A Cell-surface Phylome for African Trypanosomes
Andrew P. Jackson1,2*, Harriet C. Allison3, J. David Barry4, Mark C. Field3, Christiane Hertz-Fowler5,
1Pathogen Genomics Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, England, United Kingdom, 2Department of
Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool, England, United Kingdom, 3Department of Pathology, University of
Cambridge, Cambridge, England, United Kingdom, 4Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, Scotland, United Kingdom,
5Centre for Genomic Research, Institute of Integrative Biology, Biosciences Building, University of Liverpool, Liverpool, England, United Kingdom
The cell surface of Trypanosoma brucei, like many protistan blood parasites, is crucial for mediating host-parasite
interactions and is instrumental to the initiation, maintenance and severity of infection. Previous comparisons with the
related trypanosomatid parasites T. cruzi and Leishmania major suggest that the cell-surface proteome of T. brucei is largely
taxon-specific. Here we compare genes predicted to encode cell surface proteins of T. brucei with those from two related
African trypanosomes, T. congolense and T. vivax. We created a cell surface phylome (CSP) by estimating phylogenies for 79
gene families with putative surface functions to understand the more recent evolution of African trypanosome surface
architecture. Our findings demonstrate that the transferrin receptor genes essential for bloodstream survival in T. brucei are
conserved in T. congolense but absent from T. vivax and include an expanded gene family of insect stage-specific surface
glycoproteins that includes many currently uncharacterized genes. We also identify species-specific features and
innovations and confirm that these include most expression site-associated genes (ESAGs) in T. brucei, which are absent
from T. congolense and T. vivax. The CSP presents the first global picture of the origins and dynamics of cell surface
architecture in African trypanosomes, representing the principal differences in genomic repertoire between African
trypanosome species and provides a basis from which to explore the developmental and pathological differences in surface
architectures. All data can be accessed at: http://www.genedb.org/Page/trypanosoma_surface_phylome.
Citation: Jackson AP, Allison HC, Barry JD, Field MC, Hertz-Fowler C, et al. (2013) A Cell-surface Phylome for African Trypanosomes. PLoS Negl Trop Dis 7(3):
Editor: Christian Tschudi, Yale School of Public Health, United States of America
Received September 4, 2012; Accepted February 4, 2013; Published March 21, 2013
Copyright: ? 2013 Jackson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the Wellcome Trust Grants WT 085775/Z/08/Z, 055558/Z/98/A, and 055558/Z/98/C. The Wellcome Trust Centre for Molecular
Parasitology is supported by core funding from the Wellcome Trust (Grant 085349/Z/08/Z). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
African trypanosomes (Trypanosoma spp. section Salivaria) are
unicellular hemoparasites of vertebrates. They are transmitted by
Tsetse flies (Glossina spp.) and cause endemic disease throughout
sub-Saharan Africa. African trypanosomes include T. brucei which
causes Human African Trypanosomiasis (‘sleeping sickness’) and,
along with two related species T. congolense and T. vivax, a similar
disease in domestic and wild animals (‘nagana’). Although the
incidence of human disease has recently declined , there
remains an estimated 30,000 cases per year ; while total losses
in agricultural productivity due to animal disease across Tsetse-
infested Africa are estimated to be US$4.75 billion per annum .
The combined effects of African trypanosomes on humans and
livestock are a significant threat to public and veterinary health,
and wider socio-economic development .
The first genomic comparisons between T. brucei and related
trypansomatid parasites, T. cruzi and Leishmania major, which cause
Chagas disease and leishmaniasis in humans respectively, showed
that most genes are widespread and arranged into regions of
conserved synteny [5–7]. By contrast, it was also apparent that the
gene families likely encoding cell surfaces molecules were non-
homologous and largely lineage-specific [8–9]. In the vertebrate
host, the T. brucei surface is dominated by the Variant Surface
Glycoprotein (VSG); serial replacement of VSG (i.e. antigenic
variation) is a means of immune evasion and results in chronic
infection . African trypanosome genomes contain large VSG
gene families [11–12], but mono-allelic expression of a single gene
is ensured because transcription is restricted to telomeric VSG
expression sites (ES) [13–15]. Several other Expression Site-
Associated Genes (ESAG1-12; [16–18]) are located in the ES and
are co-transcribed with the active VSG [19–20]; all but ESAG8 are
predicted or known to be cell surface-expressed . T. cruzi and
L. major also possess multi-copy surface glycoprotein families (i.e.
mucins and amastins respectively) but these are unrelated to VSG
[8–9]. Indeed, Leishmania promastigotes have a largely non-
proteinaceous, lipophosphoglycan-based surface coat .
Hence, while T. brucei, T. cruzi and L. major have physiological
similarities associated with shared ancestry, the cell-surface
architectures are highly divergent, reflecting the evolution of
specific mechanisms for immune evasion and survival by each
parasite . A principal objective of comparative genomics is to
identify taxon-specific features that may plausibly explain such
phenotypic differences. Despite their similarities T. brucei, T. cruzi
and L. major diverged long ago; so surface features that appear
exclusive when their genomes are compared are not necessarily
species-specific, or diagnostic of the diseases they cause. In
particular, it remains to be determined if the T. brucei-specific
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surface features identified from these initial comparisons are truly
species- or disease-specific, or general features of all African
trypanosomes. Comparisons between more closely related species
are essential to resolving this issue.
We recently reported the draft genomesequences forT. congolense,
the closest known relative of T. brucei, and T. vivax, a more distantly
related species, and described the evolution of VSG genes in African
trypanosomes [12–23]. All species cause chronic animal trypano-
somiasis characterized by recurrent parasitaemia and antigenic
variation, but subtle differences are present in their pathology, life
cycle and host range. For example, T. vivax can cause hyperacute
hemorrhagic disease in cattle typically with much higher mortality
than otherspecies. Inthe Tsetse,T.bruceiand T.congolenseinfect
the midgut but then migrate to the salivary glands and proboscis
respectively prior to transmission to the vertebrate. In contrast, T.
vivax avoids the insect midgut, a feature that seems to facilitate
wholly mechanical transmission and its colonization of Tsetse-free
areas . Further, all three species infect a wide range of domestic
animals but only T. brucei has evolved human infectivity, probably
on at least two occasions in east (T. b. rhodesiense) and west Africa (T.
b. gambiense) respectively .
Cell surface-expressed gene families encode abundant proteins
at the forefront of host-parasite interactions [8–9,22,26–27]. The
major surface protease (MSP, or gp63) has multiple isoforms, one
of which (MSP-B) is responsible for cell-surface remodelling prior
to transmission into the vector [28–29]. Papain-type cysteine
peptidase B and C (also known as cathepsin-L and -B) are strongly
associated with virulence phenotypes, degrading host proteins [30–
31] and facilitating parasite transversal of the blood-brain barrier
. Other gene families encode diverse cell surface receptors, e.g.
adenylate cyclases , and membrane transporters that are
essential for normal cell physiology, e.g. transferrin receptors
(TFR) . Hence, the cell surface is an intuitive place to begin
exploring species differences and here we present phylogenetic
analyses of all gene families with predicted cell-surface roles in
African trypanosomes. Although we do not include low-copy
number features or non-protein cell-surface components, which
may be equally important in function, our detailed analysis of the
principal cell-surface gene families presents a global picture of
evolutionary change on the trypanosome cell-surface.
The African trypanosome cell surface phylome is a collection of
phylogenies for gene families with predicted cell surface expres-
sion. The approach is summarized in Figure S1. Phylogenies were
estimated from sequence data accessed through the GeneDB
portal  and extracted from four genome sequences: Trypano-
soma brucei TREU927 , T. congolense IL3000 and T. vivax Y486
 and, to provide an outgroup in phylogenetic comparisons, T.
cruzi CL Brener . Genome sequencing and annotation methods
have been described previously [6,12].
Sequence clustering and cluster refinement
All T. brucei genes with cell surface motifs, (i.e. a predicted signal
peptide, a predicted GPI anchor or a trans-membrane helix) were
extracted from the T. brucei 927 genome sequence. Genes annotated
as ‘unlikely’ or with fewer than 100 codons were removed. Homologs
to each T. brucei ‘surface’ gene were identified among all T. brucei, T.
congolense, T. vivax and T. cruzi predicted genes using wuBLAST .
Where at least four homologs occurred in at least one species, this
constituted a ‘family’ amenable to phylogenetic analysis. Surface-
expressed genes with fewer than four homologs are recorded as
singleton, paired and triplet sequences in tables available from the
CSP webpage. After removing genes already identified as homolo-
gous to T. brucei genes (i.e. widespread gene families), the BLAST
exercise was repeated for T. congolense and T. vivax genes to identify
cases absent in T. brucei. Signal peptides were predicted using SignalP
, GPI anchors were predicted using Fraganchor  and trans-
membrane helices were predicted using TMHMM . 205 ‘surface
expressed’ families were reduced to 79 by removing cases of poor
alignment (i.e. sequences that could not be aligned by eye), of mis-
annotation (i.e. non-coding sequence), of redundancy (i.e. technical
duplicates arising from alleles in the T. congolense genome that were
separately assembled), of genes with known expression in mitochon-
drial, lysosomal or other internal membranes, and by combining
families with overlapping homology. Surface-expressed families may
have been omitted because they possess signal peptides,GPI anchors,
or trans-membrane helices that cannot be reliably recognized by
current methods, or because their 59 or 39 regions are mis-specified.
Equally, spurious recognition of these domains in hypothetical
proteins (mostly T. vivax families) cannot be excluded. Each family is
given a ‘Fam’ number (0–81) as described in Table S1; note that for
historical reasons, there is no Fam48 or 68.
Evidence for transcription
Given that most species-specific genes are putative and encode
hypothetical proteins, evidence in support of their coding status
was gathered from three sources: i) transcriptomic studies of T.
brucei ; ii) Expressed Sequence Tags (EST) in multiple life
stages of T. congolense ; and iii) partial RNAseq data for
bloodstream form T. vivax  mapped against the T. vivax
genome using SMALT .
Multiple sequence alignment
Translated nucleotide sequences for each family were aligned in
ClustalW ; all multiple alignments were then manually edited
in BioEdit 7.1.3. . In most cases, the amino acid sequence
alignment was used in phylogenetic analysis to reduce homoplasy,
but nucleotide sequences were examined in cases of low sequence
divergence. The rates of synonymous (ks) and non-synonymous
The African trypanosome (Trypanosoma brucei) is a single-
celled, vector-borne parasite that causes Human African
Trypanosomiasis (or ‘sleeping sickness’) throughout sub-
Saharan Africa and, along with related species T. con-
golense and T. vivax, a similar disease in wild and domestic
animals. Together, the African trypanosomes have signif-
icant effects on human and animal health and associated
costs for socio-economic development in Africa. Genes
expressed on the trypanosome cell surface are instrumen-
tal in causing disease and sustaining infection by resisting
the host immune system. Here we compare repertoires of
genes with predicted cell-surface expression in T. brucei, T.
congolense and T. vivax and estimate the phylogeny of
each predicted cell-surface gene family. This ‘cell-surface
phylome’ (CSP) provides a detailed analysis of species-
specific gene families and of gene gain and loss in shared
families, aiding the identification of surface proteins that
may mediate specific aspects of pathogenesis and disease
progression. Overall, the CSP suggests that each trypano-
some species has modified its surface proteome uniquely,
indicating that T. brucei, T. congolense and T. vivax have
subtly distinct mechanisms for interacting with both
vertebrate and insect hosts.
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substitutions (ka) per site were calculated for each alignment using
KaKs Calculator 2.0  to estimate within-family sequence
Bayesian phylogenies were estimated using MrBayes v3.2.1 
freq=500 and default prior distribution. Nucleotide and amino
acid sequence alignments were analyzed using GTR+C and
WAG+C models respectively. Maximum likelihood phylogenies
were estimated using PHYML v3.0  under an LG+C model
 for amino acid sequences or a GTR+C model for nucleotide
sequences. Node support was assessed using 100 non-parametric
bootstrap replicates in addition to Bayesian posterior probabilities.
Trees were rooted using T. cruzi sequences, or otherwise mid-point
rooted. VSG phylogenies were estimated using alignments of
selected, full-length sequences representative of global diversity
under different conditions, as described previously .
The CSP contains phylogenies of gene families drawn from
multiple species. We caninfer historical gene duplications and losses
from comparison of gene family phylogenies with the overlying
species evolution [49–50]. For each gene family, a fully binary,
rooted gene tree was integrated across the species tree (i.e. [T. brucei,
T. congolense], T. vivax], T. cruzi]) using NOTUNG 2.6 . A
parameter r, was calculated from the ratio of speciation duplica-
tions (i.e. nodes supporting orthologs in daughter species) to
unilateral duplications (i.e. nodes supporting in-paralogs in the
same species), adjusted for gene family size. r reflects the degree of
gene family turnover (combined incidence of gene gain and loss);
high values of r indicate a phylogeny with minimal turnover, in
which most lineages are represented by orthologs in all species. Low
values indicate a phylogeny with high turnover, in which ancestral
genes are frequently lost and replaced by novel duplicates, resulting
in clades of species-specific in-paralogs and minimal orthology.
Relative rate analysis
Significant differences in evolutionary rate between two lineages
were examined using relative rates tests (RRTs; ). Nucleotide
sequence alignments combining a given lineage, its sister taxon
and an out-group (as described in Tables 1 and 2) were created
and evaluated with MEGA v5.05 . Where a test lineage
consisted of multiple paralogous genes, the average rate difference
between all comparisons is reported.
Phylogenetic incompatibility describes the presence of multiple
phylogenetic signals within a single sequence alignment and is the
historical signature of recombination. The Pair-wise Homoplasy
Index (PHI) detects incompatibility between sites and is robust in
the presence of rate heterogeneity , which might otherwise
simulate the effects of recombination. P,0.05 for PHI indicates
significant incompatibility between sites within an alignment,
consistent with recombination. For each ESAG family, the index
was calculated using PhiPack  for separate alignments of
ESAGs sensu stricto and of homologous sequences from non-ES loci
(unless these are largely absent, i.e. ESAG6/7, 8 and 12).
Analysis of gene expression
To determine mRNA expression levels for a single Fam50
family member (Tb927.7.380), quantitative real-time polymerase
chain reaction (qRT-PCR) was carried out on total RNA extracted
using RNeasy Mini Kit (QIAGEN). cDNA was generated using
SuperScript II reverse transcriptase according to the manufactur-
er’s instructions. qRT-PCR was carried out using three different
isolated mRNA samples from four life-cycle stages (in vitro cultured
bloodstream-stage and procyclic forms; in vivo cultured short
stumpy bloodstream-stage; and in vivo cultured T. brucei blood-
stream-stage). T. brucei Rab11 was used as a control to determine
relative quantities of mRNA. The relative abundance of specific
RNA was subsequently determined.
Transfection and protein localization
Fam1 (i.e. Tb927.6.1310) and Fam50 (i.e. Tb927.7.380) genes
were synthesized by Eurogentec. Tb927.6.1310 is the most
divergent of all Fam1 gene copies, so it was selected for the
benefit of targeting a single copy gene in localization experiments.
Tb927.7.380 is also one of five tandem copies, and was selected
because it was expressed to the greatest level for all paralogs in
qPCR analyses. T. brucei single marker bloodstream line cells were
cultured in HMI-9 medium as described previously . Ectopic
expression of haemagglutinin (HA) epitope-tagged Tb927.6.1310/
Tb927.7.380 at the N-terminus (following the predicted signal
peptide sequence) was carried out using pXS5/pDEX-577 
constitutive and inducible expression vectors respectively. For
Western blotting, proteins were transferred onto Immobilon
(polyvinyildene fluoride) membranes and incubated with primary
mouse anti-HA antibody (1:8,000) and subsequently with second-
ary rabbit anti-mouse peroxidase conjugate antibody (1:10,000,
Sigma). Immunofluorescence microscopy was carried out on
permeabilised and non-permeabilised transfected cells harvested at
We estimated phylogenies using Maximum Likelihood and
Bayesian methods for 79 gene families in African trypanosomes
with known or predicted cell-surface location. This cell-surface
phylome describes how these families have diversified during the
evolution of African trypanosomes, and also identifies species-
specific gene families, many of which remain uncharacterized.
Taken together, the CSP shows that the cell-surface architecture
has subsequently experienced subtle changes in individual lineages,
suggesting the adaptation of their common inheritance. The CSP is
described in a Venn diagram (Figure 1) and in Table S1.
Throughout, gene families are referred to by their ‘Fam’ number
(0–81; see methods). All sequence alignments, Hidden Markov
Models (HMMs) and phylogenetic trees can be accessed at: http://
Phylogenetic diversity in conserved cell surface-
expressed gene families
The conserved elements of the CSP, at the centre of Figure 1,
generally contain cell-surface features that have been well
described, including most known principal parasite effectors (i.e.
MSPs, cathepsins and trans-sialidases) [26–27]. By contrast, genes
at the periphery of Figure 1 are species-specific and mostly
uncharacterized, even when they have given names in T. brucei;
only 8/45 species-specific families (Fam0, 2, 3, 8, 12, 14–16) are
characterized to some extent (e.g. by cellular localization) and
function is only well known for two (VSG and ESAG6/7).
Naturally, many trypanosome cell-surface proteins perform basic
functions that are constrained by selection, resulting in small
species differences (e.g. Fam54-56, 59-60, 62-65, 69-76 and
78-81). However, a widespread family is not necessarily un-
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changed, and the phylogenies of several conserved families
involved in host-parasite interaction indicate surface proteome
differences between species that could have functional implica-
In T. vivax, whole lineages have been lost, and on multiple
occasions; for example among trans-sialidase genes (see Fam47
CSP page), there are no T. vivax orthologs to basal-branching
Tb927.2.5280, which are otherwise widespread. Similarly, there
are only three Major Facilitator Superfamily (MFS) transporters
loci in T. vivax compared with six in T. brucei (see Fam58 CSP
page), and no orthologs to the Proteins Associated with
Differentiation (PAD) genes, one of which encodes a carboxylate
transporter implicated in differentiation from vertebrate to insect
life stages in T. brucei (i.e. Tb927.7.5930; ). Such within-family
losses may coincide with the expansion of the remaining lineages.
For instance MSP-B (Fam46) is present in T. brucei, T. congolense and
the outgroup T. cruzi, but is absent from T. vivax; (a result
confirmed by searching T. vivax unassembled reads for reciprocal
BLASTx matches to MSP-B). This coincides with the evolution of
11 MSP-C genes in T. vivax, a gene that is single-copy in all other
species (see Fam46 CSP page, and Table S1).
The surface functional repertoire also diverges through gene
gain, for example among Fam61 genes (nucleoside/nucleobase
transporters), required to scavenge host purines and are function-
ally differentiated with respect to both parasite life stage and
substrate [58–61]. The Fam61 phylogeny shows that multiple gene
duplications have occurred in both T. brucei and T. congolense (see
Fam61 CSP page). However, while T. brucei has elaborated its
nucleoside transporter lineage, producing four species-specific loci
from a single-copy ancestral locus (probably Tb09.160.5480), T.
congolense instead diversified its nucleobase transporter lineage, with
18 gene copies compared with three in T. brucei and five in T. vivax.
This is not simply a difference in gene dosage, or an artifact of
sequence assembly, since seven of these T. congolense-specific
transporters (e.g. TcIL3000.0.12740) have a highly derived
predicted protein sequence, lacking ,130 amino acids from the
39 end and displaying only 39% amino acid identity with the T.
congolense chromosome 11 isoform (54% similarity), and which itself
displays 54% identity and 66% similarity with its T. brucei ortholog.
Therefore, these genes are predicted to encode proteins with signal
peptides and eight trans-membrane helices, but lack the canonical
C-terminus of the conserved nucleobase transporter including its
The combined effect of gene gains and losses, i.e. gene family
turnover, is reflected in the topology of phylogenies. Typically,
gene families predate contemporary genomes, and orthologs in
each species of each ancestral gene form a clade in the phylogeny.
Examples of this familiar pattern in trypanosomes naturally
include structural or metabolic gene families displaying little
innovation [62–64], as well as some CSP families including Fam56
(ABC transporters) and Fam65 (aldehyde dehydrogenase), al-
though the majority of these genes are likely intracellular. Many
cell surface-expressed gene families similarly originate prior to
contemporary species, but their tree topologies indicate greater
post-speciation innovation. To investigate the extent to which
species derive novel genes post-speciation, we calculated r for each
family, the ratio of orthology (DIV) to paralogy (DUP), corrected
for gene family size, and where DIV is the incidence of gene
divergence through speciation and DUP is the incidence of gene
duplication, inferred through phylogenetic reconciliation (Table
S1). Families like Fam56 (r=0.67) and Fam65 (r=0.73) possess
high r values, indicating that most loci are retained in all species;
for example, across 22 ABC transporter loci there are no unilateral
gene duplications and only 7 gene losses (2 in T. brucei/T. congolense,
1 in T. congolense and 4 in T. vivax). While these losses probably
have functionally consequence, Fam56 and similar examples have
a relatively constant gene complement.
Conversely, many familiar cell surface components have
r,0.05, indicating that gene copies cluster more by species than
by locus, i.e. recent paralogy rather than ancient orthology. Fam54
(amino acid transporters; r=0.006), Fam58 (MFS transporters;
r=0.018) and Fam61 (nucleoside transporters; r=0.01) all
display low r values due to T. brucei-specific expansions (see
individual CSP pages), which occurs against a general background
of conservation. This cannot be said for phylogenies for other
families, e.g. Fam46 (r=0.013), Fam49 (r=0.002), Fam50
(r=0.003), Fam67 (r=0.003), and Fam77 (r=0.002), which
Table 1. Examples of significant substitution rate asymmetry inferred by relative rates tests.
Relative rates test:
In-group 1In-group 2Out-group
46T. vivax-specific MSP-C genesTvY486_0023730 T. congolense orthologTcIL3000.10.2050TcCLB_505931.205 4.160.044
58T. brucei-specific MFS transporter
Tb927.7.5950T. congolense sister clade TcIL3000.7.5000Tb927.8.16508 7.11 0.045
61 T. congolense-specific nucleobase
TcIL3000.0.59630Conserved chr11 locus TcIL3000.11.3580Tb11.02.11056107.79 0.00001
61T. brucei-specific subtelomeric
nucleotide transporter genes
Tb09.v4.0106 Conserved chr9 locusTb09.160.5480TcIL3000.9.250049.590.022
67T. congolense-specific cysteine
peptidase C genes*
TcIL3000.0.48140T. brucei sister clade Tb927.6.560TvY486_060006078.11 0.0054
72T. congolense tandem gene
copies of a hypothetical protein
TcIL3000.8.6610Positional homologs in T.
75T. congolense tandem gene copies
of a hypothetical protein
TcIL3000.0.05220Positional homologs in T.
Note: results are averaged across multiple comparisons of paralogous genes (n).
*Previously described  and divided into functionally distinct variants ‘CBs’ and ‘CBc’; this significant result relates only to ‘CBs’ genes. ‘CBc’ genes returned a non-
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Table 2. Taxonomic distribution and sequence properties of ESAG gene families in African trypanosomes.
Relative rates teste:
Note: ESAG family size is given for non-ES linked (i.e. core or subtelomeric) copies.
aPlus and tilde symbols indicate the presence of orthologs and homologs respectively in T. brucei (Tb), T. congolense IL3000 (Tco), T. vivax Y486 (Tv) and T. cruzi CL Brener (Tc).
bWhile the closest relatives of ESAG2 are in T. congolense, orthologs cannot be precisely defined among the T. congolense VSG repertoire.
cReciprocal monophyly is confirmed where mutually exclusive clades of ES-linked and non-ES gene copies occur.
dAverage pair-wise sequence divergence estimated from Bayesian phylogenies using RAxML 7.0.4. .
eEstimated using MEGA v5  where appropriate out-groups are present; the number of unique differences per lineage given is given in brackets.
fEstimated using PhiPack  for separate alignments of ESAGs and related, non-ES gene copies (where non-ES copies are present).
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consist of species-specific clades of highly similar, tandem
duplicates, at one or a few conserved loci. Fam47 (trans-sialidase;
r=0.025) and Fam51 (adenylate cyclase; r=0.002) provide
examples intermediate between the first and second patterns,
with T. brucei and T. congolense possessing orthologs to conserved
loci, while all T. vivax genes are monophyletic and hence lack
orthology with other species.
Gene family diversification is a product of both gene duplication
and sequence divergence , so even where gene repertoire is
constant, significant asymmetry in nucleotide substitution rates
between ancestral and duplicated lineages may indicate that
important functional change has occurred in either lineage.
Previously, we have identified frequent rate asymmetry following
gene duplication of amino acid transporters (Fam54) in T. brucei
. Further examples are evident in the CSP. For instance,
Figure 1. The taxonomic distribution of gene families in the cell-surface phylome, displayed in a Venn diagram. Phylogenies for all
families are available through GeneDB. Each circle represents a family (i.e. .3 gene copies). The label in each circle refers to the description key, while
size reflects the number of genes it contains; for large families the absolute number is shown in parentheses. For families present in multiple species,
a pie chart is shown indicating relative gene numbers. The three tabs attending each species domain show the number of single-copy genes, pairs
and triplets also predicted to have cell surface roles and to be species-specific (e.g. 101 singletons in T. brucei).
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branch lengths among cysteine peptidase B (Fam67) genes in T.
congolense (average genetic distance=0.092, n=16) are significantly
longer than in T. brucei (0.0037, n=11, p,0.0001; t-test) or T.
vivax (0.016, n=6, p,0.0001). T. congolense cysteine peptidase B
includes structural variants with distinct catalytic functionality
, which is clearly absent from T. brucei. Table 1 records this
and other cases of rate asymmetry involving species-specific
expansions, further details of which are provided in each CSP
The transferrin receptor (TFR) gene family evolved in the
ancestor of T. brucei and T. congolense
A TFR is expressed in bloodstream form T. brucei and is
required for iron uptake . It is not homologous with its
mammalian counterpart, and they function quite differently .
The trypanosome TFR is a GPI-anchored heterodimer encoded
by paralogous gene families ESAG6 and 7 (Fam15; [68–70]).
ESAG7 is 57 amino acids shorter than ESAG6 and encodes a
protein without a GPI-anchor signal, but otherwise the genes are
very closely related . When present, ESAG6 and 7 are found in
tandem immediately downstream of the ES promoter. Outside of
the ES, genes homologous to ESAG6/7 in T. brucei 927 consist of a
single ESAG6/7 tandem pair (Tb927.7.3250/3260) at a strand-
switch region on chromosome 7, probably representing a
secondary transposition from an ES, and the Procyclin-Associated
Genes (PAG1, 2, 4 and 5; Fam14), which are adjacent to the
procyclin loci . ESAG6 and 7 and the PAGs are homologous to
the a-type VSGs (a-VSG; [69,71]), leading to the suggestion that the
TFR derives from VSG [26,73].
The T. congolense genome contains 45 genes (in Fam15) that are
homologous to ESAG6/7, plus 31 genes (in Fam14) whose closest
sequence match is to PAGs in T. brucei. We refer to both Fam14
and Fam15 as TFR-like genes. Figure 2 describes the phylogeny
for TFR-like genes and shows that the T. congolense genes are
paraphyletic, that is, there are two clades (Fam14 and 15) each
more closely related to sequences in T. brucei (PAG and ESAG6/7
respectively) than to each other. Given the homology between a-
VSG and TFR genes, this shows that Fam14 and 15 are not a-VSG
(of which T. congolense has none; ) because they are much closer
to T. brucei TFR than a-VSG. The T. vivax genome contains a-VSG-
like genes but these have an equally distant relationship to both
ESAG6/7 and a-VSG in T. brucei, and significantly are not part of
the TFR gene family of T. brucei and T. congolense . Therefore,
genes that now encode TFR proteins, and others associated with
procyclin expression sites in T. brucei, likely evolved before the
speciation of T. brucei and T. congolense, but after the separation
from T. vivax.
The essential difference between TFR genes in T. brucei and T.
congolense is genomic distribution. While ESAG6/7 are almost
exclusively found in ESs, T. congolense orthologs are distributed
widely among subtelomeres and not usually close to telomeres.
Nevertheless, phylogenetic and sequence comparisons suggest that
TFR function is conserved in T. congolense. First, like ESAG6/7,
Fam15 genes in T. congolense split into two equal-sized sister clades,
encoding proteins that differ in the prediction of a GPI anchor
(Figure 2). Second, just as ESAG6 and 7 are typically arranged in
GPI+/GPI2 tandem pairs in T. brucei, 28/45 of T. congolense genes
are also arranged in tandem pairs at subtelomeric loci, each pair
combining representatives from the GPI+ and GPI2 clades.
Finally, amino acid positions within the transferrin binding
domain  are conserved in all ESAG6/7, PAG and their T.
congolense orthologs (see Fam15 CSP page). These results suggest
that an orthologous TFR is present in T. brucei and T. congolense but
not T. vivax.
An expanded insect stage-specific surface glycoprotein
gene family conserved across African trypanosomes
In addition to procyclin and VSG, T. brucei and T. congolense
possess a third, highly abundant major surface glycoprotein
expressed during the insect stage. These are known as Brucei
Alanine-Rich Protein (BARP; ) and Glutamine Alanine-Rich
Protein (GARP; [75–78]) respectively. Although GARP was
initially thought analogous to procyclin in T. brucei , a
procyclin ortholog was subsequently identified in T. congolense 
and the CSP confirms a widespread procyclin family (Fam12).
Structural affinities between GARP, which is expressed most
strongly in epimastigotes , and BARP, which is epimastigote-
specific , have been demonstrated , and the CSP confirms
these two gene families as sister taxa (see Figure 3A). T. vivax
contains 15 genes encoding BARP/GARP-like proteins that form
three distinct subfamilies; each subfamily encodes proteins with
distinctive repetitive domains towards the N-terminus that are
absent in other species. Unfortunately, poor assembly in these
regions prevents us from discerning their genomic organization,
but at least some are arranged in tandem as in T. brucei and T.
BARP, GARP and their T. vivax homologs are part of a larger
gene family (Fam50) in the CSP. We identified conserved
sequence regions that unite these familiar families with other
insect stage-specific genes and several hypothetical or unchar-
acterized genes (Figure 3B). The region at positions 262–274
contains a ubiquitous cysteine residue, followed four positions
downstream by a VTxxSL motif in BARP and GARP, which is
present with slight variations in all family members. A single-copy
locus on chromosome 11 (i.e. Tb11.02.2370/TcIL3000.11.4860;
marked ‘i’) is the sister clade to BARP/GARP and may be present
in T. vivax also (TvY486_11149440). The Congolense Epimastigote-
Specific Protein (CESP) gene family is expressed in T. congolense
epimastigotes only, where it may have a role in adhesion to host
surfaces . Figure 3A shows that CESP has a sister clade in T.
brucei comprising a tandem gene array on chromosome 8 (marked
‘ii); notably, these genes may be preferentially expressed in insect
salivary glands , i.e. the location of T. brucei epimastigotes. An
TvY486_0016400), although the position of this gene is not robust.
In addition to GARP and CESP, the CSP identified two related
subfamilies encoding hypothetical proteins, (marked ‘iii’ and ‘iv’),
which comprise subtelomeric tandem gene arrays. Analysis of
stage-defined T. congolense EST (Figure 3C; ) proteomic
analysis  found that subfamily ‘iii’ is preferentially expressed
in T. congolense procyclic stage (see Figure 3C). Accordingly, the
single-copy ortholog to subfamily ‘iii’ in T. brucei (Tb927.5.4020) is
also preferentially expressed in procyclic cells based on transcrip-
tome data [40,84]; and a recent qRT-PCR analysis identified
transcripts corresponding to Tb927.5.4020 in the insect midgut,
although protein expression was not examined . Subfamily ‘iv’
comprises sequences on chromosome 7 in T. brucei (i.e.
Tb927.7.360) and a single-copy ortholog in T. congolense (i.e.
TcIL3000.0.02370). In transcriptomic studies of T. brucei, expres-
sion data for these genes was weak and inconclusive .
However, qRT-PCR in various insect tissues suggests significant
up-regulation of Tb927.7.360 (and paralogs) in the insect salivary
gland and in metacyclic trypomastigotes . Quantitative
proteomic analysis in T. congolense indicated 13-fold higher
expression of TcIL3000.0.02370 in epimastigotes over procyclics
. Hence, it seems likely that subfamily ‘iv’ genes are expressed
during the insect-to-vertebrate transition.
To localize expression of a single gene copy of subfamily ‘iv’ in
T. brucei, Tb927.7.380 was haemagglutinin (HA) epitope-tagged at
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the N-terminus (following the predicted signal peptide sequence)
and expressed ectopically using a pDEX-577 inducible-expression
vector. Protein expression was confirmed by Western blot
(Figure 4A), and immuno-fluorescence microscopy indicates that
Tb927.7.380 protein co-localizes with paraflagellar rod protein 2,
consistent with specific expression at, or close to, the flagellar
membrane (Figure 4B).
Expression site-associated gene families (ESAGs) evolved
uniquely in T. brucei
ESAGs have homologs outside of T. brucei [85–86], but these
may only represent distant relationships within widely conserved
protein families. With complete genome sequences for T. congolense
and T. vivax we can now examine evidence for true orthology and
therefore, the possibility that ESAG phylogenetic lineages predate
T. brucei (Table 2). Orthologous lineages of TFR genes (i.e. ESAG6/
7) are present in T. congolense and we have previously argued that
ESAG2 belongs to a widespread lineage most closely related to b-
type VSG in T. congolense . Altogether, we find evidence that the
T. congolense and T. vivax genomes contain homologous sequences
to 9 of 12 ESAG families, while ESAG9 may have homologs in T.
cruzi  (Table 2). Two trends emerge from phylogenies for each
ESAG family shown in their individual CSP pages. First, ESAGs
from multiple T. brucei strains are monophyletic and therefore,
have a single origin; and second, with the exception of ESAG6/7,
the sister clades to ESAGs are not orthologs in other species but
chromosomal-internal genes in T. brucei. We interpret this as
evidence for origins post-speciation, i.e. ESAGs are T. brucei-
specific. Examining these closest relatives outside of the expression
sites provides some indication of the origins of ESAGs, as
demonstrated by Fam51, i.e. ESAG4 and the adenylate cyclases.
Trans-membrane adenylate cyclases are conserved across
Trypanosomatids [88–89], and comprise a large gene family with
diverse roles in T. brucei [85,90–91]. ESAG4 is one lineage
expressed specifically in the bloodstream stage, and instrumental
in inhibiting host innate immunity . The T. congolense and T.
vivax genome sequences include 34 and 24 adenylate cyclase genes
respectively. The adenylate cyclase phylogeny (Figure 5) shows
that T. brucei and T. congolense lineages are paraphyletic, and in 10
cases T. brucei genes have orthologs in T. congolense that are
positionally conserved. However, there are no orthologs of ESAG4
among T. congolense homologs. Indeed, the most closely related
gene to ESAG4 is Tb11.01.8820, located at the subtelomeric
boundary of chromosome 11. This gene has an ortholog in T.
congolense (TcIL3000.11.16970), which is syntenic. Relative rates
tests show that the substitution rate of ESAG4 has accelerated
significantly compared with Tb11.01.8820 (p,0.0001; Table 2).
Comparison of Tb11.01.8820 and ESAG4 sequences (Figure S2)
shows that this remodelling has primarily affected the intracellular
domains. 245 amino acid differences are distributed preferentially
towards the C-terminal, with 69% occurring after the putative
trans-membrane helix (a portion accounting for only 35% of total
characters). Furthermore, of 54 sites where Tb11.01.8820 and
TcIL3000.11.16970 are conserved, but ESAG4 is derived (i.e.
unambiguous ESAG4 apomorphies), 41 occur in the intracellular
domain. While the adenylate cyclase catalytic domain is intracel-
lular, the evolution of ESAG4 has not altered the 8 residues
identified as important for catalytic function . Hence, ESAG4
represents a T. brucei-specific expansion of adenylate cyclase genes,
most likely initiated through the transposition of a conserved locus
to the ES, and coinciding with derivation of the protein structures
associated with signal transduction within the cell but not catalysis.
The detail presented for other ESAGs in their CSP pages
suggests that, like ESAG4 and Tb11.01.8820, ESAGs themselves
are T. brucei-specific but descended from conserved genes, typically
members of multi-copy families with subtelomeric distributions in
several species. For example, ESAG2 and ESAG6/7 were, as
previously noted, derived from VSG [12,68]. ESAG3- and ESAG5-
like loci are on T. vivax contigs containing telomeric repeats
(GenBank accessions HE578915 and HE578917), but not VSG.
ESAG8, although not surface-expressed, is most closely related to
two leucine-rich repeat protein (LRRP) genes (i.e. Tb927.1.3670
and Tb927.3.580), that are chromosome-internal and include
nuclear localization signal and RING motifs, which are diagnostic
of ESAG8 . While these two genes are T. brucei-specific, they
are more closely related to conserved LRRP genes, suggesting that
they may be progenitors of ESAG8. Finally, on the Fam3 CSP
page, a structural comparison of ESAG11 and Invariant Surface
Glycoprotein (ISG) sequences indicates that ESAG11 is homolo-
gous to ISG and so perhaps a highly modified derivative of these
widespread surface proteins . As Table 2 shows, only ESAG1
and ESAG12 appear to have no homology beyond T. brucei,
suggesting that they have evolved de novo within the ES.
Species-specific genes include a family derived from b-
type VSG and expressed in the flagellar pocket of T.
Besides ESAGs, the CSP contains various species-specific genes
(Table S1). T. brucei-specific gene families include Fam4-7
encoding hypothetical proteins with predicted signal peptides but
no similarity to known proteins. Fam4-7 genes are all adjacent to
strand-switch regions and typically arrayed in tandem; transcrip-
tomic studies suggest that they are expressed preferentially or
solely in bloodstream forms [40,84,95]. The VSG-related (VR)
genes previously identified in T. brucei  are also specific to T.
brucei, although similar in structure to canonical VSG in T. congolense
. Finally, the CSP contains another family of VSG-like genes
unique to T. brucei: Fam1.
In T. brucei 927, Fam1 comprises a polymorphic tandem array
of 5 copies (0.1–3.2% nucleotide sequence divergence) at a strand-
switch region on chromosome 6 (Figure 6A). Comparison with T.
b. gambiense 972 indicates that Fam1 copy number may differ
between strains because only a single gene (corresponding to the
divergent 59-most copy, Tb927.6.1310, Figure 6B) is present. The
gene encodes a 347 amino acid protein with a predicted signal
peptide and GPI-anchor. Fam1 genes are homologous to b-type
VSG, but lack the typical C-terminal domain of canonical VSGs
. qRT-PCR analysis indicated that Tb927.6.1310 is predom-
inantly expressed in bloodstream stages . Enrichment of
Tb927.6.1310 transcripts has been observed in metacyclic forms in
Figure 2. Bayesian phylogeny of transferrin receptor-like genes in T. brucei and T. congolense (Fam14/15). The phylogeny was estimated
from an amino acid sequence alignment of 342 characters including all ESAG6-like proteins from T. brucei 927 (light blue) and T. congolense IL3000
(green), as well as ESAG6/7 sensu stricto from T. brucei 927, 427 and T. b. gambiense 972 (dark blue). A mixed amino acid substitution strategy was
applied with default settings using MrBayes v3.2.1. The phylogeny is rooted using an outgroup of two a-type VSG protein sequences from T. brucei.
Bayesian posterior probability/non-parametric bootstrap values are provided for selected nodes. Terminal nodes that describe sequences derived
from tandem pairs in T. congolense are labeled with common symbols. Terminal nodes representing Procyclin-Associated Genes (PAG1, 2, 4 and 5) are
numbered. Throughout, the presence or absence of predicted GPI anchor signals is noted using + and 2 respectively.
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Figure 3. Bayesian phylogeny and expression of BARP/GARP-like genes (Fam50). A. The phylogram was estimated from a multiple protein
sequence alignment of 307 characters. The tree is mid-point rooted. Selected nodes are supported by posterior probability values and non-
parametric bootstraps generated from a maximum likelihood analysis using an LG model with rate heterogeneity. B. Cartoon of sequence
conservation across the Fam50 protein sequence alignment, darker shading reflects conservation. Two conserved regions are expanded to show
sequence motifs in WebLogo v2.8.2. format ; ubiquitous residues are shaded red. C. Histograms showing the number of T. congolense EST
corresponding to each of four clades in A, (CESP, GARP, subfamily ‘iii’ and subfamily ‘iv’), recovered from four life stages: procyclic (PC), metacyclic
(MC), epimastigote (EPM) and bloodstream form (BSF); data from .
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the insect salivary gland , but this remains to be verified at the
We expressed the gene product of Tb927.6.1310 using a
constitutive expression system (pXS5), and tagged at the N-
terminus of the mature protein with an HA-9 epitope
(Figure 6C). The HA epitope was placed two residues
downstream of the predicted N-terminus of the mature protein
following signal sequence processing. By Western analysis a
single band was detected migrating at ,45 kDa in whole cell
lysates. However, the predicted molecular weight of the protein
is ,39 kDa, suggesting glycosylation at either or both predicted
N-glycosylation sites. Cells were stained with a monoclonal
antibody against HA and counterstained with FITC-concanav-
alin A. At 4uC, the fusion protein clearly colocalized with conA,
conditions which block endocytosis and so retain the lectin
exclusively within the flagellar pocket, a subdomain of the
plasma membrane, and therefore demonstrating access to the
cell surface. When cells were permeabilised with detergent, it
was clear that Tb927.6.1310 protein was also present in
additional internal compartments, and based on partial overlap
of conA at 12uC (which retains conA in the flagellar pocket and
early endosomes) and HA signals, these structures likely
correspond to early and/or recycling endosomes. Hence, we
conclude that the Tb927.6.1310 gene product is present at the
parasite surface and may be restricted to the flagellar pocket,
which is frequently observed for low abundance GPI-anchored
proteins in this organism, and Tb927.6.1310 is also present
within the endosomal apparatus.
In T. congolense, Fam22 is the most abundant species-specific
gene family with .100 copies. Fam22 genes are distributed
throughout putative subtelomeric regions and are typically situated
immediately downstream of VSG. T. congolense VSG 39UTR’s are
too short, (often only 15–30 bp; ) for Fam22 to fall within
these regions. qRT-PCR analysis identified Fam22 sequences in
all life stages except bloodstream forms (J. Donelson, unpublished
data), but it is unclear whether Fam22 is a novel family of coding
sequences or a non-coding, regulatory sequence. Nevertheless,
Fam22 sequences are highly abundant. Trypanosoma vivax has
substantially more species-specific gene families (19; Fam27-45)
than either other species, which may be expected given that T.
vivax is the natural outgroup to T. brucei and T. congolense. None
have any significant similarity with known protein structures and
more transcriptomic and proteomic surveys will be required to
confirm that these sequence families genuinely encode T. vivax-
specific proteins. However, many of these putative gene families
are abundant (e.g. Fam31 and Fam34 have 38 and 34 members
Figure 4. Expression of a Fam50 gene (Tb927.7.380) in T. brucei. A. Western blot; 26107cells were sampled from either induced (+) or
uninduced (2) cells after 1-day induction with 1 mg/mL tetracycline. Markers shown in kDa. B. Immunofluorescence analysis of Tb927.7.380
expression in T. brucei, showing co-staining of non-permeabilised cells with plasma membrane protein VSG 221 (top) and PFR2 (bottom). Left: Merged
images with color combination for DAPI-stain of the nucleus and kinetoplast (blue), fluorescent stain of HA epitope tag (red) and VSG/PFR
fluorescence (green). Right: merged pictures from phase and fluorescence. Inset: co-staining of non-permeabilised and permeabilised cells with Rab11
(intracellular marker). No fluorescence was seen from non-transfected cells (data not shown). GFP expressed from pDEX-577 vector localised to the
cytoplasm (data not shown). Scale bar is 2 mm.
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respectively) and transcripts corresponding to several gene families
are among bloodstream-form RNA-seq data (Fam29-32, 34-35,
38-39; see Table S1).
The ancestor of T. brucei, T. congolense and T. vivax was very likely
a hemoparasite of vertebrates, spread by Tsetse flies, and likewise
fully exposed to the host immune response during its period in the
mammalian host. Most familiar cell-surface features – both
physiological regulators such as membrane transporters and
disease effectors such as MSP and cathepsin – were already
present in the ancestor. This is intuitive given that these features
are typically present in T. cruzi. However, the CSP shows that the
peculiar nature of the T. brucei cell surface, dominated by VSG
, BARP/GARP-like genes and procyclin (Fam12) during
Figure 5. Bayesian phylogeny of adenylate cyclase genes from African trypanosomes (Fam51). The phylogeny was estimated from an
amino acid sequence alignment of 1239 characters including all adenylate cyclase proteins from T. brucei 927, T. congolense IL3000 and T. vivax Y486,
as well as ESAG4 sensu stricto from T. brucei 927, 427 and T. b. gambiense 972. A mixed amino acid substitution strategy was applied with default
settings using MrBayes v3.2.1. The phylogeny is rooted using an outgroup of selected T. cruzi homologs that represent total diversity. Bayesian
posterior probability/non-parametric bootstrap values are provided for selected nodes. Black arrows denote the positions of previously named
‘GRESAG4’ sequences, as well as an ESAG4-like cDNA from T. congolense (Z67964; ).
Figure 6. Phylogeny and expression of a T. brucei-specific, VSG-like hypothetical protein (Fam1). A. Fam1 consists of five, non-identical
tandem gene copies at a strand-switch region on chromosome 5, which is unique to T. brucei. B. A Bayesian phylogram estimated from a multiple
nucleotide sequence alignment of 1068 characters. The tree is midpoint-rooted. Nodes are supported by posterior probability values and non-
parametric bootstraps generated from a maximum likelihood analysis using a GTR+G model. C. Immunofluorescence analysis of Tb927.6.1310
expression in T. brucei. Tb927.6.1310 was N-terminally HA epitope-tagged and expressed in bloodstream-form cells. Cells expressing
HA::Tb927.6.1310 were loaded with FITC-concanavalin-A in serum-free media and incubated at either 4uC (upper panel) or 12uC (lower panel).
ConA is restricted to the flagellar pocket at the lower temperature, whereas it is transported to, and trapped within, Rab5A positive early endosomes
at 12uC. Columns in each panel (from left to right); fluorescent stain of HA epitope tag (red); FITC-ConA fluorescence (green); merged images for
fluorescence; merged images from phase and fluorescence. DAPI-stain of the nucleus and kinetoplast is shown in blue. HA::Tb927.6.1310 co-localizes
with ConA at the flagellar pocket (indicated with arrow head) and early endosomes (indicated with a whole arrow).
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various life-stages, also appears to have originated in the ancestral
The role of the TFR on the ancestral cell-surface is more
debatable. ESAG6/7 are thought to have evolved from a-VSG
variant antigens [26,73] but we show that the sister clade to
ESAG6/7 are T. congolense Fam15 genes, which do not encode any
known variant antigens . Rather than originating from a-VSG
in T. brucei, phylogenetic analysis of all VSG-like sequences [see
Fam0 CSP pages] indicates that TFR-like sequences evolved from
an a-VSG-like gene, (and further differentiated into ESAG6- and
PAG-like genes), in the T. brucei/T. congolense ancestor, after
separation from the lineage leading to T. vivax. While there are no
TFR-like sequences in T. vivax, this does not preclude an analogous
transferrin receptor in this species, since there is a large and
structurally diverse a-VSG-like family (Fam23 ), the functional
diversity of which is unknown. In short, we predict that Fam15
genes in T. congolense also encode a heterodimeric transferrin
receptor, orthologous to the T. brucei TFR.
However, if the T. brucei/T. congolense ancestor possessed an
orthologous heterodimeric TFR comprising GPI+ and GPI2
monomers, we would expect GPI+ genes from T. brucei and T.
congolense to be sister taxa reflecting their ancestry, and likewise for
GPI2. Yet a literal interpretation of Figure 2 suggests separate
expansions of Fam15 genes in each species, and thus independent
origins of GPI+/2 isoforms. Furthermore, branches separating
ESAG6 and 7 (average genetic distance (p)=0.114, n=21) are
much shorter than distances among the T. congolense genes
(p=0.604, n=49), implying a recent origin for ESAG7 from
ESAG6 through the deletion of its C-terminus. We consider this to
reflect rapid turnover post-speciation of TFR-like genes that
evolved in the ancestor, rather than independent origins, which is
less parsimonious. Indeed, the same pattern of reciprocal
monophyly between species is seen in other phylogenies (e.g.
VSG, Fam50, Fam67), but it is clearly unparsimonious to suggest
recent origins for these widely conserved families. Gene turnover
replaces ancestral-type genes with more derived types post-
speciation resulting in concerted evolution, a process exacerbated
by recombination among tandem gene duplicates , and
causing any signature of orthology to be ‘overwritten’ . Such
processes are known to affect ESAG6/7 routinely [20,99] and
frequent transposition of Fam15 genes between T. congolense
subtelomeres is also apparent (data not shown). Given that this
molecular evolution introduces phylogenetic artefacts, the Fam15
phylogeny need not refute the most parsimonious hypothesis that a
TFR protein originated in the T. brucei/T. congolense ancestor.
While the essential character of the cell surface was established
in the ancestral trypanosome, this common inheritance has been
adapted subsequently. The evolution of ESAGs in T. brucei,
uniquely linked to the telomeric VSG expression site, is a principal
example of species-specific genomic adaptation. In some cases we
can identify the likely origin of ESAG lineages among chromo-
some-internal loci; ESAGs 3, 4, 5 and 10 are derived from
conserved loci that can be located precisely [85–86,100]. ESAGs 2
and 6/7 are derived from variant antigen genes that evolved in the
T. brucei/T. congolense ancestor . ESAGs 8, 9 and 11 have more
remote homology to conserved subtelomeric gene families, i.e.
LRRP , MASP  and ISG (see Fam3 CSP page)
respectively. This suggests a scenario in which genes with existing
subtelomeric distributions (except ESAG10) and cell-surface roles
(except ESAG8) were progressively compartmentalized into an
independently-promoted telomeric locus, perhaps to provide a
more precise regulatory environment.
Like the origin of Fam1 in T. brucei, the evolution of the ES
demonstrates how novel cell-surface genes are repeatedly derived
from existing major surface glycoproteins, whose abundance seems
to provide a reservoir of raw material for neofunctionalization.
Although ESAG functions are obscure, ESAG phylogenies suggest
that they are distinct from those of conserved genes from which
ESAGs evolved and indispensable on an evolutionary timescale.
ESAGs from different T. brucei strains are monophyletic (except
ESAG3), indicating no frequent transposition of sequences between
ES and non-ES loci. ESAG-related genes at chromosome-internal
loci are not observed in the ES and do not recombine with ESAGs,
despite very frequent recombination among ES and non-ES copies
respectively [20,99,102]. So although previous work has reported
that ESAGs are not essential in the short term [101–102], the
association between ESAG sequences sensu stricto and the telomeric
ES has been preserved by selection over the long term, suggesting
that ESAG and ESAG-like functions are distinct and non-
The CSP emphasizes dramatic cases of gene gain such as
ESAGs in T. brucei, but significant phenotypic differences, such as
life cycle variation, could be due to relatively subtle differences
in conserved gene families such as Fam50. Given that BARP,
GARP and CESP are preferentially expressed in the epimastigote
stage [74,79,81] and that transcriptome data for both T.
congolense and T. brucei indicate that subfamilies ‘iii’ and ‘iv’ are
associated with insect mid-gut and salivary gland stages
respectively , we suggest that Fam50 ranks alongside
procyclin and VSG as a major surface glycoprotein, specifically
related to the insect-to-vertebrate transition in multiple species.
This is especially interesting because of the developmental
variation among African trypanosomes during this transition.
Unlike T. brucei and T. congolense, T. vivax remains within the
insect mouthparts after feeding; this could reflect the basal-
branching position of T. vivax in the species phylogeny (i.e. T.
vivax is plesiomorphic and never evolved a mid-gut stage) or
secondary loss (i.e. a mid-gut stage is the ancestral state). T. vivax
also has a relatively small Fam50 repertoire, lacking orthologs to
three clades: BARP/GARP and subfamilies ‘iii’ and ‘iv’. These
genes might have evolved in the T. brucei/T. congolense ancestor if
T. vivax is plesiomorphic, in which case all T. vivax genes should
branch towards the root. Yet two of five Fam50 lineages in T.
vivax, (i.e. TvY486_0016400 and TvY486_1114940), are nested
among the would-be T. brucei/T. congolense gains. Reconciliation
of this topology with the species tree indicates that if
functionality is absent in T. vivax, this is due to secondary loss,
rather than T. brucei/T. congolense gain.
Having systematically analyzed protein coding sequences for
species differences, it is particularly important to remember that
the cell-surface architecture comprises much more than the
proteins encoded by the genes in the CSP and that non-
proteinaceous elements, not least the surrounding glycocalyx
composed of the carbohydrate moieties attached to membrane
glycoproteins and glycolipids, might be equally important in
determining phenotypic variation. Experimental studies of the cell-
surface demonstrate that non-protein glycoconjugates could play
an equal role in regulating host-parasite interactions, for example,
a protease-resistant surface molecule (PRS) is known to dominate
the surface of procyclic-stage T. conglolense . T. brucei expresses
various glycoconjugates on their surfaces that only become
apparent in null mutants that cannot express the major surface
glycoprotein [103–104]. Even considering the protein component,
low abundance genes not considered in the CSP may still perform
a vital role; for example, the haptoglobin-hemoglobin receptor
(Tb927.6.440; ) responsible for resistance to trypanolytic
factor by T. brucei is single-copy.
African Trypanosome Cell-surface Phylome
PLOS Neglected Tropical Diseases | www.plosntds.org14March 2013 | Volume 7 | Issue 3 | e2121
The essential character of genes expressed on African trypano-
somes cell-surfaces was largely established in the common
ancestor. Subsequently, prominent families have experienced
rapid turnover of phylogenetic diversity, indicating both functional
dynamism and redundancy. As we distinguish the functions of
family members, we should be mindful of where orthology is
absent and where it is retained; the latter, for example among MSP
subtypes, cathepsin-L and B, or ESAG6-like and PAG-like TFR
genes, is a strong indication of long-term functional differentiation
and non-redundancy among paralogs. Truly species-specific genes
represent adaptations of this shared inheritance and, in T. brucei,
include almost all ESAGs as well as various GPI-anchored
glycoproteins associated with strand-switch regions (Fam4-7). We
anticipate that with improved genome assembly, species-specific
genes, perhaps analogous to ESAGs, will be revealed in T. congolense
and T. vivax also. To this extent, comparative genomics has met its
objectives and the challenge now is to define how these unique
genes and variants influence phenotypic differences in biology and
Flowchart describing how the cell-surface phylome
in ESAG4. The figure shows an amino acid sequence alignment
Distribution of unambiguous, apomorphic characters
for four ESAG4 proteins and Tb11.01.8820, the most related non-
ES homolog (at top). Identical residues are represented with a dot.
Positions conserved in ESAG4 only are shaded red. The location
of the predicted trans-membrane helix (green) and adenyly cyclase
catalytic Pfam domain (yellow) are marked on the Tb11.01.8820
sequence. ESAG4 apomorphies, i.e. characters that have changed
in ESAG4 but remained constant in Tb11.01.8820 and its
ortholog in T. congolense (TcIL3000.11.16970), are marked with
Gene families comprising the cell surface phylome.
We thank our colleagues in the sequencing and informatics groups at the
Wellcome Trust Sanger Institute. Prof. John Donelson (University of Iowa)
provided helpful comments and preliminary results on the expression of
Fam22. The manuscript benefitted from the comments of three
Conceived and designed the experiments: APJ HCA MCF. Performed the
experiments: APJ HCA. Analyzed the data: APJ HCA. Contributed
reagents/materials/analysis tools: CHF MCF MB JDB. Wrote the paper:
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African Trypanosome Cell-surface Phylome
PLOS Neglected Tropical Diseases | www.plosntds.org 15March 2013 | Volume 7 | Issue 3 | e2121