The ATP-binding cassette proteins of the deep-branching protozoan parasite Trichomonas vaginalis.
ABSTRACT The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters.
- SourceAvailable from: cedoc.cies.edu.ni[show abstract] [hide abstract]
ABSTRACT: To update the WHO global and regional estimates of the prevalence and incidence of syphilis, gonorrhoea, chlamydia, and trichomoniasis. Prevalence estimates for syphilis, gonorrhoea, chlamydia, and trichomoniasis were generated for each of the nine UN regions for males and females between the ages of 15 and 49 in 1995 based on an extensive review of the published and unpublished medical literature since 1985. Incidence estimates were based on the prevalence figures and adjusted to take into account the estimated average duration of infection for each disease in a particular region. The latter was assumed to depend upon a number of factors including the duration of infection in the absence of treatment, the proportion of individuals who develop symptoms, the proportion of individuals treated, and the appropriateness of treatment. In 1995 there were over 333 million cases of the four major curable STDs in adults between the ages of 15 and 49--12 million cases of syphilis, 62 million cases of gonorrhoea, 89 million cases of chlamydia, and 170 million cases of trichomoniasis. Geographically, the vast majority of these cases were in the developing world reflecting the global population distribution. STDs are among the most common causes of illness in the world. Estimates of the global prevalence and incidence of these infections are limited by quantity and quality of data available from the different regions of the world. Improving global STD estimates will require more well designed epidemiological studies on the prevalence and duration of infection.Sexually Transmitted Infections 07/1998; 74 Suppl 1:S12-6. · 2.61 Impact Factor
Article: Trichomonads under Microscopy.[show abstract] [hide abstract]
ABSTRACT: Trichomonads are flagellate protists, and among them Trichomonas vaginalis and Tritrichomonas foetus are the most studied because they are parasites of the urogenital tract of humans and cattle, respectively. Microscopy provides new insights into the cell biology and morphology of these parasites, and thus allows better understanding of the main aspects of their physiology. Here, we review the ultrastructure of T. foetus and T. vaginalis, stressing the participation of the axostyle in the process of cell division and showing that the pseudocyst may be a new form in the trichomonad cell cycle and not simply a degenerative form. Other organelles, such as the Golgi and hydrogenosomes, are also reviewed. The virus present in trichomonads is discussed.Microscopy and Microanalysis 11/2004; 10(5):528-50. · 2.50 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Trichomonas vaginalis, a parasitic protozoan, is the etiologic agent of trichomoniasis, a sexually transmitted disease (STD) of worldwide importance. Trichomoniasis is the most common nonviral STD, and it is associated with many perinatal complications, male and female genitourinary tract infections, and an increased incidence of HIV transmission. Diagnosis is difficult, since the symptoms of trichomoniasis mimic those of other STDs and detection methods lack precision. Although current treatment protocols involving nitroimidazoles are curative, metronidazole resistance is on the rise, outlining the need for research into alternative antibiotics. Vaccine development has been limited by a lack of understanding of the role of the host immune response to T. vaginalis infection. The lack of a good animal model has made it difficult to conduct standardized studies in drug and vaccine development and pathogenesis. Current work on pathogenesis has focused on the host-parasite relationship, in particular the initial events required to establish infection. These studies have illustrated that the pathogenesis of T. vaginalis is indeed very complex and involves adhesion, hemolysis, and soluble factors such as cysteine proteinases and cell-detaching factor. T. vaginalis interaction with the members of the resident vaginal flora, an advanced immune evasion strategy, and certain stress responses enable the organism to survive in its changing environment. Clearly, further research and collaboration will help elucidate these pathogenic mechanisms, and with better knowledge will come improved disease control.Clinical Microbiology Reviews 05/1998; 11(2):300-17. · 17.31 Impact Factor
The ATP-Binding Cassette Proteins of the Deep-
Branching Protozoan Parasite Trichomonas vaginalis
Christopher Kay1., Katharine D. Woodward1., Karen Lawler1, Tim J. Self1, Sabrina D. Dyall1,2,
Ian D. Kerr1*
1School of Biomedical Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham, United Kingdom, 2Department of Biosciences, University
of Mauritius, Reduit, Mauritius
The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for
diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins
in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis,
gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely
among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of
some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-
frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with
confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC
Citation: Kay C, Woodward KD, Lawler K, Self TJ, Dyall SD, et al. (2012) The ATP-Binding Cassette Proteins of the Deep-Branching Protozoan Parasite Trichomonas
vaginalis. PLoS Negl Trop Dis 6(6): e1693. doi:10.1371/journal.pntd.0001693
Editor: Elodie Ghedin, University of Pittsburgh, United States of America
Received October 11, 2011; Accepted March 18, 2012; Published June 19, 2012
Copyright: ? 2012 Kay 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: CK was funded by a BBSRC Research Studentship. 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
. These authors contributed equally to this work.
Trichomonas vaginalis is a human-infective parasitic protozoan
that is the most prevalent causative agent for non-viral sexually
transmitted infections,with anestimatedannualincidenceofatleast
170 million new cases of trichomoniasis worldwide . Character-
istic of other parabasalids in the order Trichomonadida, T. vaginalis
is a flagellated, microaerophilic eukaryote which displays many
features typical of eukaryotic cells including a membrane-bound
nucleus, endoplasmic reticulum, ribosomes and the nine plus two
arrangement of microtubules . The unicellular organism
possesses five microtubule-based flagella – four located anteriorly
and one posteriorly associated with an undulating membrane, the
structure responsible for the parasite’s distinctive quivering move-
ment . An additional key morphological feature of T. vaginalis is
its axostyle, a cylindrical structure spanning the length of the cell
from an anteriorly positioned nucleus. Also composed of microtu-
bules, it is believed to play a role in anchoring the parasite to the
vaginal epithelium . In common with many unicellular
eukaryotes with apparently early evolutionary divergence, T.
vaginalis has a reduced complement of organelles in comparison
with higher eukaryotes, lacking both mitochondria and peroxi-
somes, but containing instead a hydrogen producing organelle (the
hydrogenosome) which may be a relic of an endosymbiotically
acquired mitochondrion-like organelle, or which may be the result
of a 2ndsymbiotic event (see  for a review).
Given the unusual cell biology of T. vaginalis we decided to
investigate a class of membrane proteins (ATP binding cassette
(ABC) transporters) localized to the plasma membrane and
organellar membranes of all eukaryotic cells, and examine
differences in the complement of these proteins in T. vaginalis
compared to other eukaryotes, with a longer term view to
determining the contribution of ABC transporters to the parasite’s
ATP binding cassette (ABC) systems encompass a family of
proteins found in all 3 domains of life, which are responsible for an
abundance of transport roles as well as a variety of intracellular
non-transport processes including gene regulation and DNA repair
. The vast majority of ABC transporters in eukaryotes are
exporters, whilst prokaryotes encode functioning importers too. All
proteins in this family are defined by the presence of a highly
conserved nucleotide binding domain (NBD; the eponymous ATP
binding cassette (ABC)), an ATPase domain with characteristic
motifs including a Walker A, Walker B and Signature sequences,
which contribute to the hydrolysis of ATP, the energy of which
drives the various cellular processes mentioned. The NBDs are
highly conserved among different ABC proteins, sharing typically
greater than 25% sequence identity , and a common 3-
dimensional fold .
In addition to the cytoplasmic NBDs, ABC transporters contain
transmembrane domains (TMDs), which span the membrane
numerous times via a helices (typically 4–11 helices per domain,
), and which contain binding sites for transported substrates.
The typical configuration for a functional ABC transporter is
believed to comprise 2 NBDs and 2 TMDs, although these need
not necessarily be present within the same polypeptide. For
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example, ‘‘full-transporters’’ are defined as containing all four
domains within the same polypeptide, whilst ‘‘half-transporters’’
typically comprise a single NBD and TMD within one polypeptide
and are believed to either homo- or hetero-dimerize in order to
function  . In contrast to the NBDs, the TMD components
show considerable sequence variation between unrelated transport-
ers reflecting their role in recognition and transport of substrate.
Based on the organisation of their NBDs and TMDs as well as
features such as membrane topology, sequence homology and
gene structure, eukaryotic ABC proteins have been grouped into 7
subfamilies - ABCA to ABCG [7,8], although occasionally
sequences have been identified as not fitting with this classification
and these have been annotated as ABCH and ABCI sequences
(e.g. see ). The availability of the recently completed T. vaginalis
genome sequence  enabled a database search for ABC
proteins within T. vaginalis to be conducted, as presented here,
using the sequences of known ABC proteins from other species for
comparison. A phylogenetic classification of the hypothetical ABC
proteins into subfamilies has been conducted, accompanied by
discussion regarding the putative location and function of different
transporters within the parasite. In addition we have performed
sub-cellular localization studies on selected ABC proteins to
validate our analysis. Finally, comparisons are drawn between the
ABC families of T. vaginalis and three other sequenced protists P.
falciparum, E. histolytica and G. lamblia [11,12,13].
Materials and Methods
Cell cultivation and transfection
The sequenced T. vaginalis strain G3 (ATCC PRA-98; ),
kindly provided by G. H. Coombs, was used for genomic DNA
preparation throughout this study and was grown in modified
TYM medium . Strain C1 (ATCC 30001), which is less
pathogenic, was employed for transfection and localization studies.
Transfection of T. vaginalis
Late stage cultures of T. vaginalis C1 were centrifuged (1500 g,
10 minutes, 4uC) and resuspended in supplemented Diamond’s
medium to a density of 108cells/ml. Cells (36107) were incubated
with plasmid DNA (50 mg) for 15 minutes on ice and then
electroporated 350 V, 960 mF (BioRad GenePulser) in chilled
0.4 cm spacing electroporation cuvettes (GeneFlow). Electropo-
rated cells were immediately diluted into 50 ml of complete media,
pre-warmed to 37uC and then incubated for a further 4 hours,
prior to the addition of G418 to 50 mg/ml, and left overnight,
before surviving cells (those in motile suspension) were transferred
into fresh complete, selective media and incubated for 3–21 days
until a density of ca. 26106cells/ml was obtained, whereupon
they were passaged as described above.
Extraction of nucleic acids from T. vaginalis
Genomic DNA (gDNA) was isolated from 36108T. vaginalis
cells as described previously  and resuspended in 500 ml TE
containing 10 mg/ml RNase A. Total RNA was extracted from
26109T. vaginalis cells by the single-step acid guanidinium
thiocyanate-phenol-chloroform method . 1 mg of total RNA
was processed using the PolyATtract mRNA isolation system
(Promega Corporation, Madison, WI, USA) according to manu-
facturers’ instructions to yield ca. 10 mg of enriched polyA+ RNA
that was stored at 280uC until required.
Reverse transcription and PCR
Reverse transcription and PCR of specific regions of the genes
TVAG_275410, TVAG_275420, TVAG_072410, TVAG_072420
and TVAG_470720 were carried out according to manufacturer’s
instructions using the Access RT-PCR System (Promega Corpora-
tion, Madison, WI, USA) with appropriate primers listed in Table
S1. Bands of interest were separated by agarose gel electrophoresis,
and DNA was extracted using the Qiaquick Gel Extraction Kit
(Qiagen Inc.) prior to treatment with T4 DNA polymerase (New
England Biolabs) to remove 39 A overhangs introduced by Tfl DNA
polymerase in the Access RT-PCR System. To allow for
sequencing, the blunt-ended fragments were then ligated into the
pSC-B vector using the StrataClone Blunt PCR Cloning Kit
(Stratagene). Where indicated, specific gene fragments were
amplified by the polymerase chain reaction (PCR) from T. vaginalis
gDNA using Phusion DNA polymerase (NewEngland Biolabs, Inc.)
according to manufacturer’s recommendations.
Construction of plasmids for transfection of T. vaginalis
cDNA regions comprising the open reading frames for
genes TVAG_470720, TVAG_605460 and fused TVAG_415980/
TVAG_415990 were amplified by PCR using the primer pairs
470720NdeI and 470720BamHI, 605460NdeI and 605460BamHI,
415980NdeI and 415990BamHI respectively (Table S1). The
introduced NdeI and BamHI restriction recognition sites facilitated
ligation into corresponding sites on the pTagVag2 vector, thereby
resulting in C-terminal tagging with a double haemagluttinin
epitope , (a kind gift of Professor Jan Tachezy). The resulting
plasmid constructs, pTagVag2-470720, pTagVag2-605460 and
pTagVag2-415980/90 were maintained in E. coli XL1-Blue and
plasmid DNA was purified using Qiagen maxiprep kits. The
of 10 mg/ml for transfection into T. vaginalis.
Late stage cells were centrifuged (1500 g, 10 minutes, 4uC) and
resuspended in phosphate buffered saline (PBS) at 16107/ml.
Aliquots (0.5 ml) of this suspension were layered onto silane
covered microscope slides (Sigma) and left to adhere for
The parasite Trichomonas vaginalis infects in excess of 100
million people per year, and is a contributory factor to
enhanced transmission rates of HIV, the causative virus in
AIDS. As such, T. vaginalis infection is an important public
health concern. Understanding the biology of the organ-
ism is important to determine aspects of the response to
drug treatment, host:parasite interactions and so on. We
have investigated an important family of proteins – the
ATP binding cassette transporters – which are present in
the membranes of all cells, and which contribute to a
diverse spectrum of important cellular processes. The ABC
transporters of T. vaginalis were identified by analysis of
primary amino acid sequence data, and examined by
subsequent protein and gene expression studies. Our most
important conclusion is that – uniquely amongst eukary-
otes - T. vaginalis has no ABC transporters capable of
acting as monomers. In other words, its ABC transporters
must all act by forming functional complexes with other
ABC proteins. This has implications for our understanding
not just of the parasite’s biology, but also its evolution. In
summary our analysis opens up the path for future
research of individual members of the ABC protein family
in T. vaginalis.
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30 minutes at room temperature. Non-adherent cells were
removed by washing once with PBS, and remaining cells were
fixed and permeabilized with 0.5 ml 4% w/v paraformaldehyde,
0.1% v/v Triton X-100 for 20 minutes at room temperature.
Slides were washed twice with PBS, and then incubated for
1 hour at room temperature in blocking solution (PBS supple-
mented with 0.25% w/v each BSA and fish gelatin). Slides were
then incubated with primary antibody (mouse anti-HA, 1:2500 or
rabbit anti-BiP, 1:1000 in blocking solution) for 1 hour at room
temperature, washed twice with PBS and then incubated with
secondary antibody (species specific AlexaFluor-488, 1:1000 in
blocking solution) for 1 hour at room temperature in the dark.
After further washing in PBS, slides were treated with RNAase
(100 mg/mL in PBS, 37uC, 20 minutes), washed, and nuclei
stained by addition of propidium iodide (3.3 mg/mL in PBS,
5 minutes) and then mounted with 50% v/v glycerol in PBS.
Slides were kept at 4uC in the dark until analyzed, and could be
stored for at least 2 months without loss of signal quality. Slides
were analysed on a Zeiss LSM 710 confocal laser scanning
microscope, using a 636oil-immersion objective. The fluorescent
tags were excited using a laser sources at 488 nm (Alexa488) and
561 nm (propidium iodide), and emitted light collected. Image
files were subsequently processed using the Zeiss LSM Image
The protein sequence of human ABCB1/P-glycoprotein
(AAA59575) was used as a query for a homology search of
TrichDB (http://trichdb.org/trichdb), the complete T. vaginalis
genome database, using BLASTp. Pairs of sequences considered
redundant due to greater than 95% amino acid identity were
removed prior to further analysis. All remaining sequences were
screened manually for Walker A (GxxGxGK(S/T), where x=any
amino acid), Walker B (hhhhDE, where h=hydrophobic amino
acid) and ABC signature (LSGGQ) motifs. Putative TM spanning
regions of hypothetical T. vaginalis ABC transporters was predicted
using the programs TMHMM , TopPred , and
Multiple BLASTp searches were performed on the NCBI
(National Centre for Biotechnology Information) and UNIPROT
websites to identify characterised and curated homologues of the
hypothetical ABC proteins of T. vaginalis in other species and thus
facilitate classification of the T. vaginalis proteins. Each protein
sequence was also used as a query to search the Expressed
Sequence Tag (EST) database using tBLASTn. The EST database
consists of 26,491 single pass cDNA sequences obtained from the
C1 and T1 strains of T. vaginalis (TrichDB). Protein sequences
showing greater than 97% identity to translated ESTs were
categorised as being expressed in T. vaginalis.
Consensus phylogenetic trees were constructed via a multistep
process to examine relationships between different protein
sequences. Multiple sequence alignment of the hypothetical
ABC proteins was performed on Bioedit with ClustalW_2 using
the BLOSUM-62 matrix. Alignments were manually edited to
remove internal gaps and N and C-terminal extensions where
necessary to prevent differences in sequence length affecting
protein clustering. The amended alignment was bootstrapped
with 500 replications using Seqboot, whilst Protpars generated
trees from the resulting alignments to be used by Consense in
producing a consensus. Seqboot, Protpars and Consense all form
part of the Phylip Package Version 4.0, which was accessed via
the Mobyle website (http://mobyle.pasteur.fr). Trees were
visualised using Treeview.
Results and Discussion
T. vaginalis has 98 putative ABC proteins
A BLASTp search of TrichDB  using human P-glycoprotein
 as a query sequence identified 102 predicted T. vaginalis ABC
proteins, four of which showed .95% identity to another sequence
and so were removed to avoid redundancy (TVAG_059100,
TVAG_132360, TVAG_431960 and TVAG_510260). The 98
hypothetical ABC proteins identified here exceeds the 88 originally
estimated based on the draft genome sequence  and, compared
with the number of ABC proteins identified in other species,
constitutes a significant total. Table 1 compares the ABC family of
T. vaginalis with multi- and unicellular non-parasitic species as well
as four other disease-causing parasites: P. falciparum, E. histolytica, L.
major and G. lamblia. The number of ABC genes in T. vaginalis
exceeds all but the two plant species [9,23], and indicates that the
Table 1. ABC proteins in sequence genomes.
Organism Estimate number of proteinsEstimated number of ABC proteinsReference
T. vaginalis60000 982
H. sapiens25000 482
C. elegans18400 60
A. thaliana35000 1292
O. sativa37500 121 
S. cerevisiae 6300 292
L. major850042 
P. falciparum 5300161
This work & 
E. histolytica9900 261
This work & 
G. lamblia6500 222
This work & 
E. coli 4300 792
B. subtilis4100 842
1these numbers are greater than those presented in a recent analysis of ABC transporters from protozoan parasites .
2The proportion of full length ABC transporter genes is 0% in T. vaginalis, 69% in humans, 62% in Arabidopsis, 83% in yeast, 68% in Giardia and 0% in E. coli and B.
Trichomonas ABC Transporters
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ABC family of genes has undergone considerable expansion in T.
vaginalis. Genome analysis indicates many other gene families
(including several involved in membrane trafficking and transport)
have expanded similarly . As with these, expression proteomics
under diverse growth conditions is required before the tags
‘‘putative’’ or ‘‘hypothetical’’ can be dispensed with. The large
number of ABC proteins in plants is believed to be partly due to
genome expansion and also due to functional diversification .
The putative functional diversity of T. vaginalis ABC proteins will be
The lengths of hypothetical proteins varied from 116 to 919
amino acids, although for three sequences this is difficult to verify
as three are located at the ends of unassembled sequence scaffolds
(TVAG_241640, TVAG_542450 and TVAG_542470). All 98
sequences were analysed manually for the presence of Walker A
(GxxGxGKS/T), Walker B (hhhhDE) and Signature motifs
(LSGGQ). The majority were found to contain all three although
it was common in putative ABC proteins for one of these, usually
the Signature, to be very distinct from the canonical sequences
(Table S2). This is not atypical as examination of multiple ABC
transporter families has previously shown (e.g. ). Analysis of
the hydrophobicity of the ABC proteins using TOPPRED,
TMHMM and HMMTOP resulted in predictions of between 0
to 9 transmembrane (TM) helices. Indeed 26 sequences were
identified as containing no transmembrane regions, a number far
in excess of any other non-plant, eukaryotic genome. No single
sequence appeared to encode two blocks of multiple TMHs,
separated by an NBD sequence, indicating that the T. vaginalis
genome does not encode any full length transporters, an
observation discussed further below. Based on the number and
order of NBDs and TMDs, the topology of each protein was
defined and table S2 presents an inventory of all 98 predicted
proteins with respect to length, membrane topology, motifs and
subfamily classification (explained below).
Classification of ABC proteins of T. vaginalis
Initial phylogenetic analyses (data not shown) of the T. vaginalis
ABC protein sequences resulted in a tentative assignment of the
majority to one of the major sub-families of ABC proteins
documented in eukaryotes. However, bootstrap analyses indicated
low certainty in many of the branch-points and thus three further
measures were taken to reinforce our assignment of proteins to the
ABC sub-families as listed in Table S2. Firstly, we considered the
topology of each predicted protein. For example, sequences with a
large (.250 amino acids long) predicted extracellular loop (ECL)
sandwiched between the first two predicted TM helices were
candidates for the ABCA family (e.g. TVAG_173120) as this
insertion is exclusively found in ABCA sequences . Secondly,
sequences with two consecutive NBDs and no TMDs were likely to
represent members of the ABCE or ABCF sub-families (e.g.
TVAG_385840) as again in eukaryotes this domain organization is
only found in non-transporting ABC proteins . Thirdly, for
each sequence we performed BLASTp analyses against the
GenBank non-redundant protein sequence database and used
the highest ranked sequences as a guide for sub-family allocation.
For example, in the case of the TVAG_542450 sequence, which
had been previously categorised as being the parasite’s homologue
of P-glycoprotein/ABCB1 , we found that all of the highest-
ranking 100 sequences for TVAG_542450 were classified as being
predicted or characterised members of the ABCB family. Finally,
to improve the accuracy of bootstrap analyses, we removed the
confounding factor of highly variable sequence lengths and aligned
the NBD sequences only. This analysis demonstrated clear
clustering of the ABC proteins into several sub-families, and the
removal of putative ABCH and ABCI sequences (Table S2) from
the analysis further improved the clarity of the sub-classification
(Figure 1). The final predicted numbers of sequences in each sub-
family are given in Table 1, with numbers from other eukaryotes
given for comparison. Among the findings we discuss below are the
absence of two families – namely ABCG and ABCC, the expansion
of the ABCA sub-family, and the preponderance (31 in total) of
proteins that are unclassifiable with the ABCA-ABCG proteins.
A large number of orphan abc genes in T. vaginalis
Upon examination of the chromosomal localisation of the genes
listed in Table S2, we noted in excess of 20 examples of ORFs
linked on the same loci. Several of these ORFs apparently encode
half-transporters with a complete NBD and several transmem-
brane segments, but a large proportion (see Table S2, ‘‘Others’’)
encoded only part of the NBD on one ORF, and the rest on
adjacent genes with a linked head-to-tail orientation (e.g. see
Figure 2 and 3). The intergenic regions in the latter cases were
found to be relatively small, ranging from 0 to a few hundred
bases. BLASTx searches with these intervening sequences revealed
that they are themselves highly similar to coding abc gene
sequences, but were either out of frame with the flanking partial
abc ORFs, or were in frame but interrupted by stop codons.
To audit whether these abc ORFs are genuinely partial genes or
the result of incomplete sequence data, we sought to analyse the
transcription and the genomic arrangement of a representative
subset (Figure 2). TVAG_275420 is an ORF that encodes a
predicted 478 aa protein of the ABCA subfamily that includes a
TMD, a Walker A motif and a Signature sequence shortly
followed by an in-frame TGA stop codon (Table S2, Figure 2A).
This predicted protein is 70% identical to TVAG_440500, also an
ABCA subfamily member (Table S2) of 830 aa that includes a full
NBD. Downstream of TVAG_275420 there is a linked ORF,
TVAG_275410 (Figure 2A, top panel) which is 82% identical to
the last 70 amino acids of TVAG_440500. A Blastx search of the
232 bpintergenic regionbetween
TVAG_275410 revealed that this is 88% identical to a similar
region in the predicted TVAG_440500 protein sequence. Collec-
tively, these data suggest that TVAG_275420, the intergenic
region and TVAG_275410 are all part of a single gene/
pseudogene (encoding a half ABC transporter) that is highly
related to TVAG_440500. Moreover, an EST was found that
matched the region from the 39 end of TVAG_275420, the
intergenic region and the complete sequence of TVAG_275410
(Figure 2A, open arrow). This finding suggests that there is either
an error in the genomic sequence or that bicistronic transcription
occurs at this locus. To assess this locus, we used RT-PCR to
amplify putative transcripts running from TVAG_275420 through
to TVAG_275410 (Figure 2D). Primers were designed to amplify
any transcript or gDNA fragment from position 862 on
TVAG_275420 to position 186 on TVAG_275410 (Table S1).
We successfully amplified a band of the expected size at 990 bp by
RT-PCR of polyA+ RNA (Figure 2D, lane 3). This band migrated
at a similar size to one amplified from gDNA using the same
primer set (Figure 2D, lane 2) but no such species was amplified
from the negative control (Figure 2D, lane 4), confirming that the
band in lane 2 (Figure 2B) originates from mRNA and not from
gDNA contamination of the poly A+ template.
As a positive control, we ran a parallel set of reactions on the
locus of TVAG_470720 (Figure 2C), an abc gene which is known
to be expressed, based upon both EST and protein detection (our
unpublished data). This gene encodes a complete TMD-NBD and
both its transcript and a corresponding gDNA fragment were
amplified using primers listed in Table S1 to yield bands of 1 kb
Trichomonas ABC Transporters
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(Figure 2D, lanes 8–10). The sequences of both the transcript and
the gDNA fragment were found to be identical to the
TVAG_470720 sequence from TrichDB. However, upon se-
quencing of both the gDNA and the amplified cDNA from the
TVAG_275420/410 locus, we noted that a cytosine (C) was
absent from a predicted C triplet at position 1414 to 1416 on the
TrichDB TVAG_275420 sequence. The absence of this C residue
results in an ORF of 1878 bp that runs from TVAG_275420
through to the 39 end of TVAG_275410. Thus, it appears that a
sequencing error, and not bicistronic transcription, is responsible
for the original arrangement of genes shown in Figure 2A, top
panel, and raises the possibility that the same may apply to other
loci containing split abc genes.
We therefore investigated an additional locus (Figure 2B) that
consists of TVAG_072420, an 875 bp ORF encoding a single
predicted TM helix and TVAG_072410, a 1703 bp encoding five
further TM helices followed by a complete NBD (Table S2). The
two ORFs are separated by only 5 bp and no ESTs have been
matched to either sequence. We designed primers to run from
position 658 on TVAG_072420 to position 391 on TVAG_072410
Figure 1. NBDs of 65 predicted ABC proteins of T. vaginalis. Multiple sequence alignment and boot-strapping was performed as described in
the material and methods. The boot-strap values (percentages) are displayed on all branches. The clustering of the ABC genes from families A–G is
evident. The ABCE and F families of non-transporter proteins are highlighted on a lighter background for clarity.
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(Table S1), thus expecting an amplicon of 613 bp. We were unable
to amplify a band of the correct size by RT-PCR of poly A+ RNA
butthe same primers yielded a 0.6 kb band from gDNA (Figure 2D,
lanes 5–7) which exactly matched the TrichDB sequence,
confirming that the two ORFs are indeed separated by a stop
codon and a 5 bp intergenic region.
These data collectively show that not all split abc genes can be
explained by sequencing discrepancies. Two further examples of this
DNA sequences are located in the intergenic regions between closely
separated partial ABC transporter reading frames. The two
additional examples (Two TVAG_049010 & TVAG_049020, and
TVAG_254080, TVAG_254070 & TVAG_254060) are shown in
Figure 2E and F respectively. In the second locus, it appears that an
abc gene has been split into four parts that each contain at least an
ABC motif (Figure 2F). No ESTs were found in the current
databases to match any of these genes or intergenic regions.
TVAG_049010 is predicted to encode three TM helices and is
followed by a 93 bp intergenic region and then a downstream ORF
TVAG_049020 encodes two further TM helices and a complete
NBD (Figure 2E). As observed above, the intergenic region has high
sequence identity, as detected by BLASTx, to another putative
ABCBgene TVAG_127410.Thus, inthe absence of a stop codon at
the end of TVAG_049010, this locus could potentially comprise a
complete half-transporter. We successfully amplified a fragment of
1.3 kb from genomic DNA (data not shown) using primers from
position 191onTVAG_049010 toposition 710onTVAG_0490120
(TableS1), and found the sequence of the amplicon to exactly match
that on TrichDB.
The TVAG_254080/70/60 locus (Figure 2F) contains ORF
TVAG_254080 with two predicted TMS followed by an open
intergenic region that could potentially encode 3 TMS that are 44%
identical to those of ORF TVAG_299600, a member of the ABCA
subfamily. Downstream, TVAG_254070 contains a Walker A motif,
with TVAG_254060 containing the Signature sequence and Walker
B motif, and these two ORFs are separated by a 27 bp intergenic
region. We amplified and sequenced a 2 kb region of the genomic
to position 466 on TVAG_254060 and again found the sequence to
be identical to the TrichDB sequence, confirming that the
arrangement of the partial genes depicted in Figure 2F is correct.
The absence of full length ABC transporters from T.
We further noted another category of loci where ORFs are
arranged tail to tail, separated by short intergenic regions as shown
Figure 2. Validation of ABC transporter gene sequence and transcripts from T. vaginalis. A, B, C, E, and F. The genomic organisation of 5
combinations of open reading frames (except C, where a single gene is shown) are displayed such that predicted genes are in thick, filled arrows, with
the gene identifier on a grey boxed background above, and the gene size in plain font below. The presence of intervening sequences with high
homology to other ABC genes elsewhere in the genome is denoted by blue boxes. Expressed sequence tags (ESTs) are indicated by open arrows
below the gene of interest. (D) RT-PCR of representative T. vaginalis ABC genes. Lanes 2–4: 275410/420; lanes 5–7: 072410/420; lanes 8–10: 470720.
Primers for amplification from cDNA are listed in Table S1. Controls lanes (2, 5, 8) employed genomic DNA as template for the PCR, lanes 3, 6 and 9
were from complete reverse transcription and PCR reactions, lanes 4, 7 and 10 lacked the RT enzyme. Novagen Perfect DNA markers are in lane 1.
Trichomonas ABC Transporters
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by two examples with three open reading frames each in Figure 3.
In each case, the size of the assembled ORFs could constitute a full
length ABC transporter. The locus in the top panel (Figure 3A)
consists of two ORFs, TVAG_245200 and TVAG_245210,
separated by only 27 bp and arranged head to tail. TVAG_245220
is arranged tail to tail with 245210 with an intergenic space of
258 bp. We set out to investigate whether the contigs at this locus
had been correctly assembled and sequenced to ascertain that the
T. vaginalis genome does not bear any genes for full-length ABC
transporters as suggested by the data presented in Table S2. To
do so, we used primers designed to give a positive result if in the
first case, TVAG_245220 were reversed and in the second case, if
(Figure 3A). It was found that a PCR product for the locus was
generated with a single primer, 245220R2, which bound to the
corresponding complementary strand on both TVAG_245220
and TVAG_245200 (data not shown). The sequence for this
fragment was identical to that in the TrichDB database, demon-
strating that the original ORF arrangement in Figure 3A, top panel,
was correct. TVAG_245200 is transcribed as evidenced by a
matching EST from the TrichDB database, indicating that the
ORFs in this locus are unlikely to represent pseudogenes.
The locus comprising TVAG_415970, TVAG_415980 and
TVAG_415990 represents a similar situation as the previously
described locus except that there is no intergenic region between
TVAG_415980 and TVAG_415990 that are just separated by a
TGA stop codon (i.e. TVAG_415980 and TVAG_415990 could
comprise an intact half-transporter, linked head-to-head with
another half-transporter TVAG_415970; Figure 3B). Using a
similar strategy as with the TVAG_245200-220 locus, we set out
TVAG_415980 and TVAG_415990 may be reversed or whether
the original arrangement is correct. A 2.8 kb fragment was
generated by PCR with a single primer (data not shown),
TVAG_415990R1, which similarly to the previous case bound
to opposite strands on two tail to tail ORFs. Sequencing of this
product revealed that the TrichDB arrangement was correct and
that the sequence was identical to that in the database.
either TVAG_415970 or
Figure 3. The absence of full length transporters from T. vaginalis. A, B genomic context of combinations of open reading frames that could
encode for an intact full-length ABC transporter. Formatting is as described in Figure 2, with pairs of primers used to verify the genomic organization
displayed as blue and orange arrows (Table S1). C The two genes TVAG_415980 and TVAG_415990 are separated only by an in-frame stop codon
(TGA). RT-PCR analysis of mRNA with primers (black arrows) demonstrates that transcript containing both TVAG_415980 and TVAG_415990
sequences exists (lane 5, asterisk, at the same size as the genomic DNA control). Reactions lacking the RT step (lane 3), or containing primers only
(lane 2) verify the specificity of the band in lane 5. D Confocal microscopy of T. vaginalis C1 cells. The four panels represent (left to right, scale bars
10 mm) bright-field images, detection of nuclear material by propidium iodide staining, overlay of the first pair, and finally, the lack of any anti-HA
reactive signal in C1 cells. E Confocal microscopy of T. vaginalis C1 transformed with a plasmid containing the genomic DNA of TVAG_415980 and
TVAG_415990 with the stop codon of the latter replaced by a double haemagluttinin (HA) tag. Parasites were fixed as described in the Methods,
examined with a Zeiss LSM 710 confocal microscope, and visualization of HA-tagged ABC TVAG_415980_90 followed incubation with anti-HA primary
and an Alexaflour-488 secondary antibody (green).
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Given that ORFs TVAG_415980 and TVAG_415990 are
separated just by one TGA stop codon, we pursued study of this
locus to investigate the possibility of stop codon read-through. T.
vaginalis was transfected with a plasmid construct, pTagVag2-
TVAG_415980 ORF and the TVAG_415990 ORF, including
the intervening TGA stop codon. This fragment had been cloned
upstream of a double haemagluttinin (HA) tag, such that detection
of a recombinant protein by an anti-HA antibody would only
occur if read-through happened or if the TGA stop codon were
processed post-transcriptionally. PolyA+-enriched RNA isolated
from the transfectants enabled us to verify by RT-PCR that the
gene cassette from pTagVag2-415980/90 was transcribed. We
detected amongst other smaller bands, a 0.9 kb band of the
expected size that was amplified from the reverse-transcribed
polyA+ template (Figure 3C, lane 5). This band was of the same
size as that obtained from pTagVag2-415980/90 plasmid DNA
that was used as a positive control (Figure 3C, lane 4), but not from
negative controls (Figure 3C, lane 3). To investigate translation, we
analysed transfected cells by immunofluorescence microscopy with
anti-HA antibody. A diffuse distribution of anti-HA signal was
seen in fixed transfected cells (Figure 3E) as opposed to wild-type
C1 cells (Figure 3D). These data provides tentative evidence for
stop codon read-through, although further experimental work
would be required to substantiate this.
Absence of the ABCG and ABCC subfamilies in T.
The ABCG sub-family sequences are distinguished from other
ABC proteins by their ‘‘reversed topology’’ , i.e. the NBD is
found at the N-terminus of the protein, whereas the C-terminus
contains the TMD. The family also contains only half-transporters
in organisms whose genomes have been sequenced to date (e.g. see
). Examination of the T. vaginalis genome’s complement of half
transporters reveals none with this altered topology. Furthermore,
even though many of the 31 unclassified proteins contain either a
single NBD or a single TMD, no 2 of these are genomically
arranged in a manner compatible with them forming a complete
ABCG transporter following stop codon read through or
sequencing inconsistencies explored above. Similarly, we were
unable to detect any members of the ABCC sub-family, which are
commonly identified by the presence of a large additional N-
terminal TMD containing (usually) 5 TM a-helices. This N-
terminal extension was not found in any of the Trichomonas ABC
transporter sequences, and none of these sequences more similar
to the ABCC transporters than to the ABCB transporters of other
parasite genomes (E. histolytica, P. falciparum, G. lamblia).
This absence of ABCC and ABCG transporters must reflect
biological perspectives of the organism. The absence of ABCG
proteins may correlate with the expansion of ABCA proteins.
Although both families have members that are involved in the
export of lipids and their derivatives, only the ABCA family has
members that are believed to be importers [8,29], and the
proposal is that in T. vaginalis a requirement for lipid import (see
next section) has driven the ABCA expansion. For the ABCC
family, the absence of members may be a correlate of the absence
of a glutathione system in T. vaginalis  as many characterised
eukaryotic ABCC members are either co-transporters of glutathi-
one, or even transport directly GSH-conjugated substrates. The
other members of the ABCC family function as ion channels or
ion channel regulators (CFTR/ABCC6 and SUR/ABCC8,C9
respectively) which are absence from other early diverging
Expansion of the ABCA subfamily in T. vaginalis
The ABCA subfamily was the largest identified in T. vaginalis,
with 34 putative transporters, several of which are transcribed as
evidenced by expressed sequence tags. With the exception of two
partial sequences, all have a (TMD-NBD) topology, range in
length from 478–919aa and all bar 7 members of this subfamily
have the characteristic extracellular loop (ECL) of the ABCA
subfamily between their first and second predicted TM helices
(Table S2). Trichomonas vaginalis has a severely compromised
ability to synthesis lipids , and is therefore reliant on their
import - a trait shared by G. lamblia, another species in which the
ABCA proteins form a significant proportion (68%) of the ABC
family (Table 2). Given that ABCA transporters in humans have
been implicated in the export and import of a variety of lipids and
lipid conjugates [29,33] it is plausible that some ABCA
transporters have evolved in T. vaginalis as importers of lipids
rather than exporters.
To date, ABCA transporters that have been characterised in
other eukaryotes are full-length (i.e. 2 NBDs and 2 TMDs in the
same polypeptide), unlike the transporters described here. In order
to reconstruct the phylogenetic history for the T. vaginalis ABCA
subfamily, we used sequences for TVAG_064700 and TVAG
064710 respectively as queries to search for homologues in
UniProt. These two sequences were chosen as the genes are linked
as inverted tandem repeats, and they both contain an ECL.
Moreover, TVAG_064700 has a degenerate signature motif
(LSDGD) whereas TVAG_064710 has a canonical one (LSGGQ).
To be able to properly align the half-transporters from T. vaginalis
to other eukaryotic full-length transporters, we selectively extract-
ed NBD sequences from the latter to comprise an N-terminal and
Table 2. The classified ABC proteins of T. vaginalis compared with other eukaryotes.
Number of proteins in each ABC sub-family
Total number of ABC proteins
T. vaginalis 34 2702130 3198
H. sapiens 1211 1241350 48
S. cerevisiae047215 102 31
G. lamblia150401101 22
P. falciparum17201212 16
E. histolytica27701223 26
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a C-terminal NBD region each. As shown in Figure 4, these ABCA
NBD sequences from other eukaryotes form two distinct clusters,
suggesting an early duplication event followed by divergence. It is
of note that the C-terminal NBD almost always contains a
degenerate signature motif in other eukaryotes whereas the N-
terminal NBD invariably contains the canonical LSGGQ. The T.
vaginalis ABCA single NBD sequences mimic the clustering of
NBD sequences from other eukaryotes in that they too fall into two
distinct groups termed Group I and Group II (Figure 4), with
Group I sequences invariantly bearing a canonical signature motif
and Group II sequences invariantly bearing a degenerate signature
motif. Twenty of the abca genes are linked as pairs that appear to
be inverted repeats (TrichDB and Table S2). Any one member of
each pair (except for TVAG_225860 and TVAG_225880) has
higher sequence similarity to other ORFs in the same group
than to its linked repeat. For instance, TVAG_072430 and
TVAG_064710 cluster together in a group that is distinct
from another that includes their respective linked partners
TVAG_072410 and TVAG_064700 (Figure 4). A possible
scenario consistent with this data that accounts for the history of
abca genes in eukaryotes is that ancestral abca genes existed both as
half-transporters and full-transporters, prior to the evolution of the
progenitor of T. vaginalis . This lineage lost the full-length
transporter gene, but the evolutionary forces that maintain the
paired canonical and degenerate NBDs in full-length eukaryotic
ABCA transporters are clearly still acting on the Trichomonas abca
Figure 4. ABCA proteins in T. vaginalis and other eukaryotes show conservation of paired consensus and degenerate NBDs. Protein
sequences for ABCA transporters from T. vaginalis and other eukaryotes had their TMD sequences removed, and in the case of full-length proteins,
the sequence was bisected into N- and C-terminal halves. The alignment and boot-strapping were as described in Figure 1. The N-terminal NBD
cluster contains almost entirely NBDs with consensus Walker and signature motifs, whereas the C-terminal cluster is almost always degenerate for at
least one of the 3 motifs. For T. vaginalis, there are ten pairs of ABCA genes, and in all bar one case, the two members of each pair split on sequence
into Group I (consensus) Walker/Signature) or Group 2 (degenerate Walker/Signature). T. vaginalis sequences marked with an asterisk also have an
extracellular loop of 200–500 amino acids between the 1stand 2ndpredicted TM helix as observed in many other eukaryotic ABCA proteins.
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Figure 5. Localization of ABCD transporters in T. vaginalis. Parasites were fixed, labelled and imaged as described in the legend to Figure 3.
The endoplasmic reticulum protein BiP in C1 (A) was detected with primary anti-BiP antibody, whilst the hydrogenosomal protein TOM40-3 (B) and
two ABCD transformants (TVAG_470720; C; and TVAG_605460; D) were detected by reactivity to anti-HA antibodies. The distributions of BIP,
Trichomonas ABC Transporters
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half-transporter genes. In the case of the 2258 locus, the duplicate
locus has apparently duplicated again to give rise to two sets of
inverted genes. This appears to be a recent duplication as the
sequences of corresponding gene fragments are almost identical to
T. vaginalis ABCD-type proteins are localized to the ER
T. vaginalis is the only parasite in Table 2 encoding ABCD type
transporters, with 2 half transporters, TVAG_470720 (556aa) and
TVAG_605460 (546aa), similar to the total number found in other
eukaryotic species , indicative of a lack of duplication
(conversely to the above). The hypothetical T. vaginalis ABCD
transporters are half transporters with the topology TMD-NBD, in
common with all other non-plant eukaryotic ABCD proteins .
The majority of ABCD transporters have been localised to
peroxisomes, where they are implicated in the transport of
VLCFAs and other co-enzyme A conjugates into the peroxisome
To investigate their sub-cellular localization, ABCD transport-
ers TVAG_470720 and TVAG_605460 were expressed in T.
vaginalis C1 cells with a C-terminal double haemagluttinin epitope,
and were visualised using immunofluorescence microscopy
(Figure 5). Control C1 cells were incubated with PI to detect the
nucleus, and with anti-BiP antibody to detect the diffuse ER
(Figure 5A). This contrasts directly with an alternative organelle
membrane marker - a hydrogenosomal TOM40 homologue ,
which showed dozens of internal, discrete, vesicular structures with
dimensions consistent with those of the hydrogenosome (Figure 5B;
). Both the ABCD transformants showed a highly diffuse
distribution similar to that observed with BiP (Figure 5C, D) with
TVAG_470720 also showing some additional perinuclear signal.
Attempts to demonstrate co-localization with anti-BiP were
confounded by this extreme diffuseness. Our argument for the
ABCD transporters being localized to the ER is further supported
by both the absence of peroxisomes from T. vaginalis, and by recent
localization of a subset of ABCD proteins to the ER rather than to
the peroxisome in humans and mice . Notably, both
TVAG_470720 and TVAG_605460 belong to this latter subset,
rather than to the ‘‘classical’’ peroxisomal ABCD proteins
Expansion of the ABCB subfamily in T. vaginalis
Twenty-seven proteins constitute the hypothetical ABCB
subfamily in T. vaginalis, the second largest subfamily, comprising
a significant proportion (28%) of all ABC proteins - a characteristic
shared by P. falciparum (44%) and E. histolytica (27%). Such findings
reflect the importance of ABCB transporters in these parasites and
raise the possibility of ABCB-specific gene amplification having
occurred. All hypothetical ABCB proteins have the same TMD-
NBD topology as ABCA transporters, but lack the defining
extracellular loop of the latter family, and range in length from
477–733aa. Additionally, and distinct from ABCA transporters,
the ABCB members all contain consensus signature sequences,
with a single exception.
In humans, ABCB proteins, both full transporters at the plasma
membrane and half transporters dimerising intracellularly, have
been implicated in various roles ranging from drug resistance
(ABCB1 or MDR1) to peptide transport into the ER (ABCB2 and
B3) and iron homeostasis in mitochondria (ABCB6) . In T.
vaginalis, the closest sequence to mammalian ABCB1/P-glycopro-
tein is TVAG_542450 (Tvpgp1; ), however research has not
supported an involvement of Tvpgp1 in resistance to metronidazole
. For other eukaryotic ABCB transporters, including Atm1,
convincing homologues in T. vaginalis could not be identified by
sequence analysis alone and further localization and functional
studies will be required.
The evolution of the ABCB family in T. vaginalis was examined
by constructing phylogenetic trees employing the same criteria as
applied to the ABCA sequences above, i.e. eukaryotic full length
transporters had their NBD sequence extracted and these were
then aligned and neighbour-joining trees generated using boot-
strap analysis (Figure S1). A similar conclusion to that regarding
the T. vaginalis ABCA proteins is reached, i.e. that despite the
absence of full length ABCB transporters, the proteins form two
distinct clusters which mimics the N- and C-terminal NBDs of full
length eukaryotic ABCB proteins, suggesting that evolutionary
pressure has acted on the T. vaginalis half transporters as it has on
the full transporters.
Non-transporting ABCE and ABCF proteins
ABCE sequences are absent from the eubacteria but present in
all Archaea and eukaryotes for which genomic sequencing
information is relatively complete. As expected, T. vaginalis
contains a single homologue of human ABCE, and of all the
ABC proteins the certainty that can be ascribed to TVAG_249850
as being ABCE is highest. T. vaginalis ABCE is 54–57% identical at
the amino acid sequence level to other eukaryotic ABCEs, and 39–
46% identical to those from Archaea (Figure 6). This degree of
conservation is remarkable, to date only Hsp70 has been shown to
have a similar level of conservation to homologues in both
eukaryotes and Archaea [26,38]. In spite of a structural description
of ABCE  a complete understanding of the function of ABCE
remains unresolved, although roles in translational control,
ribosome assembly, and ribosome recycling , have been
proposed. Clearly, its sequence conservation across the eukarya
and Archaea argues for a function critical to the evolution of cell
biology in these kingdoms .
T. vaginalis contains a larger number of predicted ABCF proteins
than other parasites listed in Table 2. Consistent with other
species’ ABCF proteins, the hypothetical T. vaginalis ABCF
subfamily is another group of non-transporters composed of two
fused NBD domains (NBD2) and lacking membrane-spanning
regions. Taxonomic BLAST searches with TVAG_427530
highlight the high level of conservation shown by the predicted
T. vaginalis ABCF proteins, with E values as low as 9e-112 and
identity as high as 42% with sequences from other species. Similar
analysis with TVAG_385840 indicate that this is the Trichomonas
homologue of yeast GCN20, an activator of eukaryotic transla-
tional initiation factor 2a-kinase (eIF2a-kinase) , showing 35%
sequence identity (p-value of 3.9e-88). Confirmation of the
function of Trichomonas ABCF proteins in translational control
awaits further experimentation.
Our analysis of the ABC transporters in T. vaginalis
demonstrates three key findings with broader impact for our
TVAG_605460, and TVAG_470720 are all similar with a dispersed pattern of staining. In contrast, the hydrogenosome membrane protein TOM 40-3
(B) was detected in discrete spherical organelles, consistent with the size and shape of the hydrogenosome. E the ABCD transporters of eukaryotes
for two distinct sub-families, one of which (grey boxed) has independent evidence to support endoplasmic reticulum localization, whereas the other
sub-family are peroxisomal localized.
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understanding of the parasite’s biology. The first is the absence
of full length ABC transporters. This is unique in eukaryotes for
which we have sufficient sequence data. All other species
described as early branching (e.g. mosses), and others classified
as Excavata contain full-length ABC transporter genes (see
footnote to Table 1). The absence of these from T. vaginalis,
taken together with our data here on the maintained sequence
separation of the half-transporters in the ABCA and ABCB sub-
families suggests that either the full-transporter gene was an
early loss in the evolution of T. vaginalis from a common
ancestor with other eukaryotes, or that gene fusion events that
produced full length ABC transporters in other extant eukary-
otes have not been selected for in T. vaginalis. Another finding is
the putative suppression of stop codons by Trichomonas. The
expression of the ABCA half-transporter TVAG_415980 and
TVAG_415990 as a single protein warrants further investiga-
tion of the mechanism for this suppression and its frequency.
Finally, our confocal microscopy work shows that sub-cellular
localization studies in T. vaginalis are accessible enabling further
proteomic classification of this organism.
eukaryotes show conservation of NBDs. Protein sequences
for ABCB transporters from T. vaginalis and other eukaryotes had
their TMD sequences removed, and in the case of full-length
proteins, the sequence was bisected into N- and C-terminal halves.
ABCB proteins in T. vaginalis and other
The alignment and boot-strapping were as described in Figure 1.
The N-terminal NBDs of eukaryotic full-length ABCB proteins
cluster as sequentially distinct from the C-terminal NBDs. Despite
their being no full-length ABCB proteins in T. vaginalis the ABCB
sequences also cluster into two sub-groups.
are written 59 to 39, with restriction sites encoded underlined.
List of primers used in this study. All primers
identifications are from TrichDB. The predicted length of each
primary sequence is given, in addition to predictions regarding the
number of transmembrane (TM) segments, predicted topology,
and the identification of classical ABC transporter sequence
motifs. Abbreviation used: EST – expressed sequence tag.
Predicted T. vaginalis ABC proteins. Gene
For preliminary analysis we thank Susan Jackson, Kahawalage P. P.
Senaratne, and Suraiya B. Abdul Munaff
Conceived and designed the experiments: CK SDD IDK. Performed the
experiments: CK KL KDW TJS. Analyzed the data: CK KDW SDD
IDK. Contributed reagents/materials/analysis tools: CK KL KDW TJS
SDD IDK. Wrote the paper: CK IDK.
Figure 6. The ABCE proteins of archaea and eukaroytes. Multiple sequence alignment and boot-strapping was performed as described in the
material and methods. The boot-strap values (percentages) are displayed on all branches.
Trichomonas ABC Transporters
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Trichomonas ABC Transporters
www.plosntds.org13 June 2012 | Volume 6 | Issue 6 | e1693