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RESEARCH ARTICLE
Ectopic Expression of a Neospora caninum
Kazal Type Inhibitor Triggers Developmental
Defects in Toxoplasma and Plasmodium
Zoi Tampaki
1,2☯†
, Ramadhan S. Mwakubambanya
1,2☯¤
, Evi Goulielmaki
1,2
, Sofia Kaforou
1
,
Kami Kim
3
, Andrew P. Waters
4
, Vern B. Carruthers
5
, Inga Siden-Kiamos
1
, Thanasis
G. Loukeris
1†
, Konstantinos Koussis
1
*
1Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology—Hellas,
Heraklion, Greece, 2Department of Biology, University of Crete, Heraklion, Greece, 3Department of
Medicine, Albert Einstein College of Medicine, Bronx, United States of America, 4Faculty of Biomedical &
Life Sciences, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, Scotland, United
Kingdom, 5Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor,
United States of America
†Deceased.
☯These authors contributed equally to this work.
¤Current address: Department of Biochemistry and Molecular Biology, Egerton University, P.O. Box 536-
20115, Egerton, Kenya
*kousis@imbb.forth.gr
Abstract
Regulated proteolysis is known to control a variety of vital processes in apicomplexan para-
sites including invasion and egress of host cells. Serine proteases have been proposed as
targets for drug development based upon inhibitor studies that show parasite attenuation
and transmission blockage. Genetic studies suggest that serine proteases, such as subtili-
sin and rhomboid proteases, are essential but functional studies have proved challenging
as active proteases are difficult to express. Proteinaceous Protease Inhibitors (PPIs) pro-
vide an alternative way to address the role of serine proteases in apicomplexan biology. To
validate such an approach, a Neospora caninum Kazal inhibitor (NcPI-S) was expressed
ectopically in two apicomplexan species, Toxoplasma gondii tachyzoites and Plasmodium
berghei ookinetes, with the aim to disrupt proteolytic processes taking place within the se-
cretory pathway. NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while
it had no effect on invasion and egress. Expression of the inhibitor in P.berghei zygotes
blocked their development into mature and invasive ookinetes. Moreover, ultra-structural
studies indicated that expression of NcPI-S interfered with normal formation of micronemes,
which was also confirmed by the lack of expression of the micronemal protein SOAP in
these parasites. Our results suggest that NcPI-S could be a useful tool to investigate the
function of proteases in processes fundamental for parasite survival, contributing to the ef-
fort to identify targets for parasite attenuation and transmission blockage.
PLOS ONE | DOI:10.1371/journal.pone.0121379 March 24, 2015 1/23
OPEN ACCESS
Citation: Tampaki Z, Mwakubambanya RS,
Goulielmaki E, Kaforou S, Kim K, Waters AP, et al.
(2015) Ectopic Expression of a Neospora caninum
Kazal Type Inhibitor Triggers Developmental Defects
in Toxoplasma and Plasmodium. PLoS ONE 10(3):
e0121379. doi:10.1371/journal.pone.0121379
Academic Editor: Gordon Langsley, Institut National
de la Santé et de la Recherche Médicale—Institut
Cochin, FRANCE
Received: September 25, 2014
Accepted: January 31, 2015
Published: March 24, 2015
Copyright: © 2015 Tampaki 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.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was performed in the framework
of the BIOSYS research project, Action KRIPIS,
project No. MIS-448301 (2013SE01380036) that was
funded by the General Secretariat for Research and
Technology, Ministry of Education, Greece and the
European Regional Development Fund (Sectoral
Operational Programme: Competitiveness and
Entrepreneurship, NSRF 2007–2013)/European
Commission. The funders had no role in study
Introduction
The phylum Apicomplexa comprises a number of intracellular parasites causing disease in hu-
mans and animals. Two prominent members are Plasmodium parasites causing malaria and
Toxoplasma that is the causative agent of toxoplasmosis in immunocompromised individuals.
These parasites are characterized by having both invasive and replicative forms. In T.gondii
during the asexual life cycle tachyzoites invade cells and replicate inside a parasitophorous vac-
uole in the host cytoplasm. Newly formed parasites egress from the host cell, and immediately
invade new target cells. During these events the secretory organelles of tachyzoites, micro-
nemes, rhoptries and dense granules (DG) have been found to have important roles. Plasmodi-
um parasites also have a complex life cycle. The parasite goes through a replicative cycle in the
blood of the human host, causing the pathology of the disease. Plasmodium is transmitted by
mosquitoes. After the uptake of sexual forms in a blood meal, the parasite develops into a zy-
gote in the mosquito midgut. The zygote in turn matures into the motile ookinete, which tra-
verses the midgut epithelium and forms the sporogonic oocyst. Sporozoites having developed
inside the cyst are transmitted to a new host during a blood meal. Zygote to ookinete transition
is a crucial point in the life cycle of the parasite as a failure to successfully complete this step
blocks transmission. This transition is accompanied by a radical reorganization of the cell, in-
cluding formation of the micronemes, which have an important role in motility and invasion,
and an extension of the cell through the elongation of the cytoskeleton.
Proteases have been recognized as basic components in the life cycle of apicomplexan para-
sites regulating a plethora of physiological processes such as replication, host invasion, egress
and metabolism [1–4]. Apart from increasing our understanding of the basic biology of api-
complexans, proteases comprise potential targets for drug development [5] or for interventions
aiming at parasite attenuation or transmission blockage.
Serine proteases have been identified in both Plasmodium and Toxoplasma and primarily
subtilisins and rhomboids have been studied in more detail. In T.gondii 12 genes encoding
subtilisin-like proteases have been identified, [6–9], while in Plasmodium, three subtilisin-like
proteases have been found [10–13]. In the case of rhomboids, 6 are encoded in T.gondii and 8
in Plasmodium spp [14,15]. Genetic studies have shown that many of these proteases are essen-
tial [6,8,16–20]. However, with the exception of Plasmodium SUB1, in vitro biochemical assays
to further elucidate their function, identify potential substrates and develop potential inhibitors
are either not available or technically challenging with contradictory data in some cases be-
tween in vitro and in vivo studies [21,22].
Small molecule protease inhibitors (SM-PIs) with a broad range of activity have been em-
ployed in different experimental models to uncover the significance of regulated proteolysis in
developmental and physiological processes. SM-PIs have been also tested against apicomplexan
parasites revealing the overall importance of proteolysis in host cell invasion, egress and intra-
cellular parasite replication [23–26]. Among the disadvantages of SM-PIs are that they affect in
parallel proteolytic processes taking place in different cellular compartments (including prote-
olysis in the host cells) and/or involving different types of proteases.
On the other hand Proteinaceous Protease Inhibitors (PPIs) are considered superior inhibi-
tors to SM-PIs. These molecules have evolved as one of the various self-protecting strategies
against assaulting or uncontrolled proteolysis. Firstly, PPIs are more specific inhibitors than
SM-PIs since co-evolution with their target proteases has shaped their specificity, and secondly
because PPIs are proteins their expression/activity can be restricted in a specific stage and/or in
a specific subcellular compartment.
Serine PPIs can be distinguished based on their structure and their mechanism of action to
serpins, canonical or non-canonical inhibitors [27]. A well-studied family of PPIs inhibiting
A Kazal Inhibitor Affects Apicomplexa Development
PLOS ONE | DOI:10.1371/journal.pone.0121379 March 24, 2015 2/23
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
serine proteases is that of Kazal type inhibitors. The basic domain of Kazal inhibitors has a
characteristic structure dictated by six conserved cysteines forming intra-domain disulfide
bonds [28]. Non-canonical inhibitors such as hirudin are much less abundant; they occur only
in blood sucking organisms and inhibit proteases involved in clot formation [29,30].
The distribution of PPIs in parasites varies. While Neospora and Toxoplasma have canonical
Kazal type inhibitors as well as serpins, other apicomplexan parasites such as Plasmodium and
Babesia do not possess any serine protease inhibitors. A protective role against digestive en-
zymes has been hypothesized for the four-Kazal domain inhibitors, TgPI-1 and TgPI-2, identi-
fied in Toxoplasma [31,32]. Besides putative homologues of TgPI-1 and TgPI-2, N.caninum
expresses the single Kazal domain NcPI-S that strongly inhibits bacterial subtilisins in vitro
[33,34] and it has been speculated that NcPI-S might regulate endogenous subtilisin activity in
N.caninum [34]. Apart from their difference in the number of Kazal-domains, there is no simi-
larity in the amino acid composition of the active sites of these inhibitors [34] suggesting that
they possess distinct substrate specificities.
We investigated the potential of NcPI-S to interfere with proteolytic processes within the se-
cretory apparatus, by ectopically expressing NcPI-S in Toxoplasma and Plasmodium; neither of
which express an NcPI-S homologue. Toxoplasma tachyzoites provided our study with a cellu-
lar and biochemical environment equivalent to that of the native NcPI-S, as the two species T.
gondii and N.caninum are phylogenetically very close. This strategy provided a system for a
fast evaluation of the chosen inhibitor. After confirming that active NcPI-S affected the growth
of T.gondii tachyzoites, we designed a stage specific ectopic expression of NcPI-S in the rodent
model parasite P.berghei that resulted in an arrest of zygote to ookinete transition. Our results
indicate that PPIs can be used as tools to study parasitic proteases and additionally could lead
to the discovery of novel antiparasitic agents.
Materials and Methods
Ethics Statement
All animal work has passed an ethical review process and was approved by the FORTH Ethics
Committee (FEC). All work was carried out in full conformity with Greek regulations: Presi-
dential Decree (160/91) and law (2015/92) which implement the directive 86/609/EEC from
the European Union and the European Convention for the protection of vertebrate animals
used for experimental and other scientific purposes and the new legislation Presidential Decree
56/2013. The experiments were carried out in a certified animal facility license number
EL91-BIOexp-02.
Parasites, cells, mosquitoes
Human foreskin fibroblasts (HFFs—ATCC SCRC-1041) were maintained in Dulbecco's Modi-
fied Eagles Medium (DMEM) containing 10% heat inactivated Fetal Bovine Serum (FBS, PAA
Laboratories), 2 mM Glutamine, 1% Penicillin/Streptomycin (GIBCO) (D10 Complete). The
T.gondii RH strain and the RHΔhxgprt [35] were maintained by serial passage in HFFs as de-
scribed [36]. Tachyzoites were harvested after lysis of the monolayer, unless otherwise stated,
by scraping, passage through a 26-gauge needle and a 3-μm Nucleopore membrane filter
(Whatman), and collected by centrifugation (400xg, 15 min at 4°C). P.berghei strains were
ANKA HP (also called 15cy1A), and the GFP-expressing 507cl1 clone [37]. Anopheles gambiae
G3 strain was used for mosquito infections.
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Plasmid constructions
All primers are listed in S1 Table in supplemental material.
pGra1/ssmycNcPIS. ROP1 signal sequence (ss) was amplified from pRop1myc plasmid
[38], using primers ssRop1_5’and ssRop1_3’.The myc tag was amplified using mycRI5’and
myctoxo3’primers. NcPI-S was amplified from pQE/NcPIS [34], using NcPIS_5’and
NcPIS_3’primers. The fragments were fused in frame and inserted in pGra1/GFP/Gra2-SK
[39], which contains the dhfr_5/HXGPRT/dhfr_3 selection marker, using the NsiI and PacI re-
striction sites (S1A Fig.).
pGra1/ssmycNcPISmut. NcPISmut was generated from NcPI-S by PCR-based mutagene-
sis with primers NcPIsFor and NcPISRev. The construct encoded NcPI-S with changed amino
acids P2, P1 and P1’-P4’of the Kazal inhibitory domain from SMEYDP to FASGKR. The con-
struct was verified by sequencing. For cloning in the transfection vector NcPIsmut was ampli-
fied with the primers Ncmut_5’and Ncmut_3’and subcloned into FseI-PacI digested pGra1/
ssmycNcPIS resulting in the exchange of the wild type NcPI-S with the mutant version.
p[CTRP-Sp-V5-NcPIS]DEFSSUToxo. The signal sequence of the PbSUB2 was amplified
from P.berghei gDNA with primers SPsub-2F and SPsub-2R. The V5 epitope was amplified
from the pIZ/V5-His vector (Invitrogen) using V5for and V5Rev primers. NcPI-S (excluding
the signal sequence) was amplified from the pQE/NcPIS plasmid with primers NcPIS_5’and
NcPIS-R. The fragments were joined in an intermediate vector and subsequently inserted be-
tween the CTRP promoter and P28 3’UTR in the vector pCp*S[40]. The derived [CTRP-Sp-
V5-NcPIS] cassette was finally inserted into the pDEF-TgDHFR/D-SSU (RV) transformation
vector (kindly provided by Dr. Franke Fayard).
NcPISmut. NcPI-Smut was inserted in the p[CTRP-Sp-V5-NcPIS]DEFSSUToxo using
FseI and PacI/NotI restriction enzymes. The sequences encoding the PbSUB2 signal sequence
and V5 remained the same.
Transfection, selection and cloning of stable transformants
To generate stable transformants freshly released RHΔhxgprt parasites were electroporated as
described [41] with 100 μg of Gra1/ssmycNcPIS and Gra1/ssmycNcPISmut constructs. Tachy-
zoites were transfected in presence of NotI restriction enzyme [42] and parasites were selected
with mycophenolic acid and xanthine as described [43]. Resistant parasites were cloned by lim-
iting dilution. Clones NcPI-S_6 and NcPI-S_8 from two independent transfection experiments,
and clone NcPI-Smut_16 were selected.
P.berghei schizonts were transfected using standard procedures [44]. Pulse Field Inversion
Gel Electrophoresis (PFIGE) and genomic PCR genotyping was used to characterize the trans-
genic clones (S2B, S2C Fig.). For PFIGE-Southern, total gDNA was separated on 1% agarose
gel under Pulsed-Field Inversion Gel Electrophoresis (PFIGE) conditions, transferred and hy-
bridized with P.berghei radio-labeled dhfr-ts 3’UTR probe as described [44](S2B Fig.). Geno-
mic PCR genotyping was performed using integration specific primer sets [45]: L665 and L740.
Clone purity and determination of the integration site (cor dssu locus), were verified by using
forward primers cssu specific L270 and dssu specific L260 in combination with the reverse
L740 primer common to both loci [46](S2C Fig.).
Two independent clones expressing NcPI-S were studied; NcPIS_3, in the GFP507cl1
(GFPp) recipient strain [37] and NcPIS_1, the latter derived from a transfection using the
ANKA 15cy1A reference strain as a recipient. The NcPISmut line expressed the mutated form
of NcPI-S and was derived from the GFP507cl1 (GFPp) recipient strain. The strain ANKA 2.34
was used as a WT control in oocyst experiments.
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T.gondii assays
Equal numbers of freshly released filtered tachyzoites were used to infect HFF cells during rep-
lication assays. After sedimentation and incubation for 1 h, cultures were washed and incubat-
ed for another 24 h. Coverslips were labeled with anti-SAG1 antibody and 4'-6-Diamidino-
2-phenylindole (DAPI). Vacuoles containing different numbers of parasites (1, 2, 4, 8 or 16)
were counted (total 150 vacuoles/sample). Three independent experiments in duplicates
were performed.
Invasion competence of the parasites, was determined according to previously described
protocols [47]. Freshly lysed parasites from all strains were allowed to invade HFF cell mono-
layers grown on coverslips for 10 min and the number of extracellular vs. intracellular parasites
was determined. Extracellular tachyzoites were revealed after labeling with anti-SAG1 without
permeabilization and intracellular tachyzoites with subsequent staining with anti-GRA3
after permeabilization.
The percentage of egressed vacuoles was determined after inducing egress with calcium ion-
ophore A23187. Intracellular replicating tachyzoites (36–40 h p.i.) were treated with 1 μM
A23187 or DMSO (solvent control) for 5 min [48] and egressed vacuoles were enumerated in
each sample.
P.berghei assays
P.berghei ookinete cultures and purification was performed as previously described [49]. Con-
version rates were calculated as previously described, using a mouse monoclonal antibody
(13.1), which recognizes the P28 protein on the surface of female gametes, zygotes and ooki-
netes [50]. Positive cells were counted on a Zeiss Axioscope2plus microscope (Carl Zeiss, Unit-
ed States) equipped with an AxioCam color Zeiss CCD camera. For oocyst counting,
mosquitoes were fed on anaesthesized infected mice for gametocyte feeding. Alternatively,
membrane feeders were used for feeding in vitro cultured ookinetes. Oocysts were counted on
day 10 post-feeding.
Transmission Electron Microscopy
For TEM observations, ookinetes were pelleted at 720xg and fixed in 2% glutataraldehyde/2%
paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.4 for 45 min at 4°C, post fixed in
1% osmium tetroxide, dehydrated in graded ethanol, stained with uranyl acetate 1% and finally
embedded in graded Durcupan:propylene oxide series (1:3, 1:1, 3:1, 4:0). Ultrathin-sections
(50–100nm) were taken on a Leica LKB2088 ultramicrotome and examined under JEM 100C/
JEOL/Japan Transmission Electron Microscope. Microphotographs were obtained with an
ES500W Erlangshen camera and analysed by the DigitalMicrograph software (Gatan,
Germany).
Antibodies
For immunoblotting of T.gondii the antibodies were: 9B10 anti-myc (1:5000 dilution) (Cell
Signaling Technology), anti-MIC2 (6D10) [51], anti-SAG1 (gift of Jean Francois Dubremetz),
anti-ROM4 (gift of Dominique Soldati-Favre) and anti-GRA1 (gift of Louis Weiss), all 1:1000
dilution. For Western blot experiments of P.berghei anti-V5 (Invitrogen, diluted 1:2000), rab-
bit anti-SOAP (developed in the Loukeris laboratory, diluted 1:500), the monoclonal antibody
13.1, which detects the P28 surface antigen of P.berghei ookinetes [52] (1:20000), anti-Bip
(1:2000-gift of Ellen Knuepfer, NIMR, UK) were used. For IFA of T.gondii mouse a-myc 9B10
(1:2000, Cell Signaling Technology), rabbit anti-GRA3 (1:300, gift of Jean Francois
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Dubremetz), rabbit anti-PfBip (MR4 ATTC, MRA-20 1:1000) were used. Antibodies used for
IFA of P.berghei were anti-serpin6 (1:1000, provided by G. Christophides) and PbCAP380 that
recognizes the oocyst capsule [53]. Secondary antibodies were conjugated to Alexa 555 or
Alexa 488 (Molecular Probes).
Fractionation and Immuno blot analysis
For analysis of T.gondii one infected HFF culture was left to lyse spontaneously, and extracellu-
lar tachyzoites were purified from the culture medium. For a second parallel culture, intracellu-
lar tachyzoites were force released (20–22 h p.i.) by trypsinolysis, passage through a 26-gauge
needle, and filtering through a 3 μm Nucleopore membrane filter. Tachyzoite pellets were re-
suspended in Tris-Cl pH 8.5, lysed by freeze-thaw and sonication, centrifuged for 30 min at
320xg, and the supernatant was removed (Soluble fraction, S). The pellet was resuspended in
0.1 M Na
2
CO
3
pH 11.5, incubated on ice for 1 h, and centrifuged at 320xg for 30 min in order
to remove peripheral membrane proteins. The supernatant (Salt-eluted fraction, SE) was sepa-
rated from the remaining pellet that was resuspended in PBS (Membrane fraction, M). β-mer-
captoethanol was added to the lysates and samples were separated on 15% SDS-PAGE gels and
transferred to nitrocellulose membranes.
For detection of NcPI-S in P.berghei zygotes/ookinetes, parasites were resuspended in 10
volumes of RIPA buffer (50 mM Tris-Cl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1%
Sodium Deoxycholate, 0.1% SDS) supplemented with Protease Inhibitor Cocktail (Sigma).
After incubation on ice for 30 min and centrifugation 8000xg for 30 min, the cleared lysates
were separated on SDS-PAGE gels and processed for Western blot analysis. For P.berghei frac-
tionation, parasite pellets were initially resuspended in 10 volumes of 100 mM Tris-Cl pH 8.0
and incubated on ice for 1 h. After centrifugation at 8000xg for 30 min, supernatants were col-
lected, while the pellet was resuspended in 10 volumes of RIPA buffer, loaded on SDS-PAGE
gels and processed for Western blot analysis. To detect phosphorylation of PbeIF2a in the dif-
ferent strains, parasite pellets were resuspended in 10 volumes modified RIPA buffer (50 mM
Tris-Cl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS) supplemented with Prote-
ase Inhibitor Cocktail (Sigma), 50 mM NaF and 1mM phenylmethylsulfonyl fluoride (PMSF).
Subsequent treatment was as described above.
Indirect immunofluorescence microscopy
All manipulations were carried out at room temperature. T.gondii tachyzoite-infected HFF
cells on 24-well chamber slides were fixed with 100% cold methanol for 10 min, followed by
three washes in 1xPBS. Fixed cells were blocked in 1% bovine serum albumin (BSA) (Merck,
Europe) with 0.25% TritonX-100 in PBS for 1 hour. The wells were then stained with different
primary antibodies, followed by conjugated secondary antibodies. DAPI was added to stain the
nucleus. Images were collected using a Zeiss Axioscope2plus (Carl Zeiss, United States) micro-
scope equipped with an AxioCam color Zeiss CCD camera. Images were processed using Ima-
geJ software. For P.berghei ookinete IFAs, purified ookinetes were allowed to settle on glass
slides pre-treated with poly-L-lysine for 15–30 min. The following procedures were all per-
formed at RT. Samples were fixed in 4% paraformaldehyde (PFA) for 10 min and washed with
PBS. This was followed by permeabilisation in PBS, 0.1% triton-x100 (PBT) for 30 min. The
primary antibody directed against V5 was added, after 1 h followed by PBS washes and incuba-
tion with secondary antibody for 30–45 min. Nuclei were stained with TO-PRO. Samples were
washed in PBS and mounted in Vectashield before being analyzed using a Leica TCS SP2 con-
focal laser scanning microscope. For mosquito midgut IFA, infected midguts were dissected in
ice-cold PBS, prefixed for 90 sec in ice-cold 4% PFA in PBS, transferred to ice-cold PBS and cut
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open longitudinally. After removal of the midgut content the epithelium was fixed for another
45–60 min in 4% PFA at RT. Samples were washed 3x10 min in PBS, blocked and permeabi-
lized for 1 h 30 min in 1% BSA in PBT at RT, followed by incubation overnight at 4°C with
anti-serpin6, followed by 3 washes with PBT and incubation with secondary antibodies for 1 h
at RT. Nuclei were stained with TO-PRO 3 (Molecular Probes). Midguts were washed three
times in PBT for 10 min, mounted in Vectashield and analyzed as described above.
In vitro inhibitory activity of transgenic parasite lysates
Increasing amounts of lysates (calculated as parasite number equivalents) prepared from RH
and NcPIS_6 were incubated with Bacillus licheniformis subtilisin A (Bl-subA) (3.6 nM) in re-
action buffer (100 mM Tris–HCl, 150 mM NaCl, 10 mM CaCl
2
, Triton X-100 0.25% v/v,
0.05% sodium Deoxycholate, pH 8.0) for 15 min at 37°C followed by the addition of the sub-
strate CBZ-Gly-Gly-Leu-pNA (0.15 mM). Residual hydrolytic activity (R.H.A) was measured
as substrate conversion in a spectrophotometer. No enzymatic activity was detected when ly-
sates were incubated with the substrate alone (NcPIs_6/NP) or with Bl-subA in presence of
2mM PMSF (NcPIS_6/+PMSF).
Statistical analysis
Student’s t test using SPSS v.11.5 software was employed.
Results
NcPI-S expression in bacteria, NcPI-S modifications and their validation
The Kazal serine protease inhibitor NcPI-S is a 79 amino acid peptide with a theoretical mw of
6 kDa (Fig. 1)[33]. We first expressed NcPI-S in bacteria as a recombinant NcPI-S tagged with
V5 and 6xHis (r(His-V5)/NcPI-S) (Fig. 1A). Western blot experiments using a monoclonal Ab
recognizing anti-V5 revealed the presence of a predominant band of *17 kDa in our prepara-
tion (Fig. 1B, right) which is slightly bigger than the predicted 10.7 kDa of the r(His-V5)/
NcPI-S. This is consistent with previous studies showing that NcPI-S migrates slower on Tris-
tricine SDS-PAGE gels [34] and even slower on Tris SDS-PAGE gels [33,34]. The inhibitor r
(His-V5)/NcPI-S was tested against the Bl-subA using previously reported enzymatic assays
[33,34](Fig. 1B, left) indicating that the N-amino terminal extension does not interfere with
the inhibitory activity of NcPI-S.
In parallel, we used bacterial expression to validate an NcPI-S mutant variant. Although the
inhibitory specificity of Kazals is primarily dictated by the identity of the amino acid at the P1
position, other amino acid interactions between the Reactive Site Loop (RSL) and the substrate
binding cavities may equally influence the binding of Kazals to proteases [28]. As previously
shown, mutagenesis of the P1 Met to Ala of NcPI-S does not affect its inhibitory activity [34].
Thus, we decided to change a significant proportion of the (RSL). To achieve this, we have
substituted the NcPI-S’s P2-P4’site with the sequence FASGKR (NcPI-Smut; Fig. 1A). This se-
quence corresponds to the non-prime site (P6-P1) of a cleavage site processed by the West Nile
Virus (WNV) trypsin-like serine protease [54]. Our hypothesis was to test the inhibitory profile
of a serine protease recognition site not present in P.berghei and T.gondii. The recombinant
mutant (His-V5)/NcPI-Smut, (r(His-V5)/NcPI-Smut), showed a substantial mobility shift in
respect to the r(His-V5)/NcPI-S, although the calculated size difference was only 0.2 kDa
(Fig. 1B). More importantly the NcPI-S mutant variant failed to inhibit Bl-subA in comparison
with the r(His-V5)/NcPI-S (Fig. 1C). At a high concentration of r(His-V5)/NcPI-Smut (750
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fmol) a low degree of inhibition was detected, although we never detected more than 20% inhi-
bition, compared to the complete inhibition of r(His-V5)/NcPI-S.
Ectopic expression of NcPI-S in Toxoplasma gondii tachyzoites
As a proof of concept that ectopic expression of a PPI can affect an aspect of parasite infection,
we expressed NcPI-S and the inactive variant NcPI-Smut in Toxoplasma tachyzoites. NcPI-S
and NcPI-Smut were expressed as amino-terminal fusions of the PPI by replacing its own sig-
nal peptide with the ROP1 signal sequence [55] followed by a myc epitope tag under the con-
trol of the constitutive Gra1 gene promoter [56](Fig. 2A). Transfection of the recombinant
plasmids (pGra1/ssmycNcPIS or pGra1/ssmycNcPISmut) in the RHΔhxgprt recipient strain
[35] and clonal selection resulted in several stable clones in both cases. One clone in each case,
NcPIS_6 or NcPISmut_16 respectively, was selected for further studies.
Western blot analysis of NcPIS_6 tachyzoite extracts identified a predominant (myc)/
NcPI-S band of *17 kDa (Fig. 2B left, arrow). An additional fainter band at *24 kDa, possibly
corresponding to a (myc)/NcPI-S dimer, as has been previously described [34], was also appar-
ent (Fig. 2B, asterisk). The (myc)/NcPI-Smut variant was detected as a single faster migrating
band than (myc)/NcPI-S (Fig. 2C) suggesting that the NcPI-Smut dimer might be below the
threshold of detection.
Immunofluorescence analysis (IFA) was performed on tachyzoites using a mAb recognizing
the myc epitope. These experiments revealed that (myc)/NcPI-S was secreted into the parasito-
phorous vacuole (PV) when intracellular NcPIS_6 tachyzoites were labeled (Fig. 2B, right and
Fig 1. Bacterial expression of NcPI-S. (A) Amino acid sequence of NcPI-S. The deduced signal peptide
sequence is shaded in green and the signal peptidase cleavage site is underlined. An open box indicates
amino acid sequence of Kazal inhibitory domain. The methionine (M) in position P1 is shadedin red and the
conserved cysteine residues are in bold. Also depicted are the putative intra-domain disulfide bridges
between cysteine numbers 1–5, 2–4, 3–6. Native signal peptide of NcPI-S (without the peptidase cleavage
site) was replaced by a 6xHis and a V5 tag (in the case of bacterial expression), by the signal peptide of ROP-
1 and a myc-tag (T.gondii expression) and by the signal peptide of PbSUB2 and a V5-tag (P.berghei
expression). Ectopic expression of NcPI-Smut in all cases was accomplished by exchanging NcPI-S
sequence with that of NcPI-Smut, where residues P2-P4’have been mutated. (B) Western blot analysis of
different amounts (1–10 μg) of r(His-V5)/NcPI-S and r(His-V5)/NcPI-Smut probed with anti-V5. (C) Activity
assay of recombinant NcPI-S/NcPI-Smut. Increasing amounts of r(His-V5)/NcPI-S and r(His-V5)/NcPI-Smut
were incubated with subtilisin A of Bacillus lechiniformis. Residual hydrolytic activity (R.H.A) was measured
through substrate conversion.
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Fig 2. NcPI-S expression in Toxoplasma tachyzoites. (A) Gra1/ssmycNcPIS WT or mutant construct is
shown indicating the signal sequence of ROP1, the myc-tag and the NcPIS variants. (B) (left) Western blot
analysis of lysates derived from extracellular parasites of transgenic NcPIs_6 and RHhxgprt–strains. An
arrow marks the predominant monomeric form of myc-tagged NcPI-S inhibitor (bottom), while asterisk
indicates the additional NcPIS dimeric form. SAG1 protein (top) was used as loading control. (Right)
Localization of NcPIS (red) and the DG protein GRA3 (green) in intracellular parasites, 24 h p.i. Nuclei
stained with DAPI (blue). Scale bars, 1μm. (C) (left) Western blot analysis of lysates derived from extracellular
parasites of transgenic NcPIsmut_16 (arrow) and RHhxgprt- strains. GRA1 protein (top) was used as loading
control. (right) Localization of mutant NcPI-S (green) and the chaperonin Bip protein (red) in intracellular
parasites, 24 h p.i. Nuclei stained with DAPI (blue). Scale bars, 1μm. (D) Inhibitory activity of tachyzoite
lysates against Bacillus lechiniformis (Bl) subtilisin. Tachyzoite lysates prepared from RH and NcPIS_6 were
incubated with Bl subtilisin in the presence of 0.15 mM CBZ-Gly-Gly-Leu-pNA substrate. Residual hydrolytic
activity (R.H.A) was measured. No activity was detected when lysates were incubated with the substrate
alone (NcPIs_6/NP) or in reactions in which lysates were incubated with the Bl subtilisin in the presence of
2mM PMSF (NcPIS_6/+PMSF). Error bars represent the means ±SEM. (n = 2 experiments per strain,
representative of triplicate samples). (E) Distribution of NcPI-S in soluble (S), membrane salt-eluted (SE) and
membrane (M) fractions derived from either extracellular (left) or intracellular (right) parasites. The monomeric
and dimeric forms of NcPIS (bottom) are marked with an arrow and asterisk, respectively. MIC4 micronemal
protein (upper) and GPI anchored SAG1 (middle) were used as indicators of soluble and membrane
associated proteins respectively.
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S1B Fig.), while in extracellular tachyzoites (myc)/NcPI-S exhibited a punctate staining pattern
(S1C Fig.). In contrast, no signal was detected in the intravacuolar space of the intracellular
growing NcPISmut_16 tachyzoites. Instead, IFA revealed retention of (myc)/NcPI-Smut within
the tachyzoites and specifically within the ER, which in tachyzoites is expanded around the cell
nucleus being more prominent posterior to the nucleus. The increased ER retention of (myc)/
NcPI-Smut was indicated by its co-localization with the ER-resident chaperone protein BiP
(Fig. 2C, right).
In order to verify that the myc-tagged NcPI-S retains its inhibitory potential we used the Bl-
SubA enzymatic assay to test extracts derived from either WT or NcPIS_6 extracellular tachy-
zoites. Only extracts derived from NcPIS_6 tachyzoites inhibited the Bl-SubA activity and
moreover this inhibition was directly proportional to the number of parasites that were used to
prepare the extracts (Fig. 2D).
Fractionation of extracts derived from extracellular tachyzoites (isolated from infected,
spontaneously-lysed cells) or from intracellular tachyzoites (isolated from vacuoles released by
force from infected cells 24 h post infection), revealed abundant presence of (myc)NcPI-S in
the soluble fraction (S) in both cases (Fig. 2E, lower panels). Association of the (myc)NcPI-S
with the membrane pellet (M), (insoluble after treatment with Na
2
CO
3
that removes mem-
brane associated proteins), was also observed but at a much lower degree (Fig. 2E, lower pan-
els). Importantly, both the *17 kDa (arrow) and the potential *24 kDa dimer (asterisk) of
the (myc)NcPI-S were detected in the S fractions of tachyzoites as has been previously observed
[34].
Expression of NcPI-S in T.gondii tachyzoites impairs parasite
replication
To study the effect of expressing NcPI-S and its mutated form in T.gondii tachyzoites we per-
formed phenotypic characterization. NcPIS_6 tachyzoites were compared to WT tachyzoites of
the RH and the parental RHΔhxgprt strains using well-established invasion [47], egress [48]
and growth assays. Neither invasion (Fig. 3A) nor egress assays (Fig. 3B) revealed any signifi-
cant phenotypic deviation of NcPIS_6 from the WT control RH strain (Fig. 3A and 3B). To as-
sess growth we carried out replication assays [57] where equal numbers of tachyzoites from
NcPIS_6 and WT control strains (RH and RHΔhxgprt), were used to infect HFF cells (Fig. 3C).
At 24 h p.i., vacuoles derived from control strains contained 8 tachyzoites in a percentage rang-
ing between 40–70% of the total vacuoles, indicating 3 cell divisions as was expected. In con-
trast, only *17% of NcPIS_6 vacuoles contained 8 tachyzoites (Fig. 3C), indicating impaired
replication. A clone (NcPIS_8), derived from a second independent transformation experi-
ment, showed similar growth kinetics to that of the NcPIS_6 (S1A Fig.), verifying that a replica-
tion defect of T.gondii tachyzoites is reproducibly associated with the expression of (myc)
NcPI-S. In contrast NcPISmut_16 tachyzoites showed the same growth dynamics as the paren-
tal RHΔhxgprt strain (Fig. 3D). When combined, these results suggest that active NcPI-S is ex-
pressed in tachyzoites, and interfere with proliferation of the parasite, while the mutated form
has no effect on parasite growth.
Ectopic expression of NcPI-S and NcPI-Smut in Plasmodium berghei
zygotes/ookinetes
The results from the analysis of T.gondii encouraged us to test similar constructs in Plasmodi-
um, to determine the effect of NcPI-S in these parasites. To this end, recombinant P.berghei
parasites were produced using standard genetic transfection protocols. In brief, the constructs
were designed to express V5 epitope tagged NcPI-S and NcPI-Smut fused to the pbsub2 signal
A Kazal Inhibitor Affects Apicomplexa Development
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peptide sequence [58], to ensure correct sorting in the secretory apparatus. The engineered
open reading frames (ORFs) were placed under the control of the 5’and 3’UTRs of ctrp and
p28 genes, respectively, which drive expression in the late zygote and ookinete stages [40]. The
construct was integrated in the c-ssu/d-ssu locus of the GFP507cl1 (GFPp) strain, constitutively
expressing GFP [37](S2A Fig.). We chose to restrict expression of NcPI-S and its inactive vari-
ant NcPI-Smut to the zygote/ookinete stages in P.berghei to avoid any toxic effects in the asex-
ual blood stages, in which transfection is carried out. Clonal lines were established and one
clone from each transfection (NcPIS_3 and NcPISmut) was selected for further analysis after
genotypic characterization (S2B and S2C Fig.). As expected asexual growth and gametocyto-
genesis were both unaffected in NcPIS_3 and NcPISmut parasites (data not shown). However,
when ookinete conversion was scored in in vitro cultures of the two strains, we found that the
majority of NcPIS_3 parasites were developmentally arrested after zygote formation. However,
*13% normal ookinetes were detected in each experiment. In contrast, the in vitro cultures of
the NcPISmut strain produced ookinetes at numbers comparable to the parental GFPp strain
(Fig. 4A).
Expression of (V5)/NcPI-S and (V5)/NcPI-Smut, was confirmed by Western blotting analy-
sis of in vitro cultured ookinetes. Consistent with the size of the bacterially expressed inhibitors,
the anti-V5 mAb detected a predominant *17 kDa band in extracts of the NcPIS_3 parasites,
while a *13 kDa band was detected in extracts of the NcPISmut strain (Fig. 4B). IFAs using
the V5 mAb revealed that in the developmentally arrested cells of the NcPIS_3 strain the PPI
was localized in an area proximal to the nucleus (Fig. 4C,NcPIS_3). In NcPISmut ookinetes the
inhibitor was detected throughout the entire cytosol including a perinuclear signal possibly co-
inciding with the ER, which in zygotes/ookinetes is composed of only a few tight stacks encircl-
ing the nucleus (Fig. 4D,NcPISmut). To further examine the potential localization of NcPI-S
Fig 3. Phenotypic characterization of NcPIS_6 strain. (A) Invasion competence of NcPIS_6 clone was
similar to the RH strain. BAPTA-AM treated parasites, which do not invade cells, were included as a control.
Error bars represent SEM (n = 3, each experiment was performed in duplicate and ten random fields were
screened blindly in each sample). (B) Percentage of lysed vacuoles of NcPIS_6 versus RH after inducing
egress with calcium ionophore A23187. Intracellular replicating tachyzoites (36–40 h p.i.) were treated with
1μM A23187 or DMSO (solvent control) for 5min. Percentage of induced egress from host cells was
determined. Error bars, SEM (n = 3, each experiment was performed in duplicate and, 10 fields were blindly
screened per sample). (C) Replication assay of NcPIS_6, RH and RHΔhxgprt
-
(parental) and (D)
NcPISmut_16 and RHΔhxgprt
-
. Following a 24 h interval of intracellular replication, vacuoles from all the
above strains were scored according to their parasite context. Error bars, SEM (n = 3, representative of
duplicate samples); P<0.001, Student’s t-test.
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and NcPI-Smut we have performed double staining with the ER chaperone BiP (S3 Fig.). In the
case of NcPIS_3 parasites, some partial colocalization is seen. In these retort forms though and
Fig 4. Developmentally arrested NcPIS_3 zygotes/ookinetes show gross abnormalities. (A) Ookinete
conversion in NcPIS_3 and NcPISmut parasites versus GFPp (WT). The percentage of ookinetes formed in
NcPIS_3 was significantly lower compared to WT and NcPISmut parasites. Ookinetes, macrogametes and
zygotes were counted after labeling with an antibody recognizing the Pbs21 surface antigen. Error bars
represent ±SEM, n = 3. (B) Whole protein extracts from *1x10
6
ookinetes derived from in vitro cultured
NcPIS_3,NcPISmut or GFPp (WT) parasites were examined for the presence of NcPI-S or NcPI-Smut
respectively using anti-V5 mAb. In NcPIS_3 extracts a predominant *17 kDa band was detected while a
band of *14 kDa was detected in extracts of NcPISmut. P28 was used as a loading control. (C-D) Sub-
cellular distribution of NcPI-S and NcPI-Smut in ookinetes of transgenic P.berghei parasites. Confocal
microscopy using anti-V5 MAb (red) shows the different localization of NcPIS_3 and NcPISmut in parasites.
In NcPISmut there is dispersed and perinuclear staining pattern compared to the NcPIS_3 where expression
is in close proximity to the nucleus. Nucleus is stained with TO-PRO-3 (blue). Scale bar 5 μM. (E) Trans-
mission electron micrographs (TEM) of arrested NcPIS_3, zygotes/ookinetes show gross abnormalities. The
endomembrane system is dilated. Left image shows an arrested parasite with discontinuities in the inner
membrane (red arrowheads) while release of content from the nucleus (N) to the cytoplasm is manifested
(white arrowhead).
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based on the TEM experiments, the ER might be severely rearranged. In the NcPISmut strain
there is almost complete colocalization of NcPI-Smut with BiP as has been also the case in in-
tracellular T.gondii parasites expressing NcPI-Smut (Fig. 2C).
Transmission Electron Microscopy (TEM) was used to further examine the morphological
defects of NcPIS_3 developmentally arrested ookinetes. We detected a pronounced dilation of
the endomembrane system with electron dense aggregates being observed (Fig. 4E, middle and
right, S4A Fig.). Importantly, this dilation appeared also polarized, in accordance with the
(V5)/NcPI-S localization pattern (compare Fig. 4C left and Fig. 4E middle). Other develop-
mentally arrested ookinetes showed a progressive degeneration (Fig. 4E, left) manifesting a
range of ultrastructural defects such as incomplete formation of the inner membrane (Fig. 4E,
red arrowheads). In some extreme cases of terminally degenerated ookinetes the whole cyto-
plasm was replaced with vacuoles of different sizes (Fig. 4E, middle, S4B Fig.). In conclusion,
our analysis revealed that the expression of (V5)/NcPI-S during zygote to ookinete transition
resulted in a prompt and almost total block in development.
NcPIS_3 parasites are impaired in oocyst formation
To assess the ability of NcPIS expressing parasites to transmit through mosquitoes we fed A.
gambiae female mosquitoes on mice infected with the strain NcPIS_3 and the clone NcPIS_1
derived from an independent transfection using the ANKA reference strain as a recipient. The
result revealed a 97–98% reduction of in oocyst numbers in mosquitoes infected with either
NcPIS_3 or NcPIS_1 parasites, compared to infection with the parental strains (Fig. 5A, left
and middle respectively) or in comparison to the NcPISmut strain (Fig. 5A, right). Midguts
were also dissected and stained 24 h after feeding with NcPIS_3 or NcPIS_1. For these IFAs,
ookinetes were labeled with the P28 antibody and with an antibody directed against the A.
gambiae serine protease inhibitor SRPN6 (AgSRPN6), which selectively labels invaded cells
[59]. Thus we confirmed that a reduced number of ookinetes invaded the midgut epithelium
(Fig. 5B). In addition we investigated the effect of NcPI-S on the sporogony of the few surviving
NcPIS_3 and NcPIS_1 oocysts (Fig. 5C). To determine if these oocysts develop normally, IFA
analysis was performed with the oocyst capsule protein PbCap380 [53] and with nuclear stain-
ing for the presence of sporozoites. From a total of 33 NcPIS_3 oocysts recorded in three inde-
pendent infection experiments only a single oocyst showed a normal morphology (budded
sporozoites), hence bite back experiments were not performed. The remaining oocysts fall into
two categories: smaller than the expected size, suggesting arrested growth and nuclear division
(minute oocysts, MO), or relatively normal in size with a collapsed capsule containing diffused
nuclear material (collapsed oocysts, CO) (Fig. 5C). Similarly, among twelve recorded NcPIS_1
oocysts, three had a normal morphology, four were MO and five were CO.
Microneme formation is severely reduced in NcPIS_3 ookinetes
Careful inspection of the TEM pictures revealed that the few mature NcPIS_3 ookinetes which
developed, suffered from ultra-structural malformations including a severe decrease in the
number of micronemes (Fig. 6A). To further investigate this we chose to study the micronemal
protein SOAP. We first verified that the soap transcript levels in the NcPIS_3 zygotes/ookinetes
were comparable to WT (Fig. 6B left). However, Western blot analysis showed that the micro-
nemal protein SOAP [60] was hardly detectable in NcPIS_3 zygote/ookinete extracts, com-
pared to GFPp or NcPISmut derived extracts where this protein was readily seen (Fig. 6B right).
Furthermore we repeated these experiments using the NcPIS_1 parasite clone to exclude any
effects deriving from the genetic background. The results were identical (S5 Fig.).
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In apicomplexans, insults that perturb ER homeostasis result in repression of translation
which is mediated by the increased phosphorylation of the eukaryotic Initiation Factor 2a
(elF2a) [61,62]. To investigate if such an effect was caused by the expression of NcPI-S we ex-
amined the levels of elF2a phosphorylation by using a Phospho-eIF2α(Ser51) antibody, that
was previously shown to cross-react with the phosphorylated form of the P.falciparum eIF2a
[63], on Western blots containing extracts from NcPIS_3,NcPISmut and GFPp zygote/ookinete
cultures. A significant increase in PbeIF2a phosphorylation levels was revealed in NcPIS_3 ex-
tracts as compared to extracts derived from the GFPp and NcPISmut (Fig. 6C). Since we did
not reveal any significant alteration in the transcript levels of SOAP (as well as of CTRP) in the
Fig 5. NcPIS_3 Plasmodium ookinetes fail to produce normal oocysts. (A) Oocyst formation of NcPI-S
expressing clones (NcPIS_3 and NcPIS_1) compared to that of the parental WT strains (GFPp and ANKA
respectively) and of NcPISmut compared to NcPIS_3 7–10 days post infection. Four sets of paired infection
experiments were included in the case of NcPIS_3/GFPp, two in the case of NcPISmut/NcPIS_3 and three in
the case of NcPIS_1/ANKA 2.34 paired infections. Error bars; SEM; P<0.001, Mann-Whitney test, in all three
cases. (B) Confocal sections of representative mosquito midgut epithelial sheets infected with GFPp,
NcPIS_3, ANKA or NcPIS_1 parasites, and stained with antibodies against the mosquito serpin6 (SRPN6)
(red) and the ookinete surface protein P28 (green). Nuclei are stained with TO-PRO 3 (blue). Arrows indicate
the position of ookinetes. NcPIS_1 and 3ookinetes fail to induce an epithelial response, which in contrast is
revealed in the case of ANKA and GFPp infected midguts by SRPN6 specific antibody. Scalebar 10μM. (C)
Confocal microscopy of mature oocysts. The two types of NcPIS_3 oocysts (upper panel) are shown
alongside the single oocyst that showed a normal morphology 14 days post infection. Main oocyst
morphology includes: Normal sized oocysts with diffused nuclear material and a collapsed capsule (CO) and
oocysts arrested in an early developmental stage (minute oocysts: MO). Green corresponds to GFP
expression; nuclei are stained with TO-PRO 3. (Lower panel) NcPIS_1 abnormal oocysts labeled with the
antibody recognizing the oocyst capsule protein PbCap380. Scale bar 20μM.
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NcPIS_3 strain as compared to WT (Fig. 4B), the marked decrease in the SOAP protein levels
could probably reflect the impaired translation in NcPIS_3 parasites.
Discussion
In this study we investigated the use of serine protease inhibitors of the Kazal type to interfere
with T.gondii tachyzoite egress, invasion and growth and P.berghei zygote to ookinete transi-
tion. We chose NcPI-S, a Kazal inhibitor originating from the related apicomplexan parasite N.
caninum. Firstly, we expressed NcPI-S in T.gondii in an attempt to validate the PPI in a fast
and reliable manner. After initial validation we expanded our studies by designing a strategy
chosen to restrict the expression of the inhibitor in the P.berghei zygote to ookinete transitional
Fig 6. Mature NcPI-S_3 ookinetes contain fewer micronemes. (A) Selected longitudinal sections, across
the apical part of wild type (left image) and NcPIS_3 mature ookinetes (middle and right images). All are
surrounded by the characteristic subpellicular inner membrane complex. Scarcity of micronemes (white
arrows), is a common feature in NcPIS_3 ookinetes. The ookinete-specific characteristic structure of
crystalloid (cr) is clearly visible. (B) (Left panel) Semi-quantitative RT-PCR analysis of micronemal proteins
CTRP and SOAP in NcPIS_3 and GFPp ookinetes. No alteration was observed in transcript levels. p28 was
used as a control. (Right panel) Western analysis of soluble (SF) and membrane (MF) fraction of ookinete
extracts derived from in vitro cultured NcPIS_3,NcPISmut and GFPp ookinetes, with antibodies against the
micronemal protein SOAP. P28 was used as a sample normalization control. No SOAP is detected in the
NcPIS_3 line. (C) Western blot analysis of PbeIF2a phosphorylation. Western blots loaded with lysates from
GFPp,NcPIS_3 and NcPISmut ookinete cultures were probed with antibodies against phosphorylated eIF2α.
NcPIS_3 parasites show an increase in PbeIF2a phosphorylation. The endoplasmic reticulum (ER) marker,
BiP was used as the loading control.
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stage, a crucial time in the life cycle of malaria species as failure to successfully complete this
step blocks transmission. Importantly, this timing of expression was also chosen as it circum-
vented the possible detrimental effects on viability parasites of the blood stages, in which the
genetic transfection takes place.
All three characterized apicomplexan Kazal inhibitors (TgPI-1, TgPI-2 and NcPI-S) are
dense granule (DG) constituents. The ectopically expressed NcPI-S in Toxoplasma tachyzoites
was also trafficked in DGs and secreted in the parasitophorous vacuole, similarly to the endoge-
nously expressed NcPI-S in N.caninum tachyzoites. Strikingly though, the catalytically dead
variant NcPI-Smut was retained within the tachyzoite ER. This could be explained if NcPI-S
piggybacks on its potential targets through the secretory pathway, or if RSL mutagenesis dis-
rupted a sorting signal enabling NcPI-S anterograde trafficking. Additional studies will be nec-
essary to distinguish between these possibilities. Interestingly, there are conserved domains in
the RSLs prime site (P2’to P5’) of several Kazal domains including NcPI-S, but not in any of
the Kazal domains of TgPI-1 and TgPI-2 inhibitors. The biological role of these though, if any,
is yet to be defined.
In T.gondii the ectopically expressed NcPI-S in Toxoplasma tachyzoites was secreted in the
parasitophorous vacuole, presumably via the DGs, similar to the endogenously expressed
NcPI-S in N.caninum tachyzoites [34]. Strikingly though, a catalytically dead variant NcPI-S-
mut was retained within the ER of the tachyzoite. In contrast to the native NcPI-S no dimer
was observed for the mutant form. This could be attributed to structural changes in the mutant
preventing its efficient dimerization. For their ectopic expression in each case, NcPI-S and
NcPI-Smut were fused to the same signal peptide and epitope tag sequences, differing in 6
amino acids within the RSL including the P1-P1’residues that are critical for the inhibitory ac-
tivity. Possible reasons firstly for this differential localization of NcPI-S and NcPI-Smut in
tachyzoites might be that either NcPI-S is trafficked within the secretory pathway piggybacked
on its potential targets, or that RSL mutagenesis disrupted the folding of a sorting signal
enabling NcPI-S anterograde trafficking as well as affected the ability of the protein to
homodimerize.
Fractionation of extracellular and 24 h intracellular tachyzoites expressing active NcPI-S re-
vealed its presence in the soluble fraction, with the *24 kDa dimer being more abundant in
the soluble fraction of the extracellular tachyzoites. In the study of Morris et al.[34] the endog-
enous NcPI-S in N.caninum was also shown to form a dimer, with anomalous migration
(*20 kDa) in extracellular tachyzoite extracts, which is in accordance with our observations.
Furthermore, our results imply that NcPI-S dimerization depends on the subcellular environ-
ment (dense granules versus vacuole). However, presently we do not know to which degree
dimerization influences the NcPI-S inhibitory capacity. Interestingly, phenotypic characteriza-
tion of NcPI-S and NcPI-Smut strains revealed that while the presence of NcPI-Smut in T.gon-
dii was not detrimental for tachyzoites, ectopic expression of NcPI-S significantly affected their
intracellular growth. Parasite replication was impaired, suggesting the possibility that NcPI-S
interfered with a proteolytic event during parasite replication.
Furthermore, ectopic expression of NcPI-S in P.berghei zygotes blocked their differentiation
to ookinetes. Consequently, oocyst production was severely impaired and the few surviving oo-
cysts were defective in sporulation. In contrast, ectopic expression of the inactive NcPI-Smut
did not have any effect on zygote to ookinete differentiation in vitro, and on mosquito virulence
in vivo. In TEM studies the majority of the developmentally arrested ookinetes showed a range
of morphological abnormalities indicative of a fatal toxic stress. The few morphologically ma-
ture ookinetes also exhibited severe defects with the most prominent being a drastic reduction
of the number of micronemes. In line with the reduced number of micronemes, the protein
level of the micronemal protein SOAP was significantly reduced even though the RNA levels of
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this molecule were unaffected. Additional studies, showed elF2a increased phosphorylation in
NcPIS_3 zygotes/ookinetes that is indicative of translational repression. Studies in other organ-
isms (yeast, Toxoplasma), have shown that apart from the reduced global protein synthesis due
to the eIF2a phosphorylation, preferential translation of certain proteins helps the cells to cope
with the elevated stress [64]. If such is the case also in Plasmodium, it might not be surprising
that SOAP levels are decreased, in contrast to the BiP levels, (major ER chaperone in many or-
ganisms) which remain unaffected. It is noteworthy that inhibition of translation by the use of
cycloheximide results in a block in zygote to ookinete development (Siden-Kiamos and Deli-
gianni, unpublished).
Similarly to our study in T.gondii it was recently shown that elimination of one of the Kazal
inhibitors expressed in Toxoplasma, TgPI-1 (ΔTgPI1), results in higher parasite burden in vivo
suggesting that the endogenously expressed Kazal inhibitors also have a negative effect on
growth of the parasite [65]. Furthermore, ΔTgPI1 parasites exhibited additionally increased
bradyzoite gene expression and more efficient in vitro differentiation after stress of the tachy-
zoites in low CO
2
and alkaline pH [65]. Even though the target protease of TgPI-1 hasn’t been
identified the construction of the ΔTgPI1 line led to the conclusion that PPIs can affect T.gon-
dii virulence and bradyzoite differentiation. The removal of a negative PPI regulator of prote-
ases in ΔTgPI1 parasites emphasizes the importance of the tight regulation of proteases in
apicomplexans. In contrast to TgPI-1, the function of NcPI-S in N.caninum is yet to be de-
fined. The notion, though, is that it might suppress proteolytic activity to protect resident PV
proteins from non-regulated proteolysis [34]. Similarly ectopic expression of active NcPI-S in
T.gondii, shows that addition of an exogenous PPI represents an insult against the balanced
regulation of protease activity, which is manifested by defects in parasite replication. More im-
portantly, the impaired oocyst development observed in P.berghei, the genome of which does
not encode a serine PPI, indicates that insults to the regulation of proteolysis cannot be tolerat-
ed in a system that has evolved to regulate proteolysis by PPI-independent mechanisms. Since
our efforts to generate transgenic P.berghei parasites expressing NcPI-S under a constitutive
promoter (eIF2a and hsp70) have so far been unsuccessful (Koussis and Loukeris, unpub-
lished), we assume that expression of NcPI-S in blood stages is probably detrimental for
the parasite.
A key question though that needs to be addressed in future studies is the identity of the tar-
get protease(s) of NcPI-S. The effects of NcPI-S in T.gondii tachyzoites and in P.berghei zy-
gotes/ookinetes are theoretically mediated through the disturbance or the complete inhibition
of processes involving subtilisin-like or rhomboid proteases. An impaired tachyzoite replica-
tion rate was previously reported as the main phenotypic manifestation conditionally depleting
a micronemal Toxoplasma rhomboid TgROM1, which shows residual Golgi localization in
tachyzoites [66]. Our notion though is that studies should also focus on other classes of mole-
cules. In Plasmodium spp and Toxoplasma genomes, proteins with a rhomboid fold have been
also identified (Derlins (Ders)). These are incapable of proteolysis but it is believed that they in-
teract with their client proteins in ways similar to the rhomboid proteases [67]. Plasmodium
spp. and T.gondii genomes encode for a yeast Der1p orthologue, hDer1-1, which is likely part
of the ER Associated Degradation (ERAD) machinery, and a second paralogue sDer1-1, which
is part of a duplicated ERAD-like translocon complex placed in the apicoplast [68]. This dupli-
cated ERAD-like translocon is possibly responsible for the transfer of apicoplast proteins across
the apicoplast plasma membranes. It would therefore be challenging to attempt silencing of
Ders at the zygote stage (by promoter swap) and investigate their effect on zygote to ookinete
transition and on the structural integrity of the zygote/ookinete, and compare it with the
NcPI-S line.
A Kazal Inhibitor Affects Apicomplexa Development
PLOS ONE | DOI:10.1371/journal.pone.0121379 March 24, 2015 17 / 23
Apart from genetic studies, in vitro biochemical studies could also be implemented to find
interacting partners. The recombinantly expressed NcPI-S and its mutant form can be used for
pull down assays as has been previously done with a subtilisin Kazal inhibitor expressed by the
oomycete pathogen Phytophthora infestans [69]. Unfortunately, antibodies for serine proteases
in Apicomplexa are not available in many cases and therefore one possibility would be a com-
bination of pull down assays and a comparison of the whole proteome profiles of WT parasites
and those that express exogenous PPIs. Such an approach could help to delineate the role of ex-
ogenous PPI in both parasites and identify the target proteases of NcPI-S.
The results from this study support our initial hypothesis that NcPI-S (and potentially other
subtilisin specific Kazal inhibitors) may be used as a tool to interfere with the physiology of the
secretory apparatus of apicomplexan parasites, and in this way to manipulate their viability
and growth. With our study we exploited T.gondii parasites as a system for biochemical and
phenotypical validation of the chosen PPI. More importantly, since our main focus is P.berghei
mosquito’s stages our interest is to develop tools that could lead to novel transmission blocking
strategies. Previously, studies have demonstrated the antiparasitic activity of antiretroviral pro-
tease inhibitors against P.falciparum [70], Leishmania [71] and T.gondii [72,73]. In addition a
native Kunitz-type serine protease inhibitor was recently proven to affect viability of T.cruzi
[74] and Leishmania [75]. The unique aspect of our study is that we chose to create transgenic
parasite lines with the PPI. We anticipate that by controlling the stage and the timing for
NcPI-S expression, as well as NcPI-S’s subcellular localization it may be possible to generate
parasitic strains growth attenuated in the vertebrate host, and at the same time incapable
of transmission.
Supporting Information
S1 Fig. Phenotypic analysis of NcPIS in T.gondii.A. Growth rate of NcPIs_8 clone compared
to that of RH. After 24 hrs of intracellular replication, vacuoles from the above strains were
scored according to their parasite context (1, 2, 4, 8, 16 and 32 parasites/vacuole). Error bars
represent the means ±S.E (n = 3 experiments per strain, duplicated samples/experiment); sta-
tistical significance was determined using t-test (p<0.001).B. 24hrs NcPIS_8 intracellular
tachyzoites, stained with a-myc antibody that detects NcPI-S (red) secreted into the parasito-
phorous vacuole partly co-localizing with the dense granule specific protein GRA3 (green).
Fluorescent images were collected at 100X with a Zeiss Axioscope2plus microscope equipped
with a CCD camera. Nuclei stained with DAPI (blue). Scale bars, 10μm. C. Extracellular freshly
lysed parasites 48 hrs p.i. were stained with-anti myc (red) and anti-ROM4 (Rhomboid prote-
ase 4) (green) antibodies. Nuclei were stained with DAPI (blue). Scale bars, 1μm.
(PDF)
S2 Fig. Strategy for the generation of transgenic NcPI-S expressing P.berghei parasites. A.
Schematic representation of the gene targeting strategy used for integrating NcPIS or NcPIs-
mut in the endogenous cssu-rrna locus. Double arrowhead line indicates the expected PCR
product for an intact locus. Primers used for diagnostic PCR (L665, L260, L270 and L740) are
indicated. B. PFIGE (Pulse-Field Inverted Gel Electrophoresis) analysis of transfected parasites.
(i) DNA from transfected parasites run on 1% agarose gel, showing chromosomes separated
according to their molecular size; Chromosomes 5/6 (which can’t be separated from each
other) and 7 are indicated by arrows. (ii) The blot from the same gel hybridized with the 3’
UTR sequences of Pbdhfr/ts. The probe detects the 3’UTR of the endogenous dhfr gene on
chromosome 7, and the 3’UTR of the selectable marker indicating correct integration on chro-
mosome 5. C. PCR genotypic analysis of the derived transgenic clones. Upper panel: Lane 3
corresponds to NcPIS_3 clone. The primer pair a (L665/L740) amplifies the integration
A Kazal Inhibitor Affects Apicomplexa Development
PLOS ONE | DOI:10.1371/journal.pone.0121379 March 24, 2015 18 / 23
diagnostic 2.1kb band from genomic DNA isolated from all clones. Failure to amplify a 3kb
band using the primer pair c (L270/L740) from gDNA of the two clones indicates that integra-
tion took place at the cssu-rrna locus, while presence of a 3kb band amplified by the primer set
b (L260/L740) in WT control and the two NcPI-S expressing clones indicates the integrity of
dssu-rrna locus. (Lower panel) The same primer sets were used to confirm integration of
NcPISmut and NcPIS_1 in the specified locus.
(PDF)
S3 Fig. Localization studies of NcPI-S/NcPI-Smut and Bip in ookinetes. IFA analysis of
NcPI_S3 and NcPI_Smut lines, showing expression of the PPI (red) and the ER-chaperone BiP
(green) in P.berghei ookinetes. NcPI-Smut is expressed throughout the cytoplasm and the ER,
while NcPI-S has limited colocalisation with BiP. Scale bar: 5 μM.
(TIF)
S4 Fig. TEM images of arrested zygote/ookinetes. A. Developmentally arrested zygotes ex-
hibiting the characteristic dilation of the endomembrane system alongside extensive vacuola-
tion. B. Longitudinal sections of a sgIV ookinetes, in which the cytoplasm has been entirely
replaced by electron-lucent vacuoles. Some subcellular structures, however, are still identifiable,
such as subpellicular microtubules (arrowheads) and a well-structured collar with the charac-
teristic electron-dense depositions surrounding the apical split; notice vacuole excretion from
the apical split. A nucleus remnant (N) with a persistent electron dense area is defined by a
rough envelope (red arrow). Reduced number of micronemes is also shown in one ookinete
(white arrow).
(PDF)
S5 Fig. Western analysis of extracts derived from in vitro ookinete cultures of NcPIS_1
clone. Western analysis of extracts derived from in vitro ookinete cultures of NcPIS_1 and the
parental ANKA 15cy1A (ANKA) strain, with V5 Mab (revealing NcPI-S) and antibodies against
the micronemal protein SOAP, and the late zygote/ookinete specific P28 protein (sample nor-
malization control). While the levels of P28 are similar between the parental ANKA strain and
the NcPI-S clone, a reduction in the levels of SOAP is observed in the case of NcPIS_1 derived
extracts.
(PDF)
S1 Table. Primers used for DNA construct generation and genotype analysis.
(DOCX)
Acknowledgments
The authors thank Drs Chris J. Janse and Blandine Franke-Fayard for their help in establishing
P.berghei transgenic lines, Eva Papadogiorgaki for her excellent technical assistance with TEM,
Dr. Jean Francois Dubremetz, Dr Louis Weiss, Dr Ellen Knuepfer, Prof Dominique Soldati-
Favre and Dr. George Christofides for gifts of antibodies and Dr. Dina Vlachou for the plasmid
containing the CTRP promoter.
This manuscript is dedicated to the memory of Dr Zoi Tampaki and Dr. Thanasis Loukeris.
Author Contributions
Conceived and designed the experiments: TGL K. Kim VC APW. Performed the experiments:
ZT RSM EG SK K. Koussis. Analyzed the data: ZT RSM EG K. Koussis ISK TGL. Wrote the
paper: ZT RSM K. Koussis K. Kim VC APW ISK TGL.
A Kazal Inhibitor Affects Apicomplexa Development
PLOS ONE | DOI:10.1371/journal.pone.0121379 March 24, 2015 19 / 23
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A Kazal Inhibitor Affects Apicomplexa Development
PLOS ONE | DOI:10.1371/journal.pone.0121379 March 24, 2015 23 / 23
