A short proregion of trialysin, a pore-forming protein
of Triatoma infestans salivary glands, controls activity
by folding the N-terminal lytic motif
Rafael M. Martins
1
, Rogerio Amino
2
, Katia R. Daghastanli
3
, Iolanda M. Cuccovia
3
, Maria A. Juliano
4
and Sergio Schenkman
1
1 Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo, Brazil
2 Departamento de Bioquı
´
mica, Universidade Federal de Sa˜o Paulo, Brazil
3 Departamento de Bioquı
´
mica, Instituto de Quı
´
mica, Universidade de Sa˜o Paulo, Brazil
4 Departamento de Biofı
´
sica, Universidade Federal de Sa˜o Paulo, Brazil
Hematophagous animals counteract physical and
molecular barriers such as the epidermis and the
inflammatory, hemostatic and immune systems of the
hosts to fulfill their nutritional needs [1]. Therefore,

Triatoma infestans; Trypanosoma cruzi
Correspondence
S. Schenkman, Departamento de
Microbiologia, Imunologia e Parasitologia,
Rua Botucatu 862 8
o
andar 04023-062 Sa˜o
Paulo, SP, Brazil
Fax: +55 11 5571 58 77
Tel: +55 11 5575 19 96
E-mail: [email protected]
(Received 30 August 2007, revised 3
December 2007, accepted 2 January 2008)
doi:10.1111/j.1742-4658.2008.06260.x
Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect that
transmits the protozoan parasite Trypanosoma cruzi, the etiological agent
of Chagas’ disease. Its saliva contains trialysin, a protein that forms pores
in membranes. Peptides based on the N-terminus of trialysin lyse cells and
fold into a-helical amphipathic segments resembling antimicrobial peptides.
Using a specific antiserum against trialysin, we show here that trialysin is
synthesized as a precursor that is less active than the protein released after
saliva secretion. A synthetic peptide flanked by a fluorophore and a
quencher including the acidic proregion and the lytic N-terminus of the
protein is also less active against cells and liposomes, increasing activity
upon proteolysis. Activation changes the peptide conformation as observed
by fluorescence increase and CD spectroscopy. This mechanism of activa-
tion could provide a way to impair the toxic effects of trialysin inside the
salivary glands, thus restricting damaging lytic activity to the bite site.
Abbreviations
Abz, o-aminobenzoic acid; APMSF, (4-amidinophenyl)methanesulfonyl fluoride; GST, glutathione S-transferase; LUV, large unilamellar vesicle;

and whether it prevents lysis induced by trialysin
released with the saliva. To identify the precursor and
further understand how a potent lytic molecule is syn-
thesized and controlled, we raised antibodies that react
with trialysin [205 amino acids long, the nonlytic (NL)
fragment] and tested for the presence of different
forms of the protein and their activity in T. infestans.
We found that a precursor is stored in the salivary
glands and processed only after saliva is released. A
peptide containing the precursor region and the lytic
N-terminus of trialysin was synthesized, containing a
fluorophore and a quencher at the N-terminus and
C-terminus respectively. With use of this peptide,
evidence was obtained showing that the activation
mechanism of trialysin involves conformational
changes in this segment of the protein.
Results
Generation of specific anti-trialysin rabbit serum
Trialysin cDNA predicts a signal sequence followed by
an acidic domain, shown in bold and italicized letters
in Fig. 1A, upstream of the N-terminus sequence
detected in the protein isolated from T. infestans
saliva [9]. In order to detect precursor forms and under-
stand the mechanism of activation of T. infestans
protrialysin, we raised specific antibodies capable of
recognizing salivary trialysin. Attempts to obtain the
full-length precursor and mature forms of the recombi-
nant protein in heterologous systems to immunize
animals were unsuccessful. Therefore, a C-terminal NL
fragment of trialysin corresponding to the underlined

FEBS Journal 275 (2008) 994–1002 ª 2008 The Authors Journal compilation ª 2008 FEBS 995
Protrialysin is processed after saliva is secreted
We first investigated whether mature trialysin was
already processed when saliva was ejected. Small
amounts of saliva were collected in ice and analyzed
by immunoblot. As shown in Fig. 2A, a protein larger
than 24 kDa (the size of the purified trialysin) was
detected. After incubation at room temperature for
10 min, the 26 kDa protein was converted to the size
of mature trialysin that was further processed at room
temperature, as judged by the lower molecular mass
bands that appeared at 45 and 60 min. This result indi-
cates that trialysin is processed after saliva ejection.
The precursor form released in saliva is found
in the salivary glands
To detect protrialysin stored in the salivary glands,
and determine whether it would retain lytic activity,
glands were extracted in the presence, or absence, of
APMSF, previously shown to inhibit triapsin activity,
a protease found in saliva that is proposed to be
responsible for trialysin activation [9]. The 26 kDa
precursor was mainly detected when the glands were
prepared in the presence of APMSF (Fig. 2B, lane
APMSF+), or when the glands were directly boiled in
SDS ⁄ PAGE loading buffer, whereas the 24 kDa form
was observed in the absence of the inhibitor (Fig. 2B,
lane APMSF)). Concomitantly, the lytic activity
against trypanosomes and erythrocytes was consider-
ably inhibited in the glands homogenized with APMSF
(Fig. 3). Inhibition of activity was not caused by the

ÆmL
)1
) (B) were
incubated at 37 °C for 1 h and 2.5 h respectively with different
amounts of salivary gland homogenates prepared in the presence
(d) or absence (s) of APMSF. In (A) the number of surviving cells
was determined in a hemocytometer, and the percentage of lysis
was calculated relative to the control. In (B), permeabilization per-
centage was obtained relative to hemoglobin release of control by
treating the cells with 0.2% Triton X-100.
Trialysin precursor activation R. M. Martins et al.
996 FEBS Journal 275 (2008) 994–1002 ª 2008 The Authors Journal compilation ª 2008 FEBS
To identify which form of the protein would represent
the active trialysin, a glutathione S-transferase (GST)–
protrialysin fusion protein was expressed in E. coli. This
protein contained GST, a thrombin cleavage site, and
the protrialysin from amino acid ) 33 to the C-terminus
(see Fig. 1A). It was obtained from soluble E. coli
extracts and purified by chromatography in a glutathi-
one–Sepharose column. The fusion protein was largely
unstable, and it did not show lytic activity, precipitating
as the protein concentration increased in solution. It
could be processed by thrombin, generating a 30 kDa
protein band in SDS ⁄ PAGE (Fig. 2C, lane triapsin)).
The processed recombinant protein was also unable to
promote lysis. When partially purified triapsin was
added to the thrombin-cleaved 30 kDa protein, it was
processed to a 24 kDa band (Fig. 2C, lane triapsin+).
In some experiments, lytic activity was detected,
although the resulting protein precipitated and became

panied by an increase in fluorescence over time
(Fig. 4B). A similar increase in fluorescence was
observed when triapsin or saliva was added (not
shown). As quenching of fluorogenic peptides longer
than 40 amino acids is minimal, unless they are folded
[21], this increase in fluorescence suggests that proP7 is
folded before being processed. The fact that proP7
fluorescence in 6 m guanidine hydrochloride was higher
than the fluorescence in nondenaturing conditions con-
firms this hypothesis, although part of the increase in
fluorescence may be due to the peptide hydrolysis,
which abolishes intramolecular quenching. Evidence
that proP7 is structured and that it unfolds after
Arg-C treatment was also obtained by CD spectros-
copy (Fig. 4C). ProP7 contains 36% a-helix, decreas-
ing to 7% after Arg-C treatment.
Lysis increases after cleavage of proP7
Next, the lytic activities of proP7 and Arg-C-processed
peptide were compared by using artificial liposome
membranes (20 : 80 cardiolipin ⁄ phosphatidylcholine)
containing 6-carboxyfluorescein, which is a fluorophore
that autosuppresses its fluorescence at higher concen-
trations. Upon permeabilization, these liposomes
release the entrapped quenched 6-carboxyfluorescein,
diluting the fluorophore in the sample, and fluores-
cence increases. As expected, the Arg-C-processed
peptide was more effective at promoting liposome
permeabilization than proP7 (Fig. 4D,E). Similar
results were obtained when the lysis of trypanosomes
was assayed (Fig. 4F). These results indicate that a

with phospholipid head groups on the target mem-
brane. The observed residual lytic activity for proP7,
and protrialysin, might occur because the acidic seg-
ment could not block all available cationic surfaces.
Alternatively, lysis could be inhibited by the formation
of dimers, in which the proregion of one molecule
would interact with the cationic surface of another
molecule. It has been shown in the case of cathelicidins
(stored in neutrophil granules) that impairment of
activity can be accomplished by domain-swapping, in
which two molecules fold themselves together as a
dimer [20]. In the case of trialysin, we have no
evidence for dimerization, as the precursor molecule
isolated from the gland in the presence of APMSF
behaves as a monomer in gel exclusion chromato-
graphy (not shown). We cannot exclude the possibility,
however, that when stored at high concentration in the
salivary glands, protrialysin dimerizes.
Structural data on the N-terminus of trialysin pep-
tides show that the very first amino acids are folded in
a nonrigid structure that could be part of the bending
region of the hairpin in the proposed model [10]. This
flexibility could allow the acidic domain to interact
with positive charges in the context of the protein, but
much less in the case of a short synthetic peptide, as
secondary interactions with the protein C-terminal
domain are absent, suggesting that the protein struc-
ture might also have additional roles in the inactiva-
tion of protrialysin. In fact, the increase in
fluorescence of proP7 after specific proteolysis by

trypomastigotes after 30 min of incubation
with the indicated concentrations of proP7
pretreated (d) or not preteated (s ) with
Arg-C.
Trialysin precursor activation R. M. Martins et al.
998 FEBS Journal 275 (2008) 994–1002 ª 2008 The Authors Journal compilation ª 2008 FEBS
Arg-C indicates that it is folded, as fluorescence
quenching is not possible for a long, unfolded peptide
[21]. This is confirmed by fluorescence readings
obtained from both peptides in 6 m guanidine hydro-
chloride showing that intramolecular quenching is very
low when proP7 is unstructured. The CD data support
the notion that the proregion stabilizes a folded struc-
ture at the N-terminus of protrialysin, as Arg-C-trea-
ted proP7 and the N-terminus-spanning peptides are
poorly structured in water solution. The results
obtained using phospholipid liposomes also indicate
that unfolding and activation are directly correlated
with an increase in lysis, and that no other molecules
of the parasite are necessary for its activation.
We have previously observed that small variations
in the sequence of the trialysin N-terminus peptides
can modify its specificity for target cells [10]. The
mobility of the N-terminus seems to prevent lysis of
erythrocytes, as substitution of Gly and Pro residues in
this peptide end increases activity for these cells, but
not for trypanosomes. This could explain why the
acidic portion of trialysin, interacting with the basic
amino acids in the amphipathic helix, is less effective
in inhibiting lysis of erythrocytes as compared to try-

protein toxins. For example, some pore-forming bacte-
rial toxins are synthesized as precursors with acidic
propieces: proaerolysin is synthesized and secreted by
Aeromonas hydrophila as a dimer that binds the glycan
core of glycosylphosphatidyl inositol-anchored proteins
on the cell surface, and it is processed by host pro-
teases, releasing a small C-terminal peptide, thus
enabling the toxin to oligomerize into the heptameric
channel [24]. The El Tor hemolysin of Vibrio cholerae
is also processed by many host proteases in different
sites at the acidic ⁄ apolar propiece [25].
In this work, we have provided evidence that the
acidic portion of a pore-forming protein precursor
controls the lytic activity of the mature molecule. A
synthetic peptide that mimics lysis inhibition and is
suitable for proteolytic activation might be useful in
designing regulated antimicrobial compounds.
Experimental procedures
Insects and cells
T. infestans (males and females) were maintained at room
temperature and fed twice weekly on mice anesthetized with
0.2% (w ⁄ v) ketamine chlorhydrate and 0.12% (w ⁄ v) xyla-
zine chlorhydrate in NaCl ⁄ P
i
. Trypomastigote forms of the
Y strain of Try. cruzi human erythrocytes were obtained as
previously described [9].
Saliva extraction and salivary glands extracts
Saliva was collected as previously described [9] from both
male and female insects 2 days after feeding. Salivary

(5¢-CCATATGAAGAAAGGAGCAGC-3¢) and Bam-LYS30
reverse (5¢-CGGGATCCTTAATCAATTTCAACTTC
ATC-3¢), and the protrialysin cDNA cloned in pGEM-T
Easy (Promega, Madison, WI, USA) as template [9] in
order to insert NdeI and BamHI restriction sites at the
5¢-terminus and 3¢-terminus. The amplified fragment was
inserted in the cloning vector pCR 2.1-TOPO (Invitrogen,
Carlsbad, CA, USA), and the reaction was used to trans-
form chemically competent E. coli DH5a. After sequence
confirmation, the obtained plasmid was digested with
restriction enzymes NdeI and BamHI (Fermentas Interna-
tional, Burlington, Canada), and the insert was purified
from agarose gel and ligated into pET-14b (Novagen,
EMD, Madison, WI, USA) previously digested with the
same restriction enzymes using a Rapid DNA Ligation Kit
(Promega). The ligation reaction was used to transform
E. coli DH5a, and the recovered plasmid (pET14b-NL2)
was used to transform BL21 (DE3) pLysE. The recombi-
nant protein expression was obtained in 300 mL of LB
medium cultures at 37 °C induced at A
600 nm
@ 0.6 with
0.6 mm isopropyl b-d-thioglucopyranoside (Sigma Chemical
Co., St Louis, MO, USA). Bacteria were collected after
overnight incubation by centrifugation at 3000 g for
10 min. The bacterial cell pellet was resuspended in 20 mm
Tris ⁄ HCl (pH 8.0), 6 mm MgCl
2
, and 0.1% Triton X-100,
and lysis was obtained by three freeze–thawing cycles. The

600 nm
= 1.5,
the culture was induced with 0.1 mm isopropyl b-d thioglu-
copyranoside, with subsequent growth overnight at 30 °Cat
200 r.p.m. Afterwards, the culture was centrifuged at 3000 g
for 10 min, and the cell pellet was subjected to 10 pulses
(20 s each, at maximum power) of sonication in a Branson
Sonifier 450 (Branson Ultrasonics Corporation, Danbury,
CT, USA) in 20 mm Tris ⁄ HCl (pH 8.0) and 5 mm EDTA
containing 0.1% Triton X-100 (v ⁄ v). Soluble proteins were
collected after centrifugation at 15 000 g for 20 min, and the
resulting supernatant was incubated with 1 mL of gluta-
thione–Sepharose 4B (GE) previously equilibrated in the
buffer used for cell lysis. The column was washed with
50 mL of lysis buffer, and bound proteins were eluted with
the same buffer containing 20 mm reduced glutathione after
an overnight incubation at 4 °C.
Antiserum production and immunoblotting
A suspension containing 100 lg of NL2 in 300 lLof
NaCl ⁄ P
i
was emulsified with the same volume of complete
Freund’s adjuvant (Sigma) and subcutaneously injected
throughout the dorsum of a female rabbit. Two consecu-
tive boosts in incomplete Freund’s adjuvant (Sigma) at
3 week intervals were administered, and blood was col-
lected from the ear marginal vein. For immunoblots,
SDS ⁄ PAGE gels were wet-transferred to nitrocellulose
membranes (Hybond C-extra; GE), and total blotted
proteins were visualized by Ponceau S staining. The mem-

water. The molecular mass and purity of synthesized
peptides were checked by amino acid analysis and MALDI-
TOF MS, using a Tof-Spec-E from Micromass, Manches-
ter, UK. Further purification was performed using a
lRPC C2 ⁄ C18 reverse-phase column in the A
¨
kta Purifier
system with 0.1% trifluoroacetic acid and a linear gradient
to 100% acetonitrile. Stock solutions of peptides were pre-
pared in dimethylsulfoxide ⁄ water (20 : 80), and the peptide
concentrations were determined spectrophotometrically
using a molar extinction coefficient of 17.300 m
)1
Æcm
)1
at
365 nm.
Fluorimetric measurements
Stock solutions of the peptide were diluted in the indicated
buffer solutions at 37 °C incubated with partially purified
triapsin (step 2 of [4]), or with 1 mgÆmL
)1
trypsin (type VI,
bovine; Sigma) or 5 lgÆmL
)1
Arg-C endoproteinase
(Calbiochem, EMD, San Diego, CA, USA). The proteo-
lytic cleavage of proP7 peptide was monitored by measuring
the fluorescence at k
em

and 50 mm carboxyfluorescein, previously purified [29], and
adjusted to pH 8.0. This suspension was extruded through
11 rounds in a LiposoFast (Avestin Inc., Ottawa, Canada)
system containing two polycarbonate membranes (100 nm)
and applied to a Sephadex G-25 medium column equili-
brated in 10 mm Tris ⁄ HCl (pH 8.0) and 0.3 m NaCl to
remove free carboxyfluorescein from LUVs. The phospho-
lipid content was determined according to Rouser [30].
LUVs were diluted in 1 mL of 10 mm Tris ⁄ HCl (pH 8.0)
and 0.3 m NaCl, and fluorescence was measured in an
Hitachi F-2000 (Japan) spectrofluorimeter (k
ex
= 490 nm
and k
em
= 512 nm) after addition of peptide solutions. At
the end of each experiment, total carboxyfluorescein fluores-
cence was recorded by the addition of 10% Triton X-100.
Acknowledgements
The authors would like to thank Claudio Roge
´
rio de
Oliveira for assistance with cell cultures, Dr Izaura
Ioshico Hirata for performing amino acid analysis, and
Dr Luis Juliano Neto for helpful suggestions. This
work was supported by grants from Fundac¸ a
˜
ode
Amparo a
`

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