Parasitology International 65 (2016) 175–179

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Parasitology International

journal homepage: www.elsevier.com/locate/parint

Morpholino antisense oligo inhibits trans-splicing of pre-inositol

1,4,5-trisphosphate receptor mRNA of Trypanosoma cruzi and suppresses
parasite growth and infectivity☆
Muneaki Hashimoto a,⁎, Takeshi Nara a, Toshihiro Mita a, Katsuhiko Mikoshiba b,c
a
Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
b
Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Saitama 351-0198, Japan
c
Calcium Oscillation Project, International Cooperative Research Project and Solution-Oriented Research for Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama
332-0012, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Morpholino antisense oligos (MAOs) are used to investigate physiological gene function by inhibiting gene
Received 9 October 2015 translation or construction of specific alternative splicing variants by blocking cis-splicing. MAOs are attractive
Received in revised form 15 November 2015 drug candidates for viral- and bacterial-infectious disease therapy because of properties such as in vivo stability
Accepted 6 December 2015
and specificity to target genes. Recently, we showed that phosphorothioate antisense oligos against Trypanosoma
Available online 8 December 2015
cruzi inositol 1,4,5-trisphosphate receptor (TcIP3R) mRNA inhibit the parasite host cell infection. In the present
Keyword:
study, we identified the spliced leader (SL) acceptor of pre-TcIP3R mRNA and synthesized MAO, which inhibited
Trypanosoma cruzi trans-splicing of the transcript (MAO-1). MAO-1 was found to inhibit the addition of SL-RNA to pre-TcIP3R mRNA
Morpholino antisense oligo by real-time RT-PCR analysis. Treatment of the parasites with MAO-1 significantly impaired the growth and
Inositol 1,4,5-trisphosphate receptor infectivity into host cells. These results indicate that MAO-1 is a potential novel drug for Chagas disease and
Trans-splicing that MAOs inhibiting trans-splicing can be used to investigate the physiology of trypanosomal genes leading to
the development of novel drugs.
© 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Trypanosoma cruzi is a parasitic protist that causes Chagas disease in
Latin America. Approximately 8 million people are infected with the
parasites [1]. No practical drug or vaccine is available for Chagas disease;
thus, these urgently need to be developed.
Morpholino antisense oligonucleotides (MAOs) are synthetic oligo-
mers composed of the same 4 bases as DNA, but with a modified back-
bone that makes them resistant to nuclease [2]. MAOs are designed to
inhibit translation or to prevent proper cis-splicing of mRNA [3]. MAOs
are considered ideal drugs for several diseases, including genetic dis-
eases such as Duchenne muscular dystrophy [4] and various bacterial-
[5–8] and viral-infectious diseases [9–14]. Recently, treatment of
T. cruzi infections with phosphorothioate antisense oligonucleotides
(S-oligos) against the parasite inositol 1,4,5-trisphosphate receptor
(TcIP3R) mRNA causes inhibition of trypomastigote invasion into the
host cells, suggesting that antisense therapy may also be effective on
Chagas disease [15].
The genome in trypanosomatids is constitutively transcribed into
long polycistronic primary transcripts (pre-mRNAs) followed by
processing with the spliced leader (SL) RNA (trans-splicing) and
maturation of mRNA for protein-coding genes [16,17]. Theoretically,
MAOs, which bind to the SL acceptor (AG dinucleotides located up-
stream of 5′-UTR), may prevent trans-splicing and therefore impair
mRNA maturation.
In the present study, treatment of the parasites with MAO, which
was designed to bind to the SL acceptor region of pre-TcIP3R mRNA,
inhibited the parasite growth and infectivity into host cells. This is
the first report that shows that MAOs could be used to inhibit
trans-splicing in trypanosomatids, which may provide new evidence
for developing anti-trypanosomal drugs.

2. Materials and methods

☆ All authors disclose no actual or potential conflict of interest including any financial,
personal or other relationships with other people or organizations within three years of 2.1. Parasite and host cell culture
beginning the submitted work that could inappropriately influence, or be perceived to
influence, their work.
⁎ Corresponding author.
Epimastigotes of T. cruzi Tulahuen strain were cultured as de-
E-mail addresses: muneaki@juntendo.ac.jp (M. Hashimoto), tnara@juntendo.ac.jp scribed [18]. Mammalian stages of the parasite were maintained in
(T. Nara), tmita@juntendo.ac.jp (T. Mita), mikosiba@brain.riken.jp (K. Mikoshiba). in vitro culture by using HeLa cells as previously described [19].

http://dx.doi.org/10.1016/j.parint.2015.12.001
1383-5769/© 2015 Elsevier Ireland Ltd. All rights reserved.
176 M. Hashimoto et al. / Parasitology International 65 (2016) 175–179

Fig. 1. Design of MAO inhibiting trans-splicing of pre-TcIP3R mRNA. (A) Schematic representation of pre-TcIP3R mRNA. ATG indicates the initiation codon of TcIP3R mRNA. The SL acceptor
(AG dinucleotides) is located 279 nucleotides upstream from the initiation codon. MAO for trans-splicing inhibition (MAO-1) is complementary to the region containing the 5′-UTR, SL
acceptor, and intergenic region. (B) The nucleotide sequences of the TcIP3R gene before (a) and after (b) trans-splicing. The protein coding sequences are shown in small letters, and
the intergenic region and the 5′-UTR are shown in capital letters. The start codon (atg) is underlined. (a) The SL-acceptor (AG dinucleotide) is underlined. The MAO-1 sequence
corresponds to the complementary sequence of the nucleotides shown in italics. (b) The SL sequence is shown in italics. The PCR primer binding sites for real-time RT-PCR are boxed.

2.2. Identification of the SL acceptor site of TcIP3R mRNA
Total RNA was isolated from epimastigotes by using the Total RNA
Isolation Mini Kit (Agilent Technologies, Santa Clara, CA), followed
by the synthesis of cDNA using SuperScript® III First-Strand Synthe-
sis Super Mix and random hexamers (Life Technologies, Inc., Rock-
ville, MD). RT-PCR was performed with the forward primer specific
for the SL sequence (5′-accgctattgatacagtttc-3′), reverse primer spe-
cific for the TcIP3R coding region (+ 611) (5′-caaaagcgttgatgctac-3′),
and KOD-Plus-Neo (TOYOBO Co., Ltd., Osaka, Japan). Direct sequenc-
ing was performed with the PCR product as the template and a prim-
er specific for TcIP3R gene (+ 201) (5′-cttgtgcctttgctgca-3′), using
GenomeLab™ Dye Terminator Cycle Sequencing with Quick Start
Kit and CEQ™ 8000 Genetic Analysis System (Beckman Coulter,
Inc., Brea, CA).
2.3. MAO synthesis and treatment
MAOs were synthesized by Gene Tools, LLC (Philomath, OR). To in-
hibit addition of SL-RNA to pre-TcIP3R mRNA, MAO-1 was designed
against the part containing the SL acceptor of pre-TcIP3R mRNA
(− 294 to − 270 bp) (5′-ggttgtttccttgttttttgttgtt-3′). For the negative
control, Standard Control Oligo Classic was purchased (Gene Tools).
Epimastigotes or trypomastigotes were treated with 10 μM MAOs,
which was denatured at 65 °C for 10 min and quickly chilled on ice,
for the indicated time.
 M. Hashimoto et al. / Parasitology International 65 (2016) 175–179 177

Fig. 2. Effect of MAO-1 treatment on epimastigote growth. MAO-1 or control oligo was
added to epimastigotes in LIT medium (2.5 × 105 cells/mL), and the number of parasites
was counted after 6 d. Data shown are the mean ± S.D. of 3 independent experiments.
Double asterisks represent a statistical significance of P b 0.01 compared with the control
oligo group.

2.4. Real-time RT-PCR

Epimastigotes cultured in 24 well plates (2 × 107 cells/500 μL) were

treated with 10 μM MAOs for 12 h at 26 °C followed by isolation of total
RNA and synthesis of cDNA as described above. Real-time RT-PCR was
performed using a Thermal Cycler Dice Real Time System and SYBR®
Premix Ex Taq™ (Tli RNaseH Plus) (Takara Bio Inc., Shiga, Japan).
Real-time RT-PCR was performed with SL-RNA-added TcIP3R-specific
primers, 5′-cgctattattgatacagtttctg-3′ (sense) and 5′-agcaacgaccgctatta
tgg-3′ (antisense), or TcIP3R ORF-specific primers, 5′-ggagcacttcagcaacat
ca-3′(sense) and 5′-gcagccagtcgtaggagaac-3′ (antisense), or T. cruzi
beta-tubulin-specific primers, 5′-tttgtcggcaacaacacctg-3′ (sense) and 5′-
ctagtactgctcctcctcgt-3′ (antisense).

2.5. Statistical analysis

Statistical analysis was performed with Sigma Plot ver. 1.2 software
(Systat Software, Inc., San Jose, CA) by using Student's t-test.

3. Results and discussion

3.1. Identification of the SL acceptor of pre-TcIP3R mRNA
To design a MAO that inhibits the addition of SL-RNA to pre-TcIP3R
mRNA, AG dinucleotides representing the SL acceptor were identified.
Then, RT-PCR was performed against cDNA from T. cruzi epimastigotes
as the template, with a forward primer specific for SL-RNA and reverse
primer specific for a part of TcIP3R protein-coding region. The PCR prod-
uct was sequenced, and the SL acceptor site was identified by determin-
ing the position of the SL-RNA sequence. The sequence of the PCR
product was determined by direct sequencing without cloning in a plas-
mid vector. The SL acceptor was found to be the AG dinucleotides, which
were located 279 nucleotides upstream from the initiation codon of
TcIP3R mRNA (Fig. 1). To study eukaryote cells containing introns
in these genomic DNAs, MAOs were designed to bind to the region
containing cis-splicing donor or acceptor sites, which inhibit mRNA
maturation or construction of specific alternative splicing variants [3].
We attempted to apply this methodology with MAOs to inhibit
trypanosomal trans-splicing. MAO, which is 25 bases in length and com-
plementary to the SL acceptor site of pre-TcIP3R mRNA, was synthesized
(MAO-1).
Fig. 3. Evaluation of the inhibitory effect of MAO-1 on SL-RNA addition to pre-TcIP3R
mRNA by real-time RT-PCR. Epimastigotes (4 × 107 cells/mL) were treated with MAO-1
or control oligo (10 μM) for 12 h. Real-time RT-PCR analysis of relative expression of SL-
RNA-added TcIP3R (A) and TcIP3R ORF (B) mRNA in each parasite expressed as a fold
change. Data shown are the mean ± S.D. of 3 independent experiments. Triple asterisks
represent a statistical significance of P b 0.001.
3.2. MAO-1 inhibits epimastigote growth
Previously, we reported that when the expression level of TcIP3R de-
creased to two-thirds of that of the wild type, epimastigote growth was
significantly impaired [20]. It was expected that epimastigote growth
would be inhibited when TcIP3R was knocked-down with antisense
oligo nucleotides. After MAO-1 was added to epimastigotes in the cul-
ture medium, the number of the parasites was counted (Fig. 2). As the
negative control, the number of epimastigotes treated with a pre-
made negative control morpholino oligo (control oligo) was also count-
ed. Epimastigotes (2.5 × 105 cells/mL) were treated with MAO-1 or con-
trol oligo (10 μM), and the number of parasites was compared after 6 d.
The growth of parasites treated with MAO-1 was significantly lower
178 M. Hashimoto et al. / Parasitology International 65 (2016) 175–179
Fig. 4. Effect of MAO-1 treatment on trypomastigote infectivity into host cells.
Trypomastigotes (4 × 105) were treated with 10 μM of control oligo or MAO-1, and
were infected with 4 × 104 HeLa cells for 24 h at 37 °C (multiplicity of infection
(MOI) = 10). For calculation of trypomastigote infectivity, the number of intracellular
amastigotes in 100 cells was counted after Giemsa staining. Data shown are the
mean ± S.D. of 3 independent experiments. Triple asterisks represent a statistical signifi-
cance of P b 0.001, compared with the control oligo group.
than that of parasites treated with control oligo. These data suggest that
MAO-1 inhibits maturation of TcIP3R mRNA.
3.3. MAO-1 inhibits trans-splicing of pre-TcIP3R mRNA
To investigate whether MAO-1 inhibits the addition of SL-RNA to
pre-TcIP3R mRNA, real-time RT-PCR was performed with a forward
primer specific for SL-RNA and a reverse primer specific for the 5′-UTR
of TcIP3R mRNA (Fig. 3). MAO-1 or control oligo was added to epi-
mastigotes in the LIT medium, and cDNAs were synthesized from para-
sites treated with oligos for 12 h. The amount of TcIP3R mRNA with
added SL-RNA was significantly lower in parasites treated with MAO-1
than in parasites treated with control oligo. However, real-time RT-
PCR with a primer set specific for a region in the TcIP3R ORF revealed
that TcIP3R mRNA was not knocked-down by MAO-1 (data not
shown). These results indicate that MAO-1 inhibited the maturation of
TcIP3R mRNA.
3.4. MAO-1 inhibits trypomastigote infectivity
We investigated whether MAO-1 inhibited the invasion of trypo-
mastigotes into host cells. After tissue culture, trypomastigotes were
collected from the in vitro culture system as previously described [21]
and parasites treated with MAOs were added to HeLa cells. After 24 h,
the number of amastigotes that parasitized in host cells was counted
(Fig. 4). Infectivity of parasites treated with MAO-1 was significantly
decreased compared with that of parasites treated with control oligo.
Expression levels of TcIP3R in trypomastigotes were very low and
trypomastigotes did not proliferate; hence, it is considered that effects
of antisense oligonucleotides on the knockdown of target gene ex-
pression are more significant in trypomastigotes compared with
epimastigotes [15].
Notably, we previously reported that S-oligos targeting TcIP3R mRNA
inhibit infectivity of trypomastigotes into host cells, but trypomas-
tigotes that were pre-incubated with S-oligos for 8 h were infected to
the host cells [15]. In the present study, MAOs and trypomastigotes
were added to host cells at the same time, and significant inhibition of
the parasite invasion was observed. In contrast, when S-oligos and
trypomastigotes were added to host cells at the same time, the inhibito-
ry effect on parasite invasion was not observed (unpublished data).
These data suggest that MAOs can knock down target gene expression
more efficiently compared to S-oligo antisense nucleotides in parasites.
Further, MAOs are resistant to nucleases and are more efficient in vivo
compared to S-oligo. Our data may provide insight into the develop-
ment of antisense oligo therapy with MAOs against T. cruzi.
Orrego et al. reported that a catalytic subunit of calcineurin in T. cruzi
(TcCaNA2) was important for the infectivity of trypomastigotes into
host cells and for epimastigote growth in knockdown experiments
with MAOs that inhibit translation of TcCaNA2 mRNA [22]. They used
Endo-Porter (Gene Tools, LLC), a carrier of MAOs in cultured cells, to in-
troduce MAOs into parasites. Our present data indicate that any carrier
such as Endo-Porter may not be required for introduction of MAOs into
parasites. Fluorescent microscopy revealed that lissamine-labeled
MAOs can be introduced into parasites without any carrier (unpub-
lished data). Modified MAOs, such as peptide-conjugated phospho-
rodiamidate morpholino oligomers (PPMOs), were used for efficient
introduction of MAOs into target cells (e.g., pathogens) [5]. Because
MAOs are introduced into T. cruzi without modification or carriers,
MAOs may be a promising drug for Chagas disease therapy.
In the present paper, we showed for the first time that MAOs could
inhibit trypanosome trans-splicing. Further, MAO-1 inhibited epimas-
tigote growth (see Fig. 2). Because epimastigotes actively proliferate
and this proliferation dilutes MAO concentration in parasites, knock-
down with MAOs in epimastigotes may be more difficult than that in
trypomastigotes. The disadvantage of MAO use in inhibiting trans-
splicing is that, in most cases, SL acceptors have to be identified to de-
sign MAOs. However, recently, Fiebig et al. developed software to calcu-
late SL acceptors on trypanosome pre-mRNAs [23], which may be very
useful for MAO design.
Acknowledgements
This work was supported by two grants from JSPS KAKENHI (15
K08452 [to MH] and 24390102 [to TN]), a grant from the Pharma-
cological Research Foundation, Tokyo (to MH), and in part by the Foun-
dation of Strategic Research Projects in Private Universities from the
Ministry of Education, Culture, Sport, Science, and Technology, Japan
(number S1201013) (to TN).
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