, it is established that trans-sialidases are individuals within the population show the ability to
involved in host cell attachments, and are also a key act co-operatively to restrict parasite numbers. This has
mediator of anemia, and thus, virulence [203–206]. a direct impact on virulence and parasite transmissibil­
Trans-sialidases are both expressed on the parasite sur­ ity. Virulence is affected because with unlimited popu­
face and secreted into the extracellular environment, lation growth, hosts lethally and rapidly succumb
and they are responsible for desialylation of red blood [213,214]. Correspondingly, the prolongation of host
cells, leading to erythrophagocytosis and anemia. There viability increases the probability of transmission –
is evidence that trypanotolerant N’Dama (African taur­ the essential requirement for any parasitic lifestyle.
ine) cattle exhibit reduced anemia compared to suscep­ This is particularly the case for African trypanosomes
tible indicine cattle [207], and concomitant evidence where transmission is restricted by the poor vectorial
that trans-sialidases purified from T. vivax desialylated capacity and relative scarcity of tsetse flies in compar­
indicine but not African taurine RBCs [203], indicating ison to, for example, mosquito vectors for parasites
a correlation between trans-sialidase host-specificity such as Plasmodium [215–217]. In addition to the
and parasite virulence. direct consequences of uncontrolled parasite prolifera­
Comparative proteomics analysis of T. vivax strains tion on host survival, the co-operative behavior of
with contrasting virulence revealed differential expres­ trypanosomes acts to promote transmission by driving
sion of several metabolic enzymes [208]. In that study, the generation of non-proliferative transmission
virulence and pathogenesis were interpreted as capacity adapted developmental forms of the bloodstream para­
to multiply and capacity to produce disease/mortality, sites – so-called stumpy forms [218]. These forms,
respectively. Protein expression profiles of two strains specific to T. brucei at least as a morphologically dis­
(high virulence and moderate pathogenicity vs low tinct entity, dominate the peak of acute and chronic
virulence and high pathogenicity) were compared. parasitaemias in experimental infections and predomi­
Amongst the significant differentially expressed pro­ nate in tissue reservoirs such as the skin and adipose
teins, there were also important glycolytic enzymes, tissue when quantitated using the molecular marker
pyruvate kinase, and glycerol kinase, expressed at defining this form, PAD1 [129,130,219].
higher levels in a T. vivax strain eliciting significantly Stumpy generation is a quorum sensing phenom­
more severe clinical pathogenesis, suggesting that gly­ enon whereby parasite numbers are detected and
colytic metabolism may play a role in driving symp­ responded to by individuals within the population –
toms [208]. a characteristic described in many species of social
It is worth noting that in vivo experiments focused microbe. Evidence of inter-parasite communication
on dissecting host–pathogen interactions and virulence controlling the production of stumpy forms was initi­
have been carried out almost exclusively on rodent ally provided by the analysis and modeling of parasites
models in contrast to clinically relevant models such in animal infections [220], but definitive evidence
as, in the case of animal trypanosomiasis, cattle [95]. emerged with the successful culture of parasites with
Ruminants such as cattle exhibit markedly divergent the developmental competence to generate stumpy
blood biochemistry from non-ruminants such as forms [221,222]. These are representative of tsetse-
rodents and humans [209], and this has the potential transmitted trypanosomes in the field and are termed
to impact both parasite and host metabolism during pleomorphic [223], and are distinguished from so-
infection. For example, ruminant blood contains called monomorphic forms that arise through
 VIRULENCE 13

Figure 2. Parasite metabolism and virulence. A) Trans-sialidases released by T. vivax cleave sialic acid moieties from glycoproteins on
the erythrocyte cell surface, leading to erythrophagocytosis and eventually, anaemia. B) All three species of pathogenic African
trypanosomes are known to release phospholipases that degrade phosphocholine-bound lipids. They are considered significant
virulence factors, and their action results in a build up of choline in the host bloodstream. C) T. brucei secretes multiple factors that
modulate macrophage ability to generate nitric oxide (NO), including TbKHC1, and soluble VSG (sVSG). The latter stimulates
arginase-1 activity, leading to increased usage of the available arginine pool to generate ornithine, reducing substrate availability
for NO production through nitric oxide synthase (NOS). Simultaneously, sVSG has an inhibitory effect on NOS. sVSG also interferes
with the phosphorylation of STAT1, an important transcription factor that drives pro-inflammatory responses. D) Parasite amino acid
metabolism and its effect on host responses has been studied to some degree in T. brucei. In particular the fate of hydroxyphe­
nylpyruvate (HPP), phenylpyruvate (PP) and indolepyruvate (IP), products of cASAT-catalysed conversions of L-tyrosine,
L-phenylalanine and L-tryptophan, respectively. IP is a potent modulator of pro-inflammatory responses in macrophages. Firstly,
IP interferes with HIF-1α, leading to a reduction in glycolytic capacity of macrophages. Secondly, IP inhibits induction of pro-IL-1,
a potent pro-inflammatory cytokine. Finally, more recent work has established that IP is a direct inhibitor of cyclooxygenase (COX),
leading to reduced prostaglandin (PG; mediators of inflammation) production. E) Trypanosome-derived IP as well as HPP can impact
upon dendritic cells, by stimulating Nrf2-mediated hemeoxygenase-1 (HO-1) induction, again leading to a reduced pro-inflammatory
response. Many other metabolic factors are known to be excreted by trypanosomes, but their molecular interactions with the host
environment remain to be established, and they are therefore not included in this overview figure.
14 L. J. MORRISON ET AL.

laboratory passage, or in parasite subspecies that have oligopeptides to activate the developmental signaling
lost the capacity for tsetse transmission, and are instead pathway [228]. Two peptidases have been found to
spread either by mechanical transmission by other bit­ provide a major contribution to the generation of the
ing flies (T. b. evansi) or by venereal transmission QS signal, Oligopeptidase B and metallocarboxypepti­
between equids (T. b. equiperdum) [224,225]. dase 1, and the individual or combined deletion of both
The molecular details that generate the quorum sen­ peptidases by gene knockout increases parasite viru­
sing signal and how this is detected and transduced to lence through reduced stumpy formation [227]. Other
effect development in the parasites have been recently parasite-derived peptidases are also likely to contribute,
unraveled (Figure 3). The signal that induces the para­ however, complementing the dominant activities of
site to undergo cell cycle arrest and stumpy formation TbOPB and TbMCP1, or pre-processing larger poly­
(classically described as an ill-defined “stumpy induc­ peptide substrates so that they can act as substrates for
tion factor”, SIF) is oligopeptides in the environment of these enzymes, which show specificity for substrates
the parasite [226]. These are generated by proteolytic limited in size [229,230]. Host peptidases in the para­
enzymes or peptidases that are released by the parasite site’s environment could also provide a signal to aug­
in the mammalian host, apparently through an uncon­ ment stumpy formation; this would not be dependent
ventional protein secretion pathway [227]. This allows upon parasite numbers directly, although immune
a density-dependent signal to be generated because as responses to the parasite population may involve pro­
parasite numbers increase, the abundance of the teolytic activities [231]. Although untested, the immune
released peptidases correspondingly increases and, response against the parasite could also contribute to
though their activity in the blood or tissues, produce promote quorum sensing if parasite-specific antibodies

Figure 3. Quorum sensing in Trypanosoma brucei. Schematic pathway for the quorum sensing signalling pathway in Trypanosoma
brucei. Slender form parasites release several peptidases into their environment, with two peptidases, Oligopeptidase B and
Metallocarboxypeptidase I being important contributors to the generation of the quorum sensing signal, oligopeptides.
Environmental oligopeptides can be transported into recipient parasites by the TbGPR89 surface transporter that is expressed on
slender cells but not stumpy forms. In an unknown mechanism, transported oligopeptides stimulate a signal transduction cascade
that promotes stumpy formation through the action of gene regulators (RNA binding proteins). Molecules that act to inhibit stumpy
formation (slender retainers) are inactivated. At least one kinase, TbDYRK, acts on both control arms, inhibiting slender retainers and
promoting stumpy formation. Other molecules, annotated as “Hypothetical proteins” in TryTrypdb (https://tritrypdb.Org/tritrypdb/
app) have been identified that control stumpy formation but their positions in the regulatory pathway are unknown.
 VIRULENCE 15

proximal to the parasite could provide substrates for stumpy forms. In particular, parasites were observed
trypanosome-released peptidases, potentially contribut­ to transition in the G1 phase of their cell cycle with
ing to the altered stumpy formation of intact versus no “intermediate form” transcriptome identified,
immunocompromised mice [232]. Importantly, the despite the description of these morphologically transi­
generation of oligopeptides by parasite-derived pepti­ tional forms [223]. Analysis of a parasite line defective
dases allows stumpy formation at high parasite num­ in quorum sensing through its deletion of a component
bers in the blood and also low parasite numbers where of the QS signaling pathway identified early transcript-
trypanosomes are constrained within tissues such as the level changes in gene expression as parasite initiate the
skin and adipose, such that local accumulation of their developmental QS response [243], providing a route to
activities and products can occur [226,227]. This pinpoint the molecular commitment events that define
resolves the perceived conundrum that stumpy forma­ the initiation of the decision to progress toward stumpy
tion in rodents involves large parasite numbers, formation.
whereas in livestock hosts the circulating parasite popu­ The generation of stumpy forms is a unique innova­
lation might be relatively low but stumpy forms are tion to T. brucei with limited evidence for morphologi­
prevalent [233]. cal development in either T. congolense or T. vivax.
The presence of oligopeptidases activates Nonetheless, both these species exhibit density-
a developmental signaling response. The signal is trans­ dependent growth arrest, accumulating as G1-arrested
ported by a surface molecule, TbGPR89, specific to forms at higher parastiaemias [244,245]. In
slender cells as the signal-receiving population. T. congolense, gene expression changes that accompany
Interestingly, not all oligopeptides operate equally this arrest have been analyzed, which predicts changes
effectively, with tripeptides being more active than in the expression of some surface proteins [246]. The
dipeptides, and with particular amino acid combina­ genomes of both T. congolense and T. vivax also encode
tions being more effective than others [226] - suggest­ orthologues of many of the regulators of quorum sen­
ing a specificity code. The quorum sensing signaling sing identified in T. brucei, and at least one of these
pathway has many components, originally identified via (TcIL3000.0.19510) can complement a T. brucei null
a genome-wide RNAi screen designed to isolate para­ mutant for TbHYP2 (Tb927.9.4080) to restore stumpy
sites unresponsive to a cell permeable mimic of the formation, demonstrating functional equivalence [245].
quorum sensing signal [234]. These molecules include Thus, despite the absence of morphological develop­
protein phosphatases and protein kinases as well as ment, it appears both T. congolense and T. vivax exhibit
RNA binding proteins acting as predicted gene regula­ quorum sensing to regulate their virulence in mamma­
tors and, more recently, a long non-coding RNA reg­ lian hosts and, potentially, as an adaptation for tsetse
ulator [235]. Several hypothetical proteins of unknown uptake.
function are also implicated [236]. In combination,
these components drive stumpy formation, although
Secreted factors and EVs
an analysis of their respective dependency relationships
indicated that the signal transduction pathway was not The importance of released peptidases and their role in
a simple linear hierarchy [237]. Perhaps more than one generating the signal that promotes the development
signal input contributes to ensure appropriate activa­ transition to stumpy forms has reemphasized that try­
tion of the developmental response, or perhaps there is panosomes are not passive entities in their hosts but
regulatory input or feedback from other molecular instead behave interactively to support their survival
components, including those not yet uncovered? and transmission and, potentially, to contribute to
Molecular inhibitors of stumpy formation have also virulence.
been identified [238–241], as has at least one molecule Several studies have analyzed the secretome of
that seems to act on both stimulatory and inhibitory bloodstream form trypanosomes. Secreted proteases,
arms of the process [242]. This reflects the complexity and T. brucei Cathepsin-L (TbCatL) in particular,
and stringent regulation of quorum sensing, which is have been implicated mediating extravasation of
necessary because stumpy formation represents T. brucei cells via perturbation of intracellular calcium
a terminal developmental step unless the parasites are levels in brain endothelial cells [247], and secreted
transmitted to tsetse. TbCatL has also been shown to induce spontaneous
The gene expression response to the quorum sensing depolarization events in isolated cardiomyocytes, also
signal has been analyzed by single-cell RNA sequencing via perturbation of intracellular calcium levels [248],
[243]. This identified the trajectory of the transition in which may contribute to cardiac pathology observed
terms of gene expression from the slender to the in human and animal infections. Secreted peptidases
16 L. J. MORRISON ET AL.

have been proposed to be involved in trypanosome peptidases from each of these classes being character­
pathogenicity by hydrolyzing host hormone peptides, ized in T. brucei.
hence affecting their functions [249250250, . Other Exosomes are a subset of extracellular vesicles and
studies have also described peptidases in the blood are formed from the budding of the late endosomes.
of infected mice and rodents where they remained They are cup-shaped, approximately 20–100 nm in dia­
catalytically active [251–253]. Various studies have meter [259], and are secreted through fusion with the
looked at these secreted or released peptidases and plasma membrane. In a variety of pathogens, exosomal
their substrates both in vitro and in vivo. For example, secretion has been proposed to be involved in cell-to-
oligopeptidase B (OPB), which cleaves Arg/Lys con­ cell communication, cell-to-host communications, and
taining peptides smaller than 30 amino acid residues, has also been implicated in pathogenicity and cell dif­
is released into the bloodstream of rats infected with ferentiation [260–262]. Protein release through exo­
trypanosomes. This cleaves available host hormones somes has been described in trypanosomes when the
such as atrial natriuretic factor resulting in hormonal secretome of the parasite was analyzed, revealing the
deregulation linked with trypanosome infections release of leaderless proteins as well as exosome asso­
[249,253]. Similarly, type 1 pyroglutamyl peptidase ciated proteins such as Rab proteins, clathrin heavy
(PGP) and prolyl oligopeptidase (POP) are also chain, enolase, and heat-shock protein 70 [254].
released into the blood of rats during trypanosome Extracellular vesicular secretion has also been reported
infections and remain catalytically active. PGP cleaves in T. cruzi [259], and in Leishmania it was recently
host demonstrated that genes encoding drug resistance can
gonadotropin-releasing hormone (GnRH) and thyro­ be passed between Leishmania cells via exosomes
tropin-releasing hormone (TRH) by removing the [263] – highlighting the potential importance of this
N-terminal pyroglutamic acid-blocking groups [252] phenomenon for multiple phenotypes. Molecules
while POP, which hydrolyses Pro/Ala containing sub­ involved in this type of secretion include the compo­
strates at the carboxyl end, hydrolyses type 1 collagen nents of the ESCRT complex (Endosomal Sorting
and native collagen [251]. POP has also been demon­ Complex Required for Transport) [262,264,265].
strated to hydrolyze substance P, oxytocin, vasopres­
sin, and angiotensin in vitro [251]. In all, the
Parasite-parasite interactions and coinfections
expression of peptidases, their secretion/release into
the host bloodstream and their activity in the host The extracellular release of factors into their environ­
plasma have been reported in African trypanosomes, ment open the possibility for coinfecting trypanosome
with interest in these molecules enhanced with the strains and species to interact with one another through
recent discovery of their involvement in the genera­ collaboration or competition, either directly or indir­
tion of the quorum-sensing signal for stumpy differ­ ectly. Different trypanosomes species co-circulate in
entiation [227,228]. sub-Saharan Africa, with transmission by the same
Numerous studies have looked at the secretome of vector species [266]. As a consequence, coinfections
the different species of trypanosomes, including in the have been frequently reported both in livestock infec­
different life cycle forms [160,161,254,255–258]. These tion and in trapped tsetse flies [267–269]. In several
studies have identified proteins that are involved in studies, the coinfection between different strains and
different functions, including folding and degradation, species have been found to alter the infection dynamics
nucleotide, carbohydrate and amino acid metabolism, and/or virulence in the host [57]. This was exemplified
protein synthesis, and transport [258]. These proteins in concomitant infections of livestock with virulent and
may interact with the host’s immune system, and by so less virulent strains of T congolense in livestock, where
doing, contribute to immunopathology and also the the presence of a less virulent strain suppressed the
survival of the parasites. This was demonstrated by pathology associated with a more virulent strain [270].
Garzon et al. [257], where they found a reduction in This was not linked to shared antigens or immune cross
the secretion of host immune molecules and an impair­ reactivity between the parasites in the infection, since
ment in the maturation of lipopolysaccharide (LPS)- removing one of the strains with trypanocides before
induced dendritic cells in the presence of inoculating the second strain eliminated the response.
T. b. gambiense secretome. As highlighted above, Competitive suppression has also been observed
among the identified secreted proteins in previous stu­ between strains of T. brucei in rodent infections, with
dies were peptidases. Different classes of peptidases are a more virulent strain being suppressed by a less viru­
expressed in trypanosome parasites; serine, cysteine, lent strain, enabling extended host survival and reduced
metallopeptidases, threonine, and aspartyl with pathology [271]. These phenomena are reminiscent of
 VIRULENCE 17

the interactions between closely related Theileria spe­ Future perspectives: identification of gaps,
cies in livestock, where the impact of the more patho­ priorities, and opportunities
genic Theileria parva was ameliorated by infection of
In this article, we have aimed to summarize current
the less virulent Theileria mutans, to the extent that
knowledge with respect to parasite factors that influ­
infection with the less virulent species was proposed as
ence the severity of clinical signs and disease outcome
a potential anti-virulence approach to disease control,
in the mammalian host. The complexity of disease
potentially more valuable than anti-parasitic drugs
caused by trypanosomes, with the involvement of mul­
[272]. In many cases, however, the coinfection of dif­
tiple host and multiple trypanosome species, means
ferent parasite species generates significantly worse out­
that this is inevitably an ambitious undertaking, and
comes [273,274].
covers multiple aspects of trypanosome infection biol­
Interactions between strains in coinfection are also
ogy. However, the advances in the last decades by many
observed with respect to quorum-sensing signals. As
researchers mean that we now have an impressively
highlighted earlier, T. congolense does not generate
detailed understanding of trypanosome factors that
morphologically stumpy forms but the parasite exhi­ influence host-parasite interactions. This is particularly
bits arrest in G1 in a density-dependent manner true for T. brucei, and for infections in mice, where the
[245]. To explore whether the parasites could detect understanding is particularly advanced – the detailed
the QS signals, mice with T. congolense infections knowledge we now have of the differentiation process
were superinfected with T. brucei and the ability of from long slender to stumpy life cycle stages being
the latter to generate stumpy forms compared with a prime example. In recent years, efforts in T. brucei
mice infected with T. brucei alone. The T. brucei in have also begun to significantly shift toward working
the coinfection were found to accelerate their stumpy with pleomorphic, differentiation-competent strains
formation reflective of the overall parasite load, (e.g. T. brucei EATRO 1125 AnTat 1.1) and away
rather than the contribution of T. brucei to the infec­ from the heavily laboratory-adapted monomorphic
tion alone. Furthermore, the response of the parasites Lister 427, a move that will provide data more relevant
was dependent on their intact QS signaling pathway to field infections. However, the science reviewed in
since null mutants for a component of the signaling this article highlights that we have identified relatively
path proliferated in the coinfection setting regardless few virulence factors responsible for even well charac­
of the existing T. congolense parasites [245]. Overall, terized phenotypes (see Table 1), and has also high­
this demonstrated that T. brucei responds to the lighted areas where knowledge in general is much less
presence of T. congolense and that this is mediated developed. These gaps in understanding represent
via a T. congolense derived QS signal, although opportunities for the trypanosome research community
whether there was a signal from T. brucei to going forward, and in the following section we aim to
T. congolense could not be explored in the absence outline research needs and opportunities.
of suitable molecular markers for transmission stages While the ability to work with T. brucei advances at
in the latter species. a spectacular pace, with examples such as precise and
These experiments established that different try­ scalable gene silencing and editing techniques that facil­
panosome strains and species can detect and itate high throughput phenotypic screens and highly
respond to one another in a coinfection and that detailed functional analysis [234,279–281], the rate of
this can alter their potential for virulence and trans­ progress in terms of advancement in knowledge, data,
mission. Such interactions in the field have the and capabilities lags significantly behind for
potential to select for evolutionary strategies that T. congolense and T. vivax. Partly, this is due to the
provide an advantage in a coinfection scenario for sheer difference in scale in terms of amount of research
parasites that reduce their sensitivity to the QS sig­ investment into these pathogens, as evidenced by the
nal, or occupy niches where they are less susceptible stark disparity in the number of research outputs on
to manipulation by a coinfecting strain [218]. As T. congolense and T. vivax compared to T. brucei over
discussed elsewhere, there are also potential impli­ the past decades [282]. However, there has been
cations for therapeutic strategies [275] or with spe­ a refocusing on T. congolense in recent years that has
cies-specific vaccination [109], since this has the seen the generation of foundational datasets and cap­
potential to perturb a coinfection equilibrium to abilities [40,44,72,133,283–285], and combined with
allow the emergence of parasites less sensitive to renewed interest from funders in AT, this is contribut­
QS signals and so more likely to be virulent when ing to a revival of research on this organism. Although
in a monoinfection setting. not to the same extent, there are also tools and
18 L. J. MORRISON ET AL.

Table 1. Virulence phenotypes and state of current knowledge.

Phenotype Mechanistic details Virulence factor References
Human infectivity Evasion of trypanosome cell lysis by preventing SRA (T. b. rhodesiense) [149],[276,277
lytic activity of human Apolipoprotein-1. TgsGP, TbHpHbR, KIFC1, V-ATPase-a, -c, -F & -H, [29,146,151]
V-ATPase assembly factors,Tb9297.10.12940,
Tb927.9.8000 (T. b. gambiense Group 1)
Unknown gene, probably located in
bloodstream expression site (T. b. gambiense
Group 2)
Tissue damage Modulation of NO production via influencing TbKHC1 [38]
arginase expression, leading to liver injury and
early mortality. (T. brucei; mice)
Strain-specific organomegaly, linked to arginase Locus on T. brucei chromosome 3 – gene [363737,
expression and alternative macrophage unknown
activation. (T. brucei, mice)
Cardiomyocyte depolarization and cardiac TbCatL [248278278,
arrhythmias via parasite-induced perturbation
of host cell Ca2+ signaling. (T. brucei; rats)
Hypergammaglobulinemia B-cell mitogenesis in response to virulence Proline racemase [200]
factors released by the parasite. (T. vivax)
Anaemia Desialylation of erythrocytes leading to Trans-sialidases [204–206]
erythrophagocytosis. (T. vivax & T. congolense)
Antigenic variation Evasion of host antigen-specific antibody VSG [42–45,99]
response by sequential switching of identity of
the expressed Variant Surface Glycoprotein.
(T. brucei, T. congolense, T. vivax; all
mammalian hosts)
Host B cell destruction Ablation of splenic B cells, Including memory Unknown [115–118,122]
B cells, leading to loss of adaptive immune
memory, including to non-trypanosome
antigens. (T. brucei, T. congolense, T. vivax;
mice, probably cattle & humans)
Trypanosome T. brucei – Genes identified in a genome wide Tb927.4.3620/30/40 (PP1) [142,234,237]
differentiation RNAi screen for resistance to chemical Tb927.10.5930/40/50 (NEK17) [238,239,241]
activators of the stumpy formation pathway Tb927.2.2720 (MEK) [235]
in vitro, cAMP or AMP. The listed genes have Tb927.10.15020 (DyrK) [226]
been confirmed to be involved in stumpy Tb927.11.6600 (Hyp 1) [227]
formation in vivo (mice). Tb927.9.4080 (Hyp2)
Negative regulator of stumpy formation Tb927.10.12090 (RBP7A)
Negative regulator of stumpy formation Tb927.10.12100 (RBP7B)
Negative regulator of stumpy formation Tb927.2.4020 (Nedd8 activating enzyme)
Long non coding RNA regulator of stumpy Tb927.3.4560 (AMPKa)
formation positioned between Tb927.10.12090 TbTOR4
and Tb927.10.12100. MAPK5
Oligopeptide transporter for the quorum sensing ZFK
signal SnoGRUMPY
Peptidases contributing to the generation of the TbGPR89
quorum sensing signal TbOPB, TbMCP1
T. congolense – functional orthologue of TcIL3000.0.19510 [246]
Tb927.9.4080 (45% identity, 58% similarity).
T. vivax – not known
Skin colonisation Invasion (& metabolic adaptation?) and Unknown [128,129]
establishment of skin resident populations.
(T. brucei; mice & humans)
Adipose tissue Invasion & metabolic adaptation to adipose Unknown [130]
colonisation tissue. (T. brucei; mice)
Endothelial cell adherence Strain-specific cerebral pathology linked to Unknown [133]
& sequestration differential sequestration in brain capillaries. TbCatL [202,247]
(T. congolense; mice)
Parasite-induced migration across endothelial
barriers by perturbation of host cell Ca2+
signaling. (T. brucei)
Metabolic suppression of Indolepyruvate impacting upon glycolytic Indole pyruvate (derived from cASAT; [163]
macrophage activity/ capacity through HIF-1α interference and Tb927.10.3660; predicted syntenic
response inhibition of pro-IL-1β. orthologues: TcIL3000_10_2990,
Inhibition of cyclooxygenase and downstream TvY486_1003700
prostaglandin production via indolepyruvate.
(T. brucei)
Metabolic modulation of Activation of Nrf2 and HO-1 induction impacting Indole pyruvate & hydroxyphenylpyruvate [163]
dendritic cell response upon glycolytic capacity, leading to reduced (derived from cASAT; Tb927.10.3660;
production of pro-inflammatory cytokines and predicted syntenic orthologues:
CD4+ T cell activation. (T. brucei) TcIL3000_10_2990, TvY486_1003700)
 VIRULENCE 19

resources available for T. vivax that enable work on this to translate to the bovine model – whether for reasons
pathogen that was not previously possible [45,88,94], of host size and scale, physiology, bovine-specific
which is already leading to notable, paradigm- aspects of the immune response, or to demonstrate
challenging studies [109]. However, there are key potential clinical relevance. Example phenotypes
basic capabilities where improvement would be trans­ where the interest in this translation to the cow are
formative. The ability to culture bloodstream form obvious include antigenic variation (where modelling
T. congolense and T. vivax in vitro is still limited, with has indicated host size is likely to play a substantive role
a very small number of strains for T. congolense having in VSG-population dynamics [286]), parasite-mediated
been successfully adapted to in vitro culture, and only B cell memory loss [115], cell adherence and tissue-
one for T. vivax – with a requirement in T. vivax for specific sequestration [133], coinfection and interaction
mammalian feeder cells for mid- to long-term culture. between trypanosome species [245], and the validation
Development of media formulations that supported of vaccine candidates [109]. However, this is to name
axenic growth of multiple strains for both species but a few phenotypes where analysis in the bovine host
would be a substantial breakthrough that would accel­ would prove informative; it is abundantly clear that
erate meaningful functional studies, and provide the many aspects of our understanding of trypanosome
ability to dissect the genetic diversity present in both infection biology would greatly benefit from assessment
species. Additionally, while genetic modification is in the bovine or other clinically relevant host species.
clearly possible in both species [94,283], in T. vivax Similar to increased research on the clinically rele­
this is currently restricted to the epimastigote life vant hosts, there is equally a lot of value to be gained
cycle stage, and requires differentiation through meta­ from more assessment of the extent to which laboratory
cyclics to bloodstream forms to obtain the relevant findings are recapitulated in the field. Likewise, findings
mammalian life cycle stage. The ability to directly in the field also have the obvious potential to stimulate
genetically modify T. vivax bloodstream form cells meaningful laboratory and experimental work, and
would be a major step forward, albeit this may depend there could perhaps be better integration of laboratory
on the prior development of an appropriate in vitro and field approaches across many aspects of trypano­
culturing medium. It is clear that the multiple funda­ some biology. The tissue distribution of trypanosomes
mental differences between these three species mean in the skin and adipose, and the findings that have
that if such basic capabilities are improved, there are resulted from the original studies describing these phe­
many opportunities for identifying novel and important nomena [129,130], is a prime example of the mutually
aspects of trypanosome cell and infection biology. beneficial advances that can be gained across basic and
As is evident from many of the advances outlined in applied approaches from combining field and labora­
this article, the mouse model has been and remains tory experimental work. But as noted above, there are
hugely influential and useful in providing key insights multiple aspects of trypanosome biology where integra­
into the infection biology of African trypanosomes. The tion of field approaches with laboratory approaches
mouse model provides experimental tractability and would enhance our understanding – for example, the
scalability that can make it an incredibly powerful increasing interest in coinfections, where experimental
experimental tool. However, ultimately for most traits approaches provide the ability to control for multiple
there is a need or desire to assess translation to the confounders and gain mechanistic insights, but prop­
clinically relevant host model. It should be stressed that erly designed field approaches enable the deconvolution
this is not always simply a matter of assessing clinical of the interaction of trypanosomes (or mechanism)
relevance, but that it can also be because analyzing with host, pathogen, and environmental factors.
a trait in such hosts provides genuine scientific insight There also remain some fundamental gaps in our
and interest. While this is clearly a challenge with the field knowledge of trypanosome virulence. For exam­
human disease in terms of both ethical reasons and the ple, almost all T. congolense studies (laboratory or field)
small and reducing number of clinical cases (although focus on T. congolense Savannah. We know astonish­
the extravascular skin populations of T. brucei are ingly little about the other subtypes of T. congolense,
a recent example of laboratory observations proving Forest and Kilifi, other than that they are detected in
important and useful in HAT [128,129]), for AT there cattle and wildlife across sub-Saharan Africa, a very few
is substantial scope for analysis in clinically relevant assessments of relative virulence of selected strains in
hosts such as cattle. Several facilities are now available cattle and mice, and the generation of a limited amount
that enable experimental infection of cattle, and there of genome data (for Forest). But the extent of the
are multiple phenotypes observed either in vitro or in disease caused in cattle and other livestock attributed
the mouse model that it would be of significant interest to these subtypes, and the intricacies of their
20 L. J. MORRISON ET AL.

epidemiology (e.g. tsetse transmission, interaction with ORCID

other trypanosome species and other subtypes of Liam J. Morrison http://orcid.org/0000-0002-8304-9066
T. congolense) are questions that are ripe for answer­ Pieter C. Steketee http://orcid.org/0000-0003-3677-5898
ing – similar questions pertain to several of the under­ Mabel D. Tettey http://orcid.org/0000-0001-6051-2735
studied trypanosomes, such as T. simiae, T. suis and Keith R. Matthews http://orcid.org/0000-0003-0309-9184
T. vivax-like. This lack of knowledge feeds into some of
the capability challenges outlined above (e.g. limited
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