RESEARCH ARTICLE

Identification of Neospora caninum proteins regulated

during the differentiation process from tachyzoite

to bradyzoite stage by DIGE

Virginia Marugan-Hernandez, Gema Alvarez-Garcıa, Veronica Risco-Castillo�,Javier Regidor-Cerrillo and Luis Miguel Ortega-Mora

SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, CiudadUniversitaria s/n, Madrid, Spain

Received: September 21, 2009

Revised: January 20, 2010

Accepted: January 25, 2010

Identification of differentially expressed proteins during Neospora caninum tachyzoite–bra-

dyzoite conversion processes may lead to a better knowledge of the pathogenic mechanisms

developed by this important parasite of cattle. In the present work, a differential expression

proteomic study of tachyzoite and bradyzoite stages was accomplished for the first time by

applying DIGE technology coupled with MS analysis. Up to 72 differentially expressed spots

were visualized (1.5-fold in relative abundance, po0.05, t-test). A total of 53 spots were more

abundant in bradyzoites and 19 spots in tachyzoites. MS analysis identified 26 proteins; 20 of

them overexpressed in the bradyzoite stage and 6 in the tachyzoite stage. Among the novel

proteins, enolase and glyceraldehyde-3-phosphate dehydrogenase (involved in glycolysis),

HSP70 and HSP90 (related to stress response) as well as the dense granule protein GRA9,

which showed higher abundance in the bradyzoite stage, might be highlighted. On the other

hand, isocitrate dehydrogenase 2, involved in the Krebs cycle, was found to be more abundant

in tachyzoites extract. Biological functions from most novel proteins were correlated with

previously reported processes during the differentiation process in Toxoplasma gondii. Thus,

DIGE technology arises as a suitable tool to study mechanisms involved in the N. caninumtachyzoite to bradyzoite conversion.

Keywords:

Bradyzoite / Developmentally expressed proteins / DIGE / Microbiology

Neospora caninum / Tachyzoite

1 Introduction

Neospora caninum is an obligate cyst-forming intracellular

protozoan parasite initially misdiagnosed as the closely

related Toxoplasma gondii [1]. N. caninum causes abortions or

stillbirths, as well as the birth of weak or healthy but

congenitally infected calves in cattle, which involves

important economic losses in the bovine industry.

The life cycle of N. caninum comprises three distinct

invasive stages: tachyzoites, bradyzoites located inside tissue

cysts and sporozoites contained in oocysts. N. caninum can

persist in brain and skeletal muscle through the tissue cyst,

forming the bradyzoite stage responsible for chronic infec-

tion for many years without the manifestation of clinical

signs [2]. However, the reactivation of a chronic infection in

a pregnant cow may lead to the switch of quiescent brady-

zoites into metabolically active tachyzoites, responsible for

Abbreviations: PV, parasitophorous vacuole; PVM, PV

membrane

�Current address: Dr. Veronica Risco-Castillo, Institut National de la

Sante et de la Recherche Medicale (INSERM), Unite Mixte de

Recherche S945, Paris F-75013, France

Correspondence: Dr. Gema Alvarez-Garcıa, SALUVET, Animal

Health Department, Faculty of Veterinary Sciences, Complutense

University of Madrid, Ciudad Universitaria s/n, 28040-Madrid,

Spain

E-mail: gemaga@vet.ucm.es

Fax: 134-913944095

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

1740 Proteomics 2010, 10, 1740–1750DOI 10.1002/pmic.200900664

the acute phase of infection. Tachyzoites disseminate

quickly, are able to cross the placenta and their replication in

the fetus may cause abortion or birth of congenitally infec-

ted calves [3]. Mechanisms triggering tachyzoite–bradyzoite

stage conversion and vice versa allow Neospora to be a highly

successful intracellular pathogen; however, these mechan-

isms are still unknown. It has been suggested that the

immune status of the host might play an important role in

these events, as described for T. gondii [4].

The expression of developmentally expressed antigens

has been suggested to be involved in immune system

evasion, as well as in the establishment of chronic infection

[2]. Thus, its use in vaccine formulations has been suggested

as a promising alternative, according to the results obtained

in a T. gondii vaccination assay [5]. Moreover, serological and

molecular assays based on antigenic stage-specific proteins

may offer additional information on the determination of

the phase of infection [6, 7]. Until now, Neospora stage-

specific proteins have been identified by genomic approa-

ches. In this sense, only two N. caninum bradyzoite-specific

antigens, NcSAG4 and NcBSR4, have been reported [8, 9],

but their role in parasite persistence remains unknown. On

the other hand, several tachyzoite-specific proteins have

been described, including surface antigens [10, 11] and

organelle components, such as micronemes and

dense granules [12]. In contrast, proteomic techniques

allow large-scale identification of proteins, which may

further lead to the identification of antigens with diagnostic

and vaccine value. Currently, several studies have been done

and focused on the N. caninum tachyzoite stage proteome

[13, 14].

Thus, a proteomic comparative study of N. caninumtachyzoite and bradyzoite stages was accomplished for the

first time. DIGE was coupled with MS to identify proteins

involved in the stage conversion.

2 Materials and methods

2.1 Tachyzoites and bradyzoites

N. caninum tachyzoites from Nc-Liv isolate were grown by

continuous passage in MARC-145 cell culture following

standard procedures. Tachyzoites were harvested 4 days

post-infection. In vitro stage conversion for bradyzoites was

carried out in sodium nitroprusside (Sigma, St. Louis, MO,

USA)-treated cultures [15]. Both zoite productions were

purified by disposable PD-10 desalting columns (GE

Healthcare, Buckinghamshire, UK), and microscope obser-

vations (Nikon Eclipse TS 100) were carefully carried out to

discard parasite batches with host cell contamination. Then,

parasites were counted, pelleted and stored at �801C until

use. Extracts from tachyzoites and bradyzoites were grouped

into four different batches of production of approximately

2� 108 zoites each, which is the required number of repli-

cates to carry out DIGE technology.

2.2 SDS-PAGE and immunoblotting analysis of

tachyzoite and bradyzoite extracts

Homogeneity between parasite batches and bradyzoite

conversions were checked by immunoblotting analysis. A

sample from each batch was resolved by 15% SDS-PAGE

under reducing conditions and transferred to nitrocellulose

membranes following a previously described procedure [16].

Membranes were incubated with both a polyclonal rabbit

antiserum developed against the recombinant NcSAG4

protein [8] at a 1:6000 dilution and a monoclonal mouse

antiserum raised against the intracytoplasmic bradyzoite

antigen TgBAG1 [17, 18] at a 1:2000 dilution. An anti-rabbit

(Sigma) or anti-mouse (GE Healthcare) IgG conjugated with

peroxidase was employed as the secondary antibody

(1:15 000 or 1:3000, respectively). The expression of NcSAG4

and TgBAG1 proteins was visualized by Immobilon

chemiluminescence (Millipore, Bedford, MA, USA).

2.3 Immunofluorescence analysis of tachyzoite and

bradyzoite extracts

The tachyzoite–bradyzoite conversion rate was assessed with

a double-immunofluorescence assay [15]. Coverslips with

infected monolayers were labeled with a monoclonal mouse

antibody directed against the tachyzoite surface antigen

NcSAG1 (1:1000) and a polyclonal rabbit antiserum raised

against the bradyzoite antigen TgBAG1 (1:500) [15] followed

by the appropriate goat IgG coupled with Alexa Fluor 488 or

Alexa Fluor 594 (1:1000) (Invitrogen, Carlsbad, CA, USA).

Coverslips were mounted on a glass slide with Dabco

(Sigma), and photographs were taken using a digital camera

(Nikon Digital Sight DS-L1) connected to an inverted

fluorescence microscope (model TE200 Nikon, 100� oil

immersion objective).

2.4 Preparation and labeling of tachyzoite and

bradyzoite extracts for DIGE

Parasite pellets were suspended in lysis buffer (6 M urea,

2 M thiourea, 4% CHAPS, 1 mM PMSF in 30 mM Tris-HCl,

pH 8.5) and disrupted by sonication cycles. Next, samples

were precipitated with the 2D-Clean up Kit (GE Healthcare)

and resuspended in 50mL of DIGE solution (30 mM Tris,

7 M urea, 2 M thiourea, 4% CHAPS). Protein concentration

was quantified by Bradford assay employing BSA as the

calibration standard. CyDye labeling was performed

following the manufacturer’s protocols (GE Healthcare).

Briefly, 50 mg protein per sample was labeled with 400 pmol

of Cy2, Cy3 or Cy5 fluorochromes dissolved in DMF (99.8%)

for 30 min at 41C in the dark. Then, reactions were quen-

ched with 1mL of lysine (10 mM/50mg protein) for 15 min at

41C in the dark. All DIGE gels included the internal stan-

dard, which was prepared by pooling equal amounts of

Proteomics 2010, 10, 1740–1750 1741

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

protein from each biological sample in the experiment and

labeling with Cy2 dye, so that all proteins from all samples

were represented in the internal standard [19].

2.5 DIGE

A total of 150 mg of protein containing the internal standard

(Cy2-labeled) and tachyzoite (Cy3-labeled for gels 1 and 2,

Cy5-labeled for gels 3 and 4) and bradyzoite extracts (Cy5-

labeled for gels 1 and 2, Cy3-labelled for gels 3 and 4) were

mixed, and an equivalent volume of loading buffer was

added (8 M urea, 4% CHAPS, 130 mM DTT and 2% IPG

buffer, pH 3–10).

Samples were loaded into 24 cm non-linear, pH 3–11,

IPG strips (GE Healthcare) by anodic cup loading and

placed on a manifold. Strips were previously hydrated

overnight with 0.45 mL hydration buffer (7 M urea, 2 M

thiourea, 2% CHAPS w/v, 10 mM DTE, IPG buffer, pH

3–10, and blue bromophenol traces). IEF was performed

with an IPGphor II unit (GE Healthcare) up to a total of

50–55 kVh.

Before the second dimension, strips were equilibrated

twice in 10 mL equilibration buffer (6 M urea, 30% glycerol

v/v, 2% SDS, 100 mM Tris-HCl, pH 6.8), first for 12 min

adding DTE (0.5%) and second for 5 min by adding iodo-

acetamide (4.5%). Strips were sealed with 1% agarose to

12% polyacrylamide gels and proteins were separated (17 h,

2 W/gel) in an Ettan Dalt Six unit (GE Healthcare). To

estimate pI and Mr of spots, a gel containing 2-D SDS-PAGE

Standards (Biorad) was run in parallel to DIGE gels and spot

matching was performed with the DeCyder 6.0 package (GE

Healthcare).

When insufficient amount of differentially expressed

spots was present in DIGE gels for MS, preparative 2-D gels

were run with a total of 300 mg protein and visualized with

an MS-compatible Coomassie blue staining. Prior to MS

analysis, spots from the preparative gel were matched with

those visualized in DIGE gels with DeCyder software for

accurate spot correspondence [20].

2.6 Image analysis and statistics

Image visualization of fluorochrome-labeled proteins was

generated with laser excitation at 488, 532 and 633 nm and

emission filters of 520, 580 and 670 nm for Cy2, Cy3 and

Cy5 fluorochromes, respectively, using a Typhoon 9400

fluorescence scanner (GE Healthcare). Image cropping and

filtering were carried out with Image Quant v. 5.2 software

(GE Healthcare), and image analyses for detection of

different abundance between spots from different stages

were performed with the DIA (Differential In gel Analysis)

module of the DeCyder 6.0 package (GE Healthcare). The

relative protein abundance of a spot was defined as the

normalized spot volume observed in the Cy3 or Cy5 channel

(protein from specific stage) divided by the normalized spot

volume of the same spot measured in the Cy2 channel

(protein reference pool) on the same gel [21]. This value was

used in the t-test statistical analysis. Spots exhibiting over

1.5-fold in their relative abundance with a p-value less than

0.05 in t-test between both stages were considered as

differentially expressed spots.

2.7 MS analysis (MS-MS/MS)

Differentially expressed spots were excised from gels and

proteins selected were in-gel reduced, alkylated and digested

with trypsin [22]. Briefly, spots were washed twice with

water, shrunk for 15 min with 100% ACN and dried in a

Savant SpeedVac for 30 min. Samples were then reduced

with 10 mM DTE in 25 mM ammonium bicarbonate (561C,

30 min) and alkylated with 55 mM iodoacetamide in 25 mM

ammonium bicarbonate for 20 min in the dark. Finally,

samples were digested overnight with 12.5 ng/mL sequen-

cing-grade trypsin (Roche) in 25 mM ammonium bicarbo-

nate (pH 8.5) at 371C.

After digestion, the supernatant was collected, and 1 mL

was spotted onto a MALDI target plate and allowed to air-dry

at room temperature. Matrix (0.4 mL of a 3 mg/mL solution

of CHCA (Sigma) in 50% ACN) was added to the dried

peptide digest spots and allowed again to air-dry at

room temperature. MALDI-TOF MS fingerprinting was

performed in a MALDI-TOF/TOF mass spectrometer (4700

Proteomics Analyzer; PerSeptive Biosystems) operating in

reflector mode with an accelerating voltage of 20 000 V. All

mass spectra were calibrated externally using a standard

peptide mixture (Sigma).

For protein identification monoisotopic peptide masses

were compared with NCBI non-redundant (NCBI nr),

Swiss-PROT/TrEMBL and ToxoDB 5.0 databases using

the MASCOT algorithm v2.1 (Matrix Science) through the

Global Protein Server v3.5 from Applied Biosystems. The

apicomplexan-specific ToxoDB database contains T. gondiiand N. caninum genome sequences. For MS/MS sequencing

analyses, suitable precursors were selected and fragmenta-

tion was carried out using CID. MASCOT search para-

meters were as follows: carbamidomethyl cysteine as fixed

modification and oxidized methionine as variable modifi-

cation; peptide mass tolerance 50–100 ppm; one missed

trypsin cleavage site; MS/MS fragments tolerance 0.3 Da.

The parameters for the combined search (peptide mass

fingerprint and MS/MS spectra) were as described above. In

all protein identifications, the probability scores were greater

than the score fixed by MASCOT as significant with a

p-value less than 0.05.

De novo sequencing from fragmentation spectra of

peptides was performed using the DeNovo software tool

(Applied Biosystems), and database homology searches of

the sequences were carried out by BLAST (http://

www.ncbi.nlm.nih.gov/BLAST).

1742 V. Marugan-Hernandez et al. Proteomics 2010, 10, 1740–1750

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

3 Results

3.1 Analysis of tachyzoite and bradyzoite extracts

First, double-immunofluorescence assays in cell cultures

raised around 60% of bradyzoite-positive parasitophorous

vacuoles (PVs) (Fig. 1A). Second, positive reactions with

anti-TgBAG1 and anti-NcSAG4 antibodies directed against

specific bradyzoite proteins of 30 and 18 kDa, respectively,

were obtained by immunoblotting (Fig. 1B). Neither anti-

body showed any reaction with pure tachyzoite extracts (data

not shown).

3.2 Differentially expressed proteins detected by

DIGE analysis

More than 2000 spots were detected for each gel, and spots

with a significant increase (or decrease) in their relative

abundance were considered as developmentally expressed

proteins. After bioinformatics analysis, a total of up to 72

differentially expressed proteins were obtained: 53 were

more abundant in the bradyzoite stage and 19 in the

tachyzoite stage (Fig. 2A).

3.3 Protein identification

A total of 31 spots were excised for MS analysis (Fig. 2B).

Only four proteins were identified as Toxoplasma proteins

when spectra data were compared against GenBank or

SwissProt non-redundant databases (data not shown).

Identifications notably increased when the ToxoDB 5.0

database was employed, and 23 spots were identified. Mass

fingerprint identified 11, and MS/MS combined with MS

identified 15 (Table 1). Of these spots, three showed double

protein identification so that 25 different Neospora proteins

were identified. In addition, a host cell protein named

vimentin was also identified, and spectra from eight addi-

tional spots did not correspond to any annotated protein.

In total, 20 out of 26 novel proteins were more abundant

in the bradyzoite stage, whereas six proteins were detected

as over-expressed in tachyzoites (Figs. 2B; Table 1).

The three double identifications were as follows: glycer-

aldehyde-3-phosphate dehydrogenase – mitochondrial

processing peptidase a subunit, DNA topoisomerase I –

myosin G in bradyzoites, and a hypothetical protein – DNA-

directed RNA polymerase III subunit in tachyzoites.

Different isoforms were detected for enolase, glycer-

aldehyde-3-phosphate dehydrogenase and GRA9 (Table 1).

4 Discussion

Tissue cyst formation containing bradyzoites is thought to

be a key factor in immune evasion by N. caninum, which

ensures its persistence in the host and makes possible

further transmission to newborn animals. During the

development of an acute infection, tachyzoites are partially

cleared by the immune response developed by the host. The

surviving parasites switch into metabolically quiescent and

low-dividing bradyzoites gathered in tissue cysts. In this

way, the parasite maintains a balance between multi-

plication and survival in the host and succeeds in persisting

[23].

Mechanisms triggering N. caninum tachyzoite–brady-

zoite conversion in apicomplexa are still unknown. In the

present study, N. caninum developmentally expressed

proteins in both tachyzoite and bradyzoite stages have been

identified for the first time by DIGE technology coupled to

MS analysis, a powerful tool to quantitatively detect devel-

opmentally expressed proteins [24].

Currently, few proteomic studies have been developed in

N. caninum, and these were only focussed on the tachyzoite

stage [13, 14, 25]. In addition, no attempt has ever been

made to identify bradyzoite-specific proteins by means of

proteomic tools in cyst-forming coccidia. This proteomic

approach has been successfully employed in other apicom-

plexan parasites to study parasite–host cell interactions

[26, 27]. Furthermore, large-scale studies on tachyzoite–

bradyzoite conversion have only been accomplished in

T. gondii by means of genomic tools [28–30].

Figure 1. Analysis of bradyzoite extracts. (A) Immuno-

fluorescence of N. caninum bradyzoite-positive parasitophorous

vacuoles in cell culture at day 7 post-treatment with 70 mM of

sodium nitroprusside. Panel 1: cell nuclear material detected by

DAPI staining. Panel 2: tachyzoite recognition by polyclonal anti-

NcSAG1 (red). Panel 3: bradyzoite recognition by monoclonal

anti-TgBAG1 (green). Panel 4: merge. (B) Immunoblotting

analysis of bradyzoite extracts with antibodies directed against

the bradyzoite stage-specific proteins NcSAG4 (18 kDa) and

TgBAG1 (30 kDa). Lane number indicates the bradyzoite batch

number for each DIGE gel replicate.

Proteomics 2010, 10, 1740–1750 1743

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Studying the Neospora bradyzoite stage is still a challen-

ging task due to the difficulty in obtaining a high amount of

material. Neospora bradyzoites can be obtained by in vivo [31]

and in vitro [15, 32] assays. The success of the in vivoapproach depends on the isolate and mouse strain

employed. In addition, after a purification process, a low

amount of cysts are obtained. On the other hand, low

bradyzoite conversion rates are obtained in cell cultures.

Despite this, bradyzoites can be easily purified without host

cell contamination for proteomic studies. This in vitroprocedure mimics early events of the tachyzoite–bradyzoite

switch since mixed PVs composed of both tachyzoites and

bradyzoites are obtained [15]. This could probably explain

the lower number of novel overexpressed proteins in

tachyzoites compared to that in bradyzoites in the present

study.

Variations in energy metabolism and alterations in the

parasite’s mitochondrial functions have been reported to be

correlated with stage conversion in Toxoplasma [33, 34]. In

this sense, isocitrate dehydrogenase, involved in the Krebs

cycle, showed higher abundance in the tachyzoite stage. The

Krebs cycle is an important key in tachyzoite metabolism

and appears to be impaired after bradyzoite differentiation

[35]. On the other hand, it has been suggested that the

Toxoplasma bradyzoite stage might rely predominantly on

anaerobic glycolysis [36]. In accordance with this, in the

present study, three glycolytic enzymes were more abundant

in the Neospora bradyzoite stage: glyceraldehyde-3-phos-

phate dehydrogenase, fructose-1,6-biphosphate aldolase and

enolase. Furthermore, two differentially expressed isoforms

of enolase, ENO2 and ENO1, have been described in Toxo-plasma tachyzoites and bradyzoites, respectively [36]. In

particular, Neospora enolase identified here showed homol-

ogy with the bradyzoite isoform of Toxoplasma (ENO1).

On the other hand, stress conditions, which are asso-

ciated with bradyzoite development [37], induce the expres-

sion of HSPs in Toxoplasma. In the present work, two HSPs

were identified (HSP70 and HSP90). Both proteins have

been reported to be expressed in the early events of brady-

zoite differentiation in response to stress conditions in

Figure 2. DIGE gels of N. caninum tachyzoite and bradyzoite proteins. (A) Overlay of images of gel 4 showing Cy3-labeled (tachyzoite

extract) and Cy5-labeled (bradyzoite extract) parasite proteins showing changes in the proteomes of N. caninum bradyzoites and/or

tachyzoites. Proteins were separated in the first dimension along a non-linear pH gradient (pH 3–11, 24 cm) and on a 12% polyacrylamide

gel in the second dimension. Spots that showed a significant difference in abundance are highlighted with red arrows for a significant

increase in the bradyzoite stage and with green arrows for a significant increase in the tachyzoite stage. (B) Silver-stained 2-D gel with

internal standard. Excised proteins for MS are indicated with arrows: in red for those overexpressed in the bradyzoite stage and in green

for those overexpressed in the tachyzoite stage. Identified proteins (Table 1) are annotated with abbreviated names; IDH2: isocitrate

dehydrogenase 2; 60S RP P0: 60S acidic ribosomal protein P0; CP CT: cholinephosphate cytidylyltransferase; CAP: cytosol aminopepti-

dase; DNA dRNA PIII: DNA-directed RNA polymerase III subunit; ENO: enolase; F1,6BPA: fructose-1,6-bisphosphate aldolase; G3PDH:

glyceraldehyde-3-phosphate dehydrogenase; MPP: mitochondrial processing peptidase alpha subunit; HSP90: heat shock protein 90;

HSP70: heat shock protein 70; DNA TPI I: DNA topoisomerase I; MyoG: myosin G; GRA9: GRA9 protein; VIM: vimentin; NLI: NLI interacting

factor-like phosphatase domain-containing protein; poly(A) BP: polyadenylate-binding protein; p36: p36 protein; HP: hypothetical

proteins; NI: non-identified proteins. To distinguish isoforms and hypothetical proteins, the spot number is also indicated. Mr and pI are

indicated in both panels.

1744 V. Marugan-Hernandez et al. Proteomics 2010, 10, 1740–1750

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Tab

le1.

N.

can

inu

mst

ag

e-e

xp

ress

ed

pro

tein

sid

en

tifi

ed

by

MS

an

d/

or

MS

/MS

Pro

tein

nam

eS

po

t

no

.a)

Avera

ge

rati

ob

)

t-T

est

valu

eO

ver-

exp

ress

ed

stag

e

Th

eo

reti

cal/

measu

red

pI

Th

eo

reti

cal/

measu

red

Mr

(kD

a)

Sco

rec)

No

.o

fm

atc

hed

pep

tid

esd

)

or

am

ino

aci

dse

qu

en

ce

of

pep

tid

ese

)

Seq

uen

ce

covera

ge

(%)f)

Acc

ess

ion

no

.Fu

nct

ion

al

cate

go

ry

Gly

cera

ldeh

yd

e-

3-p

ho

sph

ate

deh

yd

rog

en

ase

h)

1655

1.7

0.0

11

Bra

dyzo

ite

6.8

3/7

36.8

/41.1

52

35/7

d)

28

psu

|NC

_LIV

_

112720

Gly

coly

tic

en

zym

e

Gly

cera

ldeh

yd

e-

3-p

ho

sph

ate

deh

yd

rog

en

ase

g)

1597

2.3

10.0

11

Bra

dyzo

ite

6.8

3/7

.41

36.8

/42.0

79

R.S

AG

VN

IIP

AS

TG

AA

K.A

(47)e

)25

psu

|NC

_LIV

_

112720

Gly

coly

tic

en

zym

e

Gly

cera

ldeh

yd

e-

3-p

ho

sph

ate

deh

yd

rog

en

ase

h)

1648

mix

1.5

80.0

12

Bra

dyzo

ite

6.8

3/6

.73

36.8

/41.2

223

R.S

AG

VN

IIP

AS

TG

AA

K.A

(57)e

)

R.L

VE

LA

HY

MS

VQ

DG

A.-

(88)e

)

47

psu

|NC

_LIV

_

112720

Gly

coly

tic

en

zym

e

Fru

cto

se-1

,6-b

isp

ho

sph

ate

ald

ola

seh

)

1464

1.7

90.0

11

Bra

dyzo

ite

7.4

9/6

.48

89.4

/45.1

234

K.G

KP

SN

LS

IIE

VA

HG

LA

R.Y

(41)e

)

R.Y

AA

ICQ

AN

R.L

(32)e

)

22

psu

|NC

_LIV

_

124440

Gly

coly

tic

en

zym

e

K.Q

SS

HE

EV

AFY

TV

R.S

(75)e

)

En

ola

se,

pu

tati

ve

1139

3.4

60.0

11

Bra

dyzo

ite

6.1

/6.1

648.9

/50.8

102

52/1

2d

)37

psu

|NC

_LIV

_

105560

Gly

coli

tic

en

zym

e

En

ola

se,

pu

tati

ve

g)

1191

2.7

50.0

12

Bra

dyzo

ite

6.1

/6.5

948.9

/50.3

30

R.A

AV

PS

GA

ST

GIY

EA

LE

LR

.D(3

0)e

)1

psu

|NC

_LIV

_

105560

Gly

coly

tic

en

zym

e

Heat

sho

ckp

rote

in70,

pu

tati

ve

573

5.1

0.0

11

Bra

dyzo

ite

5.0

7/4

.49

73.1

/61.7

49

65/1

2d

)21

psu

|NC

_LIV

_

102920

Ch

ap

ero

ne,

stre

ssre

spo

nse

Heat

sho

ckp

rote

in90,

pu

tati

ve

428

1.7

70.0

18

Bra

dyzo

ite

5.9

1/5

.23

91.2

/65.5

78

K.E

LS

EA

EA

EA

AG

LK

R.G

(19)e

)

K.G

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Proteomics 2010, 10, 1740–1750 1745

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Tab

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1746 V. Marugan-Hernandez et al. Proteomics 2010, 10, 1740–1750

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Toxoplasma [37, 38]. Moreover, one bradyzoite overexpressed

hypothetical protein showed high homology (87% identity)

with a T. gondii bradyzoite-specific HSP (HSP21) when

BLAST was performed. Even more, it exhibited similar Mr

in a DIGE gel to TgHSP21 (data not shown). These stress

conditions could result in DNA damage in the slow repli-

cating bradyzoite stage [36], which may inhibit cell replica-

tion and delay a single cell’s entry into mitosis. Different

enzymes involved in DNA damage and repair have been

detected overexpressed in encysted bradyzoites in Toxo-plasma [39, 40]. Concerning this issue, a DNA topoisome-

rase I was found to be more abundant in the Neosporabradyzoite stage.

Other events associated with stage conversion in Toxo-plasma are changes in the main surface antigens [41]. In this

sense, the ribosomal protein P0 was found overexpressed in

tachyzoites. This protein is detected on the surface of N.caninum tachyzoites, though no assays have been performed

in bradyzoites, and seems to be directly implicated in host

cell invasion [42].

Moreover, several changes take place in the PV

membrane (PVM) after parasite invasion, with the subse-

quent formation of a thicker structure to protect bradyzoites

from environmental conditions and communicate with host

cells. Within the vacuolar compartment, Toxoplasma rhoptry

proteins are involved in PV formation, and some of them are

targeted into the host cell nucleus. On the other hand, dense

granule proteins associate with the tubular network deli-

miting membrane [43]. In our study, we report the over-

expression of ROP9 and GRA9 in the bradyzoite stage.

Conversely, in T. gondii, ROP9 has been reported as the only

tachyzoite-specific rhoptry protein [44], and GRA9 has been

described in both stages [45]. According to this finding, the

higher level of GRA9 expression in bradyzoites may give

evidence for its secretion in the PV and its implication in the

network of membranous tubules in the latest moment of

vacuole formation.

On the other hand, it is also known that tissue cysts are

wrapped into host cell filaments [46]. Interestingly, one host

cell intermediate filament, vimentin, was also more abun-

dant in bradyzoites. An explanation for this finding is the

association between vimentin and bradyzoite proteins

involved in the matrix conformation of PVs. Indeed,

vimentin is an intermediate filament that arises from the

host cell nuclear surface and progressively rearranges

around the enlarging PV compartment. In this way, the host

filament network probably serves to dock the parasite

compartment to the host cell nuclear surface [46].

Two additional identifications might be associated with

the tachyzoite–bradyzoite switch, although no previous data

have been reported in any apicomplexan parasite. First, NLI

interacting factor-like phosphatase domain-containing

protein may be involved in differentiation processes since it

plays an essential role in the development of neuronal

lineages and embryonic development [47]. Second, poly(A)

tail metabolism driven by a putative polyadenylate-binding

protein might be one of numerous cellular processes that

normally protect cells from damaging effects of high stress

conditions [48]. No biological function has been associated

with the mitochondrial processing peptidase a subunit in

apicomplexan parasites.

The myosin G heavy chain, belonging to class XXIII, has

been previously described in Toxoplasma and has an ortho-

logue in Eimeria tenella [49]. It remains unclear why myosin

G heavy chain is more abundant in the bradyzoite stage, due

to the low replication and motility found when the differ-

entiation process occurs. The only feasible explanation is its

involvement in organelle redistribution.

Our findings in tachyzoites were mainly related with the

replication process. In particular, cholinephosphate cytidy-

lyltransferase is necessary for the synthesis of phosphati-

dylcholine, which is essential for rapid parasite replication

when significant biogenesis of parasite membranes and the

concomitant enlargement of the PVM occur in T. gondii [50].

On the other hand, the overexpression of cytosolic amino-

peptidase in tachyzoites indicates that this enzymatic activity

increases during the stage when the parasite is most meta-

bolically active. Finally, DNA-dependent RNA polymerase

was also more abundant in the tachyzoite stage, probably

associated with a higher multiplication rate.

Several novel proteins showed differences between their

theoretical and measured molecular weight in DIGE gels. In

the case of isocitrate dehydrogenase 2, NLI interacting

factor-like phosphatase domain-containing protein and a

conserved hypothetical protein, these differences could be

explained by the annotation of Neospora sequences in the

database. When BLAST was performed against Toxoplasmadatabase, the theoretical molecular weight of the Neosporaprotein corresponded with two different and consecutive

Toxoplasma proteins. However, Neospora protein exhibited

homology with just one of the Toxoplasma proteins, whose

molecular weight was similar to that one exhibited by the

Neospora protein in DIGE gels. On the contrary, the theo-

retical molecular weight of myosin G, DNA topoisomerase I

and DNA-directed RNA Polymerase III subunit were iden-

tical to their Toxoplasma homologues. Thus, differences

between theoretical and measured molecular weights could

be explained by a protein processing.

Two SAG1 related sequence-family proteins, NcSAG4

and NcBSR4, have been previously identified as devel-

opmentally expressed in the Neospora bradyzoite stage [8, 9].

However, they were not found in the present study, probably

due to two possible explanations: the poor resolution of

hydrophobic proteins in 2-D PAGE and, in particular,

NcBSR4 seems to be expressed late during the tachyzoite to

bradyzoite differentiation process [9].

Furthermore, our data show that most spots are shared

between both developmental stages and do not show

differential expression, indicating that most visualized

proteins belong to housekeeping processes. As DIGE

analysis does not allow the identification of stage-specific

proteins, it is likely that several proteins detected

Proteomics 2010, 10, 1740–1750 1747

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

in the present study, such as one enolase and

glyceraldehyde-3-phosphate dehydrogenase isoforms, poly-

adenylate-binding protein, DNA topoisomerase I, myosin G

and vimentin, seem to be bradyzoite-specific proteins

according to DeCyder analysis (data not shown).

It is worthwhile mentioning that several of novel proteins

identified in the present study have been proposed as ther-

apeutic targets for diseases caused by apicomplexan parasites.

Apicomplexan glycolytic enzymes, which are plant homo-

logues, have been suggested as novel chemotherapeutic

targets [51]. Similarly, disruption of phospholipid metabolic

pathways was suggested to combat parasite growth in Toxo-plasma [50] and Plasmodium [52]. Inhibitors against amino-

peptidases [53] and type I topoisomerases [54] seem to be

potential drugs for the treatment of human parasitic diseases,

such as malaria or leishmaniasis, together with drugs block-

ing HSP90 functions that affect invasion and conversion of

the parasite [38]. Concerning vaccine development, antigens

that are known to be important for parasite survival should be

considered for inclusion in a N. caninum vaccine [55].

Consequently, the novel proteins involved in membrane

surface (ribosomal protein P0) and PVM modification (ROP9,

GRA9) might be candidates for vaccine development.

Recently, several authors have suggested the importance

of proteome and genome integration data, since surprising

discrepancies between protein abundances and transcript

expression data have been shown in Toxoplasma [56]. Our

novel proteins differ from previously described ones in those

genomic approaches, but they are certainly involved in the

same processes. In this way, determining both absolute

protein expression and post-translational events will be a key

factor in gaining a more complete understanding of the

biology of these pathogenic organisms.

In conclusion, the present results have evidenced for the

first time in Neospora the previously reported developmental

strategies in Toxoplasma during stage conversion. This study

opens the gate to further studies on tachyzoite–bradyzoite

conversion mechanisms and sets the basis for further studies

in potential targets for drug and vaccine development.

We thank Louis M. Weiss (Albert Einstein College of Medi-cine, New York) for the monoclonal anti-recombinant TgBAG1serum, Andrew Hemphill (Institute for Parasitology, Universityof Berne, Switzerland) for providing the polyclonal rabbit anti-recombinant NcSAG1 serum, and Diana Williams (LiverpoolSchool of Tropical Medicine, Liverpool, UK) for the N. caninumNc-Liv isolate. We also thank Aida Pitarch and MonserratMartınez (Pharmacy Faculty, Complutense University ofMadrid, Spain) for carefully reading the manuscript. Theproteomics work was done at the Proteomics Facility UCM-PCM, a member of ProteoRed network, funded by GenomaEspana. VMH was supported by a fellowship from the Ministryof Science and Innovation of Spain. This work was financiallysupported by AGL 2007–60132/GAN.

These authors have declared no conflict of interest.

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