Immunological Dominance of Trypanosoma cruzi Tandem Repeat Proteins

Abstract

Proteins with tandem repeat (TR) domains have been found in various protozoan parasites, often acting as targets of B-cell
responses. However, the extent of the repeats within Trypanosoma cruzi, the causative agent of Chagas’ disease, has not been examined well. Here, we present a systematic survey of the TR genes
found in T. cruzi, in comparison with other organisms. Although the characteristics of TR genes varied from organism to organism, the presence
of genes having large TR domains was unique to the trypanosomatids examined, including T. cruzi. Sequence analyses of T. cruzi TR genes revealed their divergency; they do not share such characteristics as sequence similarity or biased cellular location
predicted by the presence of a signal sequence or transmembrane domain(s). In contrast, T. cruzi TR proteins seemed to possess significant antigenicity. A number of previously characterized T. cruzi antigens were detected by this computational screening, and several of those antigens contained a large TR domain. Further
analyses of the T. cruzi genome demonstrated that previously uncharacterized TR proteins in this organism may also be immunodominant. Taken together,
T. cruzi is rich in large TR domain-containing proteins with immunological significance; it is worthwhile further analyzing such characteristics
of TR proteins as the copy number and consensus sequence of the repeats to determine whether they might contribute to the
biological variability of T. cruzi strains with regard to induced immunological responses, host susceptibility, disease outcomes, and pathogenicity.

Full text

INFECTION AND IMMUNITY, Sept. 2008, p. 3967–3974 Vol. 76, No. 9
0019-9567/08/$08.00⫹0 doi:10.1128/IAI.00604-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Immunological Dominance of Trypanosoma cruzi Tandem Repeat Proteins
䌤
Yasuyuki Goto,
1
* Darrick Carter,
1,2
and Steven G. Reed
1
Infectious Disease Research Institute
1
and Protein Advances Inc.,
2
Seattle, Washington
Received 16 May 2008/Returned for modification 20 June 2008/Accepted 30 June 2008
Proteins with tandem repeat (TR) domains have been found in various protozoan parasites, often acting as
targets of B-cell responses. However, the extent of the repeats within Trypanosoma cruzi, the causative agent of
Chagas’ disease, has not been examined well. Here, we present a systematic survey of the TR genes found in
T.cruzi, in comparison with other organisms. Although the characteristics of TR genes varied from organism
to organism, the presence of genes having large TR domains was unique to the trypanosomatids examined,
including T.cruzi. Sequence analyses of T.cruzi TR genes revealed their divergency; they do not share such
characteristics as sequence similarity or biased cellular location predicted by the presence of a signal sequence
or transmembrane domain(s). In contrast, T.cruzi TR proteins seemed to possess significant antigenicity. A
number of previously characterized T.cruzi antigens were detected by this computational screening, and
several of those antigens contained a large TR domain. Further analyses of the T.cruzi genome demonstrated
that previously uncharacterized TR proteins in this organism may also be immunodominant. Taken together,
T.cruzi is rich in large TR domain-containing proteins with immunological significance; it is worthwhile
further analyzing such characteristics of TR proteins as the copy number and consensus sequence of the
repeats to determine whether they might contribute to the biological variability of T.cruzi strains with regard
to induced immunological responses, host susceptibility, disease outcomes, and pathogenicity.
Chagas’ disease results from infection, generally via contam-
inated blood or the bite of an infected insect, by the protozoan
parasite Trypanosoma cruzi, which can live in a variety of tis-
sues in the mammalian host. According to the World Health
Organization Special Programme for Research and Training in
Tropical Disease Report 2002, the disease is endemic in 18
countries in Central and South America. It is estimated that 16
to 18 million individuals are infected with T.cruzi, with 300,000
new cases per year, and the infection causes 21,000 deaths
annually. Chagas’ disease has an acute phase and a chronic
phase. Manifestations in the acute phase include swelling at
the infection site, fever, and hepatosplenomegaly. Following
an asymptomatic period after the acute phase, an estimated
32% of infected individuals develop chronic Chagas’ disease,
often leading to fatal damage to the heart and digestive tract.
Genes encoding proteins with tandem repeat (TR) domains,
defined here as two or more copies of an amino acid pattern,
have been found in a variety of organisms, from prokaryotes to
higher animals. TRs appear to be diverse and provide regular
arrays of spatial and functional groups (38). They are conspic-
uous in structural and cell surface proteins in some organisms
(38, 60). In contrast, previously characterized T.cruzi TR pro-
teins include trans-sialidase, ribosomal protein, flagellar pro-
tein FRA, and cytoplasmic protein CRA (14, 42, 49), which do
not seem to share functional characteristics. Also, the func-
tions of many T.cruzi TR proteins remain unknown due to a
lack of systematic characterization. Although the functions of
TR proteins are disparate and not confined to a single type of
protein, and a common time of expression or cellular localiza-
tion is not consistently observed, one feature appears to be
shared: they are often potent B-cell antigens. The immunolog-
ical significance of TR proteins during bacterial infections has
been reported (3, 31), and even some cancer antigens to which
patients show antibody responses contain TR domains (41, 47).
In some organisms, having a variety of TRs within a given
protein may play an important role in generating variability in
cell surface immunogens and adhesion molecules, thereby
evading the immune system or enhancing pathogenicity (27,
37, 43, 44, 60). In protozoan parasites, TR proteins often serve
as targets of B-cell responses (39, 54). Antibody responses to
TR proteins have been found in Chagas’ disease (14, 28, 34)
and other parasitic diseases such as leishmaniasis (10, 13) and
malaria (16, 17, 40). However, because the immunological
dominance of TR proteins is not restricted to protozoan par-
asites, systematic analyses of TR genes and proteins are re-
quired to see (i) if T.cruzi has more or fewer TR proteins than
other pathogens or organisms do and (ii) whether these TR
proteins have sequence similarity, a biased cellular location, or
shared immunological recognition motifs. For example, a ge-
nome scale analysis of Saccharomyces cerevisiae has revealed
that most genes containing TRs encode cell wall proteins (60).
In a previous study, we have demonstrated that TR proteins of
Leishmania infantum share immunological dominance (26).
This is the only systematic study of the immunological prop-
erties of protozoan parasite TR proteins, and it still remains
unclear whether other protozoan parasites, including T.cruzi,
possess TR proteins with such characteristics.
Here we performed a computational search for TR genes in
T.cruzi, in comparison with various parasitic protozoan (Leish-
mania major,L.infantum,Trypanosoma brucei,Plasmodium
falciparum,Toxoplasma gondii, and Entamoeba histolytica),
fungal (Candida albicans), bacterial (Salmonella enterica and
Mycobacterium tuberculosis), and human genomes. The analy-
sis revealed no biochemical but immunological characteristics
common in the T.cruzi TR proteins. As an indication of its
* Corresponding author. Mailing address: Infectious Disease Re-
search Institute, 1124 Columbia St., Suite 400, Seattle, WA 98104.
Phone: (206) 330-2519. Fax: (206) 381-3678. E-mail: ygoto@idri.org.
䌤
Published ahead of print on 14 July 2008.
3967

detection sensitivity, this computational method captured a
number of previously characterized antigens from T.cruzi sug-
gesting the immunological dominance of TR proteins. To fur-
ther validate the immunological significance of TR proteins
from protozoan parasites, we evaluated the antigenicity of pre-
viously uncharacterized T.cruzi TR proteins. The results dem-
onstrated that immunological recognition was a feature com-
mon to the T.cruzi TR proteins, whereas there were no
apparent similarities or biases in their sequences or predicted
cellular locations.
MATERIALS AND METHODS
Bioinformatic screening of TR genes. We obtained DNA sequence data for P.
falciparum 3D7 CDS (coding sequence) version 2.1.4. (without pseudogenes)
(22), L.major CDS version 5.2 (35), L.infantum CDS version 3.0 (51), and T.
brucei Tb927_CDSs_v4_nopseudo (9) from GeneDB (www.genedb.org) (30);
Trypanosoma cruzi Annotated CDS Release 5.1 (20) from TcruziDB (www
.tcruzidb.org/tcruzidb) (2); T.gondii Annotated CDS Release 4.2 from ToxoDB
(www.toxodb.org/toxo) (21); C.albicans open reading frame coding assembly 21
(36, 58) from The Candida Genome Database (www.candidagenome.org) (5); M.
tuberculosis Release R7 (15) from TubercuList (http://genolist.pasteur.fr/TubercuList/);
S.enterica serovar Typhi CT18 (50); and Homo sapiens (59) Hs36.2 CCDS
nucleotide 20070227 from the NCBI database (www.ncbi.nlm.nih.gov/projects
/CCDS/). Tandem Repeats Finder, a program to locate and display TRs in
DNA sequences, was used for these analyses (http://tandem.bu.edu/trf/trf
.html) (8). The program calculates the score according to selected character-
istics of the TR genes such as the period size of the repeat (i.e., the length of
the repeat unit), the number of copies aligned with the consensus pattern, and
the overall percentage of matches between adjacent copies of a pattern. Most
likely, a high score indicates that the gene possesses a large TR domain. In
this study, the genes were regarded as TR genes if the scores from the
Tandem Repeats Finder analysis were 150 or higher. The cutoff value of 150
is likely to eliminate genes with repeat domains shorter than 75 bp. When
more than one TR domain was found within a gene, only the domain with the
highest score was listed or used for further analyses and protein production.
Analyses of the TR genes of T.cruzi.The biochemical properties of each of the
bioinformatically selected T.cruzi TR proteins were further analyzed virtually for
(i) a protein’s molecular mass, isoelectric point, and hydrophobicity and the
presence of a signal sequence and a transmembrane domain; (ii) its known
antigenicity and/or functions by BLAST searches with both DNA and deduced
amino acid sequences against the NCBI database; and (iii) a mass spectrometry-
evidenced protein expression profile (6), available through the database Tcruz-
iDB. Biochemical characteristics such as average hydrophobicity, isoelectric
point, and molecular weight were calculated with the ProteinMachine software
package from Protein Advances Inc., Seattle, WA. To analyze the entire data-
base, a software interface programmed in C# created protein data files as
comma-separated values for export to Excel. Average hydrophobicity or hydro-
philicity plots of each sequence were determined with a modified Kyte-Doolittle
algorithm with scores ranging from 0.6 (most hydrophilic score possible) to 9.0
(most hydrophobic score possible). T.cruzi TR genes were analyzed for their
specificity for T.cruzi, i.e., whether a homologous gene or protein is found in
Leishmania or other organisms, by blasting the DNA and deduced amino acid
sequences against the NCBI database and GeneDB.
Expression of T.cruzi TR proteins. Partial TR domains containing multiple
repeat units were either PCR amplified or synthesized. Partial TR domains of
Tc00.1047053510827.40 (designated Tc2 in this study), Tc00.1047053511821.179
(Tc3), Tc00.1047053509157.120 (Tc4), and Tc00.1047053508119.200 (Tc6) were
amplified by PCR with T.cruzi total DNA and the following primer sets: Tc2, 5⬘
CAA TTA CAT ATG AGC GCG AGC ACC GCC TGG and 3⬘CAA TTA
AAG CTT CTA GTC GCT CAA CAA CCG CAT G; Tc3, 5⬘CAA TTA CAT
ATG GAG AAC GAG GAG CTG CGT G and 3⬘CAA TTA AAG CTT CTA
CGC ACG AAG CTC CTC CAG; Tc4, 5⬘CAA TTA CAT ATG CCG GAG
ACA GCC TCA GTC and 3⬘CAA TTA AAG CTT CTA CGC GTG ACC GTC
CTC GTC; Tc6, 5⬘CAA TTA CAT ATG GCA ACG GAC GAG TTG and 3⬘
CAA TTA AAG CTT CTA GAG CGC AGT CGC ATC CCT G. Partial TR
domains of Tc00.1047053511557.50 (Tc1), Tc00.1047053510217.10 (Tc8), Tc00.
1047053504019.3 (Tc9), Tc00.1047053506495.40 (Tc10), Tc00.1047053506491.20
(Tc12), Tc00.1047053506559.559 (Tc13), and Tc00.1047053507049.119 (Tc15)
were synthesized by Blue Heron Biotechnology, Inc. (Bothell, WA). The ampli-
fied PCR products or synthesized oligonucleotides were inserted in frame with
the six-His tag of vector pET-28a. The vectors were then transformed into the
Escherichia coli Rosetta strain. The transformed E.coli cells were grown in 2⫻
yeast extract-Tryptone medium, and expression of the recombinant proteins was
induced by cultivation with 1 mM isopropyl-␤-D-thiogalactopyranoside for 3 h.
After lysing cells by sonication and centrifuging them at 10,000 ⫻g, the super-
natants were used for purifying the proteins as six-His-tagged proteins with
Ni-nitrilotriacetic acid agarose (Qiagen Inc., Valencia, CA). Proteins were bound
to the resin, washed with sodium deoxycholate-containing buffer, and eluted with
buffer containing 250 ␮M imidazole. The eluted protein was dialyzed against
phosphate-buffered saline (pH 7.4), and the concentration of the purified protein
was measured by the bicinchoninic acid protein assay (Pierce Biotechnology Inc.,
Rockford, IL). The purity of the proteins was assessed by Coomassie blue
staining following sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Antibody ELISA. The expressed T.cruzi TR proteins were analyzed for sero-
reactivity with sera from Brazilian or Ecuadorian Chagas’ disease patients (n⫽
24). Sera from Brazilian visceral leishmaniasis (VL) patients (n⫽16) and
healthy Brazilian people were used as controls. Proteins were diluted in enzyme-
linked immunosorbent assay (ELISA) coating buffer, and 96-well plates were
coated with 200 ng of individual recombinant antigens, followed by blocking with
phosphate-buffered saline containing 0.05% Tween 20 and 1% bovine serum
albumin. Plates were incubated sequentially with human serum samples (1:200
dilution) and with horseradish peroxidase-conjugated anti-human immunoglob-
ulin G (Rockland Immunochemicals, Inc., Gilbertsville, PA). The plates were
developed with tetramethylbenzidine peroxidase substrate (Kirkegaard & Perry
Laboratories, Gaithersburg, MD) and scanned with a microplate reader at 450
nm (570-nm reference). Three additional recombinant proteins were tested as
controls: T.cruzi sterol 24-c methyltransferase (TcSMT) as a conserved antigen
between Trypanosoma and Leishmania species (24), rK39 as a Leishmania-spe-
cific TR antigen (13), and CRA as a T.cruzi-specific TR antigen (42). Statistical
analyses were performed to compare the reactivity of Chagas’ disease patient
sera to individual antigens with that of VL patients or healthy donors by either
unpaired ttest or Mann-Whitney test based on whether the data sets have a
Gaussian distribution.
RESULTS
Search of TR genes from the T.cruzi genome database. With
the exception of S.enterica, all of the organisms examined in
this study had TR genes comprising ⬎1% of their genomes
(Table 1). A markedly higher prevalence of TR genes was
found in P.falciparum and T.gondii (24.61 and 5.70%, respec-
tively). In contrast, the prevalence of TR genes in the trypano-
somatid parasites was no greater than in E.histolytica,C.al-
bicans,M.tuberculosis, and H.sapiens. TR genes with a score
of ⱖ2,000 are likely to have a TR domain ⱖ1,000 bp long. The
prevalence of these genes in the whole genomes was higher in
the trypanosomatid and the apicomplexan parasites than in the
other examined pathogens or H.sapiens. The trypanosomatid
parasites, including T.cruzi, had a higher prevalence of TR
genes scoring ⱖ2,000 in the whole genomes than the apicom-
plexans, although the apicomplexan parasites, especially P.fal-
ciparum, were richer in total TR genes. The trypanosomatid
parasites seemed to have a preference for such large TR genes
according to a higher prevalence of TR genes, scoring ⱖ2,000
in all TR genes, and higher mean and median TR scores in
these species compared with others examined in this study.
The high prevalence of large genes in the trypanosomatid
and the apicomplexan parasites may reflect the high preva-
lence of large TR regions in the genes of those parasites. S.
enterica and M.tuberculosis had average gene sizes smaller
than those of the others examined in this study (Fig. 1). C.
albicans and H.sapiens showed gene size distributions over-
lapping those of the trypanosomatid and the apicomplexans,
up to 10,000 bp. The ratios of genes over 10,000 bp, however,
were much higher in the trypanosomatid and the apicomplexan
3968 GOTO ET AL. INFECT.IMMUN.

parasites (0.75 to 1.95%) than any other species, including C.
albicans and H.sapiens (0.01 to 0.05%).
The period size of the TR refers to the length of the repeat
unit. The distribution of the TR period sizes varied among
these organisms (Fig. 2). TR genes in L.infantum were diver-
gent in their repeat motifs, as their period sizes ranged widely,
from 3 to 498, and no particular period size dominated (i.e.,
constituted more than 10%) in any of the TR genes. A similar
pattern was found in L.major and T.brucei (data not shown).
T.cruzi also had a wide distribution of period sizes, although
the overall distribution was shifted to the left compared to L.
infantum, indicating that T.cruzi TR genes are also divergent.
In contrast, 76.2% of the P.falciparum TR genes had period
sizes of ⱕ36 bp and 93.4% had period sizes of ⱕ72 bp. Al-
though ⬎10% of the TR genes in C.albicans had a period size
of 51 (single peak, Fig. 2), the period sizes in other TR genes
were widely divergent, as seen in Leishmania and Trypano-
soma. There was a more restricted pattern in the period sizes
of human TR genes, which will be discussed below.
Search for functional similarity in T.cruzi TR proteins. We
examined TR genes in T.cruzi,C.albicans,M.tuberculosis, and
H.sapiens for functional similarities. There were 87 TR genes
identified in C.albicans, 25 (28.7%) of which encoded cell
FIG. 1. Gene size distribution of protozoan parasites and other
organisms. All genes in the genome of were sorted by their sizes with
100-bp intervals.
FIG. 2. Unique patterns in period sizes of TR genes. The yaxis
shows the prevalence of TR genes having a particular period size
among all of the TR genes in the same species.
TABLE 1. Numbers of TR genes in selected pathogens and H.sapiens
Species No. of
genes
No. with TR score of: Total TR genes Genes with TR score of ⱖ2,000 Mean TR
score
Median TR
Score
150–499 500–999 1,000–1,999 2,000–4,999 5,000–9,999 ⱖ10,000 No. % of total
genes No. % of total
genes
% of total
TR genes
L.major 9,218 44 15 16 15 10 3 103 1.12 28 0.30 27.20 2,846 715
L.infantum 8,184 35 9 12 21 7 7 91 1.11 35 0.43 38.50 2,651 1,055
T.brucei 8,161 39 18 21 30 9 5 122 1.49 44 0.54 36.10 2,482 1,122
T.cruzi 19,605 154 48 83 69 3 0 357 1.82 72 0.37 20.20 1,183 717
P.falciparum 5,387 1,153 135 29 7 2 0 1,326 24.61 9 0.17 0.70 356 277
T.gondii 7,793 340 53 34 17 0 0 444 5.70 17 0.22 3.80 481 253
E.histolytica 9,905 180 37 2 0 0 0 219 2.21 0 0.00 0.00 342 273
C.albicans 6,107 72 7 5 3 0 0 87 1.42 3 0.05 3.40 448 226
S.enterica 4,395 5 1 1 0 0 0 7 0.16 0 0.00 0.00 594 479
M.tuberculosis 4,005 43 4 2 0 0 0 49 1.22 0 0.00 0.00 334 230
H.sapiens 17,751 235 141 46 6 1 1 430 2.42 8 0.05 1.90 608 433
a
Number of genes with TR scores of ⱖ2,000.
VOL. 76, 2008 T.CRUZI TANDEM REPEAT ANTIGENS 3969

surface proteins such as the agglutinin-like sequence family
(33). C.albicans TR genes with higher scores were more likely
to encode cell surface proteins: 10 of 15 TR genes with a score
of ⱖ500 and 7 of 8 TR genes with a score of ⱖ1,000 encoded
cell surface proteins (the one exception was polyubiquitin).
Such a high prevalence of TR genes encoding cell surface
proteins has been shown in another fungal species, S.cerevisiae
(60). In M.tuberculosis, 49 genes were identified as containing
TRs. Thirty-five (71.4%) of those were categorized in either
the PE family polymorphic GC-rich repetitive sequence (23
genes, 46.9%) or the PPE family (12 genes, 24.5%), values that
were considerably higher than the prevalence in all M.tuber-
culosis genes (1.6 and 1.7% for the PE family polymorphic
GC-rich repetitive sequence and the PPE family, respectively).
The PE and PPE families, the major source of divergence
between the genomes of M.tuberculosis and M.bovis, which
are otherwise ⬎99% similar, often have multiple copies of
repeat motifs and participate in antigenic variation and intra-
macrophage survival of M.tuberculosis (15, 29, 45, 52). As
described above, 215 (50%) of 430 TR genes of H.sapiens had
period sizes of 84 bp or multiples of 84 bp (164, 252, 368, and
420 bp). It was revealed that most of these (209 genes, 48.6%
of all H.sapiens TR proteins) were zinc finger proteins, one of
the largest families of human proteins, composing ⬃2% of the
human proteome. The zinc finger proteins have the character-
istic, approximately 30-amino-acid-long, zinc finger motifs as
sites of binding to typically DNA, often repeated motifs within
a molecule (7, 59).
In contrast, such functional similarities as seen in C.albi-
cans,M.tuberculosis,orH.sapiens were not evident in TR
proteins of T.cruzi, especially in those with high TR scores. Of
357 TR genes identified from T.cruzi (Table 1), 14 were ap-
parently not full length, as they lacked either a start or a stop
codon, and were not analyzed further. The mean and median
CDS lengths of the remaining 343 T.cruzi TR genes were 2,220
and 1,920 bp, respectively, and were larger than those of the
average for all T.cruzi genes (1,513 and 1,152 bp, respectively
(20). The prevalences of proteins having predicted signal se-
quences or transmembrane domains among these T.cruzi TR
proteins were 27.4 and 31.1%, respectively, and were slightly
higher than those in all T.cruzi proteins (16.0 and 26.4%,
respectively [data obtained from TcruziDB]). Higher preva-
lences of TR proteins with predicted signal sequences (41.3%)
or transmembrane domains (40.1%) were found in those hav-
ing scores of ⬍500, but they were not apparent in those with
scores of ⱖ500 (16.6 and 23.8%, respectively). As shown in
Table 2, predicted trans-sialidase, mucins, and mucin-associ-
ated surface proteins, the three largest gene families in T.cruzi
(20), constituted ⬎40% of the T.cruzi TR proteins with a score
of ⬍500. In contrast, the category of TR proteins scoring ⱖ500
did not have a high prevalence of any particular family and the
majority was categorized as hypothetical proteins lacking
known functional domains. Unlike TR proteins with lower TR
values (⬍500), those with a ⱖ500 score had a higher mean
hydrophilicity compared with that of all T.cruzi proteins (Ta-
ble 2, P⬍0.0001 by Mann-Whitney test).
Immunological dominance of T.cruzi TR. For immunolog-
ical analyses of T.cruzi TR, we focused on the 203 (1.04%)
genes with a score of ⱖ500. Because the nearly 20,000 T.cruzi
genes analyzed in this study are from its diploid genome (a
haploid T.cruzi genome is estimated to have ⬃12,000 protein-
coding genes) (20), many of the genes, TR genes included, are
represented twice in the pool of analyzed genes. After consol-
idating TR genes with 70% or greater identity, 106 genes with
different TR domains were identified (the top 20 sequences are
shown in Table 3). Of the 106 genes, 10 encoded previously
characterized antigenic repeat motifs: clone 36, CRA, TcD,
B12, B13, SAPA, FRA, TcLo1.2, TcE, and antigen 38 (1, 14,
18, 28, 32, 34, 42). The remaining 96 genes are previously
uncharacterized as encoding antigens.
To examine whether previously uncharacterized T.cruzi TR
proteins are also antigenic and potentially useful as diagnos-
tics, we cloned and expressed recombinant forms of nine of the
remaining proteins listed in Table 3. Two with homology to
Leishmania proteins were excluded to avoid cross-reactivity
with antibody from leishmaniasis patients. Eight of these de-
tected antibodies in Chagas’ disease patient sera, and the re-
sponses were disease specific; the antibody recognition pat-
terns by these antigens were similar to that of CRA and
contrast with that of TcSMT or rK39 (Fig. 3). Therefore, at
least 13 of the top 20 TR proteins, including the five previously
characterized antigens, are antigenic.
DISCUSSION
TR proteins have been implicated in the ability to influence
host immune responses to protozoan parasites and possibility
to contribute to parasite survival. We previously described the
antigenicity of TRs in Leishmania (26). In this study, we dem-
onstrated that TR proteins from the related trypanosomatid
parasite T.cruzi are also immunodominant, while there is little
or no sequence similarity or apparent bias in cellular location.
These features of TR proteins in the trypanosomatid parasites
contrast with those in C.albicans,M.tuberculosis, and H.sa-
piens, which have biased cellular locations or belong to func-
tional protein families. The immunological dominance of TR
proteins in Leishmania and Trypanosoma parasites is sup-
ported by the fact that antigens of these parasites identified
through serological screening of expression libraries are en-
riched for such proteins relative to the entire proteome. Forty-
TABLE 2. Characteristics of T.cruzi TR proteins
Parameter
No. of proteins
(% of total) with: Total no. of
T.cruzi proteins
(% of total)
c
150–499
TRs
a
ⱖ500 TRs
b
Presence of:
Signal peptide 62 (41.3) 32 (16.6) 3,141 (16.0)
Transmembrane
domain(s)
61 (40.1) 46 (23.8) 5,169 (26.4)
Gene products:
trans-Sialidase 20 (13.3) 14 (7.3) 735 (3.7)
MASP
d
24 (16.0) 3 (1.6) 938 (4.8)
Mucin 22 (14.7) 2 (1.0) 662 (3.4)
Hypothetical protein 38 (25.3) 131 (67.9) 11,171 (57.0)
a
No. of genes, 150. Mean hydrophobicity score, 4.124.
b
No. of genes, 193. Mean hydrophobicity score, 3.868 (P⬍0.0001 by Mann-
Whitney test compared with hydrophobicity of all T.cruzi proteins).
c
No. of genes, 19,605. Mean hydrophobicity score, 4.177.
d
MASP, mucin-associated surface proteins.
3970 GOTO ET AL. INFECT.IMMUN.

four percent of Leishmania antigens identified by serological
screening of the expression library were TR proteins, whereas
such proteins compose only 1% of the proteome (25, 26).
There are 37 T.cruzi proteins cited as defined serological
antigens in the review article by da Silveira et al. (19), and 9
(24%) of them are TR proteins. When the prevalence of TR
proteins in the T.cruzi proteome is considered (⬍2%), the
likelihood that such proteins are antigenic is significantly
FIG. 3. Antigenic properties of T.cruzi TR proteins. Newly identified TR proteins and previously characterized Leishmania-specific antigen
rK39, T.cruzi-specific antigen CRA, and conserved antigen TcSMT were evaluated by ELISA for reactivity with sera from VL patients (n⫽16),
Chagas’ disease patients (n⫽24), and healthy controls (n⫽8). OD, optical density; ns, not significant; *,P⬍0.05; **,P⬍0.01; ***,P⬍0.001;
****,P⬍0.0001 (by either unpaired ttest or Mann-Whitney test).
TABLE 3. Top 20 TR genes of T.cruzi
Gene
no. Identity
No. of
similar
TR genes
Size
(kDa)
PS
a
(bp) CN
b
TR
score %TR
c
MS
d
expression
Export
property
e
Seroreactivity
f
Leishmania
homolog
g
Name
h
Reference
1 Tc00.1047053510217.10 11 163 195 21.7 7,161 100 No No Unknown No Tc8
2 Tc00.1047053511557.50 3 284 126 36.4 6,969 61 No TM Unknown No Tc1
3 Tc00.1047053504019.3 1 206 42 103.4 6,904 80 T No Unknown No Tc9
4 Tc00.1047053511633.79 1 126 114 22 4,849 77 AEMT SP Clone 36 No 34
5 Tc00.1047053510827.40 0 158 486 7.1 4,802 88 No No Unknown No
6 Tc00.1047053506495.40 3 106 45 65.1 4,613 93 No SP, TM Unknown No Tc10
7 Tc00.1047053506303.80 1 141 126 24.6 4,569 77 No No CRA No 42
8 Tc00.1047053509265.110 4 185 15 178 4,386 51 No TM TcD No 14
9 Tc00.1047053506491.20 2 399 126 71.2 4,384 41 No No Unknown No Tc12
10 Tc00.1047053506559.559 0 NA 72 30.9 4,017 29 M NA
i
Unknown No Tc13
11 Tc00.1047053508831.150 1 171 60 59.1 3,858 71 No No B12 Yes 28
12 Tc00.1047053511671.60 1 125 36 72.4 3,842 69 No No B13 No 28
13 Tc00.1047053511821.179 1 144 105 21.2 3,753 60 AEMT No Unknown No Tc3
14 Tc00.1047053509157.120 1 116 126 15.5 3,555 62 No No Unknown No Tc4
15 Tc00.1047053503617.20 1 218 30 99.9 3,524 51 No No Unknown Yes
16 Tc00.1047053511805.20 1 83 318 5.5 3,477 85 No No Unknown No
17 Tc00.1047053509269.4 1 150 27 111.1 3,392 73 No No Unknown Yes
18 Tc00.1047053506777.110 1 157 117 15.3 3,267 44 A No Unknown No
19 Tc00.1047053508119.200 0 103 117 14.1 3,191 55 No No Unknown No Tc6
20 Tc00.1047053507049.119 1 128 60 30.5 3,160 49 No No Unknown No Tc15
a
PS, period size.
b
CN, copy number.
c
Percentage of TR domains in nucleotide sequence of entire gene.
d
MS, mass spectroscopy-based protein expression evidence. A, amastigote; E, epimastigote; M, metacyclic trypomastigote; T, trypomastigote.
e
Presence of predicted signal sequence (SP) or transmembrane domain(s) (TM).
f
Unknown: serological reactivity not reported before; other entries are the names of previously characterized antigens.
g
Presence of Leishmania proteins with homologous repeat motifs with 60% amino acid sequence identity as the cutoff.
h
Names of recombinant proteins designated for this study.
i
NA, no data available.
VOL. 76, 2008 T.CRUZI TANDEM REPEAT ANTIGENS 3971

higher than that observed for non-TR proteins (P⬍0.0001 by
Fisher’s exact test). The role of such proteins in parasite sur-
vival remains to be defined. A2, one of the Leishmania TR
proteins, was shown to be a virulence factor of L.donovani, the
causative agent of VL; it has more than 40 copies of a 10-
amino-acid repeat, whereas A2 in L.major, which causes cu-
taneous leishmaniasis, has only 1 copy of the repeat, suggesting
that multiple repeats in the A2 protein may play a role in the
visceralization of the parasites (62, 63). We have found other
pairs of TR genes in these Leishmania species; overall se-
quences are highly conserved in both non-TR and TR do-
mains, with the major difference being in the copy number of
the repeat (data not shown), suggesting that the chromosome
bias was set before the divergence of these species or selective
pressure on certain alleles caused the expansion or loss of TR
regions in ancestor genes. Genetically distinct strains of T.cruzi
are responsible for different clinical syndromes in humans (12,
46, 48). For example, the T.cruzi Z12 zymodeme tends to
cause the acute form of Chagas’ disease, whereas the Z1 zy-
modeme preferentially causes chronic disease in humans (48).
It is not known whether variability in TR genes is a primary
factor giving different T.cruzi strains or zymodemes divergent
characteristics relating to clinical outcomes, disease-induced
immune responses, and preference for species of the insect
vector reduviid bugs. Taken together, it is worthwhile analyz-
ing characteristics of TR proteins such as the copy number and
consensus sequence of the repeat(s) to determine whether they
might explain certain biological properties of the parasite and
possibly susceptibility to drugs.
Computational searches for such TR proteins may facilitate
identifying novel antigens from parasite genomes. Identifica-
tion of B-cell antigens from pathogens may facilitate the de-
velopment of diagnostic tests or vaccines. Diagnostic methods
for VL and Chagas’ disease often rely on the detection of
parasite-specific antibodies (53, 56, 57). In Plasmodium,TR
antigens including circumsporozoite protein and merozoite
surface antigen 1 are promising malaria vaccine candidates (23,
55). Traditionally, such targets of B-cell responses have been
identified from parasites through serological screening of an
expression library or by immunoblotting of crude lysate sepa-
rated by two-dimensional gel electrophoresis. In contrast, only
a few attempts have been made to computationally predict
serological antigens of pathogens from the proteome based on
their sequences, such as predicting secreted or surface proteins
(4, 11) and identifying proteins with ␣-helical coiled-coil do-
mains (61). Although the prediction of secreted or surface
proteins has shown some promise in identifying antigens from
T.cruzi (11), it may not be powerful enough to reduce the
number of candidates to a practical level when dealing with the
whole genome. There are 3,141 T.cruzi genes containing se-
quences encoding predicted signal peptides, 5,169 with trans-
membrane domains, and 1,776 containing both. Because a
signal sequence is either present or not, it is impossible to
further prioritize these potentially secreted proteins. There-
fore, new bioinformatic tools to screen sequences for antigen-
encoding genes are needed and searching for TRs may serve as
a powerful method of discovering novel antigens in combina-
tion with other computational methods.
There is a real need for improved diagnostics for Chagas’
disease. T.cruzi parasites are not readily detected during
chronic infection. Therefore, indirect methods are required for
the diagnosis of Chagas’ disease patients and screening for T.
cruzi-contaminated samples. Serological tests for Chagas’ dis-
ease include the indirect fluorescent-antibody test and an
ELISA with whole-cell or recombinant antigens. The Ortho T.
cruzi ELISA Test System (www.orthoclinical.com/chagas
/elisaTestSystem.aspx) is the first such test approved by the
FDA and is used for blood screening. However, serological
tests that use T.cruzi whole-cell lysate have cross-reactivity
with leishmaniasis and other patient sera. This is likely because
a number of proteins are conserved between the related ki-
netoplastid T.cruzi and Leishmania parasites. Leishmania is
endemic in South America, often overlapping in distribution
with T.cruzi. For this reason, we focused on T.cruzi TR
proteins without homology to Leishmania proteins, and such
antigenic proteins will be useful in the development of more
accurate diagnostic tests for Chagas’ disease.
ACKNOWLEDGMENTS
We thank Randy Howard for critical comments and Raodoh Mo-
hamath and Alex Picone for technical assistance.
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Editor: W. A. Petri, Jr.
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