# Protein characterization of the IgM triplet involved in the diagnosis of congenital toxoplasmosis

Maria Carreno, Odile Villard, Isabelle Villena, Lucie Peyclit, Helene Diemer, Christine Schaeffer-Reiss, Coralie L'ollivier

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Maria Carreno, Odile Villard, Isabelle Villena, Lucie Peyclit, Helene Diemer, et al. Protein characterization of the IgM triplet involved in the diagnosis of congenital toxoplasmosis. Parasite, 2025, 32, pp.72. 10.1051/parasite/2025065. hal-05443649

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# Protein characterization of the IgM triplet involved in the diagnosis of congenital toxoplasmosis

Maria Carreno<fnref n="1" />, Odile Villard<fnref n="2" />,<fnref n="3" />, Isabelle Villena<fnref n="4" /> ID, Lucie Peyclit<fnref n="1" />, Helene Diemer<fnref n="5" />,<fnref n="6" />, Christine Schaeffer-Reiss<fnref n="5" />,<fnref n="6" />, and Coralie L'Ollivier<fnref n="1" />,<fnref n="7" />,* ID

Received 16 March 2025, Accepted 30 October 2025, Published online 26 November 2025

**Abstract** – Congenital toxoplasmosis is a fetal infection resulting from the transplacental transmission of *Toxoplasma gondii* in mothers who seroconvert during pregnancy. Neonatal diagnosis has recently been improved through the identification by L'Ollivier *et al.* (2012) and Peyclit *et al.* (2023) of a pathognomonic marker for congenital toxoplasmosis: the IgM triplet, corresponding to three high molecular weight bands of 75, 90, and 100 kDa, respectively found on the mother-child immunoblot pair profile. This is a new concept, as these three IgM bands reflect an immune response targeting proteins involved in vertical transmission of *T. gondii*. These proteins may be *T. gondii* secreted or non-secreted effectors implicated in host cell invasion, immune modulation, and parasite virulence. In this study, immunoproteomic techniques allowed us to identify thirty-two relevant protein spots on immunoblot, including four specifically associated with the IgM triplet. Protein identification by LC-MS/MS revealed several *T. gondii* proteins as strong candidates for the IgM triplet. Each of these proteins is, directly or indirectly, involved in cellular invasion and may also play a role in transplacental transmission of *T. gondii*. Identifying these proteins opens several avenues of therapeutic research that could improve the management of congenital toxoplasmosis.

**Key words:** *Toxoplasma gondii*, Congenital toxoplasmosis, IgM triplet, Immunoproteomics, 2-DE Immunoblotting, LC-MS/MS.

**Résumé** – Caractérisation protéique de la triplette IgM impliquée dans le diagnostic de la toxoplasmose congénitale. La toxoplasmose congénitale est une infection fœtale résultant de la transmission transplacentaire de *Toxoplasma gondii* chez des mères séroconverties pendant la grossesse. Le diagnostic néonatal a récemment été amélioré grâce à l'identification par L'Ollivier *et al.* (2012) et Peyclit *et al.* (2023) d'un marqueur pathognomonique de la toxoplasmose congénitale : la triplette IgM, correspondant à trois bandes de haut poids moléculaire de 75, 90 et 100 kDa respectivement, présentes sur le profil de paires d'immunoblots mère-enfant. Il s'agit d'un concept nouveau, car ces trois bandes IgM reflètent une réponse immunitaire ciblant des protéines impliquées dans la transmission verticale de *T. gondii*. Ces protéines pourraient être des effecteurs sécrétés ou non par *T. gondii* impliqués dans l'invasion des cellules hôtes, la modulation immunitaire et la virulence du parasite. Dans cette étude, les techniques d'immunoprotéomique nous ont permis d'identifier trente-deux spots protéiques pertinents sur immunoblot, dont quatre spécifiquement associés à la triplette IgM. Pour ces spots, l'identification des protéines par LC-MS/MS a révélé que plusieurs protéines de *T. gondii* étaient de bonnes candidates pour la triplette IgM. Chacune de ces protéines est impliquée, directement ou indirectement, dans l'invasion cellulaire et pourrait également jouer un rôle dans la transmission transplacentaire de *T. gondii*. L'identification de ces protéines ouvre de nouvelles perspectives thérapeutiques susceptibles d'améliorer la prise en charge de la toxoplasmose congénitale.

<footnote n="1">¹ IHU Méditerranée Infection, 13005 Marseille, France</footnote>

<footnote n="2">² Laboratoire de Parasitologie et Mycologie Médicale, Laboratoire Associé CNR de la Toxoplasmose, Les Hôpitaux Universitaires de Strasbourg, 67091 Strasbourg, France</footnote>

<footnote n="3">³ Institut de Parasitologie et Pathologie Tropicale, UR7292 Dynamique des interactions hôte pathogène, Fédération de Médecine Translationnelle, Université de Strasbourg, 67091 Strasbourg, France</footnote>

<footnote n="4">⁴ UR ESCAPE, Université Reims-Champagne-Ardenne, Service de Parasitologie-Mycologie, Centre National de Référence de la Toxoplasmose, Centre Hospitalier Universitaire (CHU) Reims, 51092 Reims, France</footnote>

<footnote n="5">⁵ Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR7178, 67087 Strasbourg, France</footnote>

<footnote n="6">⁶ Infrastructure Nationale de Protéomique ProFI, UAR2048, Strasbourg, France</footnote>

<footnote n="7">⁷ Aix Marseille Univ, IRD, AP-HM, SSA, VITROME, 13005 Marseille, France</footnote>
## Introduction

Toxoplasma gondii (*T. gondii*) is a ubiquitous pathogen that can infect a wide range of hosts, such as the majority of birds and mammals (including humans). In humans, infection is usually asymptomatic or causes a simple flu-like illness in immunocompetent patients, but can cause severe disease in immunocompromised patients and complications in newborns (NB) if the mother seroconverts during pregnancy (congenital toxoplasmosis [CT]) [13]. Indeed, CT can lead to serious complications in the fetus and NB, including abnormal psychomotor development, ocular lesions such as chorioretinitis, and neurological disorders [19].

Diagnosis of CT is based on prenatal screening and/or the detection of neosynthesized IgG, IgM, or IgA in the NB, and/or IgG persisting for 12 months after birth. Treatment of the infected infant should be initiated as early as possible to minimize clinical sequelae. It is recognized that the IgG/IgM mother-infant immunoblot pair profiles (IbPP) help in early neonatal diagnosis. L'Ollivier et al. [14] and Peyclit et al. have also demonstrated that the presence of three IgM bands in the infant profile, at 75, 90, and 100 kDa, is pathognomonic for the diagnosis of CT (Supplementary data S1) [14, 21]. Proteomic characterization of this IgM triplet would enhance our understanding of its significance in CT diagnosis and provide insights into the pathophysiological mechanisms underlying transplacental transmission. [2].

The precise mechanisms of *T. gondii* vertical transmission remain poorly understood due to the lack of laboratory models reflecting the complexity of the maternal-fetal interface [1]. Nevertheless, based on literature data, assumptions can be made regarding the parasite's ability to reach the intra-amniotic compartment. It has to overcome both mechanical barriers, with the syncytiotrophoblasts as the main layer to be crossed, and immunological barriers from both the maternal and fetal immune systems. As a result, *T. gondii* has developed a complex set of molecular mechanisms involving secreted and non-secreted proteins to invade and manipulate host cells, thereby contributing to its virulence. These proteins play a role throughout the parasite's lytic cycle and may be involved in maternal-fetal transmission of *T. gondii* across the placental barrier [2].

In this study, we used immunoproteomics techniques (two-dimensional electrophoresis [2-DE], immunoblotting, and liquid chromatography coupled to tandem mass spectrometry [LC-MS/MS]) to identify the proteins targeted by the IgM triplet using sera from NBs with confirmed CT who exhibited this triplet in their mother-infant IbPP.

## Materials and methods

### Ethics approval

The study was conducted in accordance with the tenets of the Declaration of Helsinki and approved by the Ethics Committee of the Assistance Publique des Hôpitaux de Marseille, France (protocol code AC-2009-1031 of July 27, 2010, and protocol code 2019-73 of May 29, 2019).

### IgM triplet control sera

The selected serum or cord blood samples were obtained from the routine diagnostic activity of a parasitology-mycology laboratory (IHU Méditerranée, Marseille, France). Sera were stored at -20 °C and selected between 2010 and 2021. Sixteen serum samples from NBs with proved CT were selected based on the presence of the IgM triplet on the IbPP performed at birth (NNTPos group). Three serum or cord blood samples from NBs with proven CT and positive IgM, but without the IgM triplet on the IbPP at birth were selected as negative controls (NNTNeg group).

## Preparation of *T. gondii* lysate antigen

Tachyzoites of the RH strain of *T. gondii* were maintained in Swiss Webster mice (CERJ, Cergy-Pontoise, France) by intraperitoneal inoculation. Three days later, the mice were sacrificed by inhalation of isoflurane (Forène®, Abbott France, Rungis, France), and the tachyzoites were recovered by instilling 5 mL of sterile 0.9% NaCl solution into the peritoneal cavity. Parasites were then washed twice in phosphate-buffered saline (PBS) and used to prepare the *T. gondii* lysate antigen (TLA) with deoxycholate 2.5% (DOC) and sodium dodecyl sulfate 2.5% (SDS).

TLA was supplemented with a protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and stored at -80 °C until purification. TLA was purified using a 2-D Clean Up Kit (GE Healthcare Bioscience, Chalfont St. Giles, United Kingdom), according to the supplier's protocol. Proteins were quantified using the Bradford assay (Bio-Rad, Inc., Hercules, CA, USA).

## Two-dimensional electrophoresis of TLA

An amount of 20 µg of purified TLA was loaded onto each Immobiline™ Drystrip 7 cm pH 3–10 gels (IPG strips) (GE Healthcare, Uppsala, Sweden) after strip rehydration into TS buffer (7M urea, 2M thiourea, 4% CHAPS) containing 60 mM dithiothreitol (DTT) and 0.5% immobilized pH gradient buffer (pH 3-10, GE Healthcare, Uppsala, Sweden). Isoelectric focusing (IEF) was performed according to a multi-step protocol: 300 V for 30 min in linear voltage mode, 1000 V for 30 min, 5000 V for 80 min, then 5000 V for 40 min in rapid voltage mode at 20 °C using an Ettan IPG Phor II IEF system (GE Healthcare). Upon completion of the IEF, the IPG strips were removed from the device and stored at -20 °C before the next step. IPG strips were then equilibrated for 15 min in equilibration buffer (1.5 M Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 0.04% bromophenol blue) containing 65 mM DTT, then for 15 min in the same buffer used for DTT plus 100 mM iodoacetamide. For two-dimensional electrophoresis (2-DE), proteins were separated by SDS-PAGE electrophoresis on 12% resolving gels. Electrophoresis was performed at 30 mA for 70 min. Next, 2-DE gels were fixed in 40% ethanol and 10% glacial acetic acid for 12 h and then stained with silver nitrate to visualize all the proteins of *T. gondii*. Silver-stained gels were scanned using ImageScanner
III (EPSON Scan) and Image Ready software (v 7.0.1, Adobe Systems).

Thirty-eight 2-DE gels were reserved for IgM triplet revela-
tion using the selected NB sera. The 16NNTPos sera and the 3
NNTNeg sera were for immunoblot analysis in duplicate.

Immunoblot analysis using selected sera

Protein on the 2-DE gels were transferred to nitrocellulose
membranes (Fisher Scientific, Illkirch, France) previously acti-
vated with 20% ethanol for optimal protein transfer. Transfer
was performed at 100 V for 90 min at 4 °C. After transfer,
the membranes were blocked with a saturation buffer contain-
ing 5% skim milk in PBS with 0.3% Tween 20 (PBST) for
1 h at room temperature. The membranes were then incubated
overnight at 4 °C with NNTPos or NNTNeg sera diluted at
1:50.

After three 10-minute washes in PBST, the transfers were incubated with peroxidase-conjugated goat anti-human IgM secondary antibodies (Jackson ImmunoResearch, Philadelphia, PA, USA) diluted 1:5000 in PBST-5% skim milk for 1 h at room temperature. After three 10-minute washes in PBST, protein spots were revealed using the Clarity Western ECL Substrate Kit (Bio-Rad, Hercules, CA, USA) via the Fusion FX digital imaging system (Vilber Lourmat, Collégien, France). The images obtained were analyzed using Photoshop software (v 7.0.1, Adobe Systems).

Determination of IgM triplet-specific proteins by
liquid chromatography-mass spectrometry

The two protein profiles of silver nitrate-stained tachyzoites
from the *T. gondii* RH strain were superimposed on the 32
transfers obtained from the 16 NNTPos sera analyzed in dupli-
cate to identify any spots specific to the IgM triplet. The two
protein profiles were also superimposed on the six transfers
obtained from the three NNTNeg sera analyzed in duplicate
to eliminate spots not specific to the IgM triplet. The spots con-
sistently containing the immunoreactive proteins detected in the
IgM triplet zone (between 75 and 100 kDa) only in NNTPos
sera were excised from the two protein profiles of *T. gondii*
RH strain tachyzoites stained with silver nitrate. The gel spots
were destained by two washes with 50 µL of 15 mM potassium
ferricyanide and 50 mM sodium thiosulfate. After three washes
with water, acetonitrile, and 25 mM ammonium hydrogen car-
bonate (NH₄HCO₃), the gel plugs were dehydrated with ace-
tonitrile. Cysteine residues were reduced with 50 µL of
10 mM dithiothreitol at 57 °C and alkylated with 50 µL of
55 mM iodoacetamide. After two washes with NH₄HCO₃
and acetonitrile, the gel plugs were dehydrated with acetonitrile.
The digestion of proteins was done in gel with 10 µL of 7 ng/
µL modified porcine trypsin (Promega, Madison, WI, USA) in
25 mM NH₄HCO₃. Digestion was performed overnight at room
temperature. The generated peptides were extracted with 40 µL
of 60% acetonitrile in 0.1% formic acid. Acetonitrile was evap-
orated under vacuum prior to MS analysis.

Mass spectrometry analysis

NanoLC-MS/MS analysis was performed using a nanoAC-
QUITY Ultra-Performance-LC (Waters Corporation, Milford,
MA, USA) coupled to a Q-Exactive Plus mass spectrometer
(Thermo Fisher Scientific, Bremen, Germany). The entire
system was fully controlled by Thermo Scientific™ Xcalibur™
software (v3.1). Peptides were trapped on a NanoEase™ M/Z
Symmetry C18 precolumn (100 Å, 5 µm, 180 µm ×
20 mm, Waters Corporation) using 99% solvent A (0.1% for-
mic acid in water) and 1% solvent B (0.1% formic acid in ace-
tonitrile) at a flow rate of 5 µL/min for 3 min. A solvent
gradient from 1 to 6% of B in 0.5 min and then from 6–40%
of B in 44 min was used for peptide elution, which was
performed at a flow rate of 350 nL/min using a NanoEase™
M/Z BEH C18 column (130 Å, 1.7 µm, 75 µm × 250 mm,
Waters Corporation) maintained at 60 °C. The Q-Exactive Plus
was operated in positive ion mode with a source temperature of
250 °C and a spray voltage of 1.8 kV. Full scan MS spectra
(300–1800 m/z) were acquired at a resolution of 70,000 at m/
z 200.

MS parameters were set as follows: maximum injection
time of 50 ms, automatic gain control (AGC) target value of
3e6 ions, lock mass option enabled (polysiloxane,
445.12002 m/z), selection of up to 10 most intense precursor
ions (doubly charged or more) per full scan for subsequent iso-
lation using a 2 m/z window, fragmentation using higher-
energy collisional dissociation (HCD, normalized collision
energy of 27), dynamic exclusion of already fragmented precur-
sors set to 60 s. MS/MS spectra (200–2000 m/z) were acquired
with a resolution of 17,500 at m/z 200. MS/MS parameters
were set as follows: maximum injection time of 100 ms,
AGC target value of 5e3 ions, peptide match selection option
turned on. Raw data were converted into mgf files using the
MSConvert tool from ProteomeWizard (v3.0.6090; http://prote-
owizard.sourceforge.net/).

Protein identification

For protein identification, MS/MS data were searched using
a local Mascot server (v2.6.2; Matrix Science, London, United
Kingdom) against a database containing the sequences of all
*T. gondii* ME49 entries as found in https://toxodb.org/
(ToxDB-10.0_TgondiiME49_AnnotatedProteins.fasta, 8,322
sequences) and the corresponding 8,322 reverse sequences.
Spectra were searched with a mass tolerance of 10 ppm for
MS and 0.05 Da for MS/MS data, allowing a maximum of
one missed trypsin cleavage. Carbamidomethylation of cysteine
residues and oxidation of methionine residues were specified as
variable modifications. Identification results were imported into
Proline v2.1 (www.pro脂proteomics.fr/proline) for validation
with the following criteria: peptide spectrum matches (PSM)
with pretty rank equal to 1 and a minimum sequence length
of 7 were retained and the false discovery rate was then opti-
mized to be less than 1% at the PSM level using the Mascot
adjusted E-value and less than 1% at the protein level using
the Mascot score.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [20] partner repository with the dataset identifier PXD060424 and DOI: https://doi.org/10.6019/PXD060424.

# Results

## Delineation of the IgM triplet zone

Using the two protein profiles of the *T. gondii* RH strain obtained from 2-DE gels and stained with silver nitrate, 32 spots were identified in the IgM triplet zone between 75 and 100 kDa (Fig. 1). These spots appeared as brown and of varying intensities. Some spots were isolated (spots 1, 2, 3, 4, 5, 9, and 12), while others were clustered (spots 6–8, spots 10–11, spots 13–14, spots 15–16, spots 17–20, spots 21–24, spots 25–26, spots 27–30, and spots 31–32). Clustered spots may represent different proteins or the same protein with post-translational modifications.

## IgM triplet revelation using NNTPos and NNTNeg sera

A total of 32 transfers were performed for IgM triplet rev- elation using NNTPos sera ($n = 16$) each analyzed in duplicate. An additional six transfers were performed to exclude the spots not specific to the IgM triplet using NNTNeg sera ($n = 3$) also analyzed in duplicate (Fig. 2).

After superimposing the silver-stained protein profiles of *T. gondii* RH strain and transfers obtained with NNTPos sera using Photoshop software (v 7.0.1, Adobe), six spots previously numbered 21–26 were found to match, corresponding to one or more potential target proteins of the IgM triplet.

After superimposing the same protein profiles and patient transfers obtained with NNTNeg sera using Photoshop soft- ware (v 7.0.1, Adobe), spots 25 and 26 were excluded. Ultimately, four protein spots (21–24) were found only with the NNTPos sera (Fig. 2). The immunoblots from the 19 patients (16 NNTPos and 3 NNTNeg) are shown in Supplementary data S2.

## Identification of IgM triplet proteins by LC-MS/MS

Protein spots to be analyzed were excised from the two silver nitrate-stained protein profiles of the *T. gondii* RH strain. A total of eight spots were collected, four from each gel as mirror images, in order to identify as many proteins as possible present in the IgM triplet zone. In the selected spots (21–24) a total of fifty-three unique proteins each with at least one matching peptide was identified by LC-MS/MS analysis. Forty proteins were identified for spot 21, twenty-nine for spot 22, thirty-three for spot 23, and thirty for spot 24 (Tables 1–4).

Some proteins were only detected on one of the two silver-stained protein profiles of the *T. gondii* RH strain and were therefore excluded. As a reminder, the IgM triplet is made up of three high molecular weight (MW) bands of 75, 90, and 100 kDa. Proteins identified for each spot (21–24) were

grouped according to their MW, to match the three IgM triplet bands (75, 90, and 100 kDa) as closely as possible (Table 5). Low MW proteins (less than 65 kDa) were excluded, as the first IgM triplet band appeared at 75 kDa. On the other hand, high MW proteins (greater than 100 kDa) were associated with the last IgM triplet band at 100 kDa, as resolution is limited in this region, making it impossible to exclude them.

A total of eight proteins (EMP24/GP25L/p24 family protein; Vacuolar ATP synthase subunit A, putative; Alveolin domain containing intermediate filament IMC1 [ALV1]; Heat Shock Protein 70 [HSP70]; Chaperonin Protein BiP; hypothetical protein; Tryptophanyl-tRNA synthetase [TrpRS2]; and Heat Shock Protein family [HSP]) identified across the four selected spots were considered potential components of the 75 kDa IgM triplet band. Two proteins found in at least three of the four selected spots (Heat Shock Protein 90 [HSP90] and hypothetical protein) appeared to be good candidates for the 90 kDa IgM triplet band. Three proteins found in at least three of the four selected spots (Aconitate hydratase ACN/IRP; Cytosolic tRNA-Ala synthetase; and Toxolysin 4 [TLN4]) appeared to be good candidates for the 100 kDa IgM triplet band (Table 5).

# Discussion

The aim of this work was to identify the proteins involved in the immunogenic recognition marked by the IgM triplet, which is pathognomonic for CT [14]. This IgM triplet, represented by three high MW bands of 75, 90, and 100 kDa on the mother-infant IbPP, is thought to target proteins involved in maternal-fetal transmission of *T. gondii*. Identification of these proteins requires proteomic analysis.

Proteomics has evolved tremendously over the last few decades, thanks to technological and methodological advances that have notably made it possible to map most of the *T. gondii* proteome [4]. For example, in 2008, Xia *et al.* described almost one-third of the proteins in the tachyzoite stage of *T. gondii*, i.e., approximately 2,252 proteins (including 2,477 intron-spanning peptides) using three different approaches (2-DE, LC-MS/MS, and multidimensional protein identification technology – MudPIT) [27]. In 2011, Che *et al.* similarly characterized 2,241 membrane proteins from the tachyzoite stage of *T. gondii* using one-dimensional gel electrophoresis LC-MS/MS, biotin labeling in conjunction with one-dimensional gel LC-MS/MS analysis, and a novel strategy that combines three-layer “sandwich” gel electrophoresis with multidimensional protein identification technology [3]. In 2017, Wang *et al.* identified by LC-MS/MS analysis some 6,285 proteins in *T. gondii* out of the 6,000 to 8,000 predicted by genomic analysis, these proteins being expressed differently depending on the parasite stage (tachyzoite, bradyzoite, or sporozoite) [3, 26]. All these advances in proteomic analysis have given rise to the *ToxoDB* database. The identification of these proteins provides a better understanding of their role in the various biological processes of the parasite cycle, as well as in the host-parasite interactions in which they are involved [4]. This actually makes it possible to characterize the proteins involved in the mechanisms used by *T. gondii* to invade, replicate, and manipulate the host’s metabolism, and establish its virulence. In fact, *T. gondii*
<figure><img src="image_3.png" /><figcaption>**Figure 1.** 2-DE gel obtained with *T. gondii* lysate antigen after silver nitrate staining. A. All proteins from *T. gondii* RH strain tachyzoites were separated by isoelectric focusing (pH 3–10), followed by SDS-PAGE electrophoresis on polyacrylamide gel (12% acrylamide), and then revealed by silver nitrate staining. B. Delineation of the IgM triplet zone between 75 and 100 kDa and numbering of all protein spots present. C. Close-up of the protein spots in the IgM triplet zone.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 2.** 2D immunoblot of *T. gondii* RH strain proteins. A and B. Obtained after incubation with two NNTPos sera (patients 5BB (A) and 8BB (B)) and revelation with peroxidase-conjugated goat anti-human IgM antibodies. C and D. Obtained after incubation with two NNTNeg sera (patients 17BB (C) and 19BB (D)) and revelation with peroxidase-conjugated goat anti-human IgM antibodies. These immunoblots reveal the IgM triplet spots of interest (21–24) present only in NNTPos sera and identified by superposition with the protein profile of the *T. gondii* RH strain (black box). These immunoblots also make it possible to exclude the spots that are not specific to the IgM triplet (25 and 26) present in NNTPos and NNTNeg sera and identified by superposition with the protein profile of the *T. gondii* RH strain (NNTPos: newborns positive for the IgM triplet; NNTNeg: newborns negative for the IgM triplet).</figcaption></figure>
<table><caption>Table 1. Proteins of *Toxoplasma gondii* RH strain identified by LC-MS/MS for the first IgM triplet spot of interest (spot 21) (ND: not detected)</caption><thead><tr><th>Protein name</th><th>Known or predicted function</th><th>Location</th><th>Accession</th><th>Organism</th><th>Molecular weight (Da)</th><th>Peptide count on protein profile No. 1</th><th>Peptide count on protein profile No. 2</th></tr></thead><tbody><tr><td>Heat Shock Protein (HSP)</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system</td><td>TGME49_chrXII:5582774-5587740(–)</td><td>TGME49_251780</td><td>Toxoplasma_gondii_ME49</td><td>78251</td><td>56</td><td>35</td></tr><tr><td>Heat Shock Protein 70 (HSP70)</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system (modulation of nitric oxide production and reduction of macrophages phagocytosis capacity)</td><td>TGME49_chrVIII:3289396-3291420(–)</td><td>TGME49_273760</td><td>Toxoplasma_gondii_ME49</td><td>72880</td><td>53</td><td>51</td></tr><tr><td>Chaperonin protein BiP</td><td>Molecular chaperone involved in protein folding and exit from the endoplasmic reticulum</td><td>TGME49_chrXI:2225378-2228753(–)</td><td>TGME49_311720</td><td>Toxoplasma_gondii_ME49</td><td>73253</td><td>43</td><td>26</td></tr><tr><td>Heat Shock Protein 90 (HSP90)</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system</td><td>TGME49_chrIX:2483404-2487798(+)</td><td>TGME49_288380</td><td>Toxoplasma_gondii_ME49</td><td>81933</td><td>32</td><td>30</td></tr><tr><td>Alveolin domain containing intermediate filament IMC1 (ALV1)</td><td>Inner membrane complex protein associated with the membrane cytoskeleton of Toxoplasma gondii, involved in maintaining parasite morphology and motility</td><td>TGME49_chrVIII:1516857-1519632(+)</td><td>TGME49_231640</td><td>Toxoplasma_gondii_ME49</td><td>69596</td><td>25</td><td>14</td></tr><tr><td>Peptidase M16 inactive domain-containing protein</td><td>Member of the M16A metalloprotease family, no function described</td><td>TGME49_chrX:716364-726488(+)</td><td>TGME49_227948</td><td>Toxoplasma_gondii_ME49</td><td>142764</td><td>13</td><td>2</td></tr><tr><td>Tryptophanyl-tRNA synthetase (TrpRS2)</td><td>Apicoplast protein involved in the aminoacylation of tRNA during proteins synthesis</td><td>TGME49_chrIX:2473710-2478137(+)</td><td>TGME49_288360</td><td>Toxoplasma_gondii_ME49</td><td>77058</td><td>10</td><td>9</td></tr><tr><td>Toxolysin 4 (TLN4)</td><td>Member of the M16A metalloprotease family, involved in Toxoplasma gondii egress from host cells and maturation of microneme proteins</td><td>TGME49_chrVIIa:903405-917753(+)</td><td>TGME49_206510</td><td>Toxoplasma_gondii_ME49</td><td>246741</td><td>10</td><td>2</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrVIIa:3523795-3527924(–)</td><td>TGME49_202200</td><td>Toxoplasma_gondii_ME49</td><td>87043</td><td>10</td><td>ND</td></tr><tr><td>Cytosolic tRNA-Ala synthetase</td><td>Cytosolic protein involved in the aminoacylation of tRNA during proteins synthesis</td><td>TGME49_chrXII:862344-876041(–)</td><td>TGME49_219540</td><td>Toxoplasma_gondii_ME49</td><td>139516</td><td>7</td><td>4</td></tr><tr><td>Vacuolar ATP synthase subunit A, putative</td><td>Proton-transporting ATP synthase activity contributing to endomembrane compartments acidification, organelle maturation, and intracellular pH regulation</td><td>TGME49_chrVIb:4315661-4325274(–)</td><td>TGME49_256970</td><td>Toxoplasma_gondii_ME49</td><td>68122</td><td>6</td><td>3</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrVIII:2878595-2883425(–)</td><td>TGME49_233890</td><td>Toxoplasma_gondii_ME49</td><td>73697</td><td>6</td><td>2</td></tr><tr><td>Membrane Occupation and Recognition Nexus protein 2 (MORN2)</td><td>No function described, hypothetical role in cytoskeleton organization</td><td>TGME49_chrIX:4562922-4564319(+)</td><td>TGME49_292120</td><td>Toxoplasma_gondii_ME49</td><td>49236</td><td>6</td><td>ND</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrII:644118-650964(+)</td><td>TGME49_221870</td><td>Toxoplasma_gondii_ME49</td><td>144135</td><td>6</td><td>ND</td></tr><tr><td>Heat Shock Protein 90, putative</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system</td><td>TGME49_chrVI:3356828-3362144(+)</td><td>TGME49_244560</td><td>Toxoplasma_gondii_ME49</td><td>96823</td><td>5</td><td>1</td></tr><tr><td>Aconitate hydratase ACN/IRP</td><td>Mitochondrial/apicoplast enzyme involved in the energy metabolism of Toxoplasma gondii (Krebs cycle) and regulation of host immune system (modulation of nitric oxide production)</td><td>TGME49_chrX:1400984-1408155(–)</td><td>TGME49_226730</td><td>Toxoplasma_gondii_ME49</td><td>114885</td><td>5</td><td>1</td></tr><tr><td>SAG-related sequence SRS29B (SRS29B)</td><td>Surface protein involved in host cell attachment</td><td>TGME49_chrVIII:2664481-2665491(+)</td><td>TGME49_233460</td><td>Toxoplasma_gondii_ME49</td><td>34732</td><td>4</td><td>4</td></tr><tr><td>DnaK family protein</td><td>ATP-dependent chaperone involved in protein folding</td><td>TGME49_chrX:1311834-1315968(–)</td><td>TGME49_226830</td><td>Toxoplasma_gondii_ME49</td><td>103193</td><td>4</td><td>2</td></tr><tr><td>Tetratricopeptide repeat-containing protein</td><td>ATP-dependent chaperone involved in protein folding</td><td>TGME49_chrIa:1252410-1260641(–)</td><td>TGME49_294898</td><td>Toxoplasma_gondii_ME49</td><td>88633</td><td>4</td><td>2</td></tr><tr><td>Actin 1 (ACT1)</td><td>Glideosome protein involved in gliding movement of Toxoplasma gondii, organelles distribution during cell division and secretory vesicles movement within the parasite</td><td>TGME49_chrIb:998462-1000024(+)</td><td>TGME49_209030</td><td>Toxoplasma_gondii_ME49</td><td>41908</td><td>3</td><td>3</td></tr><tr><td>Alpha Tubulin A1 (TUBA1)</td><td>Microtubule-associated protein involved in maintaining Toxoplasma gondii's shape, intracellular transport, and apical complex organization</td><td>TGME49_chrXI:5274572-5276593(+)</td><td>TGME49_316400</td><td>Toxoplasma_gondii_ME49</td><td>50114</td><td>3</td><td>2</td></tr><tr><td>HEAT repeat-containing protein</td><td>Protein involved in intracellular transport, chromosome segregation, and signal transduction</td><td>TGME49_chrXI:6061226-6067819(–)</td><td>TGME49_216590</td><td>Toxoplasma_gondii_ME49</td><td>111196</td><td>3</td><td>ND</td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 1. (Continued)</caption><thead><tr><th>Protein name</th><th>Known or predicted function</th><th>Location</th><th>Accession</th><th>Organism</th><th>Molecular weight (Da)</th><th>Peptide count on protein profile No. 1</th><th>Peptide count on protein profile No. 2</th></tr></thead><tbody><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49 chrVIIa:2062064-2074174(+)</td><td>TGME49_203980</td><td>Toxoplasma gondii ME49</td><td>201280</td><td>3</td><td>ND</td></tr><tr><td>Chaperonin cpn60, putative</td><td>ATP-dependent chaperone involved in protein folding</td><td>TGME49 chrVI:1335457-1339036(+)</td><td>TGME49_240600</td><td>Toxoplasma gondii ME49</td><td>72237</td><td>3</td><td>ND</td></tr><tr><td>Protein disulfide isomerase</td><td>Endoplasmic reticulum enzyme involved in protein folding and redox homeostasis contributing to maturation and quality control of secretory and membrane proteins</td><td>TGME49 chrIV:1626551-1628375(+)</td><td>TGME49_211680</td><td>Toxoplasma gondii ME49</td><td>52802</td><td>2</td><td>3</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49 chrXI:4609959-4617338(-)</td><td>TGME49_315270</td><td>Toxoplasma gondii ME49</td><td>84600</td><td>2</td><td>2</td></tr><tr><td>Histone lysine methyltransferase, SET, putative</td><td>Histone-lysine N-methyltransferase activity involved in epigenetic regulation</td><td>TGME49 chrVIIb:1042995-1044560(-)</td><td>TGME49_262750</td><td>Toxoplasma gondii ME49</td><td>53633</td><td>2</td><td>1</td></tr><tr><td>EMP24/GP25L/p24 family protein</td><td>Family of proteins involved in protein transport between endoplasmic reticulum, Golgi, and specialized organelles (micronemes or rhoptries)</td><td>TGME49 chrXI:1568699-1573498(+)</td><td>TGME49_310750</td><td>Toxoplasma gondii ME49</td><td>64977</td><td>2</td><td>1</td></tr><tr><td>D-3-phosphoglycerate dehydrogenase</td><td>Enzyme contributing to amino acid metabolism and providing precursors for nucleotide and lipid biosynthesis</td><td>TGME49 chrVI:939130-947919(-)</td><td>TGME49_239820</td><td>Toxoplasma gondii ME49</td><td>65194</td><td>2</td><td>ND</td></tr><tr><td>Cell division protein C48CY (CDC48CY)</td><td>AAA + ATPase protein involved in protein unfolding, degradation, membrane fusion, and regulation of turnover during cell cycle transitions</td><td>TGME49 chrVIII:3664539-3673736(-)</td><td>TGME49_273090</td><td>Toxoplasma gondii ME49</td><td>89397</td><td>2</td><td>ND</td></tr><tr><td>Microneme protein 1 (MIC1)</td><td>Protein secreted from apical micronemes during initial stages of host cell entry, forming a complex with MIC4 and MIC6 to mediate adhesion to host receptors</td><td>TGME49 chrIX:4395345-4397688(-)</td><td>TGME49_291890</td><td>Toxoplasma gondii ME49</td><td>48629</td><td>1</td><td>2</td></tr><tr><td>Inner Membrane Complex protein IMC2A (IMC2A)</td><td>Inner membrane complex protein contributing to the structural integrity and organization of the parasite pellicle</td><td>TGME49 chrX:520228-525722(-)</td><td>TGME49_228170</td><td>Toxoplasma gondii ME49</td><td>170816</td><td>ND</td><td>11</td></tr><tr><td>Cathepsin B</td><td>Cysteine protease localized to the endolysosomal system, involved in proteolytic processing, digestion of host macromolecules, and protein turnover</td><td>TGME49 chrXII:4721208-4726837(-)</td><td>TGME49_249670</td><td>Toxoplasma gondii ME49</td><td>62477</td><td>ND</td><td>8</td></tr><tr><td>Nuclear Factor 4 (NF4)</td><td>Putative transcription factor regulating gene expression</td><td>TGME49 chrVIIa:1293654-1297697(+)</td><td>TGME49_205580</td><td>Toxoplasma gondii ME49</td><td>51221</td><td>ND</td><td>7</td></tr><tr><td>Alveolin domain containing intermediate filament IMC4 (ALV4)</td><td>Inner membrane complex protein contributing to the cytoskeletal scaffold that provides mechanical strength and structural support to the parasite</td><td>TGME49 chrVIII:1513141-1515301(-)</td><td>TGME49_231630</td><td>Toxoplasma gondii ME49</td><td>50172</td><td>ND</td><td>4</td></tr><tr><td>Ubiquitin family protein</td><td>Regulator of protein turnover through the ubiquitin-proteasome system</td><td>TGME49 chrV:633282-639366(+)</td><td>TGME49_212820</td><td>Toxoplasma gondii ME49</td><td>119003</td><td>ND</td><td>3</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49 chrXI:1880862-1900195(+)</td><td>TGME49_311230</td><td>Toxoplasma gondii ME49</td><td>504758</td><td>ND</td><td>2</td></tr><tr><td>AMP-binding enzyme domain-containing protein</td><td>Protein involved in metabolic pathways such as fatty acid activation, amino acid adenylation, and secondary metabolite biosynthesis, contributing to parasite energy metabolism and adaptation</td><td>TGME49 chrXI:1092557-1096957(-)</td><td>TGME49_310150</td><td>Toxoplasma gondii ME49</td><td>95937</td><td>ND</td><td>2</td></tr><tr><td>NTPase I</td><td>Enzyme involved in energy metabolism, signal transduction, and regulation of nucleotide hydrolysis-dependent cellular processes</td><td>TGME49 chrXII:6699033-6699642(-)</td><td>TGME49_277240</td><td>Toxoplasma gondii ME49</td><td>22641</td><td>ND</td><td>2</td></tr><tr><td>Dynamin-Related Protein B (DRPB)</td><td>Member of the dynamin superfamily of GTPases involved in membrane remodeling processes</td><td>TGME49 chrIb:1689989-1700172(+)</td><td>TGME49_321620</td><td>Toxoplasma gondii ME49</td><td>95931</td><td>ND</td><td>2</td></tr></tbody></table>
<table><caption>**Table 2.** Proteins of *Toxoplasma gondii* RH strain identified by LC-MS/MS for the second IgM triplet spot of interest (spot 22) (ND: not detected).</caption><thead><tr><th>Protein name</th><th>Known or predicted function</th><th>Location</th><th>Accession</th><th>Organism</th><th>Molecular weight (Da)</th><th>Peptide count on protein profile No. 1</th><th>Peptide count on protein profile No. 2</th></tr></thead><tbody><tr><td>Heat Shock Protein (HSP)</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system</td><td>TGME49_chrXII:5582774-5587740(–)</td><td>TGME49_251780</td><td>Toxoplasma gondii_ME49</td><td>78251</td><td>46</td><td>60</td></tr><tr><td>Heat Shock Protein 70 (HSP70)</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system (modulation of nitric oxide production and reduction of macrophages phagocytosis capacity)</td><td>TGME49_chrVIII:3289396-3291420 (–)</td><td>TGME49_273760</td><td>Toxoplasma gondii_ME49</td><td>72880</td><td>43</td><td>42</td></tr><tr><td>Heat Shock Protein 90 (HSP90)</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system</td><td>TGME49_chrIX:2483404-2487798(+)</td><td>TGME49_288380</td><td>Toxoplasma gondii_ME49</td><td>81933</td><td>27</td><td>25</td></tr><tr><td>Chaperonin protein BiP</td><td>Molecular chaperone involved in protein folding and exit from the endoplasmic reticulum</td><td>TGME49_chrXI:2225378-2228753(–)</td><td>TGME49_311720</td><td>Toxoplasma gondii_ME49</td><td>73253</td><td>26</td><td>40</td></tr><tr><td>Alveolin domain containing intermediate filament IMC1 (ALV1)</td><td>Inner membrane complex protein associated with the membrane cytoskeleton of *Toxoplasma gondii*, involved in maintaining parasite morphology and motility</td><td>TGME49_chrVIII:1516857-1519632(+)</td><td>TGME49_231640</td><td>Toxoplasma gondii_ME49</td><td>69596</td><td>15</td><td>25</td></tr><tr><td>Cytosolic tRNA-Ala synthetase</td><td>Cytosolic protein involved in the aminoacylation of tRNA during proteins synthesis</td><td>TGME49_chrXII:862344-876041(–)</td><td>TGME49_219540</td><td>Toxoplasma gondii_ME49</td><td>139516</td><td>6</td><td>7</td></tr><tr><td>Vacuolar ATP synthase subunit A, putative</td><td>Proton-transporting ATP synthase activity contributing to endomembrane compartments acidification, organelle maturation, and intracellular pH regulation</td><td>TGME49_chrVIIB:4315661-4325274 (–)</td><td>TGME49_256970</td><td>Toxoplasma gondii_ME49</td><td>68122</td><td>4</td><td>13</td></tr><tr><td>Protease inhibitor 1 (PI1)</td><td>Protein involved in modulation of protease activity by inhibiting cysteine or serine proteases</td><td>TGME49_chrXII:1832043-1834438(–)</td><td>TGME49_217430</td><td>Toxoplasma gondii_ME49</td><td>47782</td><td>4</td><td>3</td></tr><tr><td>Nuclear Factor 4 (NF4)</td><td>Putative transcription factor regulating gene expression</td><td>TGME49_chrVIIa:1293654-1297697 (+)</td><td>TGME49_205580</td><td>Toxoplasma gondii_ME49</td><td>51221</td><td>4</td><td>ND</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrVIII:2878595-2883425 (–)</td><td>TGME49_233890</td><td>Toxoplasma gondii_ME49</td><td>73697</td><td>3</td><td>7</td></tr><tr><td>Toxolysin 4 (TLN4)</td><td>Member of the M16A metalloprotease family, involved in *Toxoplasma gondii* egress from host cells and maturation of microneme proteins</td><td>TGME49_chrVIIa:903405-917753(+)</td><td>TGME49_206510</td><td>Toxoplasma gondii_ME49</td><td>246741</td><td>3</td><td>6</td></tr><tr><td>Tryptophanyl-tRNA synthetase (TrpRS2)</td><td>Apicoplast protein involved in the aminoacylation of tRNA during proteins synthesis</td><td>TGME49_chrIX:2473710-2478137(+)</td><td>TGME49_288360</td><td>Toxoplasma gondii_ME49</td><td>77058</td><td>3</td><td>6</td></tr><tr><td>Lysine-tRNA ligase</td><td>Enzyme ensuring the fidelity of lysine incorporation into nascent polypeptides, playing a key role in protein synthesis and cellular function</td><td>TGME49_chrVIIa:1202972-1207953 (+)</td><td>TGME49_205710</td><td>Toxoplasma gondii_ME49</td><td>74395</td><td>3</td><td>ND</td></tr><tr><td>SAG-related sequence SRS29B (SRS29B)</td><td>Surface protein involved in host cell attachment</td><td>TGME49_chrVIII:2664481-2665491(+)</td><td>TGME49_233460</td><td>Toxoplasma gondii_ME49</td><td>34732</td><td>2</td><td>2</td></tr><tr><td>Actin 1 (ACT1)</td><td>Glidesosome protein involved in gliding movement of *Toxoplasma gondii*, organelles distribution during cell division and secretory vesicles movement within the parasite</td><td>TGME49_chrIb:998462-1000024(+)</td><td>TGME49_209030</td><td>Toxoplasma gondii_ME49</td><td>41908</td><td>2</td><td>2</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrXI:4609959-4617338(–)</td><td>TGME49_315270</td><td>Toxoplasma gondii_ME49</td><td>84600</td><td>2</td><td>2</td></tr><tr><td>Histone lysine methyltransferase, SET, putative</td><td>Histone-lysine N-methyltransferase activity involved in epigenetic regulation</td><td>TGME49_chrVIIB:1042995-1044560 (–)</td><td>TGME49_262750</td><td>Toxoplasma gondii_ME49</td><td>53633</td><td>2</td><td>1</td></tr><tr><td>Alveolin domain containing intermediate filament IMC4 (ALV4)</td><td>Inner membrane complex protein contributing to the cytoskeletal scaffold that provides mechanical strength and structural support to the parasite</td><td>TGME49_chrVIII:1513141-1515301 (–)</td><td>TGME49_231630</td><td>Toxoplasma gondii_ME49</td><td>50172</td><td>2</td><td>1</td></tr><tr><td>Ulp1 protease family, C-terminal catalytic domain-containing protein</td><td>Enzyme involved in the maturation of ubiquitin-like proteins such as SUMO, contributing to post-translational modification regulation</td><td>TGME49_chrIX:1711745-1725039(+)</td><td>TGME49_265190</td><td>Toxoplasma gondii_ME49</td><td>329629</td><td>2</td><td>1</td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 2. (Continued)**</caption><thead><tr><th>Protein name</th><th>Known or predicted function</th><th>Location</th><th>Accession</th><th>Organism</th><th>Molecular weight (Da)</th><th>Peptide count on protein profile No. 1</th><th>Peptide count on protein profile No. 2</th></tr></thead><tbody><tr><td>Inner Membrane Complex protein 2A (IMC2A)</td><td>Inner membrane complex protein contributing to the structural integrity and organization of the parasite pellicle</td><td>TGME49_chrX:520228-525722(–)</td><td>TGME49_228170</td><td>Toxoplasma gondii_ME49</td><td>170816</td><td>2</td><td>1</td></tr><tr><td>EMP24/GP25L/p24 family protein</td><td>Family of proteins involved in protein transport between endoplasmic reticulum, Golgi, and specialized organelles (micronemes or rhoptries)</td><td>TGME49_chrXI:1568699-1573498(+)</td><td>TGME49_310750</td><td>Toxoplasma gondii_ME49</td><td>64977</td><td>2</td><td>1</td></tr><tr><td>Chaperonin cpn60, putative</td><td>ATP-dependent chaperone involved in protein folding</td><td>TGME49_chrVI:1335457-1339036(+)</td><td>TGME49_240600</td><td>Toxoplasma gondii_ME49</td><td>72237</td><td>ND</td><td>7</td></tr><tr><td>Peptidase M16 inactive domain-containing protein</td><td>Member of the M16A metalloprotease family, no function described</td><td>TGME49_chrX:716364-726488(+)</td><td>TGME49_227948</td><td>Toxoplasma gondii_ME49</td><td>142764</td><td>ND</td><td>5</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrVIIa:3523795-3527924 (–)</td><td>TGME49_202200</td><td>Toxoplasma gondii_ME49</td><td>87043</td><td>ND</td><td>4</td></tr><tr><td>Membrane Occupation and Recognition Nexus protein 2 (MORN2)</td><td>No function described, hypothetical role in cytoskeleton organization</td><td>TGME49_chrIX:4562922-4564319(+)</td><td>TGME49_292120</td><td>Toxoplasma gondii_ME49</td><td>49236</td><td>ND</td><td>4</td></tr><tr><td>Heat Shock Protein 90, putative</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system</td><td>TGME49_chrVI:3356828-3362144(+)</td><td>TGME49_244560</td><td>Toxoplasma gondii_ME49</td><td>96823</td><td>ND</td><td>2</td></tr><tr><td>Domain K- type RNA binding proteins family protein</td><td>Proteins involved in post-transcriptional regulation, including RNA stability, splicing, transport, and translation</td><td>TGME49_chrX:5186140-5192433(+)</td><td>TGME49_235930</td><td>Toxoplasma gondii_ME49</td><td>64265</td><td>ND</td><td>2</td></tr><tr><td>Aconitate hydratase ACN/IRP</td><td>Mitochondrial/apicoplast enzyme involved in the energy metabolism of Toxoplasma gondii (Krebs cycle) and regulation of host immune system (modulation of nitric oxide production)</td><td>TGME49_chrX:1400984-1408155(–)</td><td>TGME49_226730</td><td>Toxoplasma gondii_ME49</td><td>114885</td><td>ND</td><td>2</td></tr><tr><td>D-3-phosphoglycerate dehydrogenase</td><td>Enzyme contributing to amino acid metabolism and providing precursors for nucleotide and lipid biosynthesis</td><td>TGME49_chrVI:939130-947919(–)</td><td>TGME49_239820</td><td>Toxoplasma gondii_ME49</td><td>65194</td><td>ND</td><td>2</td></tr></tbody></table>
<table><caption>**Table 3.** Proteins of *Toxoplasma gondii* RH strain identified by LC-MS/MS for the third IgM triplet spot of interest (spot 23) (ND: not detected).</caption><thead><tr><th>Protein name</th><th>Known or predicted function</th><th>Location</th><th>Accession</th><th>Organism</th><th>Molecular weight (Da)</th><th>Peptide count on protein profile No. 1</th><th>Peptide count on protein profile No. 2</th></tr></thead><tbody><tr><td>Heat Shock Protein (HSP)</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system</td><td>TGME49_chrXII:5582774-5587740(–)</td><td>TGME49_251780</td><td>Toxoplasma_gondii_ME49</td><td>78251</td><td>55</td><td>61</td></tr><tr><td>Chaperonin protein BiP</td><td>Molecular chaperone involved in protein folding and exit from the endoplasmic reticulum</td><td>TGME49_chrXI:2225378-2228753(–)</td><td>TGME49_311720</td><td>Toxoplasma_gondii_ME49</td><td>73253</td><td>27</td><td>43</td></tr><tr><td>Heat Shock Protein 70 (HSP70)</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system (modulation of nitric oxide production and reduction of macrophages phagocytosis capacity)</td><td>TGME49_chrVIII:3289396-3291420 (–)</td><td>TGME49_273760</td><td>Toxoplasma_gondii_ME49</td><td>72880</td><td>24</td><td>30</td></tr><tr><td>Vacuolar ATP synthase subunit A, putative</td><td>Proton-transporting ATP synthase activity contributing to endomembrane compartments acidification, organelle maturation, and intracellular pH regulation</td><td>TGME49_chrVIIB:4315661-4325274 (–)</td><td>TGME49_256970</td><td>Toxoplasma_gondii_ME49</td><td>68122</td><td>20</td><td>24</td></tr><tr><td>Alveolin domain containing intermediate filament IMC1 (ALV1)</td><td>Inner membrane complex protein associated with the membrane cytoskeleton of Toxoplasma gondii, involved in maintaining parasite morphology and motility</td><td>TGME49_chrVIII:1516857-1519632(+)</td><td>TGME49_231640</td><td>Toxoplasma_gondii_ME49</td><td>69596</td><td>17</td><td>21</td></tr><tr><td>Cytosolic tRNA-Ala synthetase</td><td>Cytosolic protein involved in the aminoacylation of tRNA during proteins synthesis</td><td>TGME49_chrXII:862344-876041(–)</td><td>TGME49_219540</td><td>Toxoplasma_gondii_ME49</td><td>139516</td><td>12</td><td>24</td></tr><tr><td>Heat Shock Protein 90 (HSP90)</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system</td><td>TGME49_chrIX:2483404-2487798(+)</td><td>TGME49_288380</td><td>Toxoplasma_gondii_ME49</td><td>81933</td><td>11</td><td>20</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrVIII:2878595-2883425 (–)</td><td>TGME49_233890</td><td>Toxoplasma_gondii_ME49</td><td>73697</td><td>9</td><td>13</td></tr><tr><td>Chaperonin cpn60, putative</td><td>ATP-dependent chaperone involved in protein folding</td><td>TGME49_chrVI:1335457-1339036(+)</td><td>TGME49_240600</td><td>Toxoplasma_gondii_ME49</td><td>72237</td><td>6</td><td>12</td></tr><tr><td>Tryptophanyl-tRNA synthetase (TrpRS2)</td><td>Apicoplast protein involved in the aminoacylation of tRNA during proteins synthesis</td><td>TGME49_chrIX:2473710-2478137(+)</td><td>TGME49_288360</td><td>Toxoplasma_gondii_ME49</td><td>77058</td><td>4</td><td>13</td></tr><tr><td>Aconitate hydratase ACN/IRP</td><td>Mitochondrial/apicoplast enzyme involved in the energy metabolism of Toxoplasma gondii (Krebs cycle) and regulation of host immune system (modulation of nitric oxide production)</td><td>TGME49_chrX:1400984-1408155(–)</td><td>TGME49_226730</td><td>Toxoplasma_gondii_ME49</td><td>114885</td><td>3</td><td>9</td></tr><tr><td>Actin 1 (ACT1)</td><td>Glideosome protein involved in gliding movement of Toxoplasma gondii, organelles distribution during cell division and secretory vesicles movement within the parasite</td><td>TGME49_chrIb:998462-1000024(+)</td><td>TGME49_209030</td><td>Toxoplasma_gondii_ME49</td><td>41908</td><td>2</td><td>5</td></tr><tr><td>SAG-related sequence SRS29B (SRS29B)</td><td>Surface protein involved in host cell attachment</td><td>TGME49_chrVIII:2664481-2665491(+)</td><td>TGME49_233460</td><td>Toxoplasma_gondii_ME49</td><td>34732</td><td>2</td><td>4</td></tr><tr><td>ATPase, AAA family protein</td><td>AAA + ATPase family protein involved in protein unfolding, degradation, membrane fusion, and regulation of turnover during cell cycle transitions</td><td>TGME49_chrX:4365489-4372954(–)</td><td>TGME49_234420</td><td>Toxoplasma_gondii_ME49</td><td>66807</td><td>2</td><td>3</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrXI:4609959-4617338(–)</td><td>TGME49_315270</td><td>Toxoplasma_gondii_ME49</td><td>84600</td><td>2</td><td>2</td></tr><tr><td>Cell Division protein C48CY (CDC48CY)</td><td>AAA + ATPase protein involved in protein unfolding, degradation, membrane fusion, and regulation of turnover during cell cycle transitions</td><td>TGME49_chrVIII:3664539-3673736 (–)</td><td>TGME49_273090</td><td>Toxoplasma_gondii_ME49</td><td>89397</td><td>2</td><td>2</td></tr><tr><td>Alveolin domain containing intermediate filament IMC3 (ALV3)</td><td>Structural component of the inner membrane complex involved in maintaining parasite morphology and mechanical integrity, serves as a scaffold during daughter cell formation</td><td>TGME49_chrXI:6475497-6477113(+)</td><td>TGME49_216000</td><td>Toxoplasma_gondii_ME49</td><td>58000</td><td>2</td><td>1</td></tr><tr><td>EMP24/GP25L/p24 family protein</td><td>Family of proteins involved in protein transport between endoplasmic reticulum, Golgi, and specialized organelles (micronemes or rhoptries)</td><td>TGME49_chrXI:1568699-1573498(+)</td><td>TGME49_310750</td><td>Toxoplasma_gondii_ME49</td><td>64977</td><td>1</td><td>4</td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 3. (Continued)**</caption><thead><tr><th>Protein name</th><th>Known or predicted function</th><th>Location</th><th>Accession</th><th>Organism</th><th>Molecular weight (Da)</th><th>Peptide count on protein profile No. 1</th><th>Peptide count on protein profile No. 2</th></tr></thead><tbody><tr><td>Protein disulfide isomerase</td><td>Endoplasmic reticulum enzyme involved in protein folding and redox homeostasis contributing to maturation and quality control of secretory and membrane proteins</td><td>TGME49_chrIV:1626551-1628375(+)</td><td>TGME49_211680</td><td>Toxoplasma_gondii_ME49</td><td>52802</td><td>1</td><td>3</td></tr><tr><td>Dynamin-Related Protein B (DRPB)</td><td>Member of the dynamin superfamily of GTPases involved in membrane remodeling processes</td><td>TGME49_chrIb:1689989-1700172(+)</td><td>TGME49_321620</td><td>Toxoplasma_gondii_ME49</td><td>95931</td><td>1</td><td>3</td></tr><tr><td>Microneme protein 1 (MIC1)</td><td>Protein secreted from apical micronemes during initial stages of host cell entry, forming a complex with MIC4 and MIC6 to mediate adhesion to host receptors</td><td>TGME49_chrIX:4395345-4397688(-)</td><td>TGME49_291890</td><td>Toxoplasma_gondii_ME49</td><td>48629</td><td>1</td><td>2</td></tr><tr><td>Toxolysin 4 (TLN4)</td><td>Member of the M16A metalloprotease family, involved in Toxoplasma gondii egress from host cells and maturation of microneme proteins</td><td>TGME49_chrVIIa:903405-917753(+)</td><td>TGME49_206510</td><td>Toxoplasma_gondii_ME49</td><td>246741</td><td>1</td><td>2</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrVIIa:3523795-3527924 (-)</td><td>TGME49_202200</td><td>Toxoplasma_gondii_ME49</td><td>87043</td><td>ND</td><td>7</td></tr><tr><td>Peptidase M16 inactive domain-containing protein</td><td>Member of the M16A metalloprotease family, no function described</td><td>TGME49_chrX:6310793-6320102(-)</td><td>TGME49_214490</td><td>Toxoplasma_gondii_ME49</td><td>148868</td><td>ND</td><td>5</td></tr><tr><td>FUSE-Binding Protein 2 / KH-type splicing regulatory protein (FUBP2)</td><td>Protein involved in transcriptional regulation and post-transcriptional control, including mRNA splicing, stabilization, and localization</td><td>TGME49_chrXI:5969861-5973900(-)</td><td>TGME49_216670</td><td>Toxoplasma_gondii_ME49</td><td>100094</td><td>ND</td><td>4</td></tr><tr><td>D-3-phosphoglycerate dehydrogenase</td><td>Enzyme contributing to amino acid metabolism and providing precursors for nucleotide and lipid biosynthesis</td><td>TGME49_chrVI:939130-947919(-)</td><td>TGME49_239820</td><td>Toxoplasma_gondii_ME49</td><td>65194</td><td>ND</td><td>4</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrIII:1831237-1844046(-)</td><td>TGME49_254570</td><td>Toxoplasma_gondii_ME49</td><td>221185</td><td>ND</td><td>4</td></tr><tr><td>RNA recognition motif-containing protein</td><td>Protein involved in various aspects of RNA metabolism including splicing, transport, stability, and translation</td><td>TGME49_chrIX:4405615-4411375(+)</td><td>TGME49_291930</td><td>Toxoplasma_gondii_ME49</td><td>73118</td><td>ND</td><td>3</td></tr><tr><td>Domain K-type RNA binding proteins family protein</td><td>Proteins involved in post-transcriptional regulation, including RNA stability, splicing, transport, and translation</td><td>TGME49_chrX:5186140-5192433(+)</td><td>TGME49_235930</td><td>Toxoplasma_gondii_ME49</td><td>64265</td><td>ND</td><td>2</td></tr><tr><td>Apical Membrane Antigen 1 (AMA1)</td><td>Microneme protein secreted onto the parasite surface during host cell invasion, plays a key role in moving junction formation by interacting with rhoptry neck proteins</td><td>TGME49_chrVIIb:4948067-4952962 (+)</td><td>TGME49_255260</td><td>Toxoplasma_gondii_ME49</td><td>63021</td><td>ND</td><td>2</td></tr><tr><td>Peptidase M16 inactive domain-containing protein</td><td>Member of the M16A metalloprotease family, no function described</td><td>TGME49_chrX:716364-726488(+)</td><td>TGME49_227948</td><td>Toxoplasma_gondii_ME49</td><td>142764</td><td>ND</td><td>2</td></tr><tr><td>Membrane Occupation and Recognition Nexus protein 2 (MORN2)</td><td>No function described, hypothetical role in cytoskeleton organization</td><td>TGME49_chrIX:4562922-4564319(+)</td><td>TGME49_292120</td><td>Toxoplasma_gondii_ME49</td><td>49236</td><td>ND</td><td>2</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrII:644118-650964(+)</td><td>TGME49_221870</td><td>Toxoplasma_gondii_ME49</td><td>144135</td><td>ND</td><td>2</td></tr></tbody></table>
<table><caption>**Table 4.** Proteins of *Toxoplasma. gondii* RH strain identified by LC-MS/MS for the fourth IgM triplet spot of interest (spot 24) (ND: not detected).</caption><thead><tr><th>Protein name</th><th>Known or predicted function</th><th>Location</th><th>Accession</th><th>Organism</th><th>Molecular weight (Da)</th><th>Peptide count on protein profile No. 1</th><th>Peptide count on protein profile No. 2</th></tr></thead><tbody><tr><td>Heat Shock Protein (HSP)</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system</td><td>TGME49_chrXII:5582774-5587740(–)</td><td>TGME49_251780</td><td>Toxoplasma_gondii_ME49</td><td>78251</td><td>51</td><td>45</td></tr><tr><td>Chaperonin protein BiP</td><td>Molecular chaperone involved in protein folding and exit from the endoplasmic reticulum</td><td>TGME49_chrXI:2225378-2228753(–)</td><td>TGME49_311720</td><td>Toxoplasma_gondii_ME49</td><td>73253</td><td>33</td><td>30</td></tr><tr><td>Cytosolic tRNA-Ala synthetase</td><td>Cytosolic protein involved in the aminoacylation of tRNA during proteins synthesis</td><td>TGME49_chrXII:862344-876041(–)</td><td>TGME49_219540</td><td>Toxoplasma_gondii_ME49</td><td>139516</td><td>29</td><td>21</td></tr><tr><td>Vacuolar ATP synthase subunit A, putative</td><td>Proton-transporting ATP synthase activity contributing to endomembrane compartments acidification, organelle maturation, and intracellular pH regulation</td><td>TGME49_chrVIb:4315661-4325274 (–)</td><td>TGME49_256970</td><td>Toxoplasma_gondii_ME49</td><td>68122</td><td>28</td><td>17</td></tr><tr><td>Tryptophanyl-tRNA synthetase (TrpRS2)</td><td>Apicoplast protein involved in the aminoacylation of tRNA during proteins synthesis</td><td>TGME49_chrIX:2473710-2478137(+)</td><td>TGME49_288360</td><td>Toxoplasma_gondii_ME49</td><td>77058</td><td>24</td><td>10</td></tr><tr><td>Chaperonin cpn60, putative</td><td>ATP-dependent chaperone involved in protein folding</td><td>TGME49_chrVI:1335457-1339036(+)</td><td>TGME49_240600</td><td>Toxoplasma_gondii_ME49</td><td>72237</td><td>23</td><td>11</td></tr><tr><td>Alveolin domain containing intermediate filament IMC1 (ALV1)</td><td>Inner membrane complex protein associated with the membrane cytoskeleton of *Toxoplasma gondii*, involved in maintaining parasite morphology and motility</td><td>TGME49_chrVIII:1516857-1519632(+)</td><td>TGME49_231640</td><td>Toxoplasma_gondii_ME49</td><td>69596</td><td>14</td><td>12</td></tr><tr><td>Heat Shock Protein 70 (HSP70)</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system (modulation of nitric oxide production and reduction of macrophages phagocytosis capacity)</td><td>TGME49_chrVIII:3289396-3291420 (–)</td><td>TGME49_273760</td><td>Toxoplasma_gondii_ME49</td><td>72880</td><td>13</td><td>12</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrVIII:2878595-2883425 (–)</td><td>TGME49_233890</td><td>Toxoplasma_gondii_ME49</td><td>73697</td><td>11</td><td>7</td></tr><tr><td>Aconitate hydratase ACN/IRP</td><td>Mitochondrial/apicoplast enzyme involved in the energy metabolism of *Toxoplasma gondii* (Krebs cycle) and regulation of host immune system (modulation of nitric oxide production)</td><td>TGME49_chrX:1400984-1408155(–)</td><td>TGME49_226730</td><td>Toxoplasma_gondii_ME49</td><td>114885</td><td>10</td><td>3</td></tr><tr><td>Cell Division protein C48CY (CDC48CY)</td><td>AAA + ATPase protein involved in protein unfolding, degradation, membrane fusion, and regulation of turnover during cell cycle transitions</td><td>TGME49_chrVIII:3664539-3673736 (–)</td><td>TGME49_273090</td><td>Toxoplasma_gondii_ME49</td><td>89397</td><td>7</td><td>4</td></tr><tr><td>FUSE-Binding Protein 2 / KH-type splicing regulatory protein (FUBP2)</td><td>Protein involved in transcriptional regulation and post-transcriptional control, including mRNA splicing, stabilization, and localization</td><td>TGME49_chrXI:5969861-5973900(–)</td><td>TGME49_216670</td><td>Toxoplasma_gondii_ME49</td><td>100094</td><td>7</td><td>1</td></tr><tr><td>Toxolysin 4 (TLN4)</td><td>Member of the M16A metalloprotease family, involved in *Toxoplasma gondii* egress from host cells and maturation of microneme proteins</td><td>TGME49_chrVIIa:903405-917753(+)</td><td>TGME49_206510</td><td>Toxoplasma_gondii_ME49</td><td>246741</td><td>6</td><td>ND</td></tr><tr><td>ATPase, AAA family protein</td><td>AAA + ATPase family protein involved in protein unfolding, degradation, membrane fusion, and regulation of turnover during cell cycle transitions</td><td>TGME49_chrX:4365489-4372954(–)</td><td>TGME49_234420</td><td>Toxoplasma_gondii_ME49</td><td>66807</td><td>5</td><td>1</td></tr><tr><td>RNA recognition motif-containing protein</td><td>Protein involved in various aspects of RNA metabolism including splicing, transport, stability, and translation</td><td>TGME49_chrIX:4405615-4411375(+)</td><td>TGME49_291930</td><td>Toxoplasma_gondii_ME49</td><td>73118</td><td>5</td><td>1</td></tr><tr><td>SAG-related sequence SRS29B (SRS29B)</td><td>Surface protein involved in host cell attachment</td><td>TGME49_chrVIII:2664481-2665491(+)</td><td>TGME49_233460</td><td>Toxoplasma_gondii_ME49</td><td>34732</td><td>4</td><td>2</td></tr><tr><td>Actin 1 (ACT1)</td><td>Glideosome protein involved in gliding movement of *Toxoplasma gondii*, organelles distribution during cell division and secretory vesicles movement within the parasite</td><td>TGME49_chrIb:998462-1000024(+)</td><td>TGME49_209030</td><td>Toxoplasma_gondii_ME49</td><td>41908</td><td>4</td><td>2</td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 4. (Continued)**</caption><thead><tr><th>Protein name</th><th>Known or predicted function</th><th>Location</th><th>Accession</th><th>Organism</th><th>Molecular weight (Da)</th><th>Peptide count on protein profile No. 1</th><th>Peptide count on protein profile No. 2</th></tr></thead><tbody><tr><td>Heat Shock Protein 90 (HSP90)</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system</td><td>TGME49_chrIX:2483404-2487798(+)</td><td>TGME49_288380</td><td>Toxoplasma_gondii_ME49</td><td>81933</td><td>4</td><td>1</td></tr><tr><td>D-3-phosphoglycerate dehydrogenase</td><td>Enzyme contributing to amino acid metabolism and providing precursors for nucleotide and lipid biosynthesis</td><td>TGME49_chrVI:939130-947919(-)</td><td>TGME49_239820</td><td>Toxoplasma_gondii_ME49</td><td>65194</td><td>4</td><td>1</td></tr><tr><td>Dynamin-Related Protein B (DRPB)</td><td>Member of the dynamin superfamily of GTPases involved in membrane remodeling processes</td><td>TGME49_chrlb:1689989-1700172(+)</td><td>TGME49_321620</td><td>Toxoplasma_gondii_ME49</td><td>95931</td><td>4</td><td>1</td></tr><tr><td>Microneme protein 1 (MIC1)</td><td>Protein secreted from apical micronemes during initial stages of host cell entry, forming a complex with MIC4 and MIC6 to mediate adhesion to host receptors</td><td>TGME49_chrIX:4395345-4397688(-)</td><td>TGME49_291890</td><td>Toxoplasma_gondii_ME49</td><td>48629</td><td>4</td><td>ND</td></tr><tr><td>EMP24/GP25L/p24 family protein</td><td>Family of proteins involved in protein transport between endoplasmic reticulum, Golgi, and specialized organelles (micronemes or rhoptries)</td><td>TGME49_chrXI:1568699-1573498(+)</td><td>TGME49_310750</td><td>Toxoplasma_gondii_ME49</td><td>64977</td><td>3</td><td>1</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrVIIa:3523795-3527924 (-)</td><td>TGME49_202200</td><td>Toxoplasma_gondii_ME49</td><td>87043</td><td>3</td><td>ND</td></tr><tr><td>Protein disulfide isomerase</td><td>Endoplasmic reticulum enzyme involved in protein folding and redox homeostasis contributing to maturation and quality control of secretory and membrane proteins</td><td>TGME49_chrIV:1626551-1628375(+)</td><td>TGME49_211680</td><td>Toxoplasma_gondii_ME49</td><td>52802</td><td>2</td><td>1</td></tr><tr><td>Heat Shock Protein 90, putative</td><td>Molecular chaperone involved in protein folding, cellular stress responses, and regulation of host immune system</td><td>TGME49_chrVI:3356828-3362144(+)</td><td>TGME49_244560</td><td>Toxoplasma_gondii_ME49</td><td>96823</td><td>2</td><td>ND</td></tr><tr><td>Ubiquitin specific protease 39 isoform 2, putative</td><td>Protease involved in spliceosome assembly, regulation of pre-mRNA splicing and proper gene expression</td><td>TGME49_chrla:840906-848857(+)</td><td>TGME49_294360</td><td>Toxoplasma_gondii_ME49</td><td>64190</td><td>2</td><td>ND</td></tr><tr><td>Domain K- type RNA binding proteins family protein</td><td>Proteins involved in post-transcriptional regulation, including RNA stability, splicing, transport, and translation</td><td>TGME49_chrX:5186140-5192433(+)</td><td>TGME49_235930</td><td>Toxoplasma_gondii_ME49</td><td>64265</td><td>2</td><td>ND</td></tr><tr><td>Apical Membrane Antigen 1 (AMA1)</td><td>Microneme protein secreted onto the parasite surface during host cell invasion, plays a key role in moving junction formation by interacting with rhoptry neck proteins</td><td>TGME49_chrVIIb:4948067-4952962 (+)</td><td>TGME49_255260</td><td>Toxoplasma_gondii_ME49</td><td>63021</td><td>2</td><td>ND</td></tr><tr><td>Alpha Tubulin A1 (TUBA1)</td><td>Microtubule-associated protein involved in maintaining Toxoplasma gondii's shape, intracellular transport, and apical complex organization</td><td>TGME49_chrXI:5274572-5276593(+)</td><td>TGME49_316400</td><td>Toxoplasma_gondii_ME49</td><td>50114</td><td>2</td><td>ND</td></tr><tr><td>Hypothetical protein</td><td>No function described</td><td>TGME49_chrXI:4609959-4617338(-)</td><td>TGME49_315270</td><td>Toxoplasma_gondii_ME49</td><td>84600</td><td>2</td><td>ND</td></tr></tbody></table>
<table><caption>**Table 5.** Proteins identified by LC-MS/MS for spots 21–24 classified according to their molecular weight and assigned to the IgM triplet band with the closest molecular weight. Proteins with too low molecular weight (below 65 kDa) were not considered to belong to the IgM triplet. High molecular weight proteins (over 100 kDa) were integrated into the IgM triplet, as protein resolution is lower in this zone (ND: not detected). Lines in bold refer to the thirteen main candidates for the IgM triplet.</caption><thead><tr><th></th><th>Protein name for spot No. 21</th><th>Protein name for spot No. 22</th><th>Protein name for spot No. 23</th><th>Protein name for spot No. 24</th></tr></thead><tbody><tr><td rowspan="10">Low molecular weight</td><td>SAG-related sequence SRS29B (SRS29B)</td><td>SAG-related sequence SRS29B (SRS29B)</td><td>SAG-related sequence SRS29B (SRS29B)</td><td>SAG-related sequence SRS29B (SRS29B)</td></tr><tr><td>Actin 1 (ACT1)</td><td>Actin 1 (ACT1)</td><td>Actin 1 (ACT1)</td><td>Actin 1 (ACT1)</td></tr><tr><td>ND</td><td>Protease Inhibitor 1 (PI1)</td><td>ND</td><td>ND</td></tr><tr><td>Microneme protein 1 (MIC1)</td><td>ND</td><td>Microneme protein 1 (MIC1)</td><td>ND</td></tr><tr><td>Alpha Tubulin A1 (TUBA1)</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>ND</td><td>Alveolin domain containing intermediate filament IMC4 (ALV4)</td><td>ND</td><td>ND</td></tr><tr><td>Protein disulfide isomerase</td><td>ND</td><td>Protein disulfide isomerase</td><td>Protein disulfide isomerase</td></tr><tr><td>Histone lysine methyltransferase, SET, putative</td><td>Histone lysine methyltransferase, SET, putative</td><td>ND</td><td>ND</td></tr><tr><td>ND</td><td>ND</td><td>Alveolin domain containing intermediate filament IMC3 (ALV3)</td><td>ND</td></tr><tr><td rowspan="10">75 kDa band</td><td>EMP24/GP25L/p24 family protein</td><td>EMP24/GP25L/p24 family protein</td><td>EMP24/GP25L/p24 family protein</td><td>EMP24/GP25L/p24 family protein</td></tr><tr><td>ND</td><td>ND</td><td>ND</td><td>D-3-phosphoglycerate dehydrogenase</td></tr><tr><td>ND</td><td>ND</td><td>ATPase, AAA family protein</td><td>ATPase, AAA family protein</td></tr><tr><td>Vacuolar ATP synthase subunit A, putative</td><td>Vacuolar ATP synthase subunit A, putative</td><td>Vacuolar ATP synthase subunit A, putative</td><td>Vacuolar ATP synthase subunit A, putative</td></tr><tr><td>Alveolin domain containing intermediate filament IMC1 (ALV1)</td><td>Alveolin domain containing intermediate filament IMC1 (ALV1)</td><td>Alveolin domain containing intermediate filament IMC1 (ALV1)</td><td>Alveolin domain containing intermediate filament IMC1 (ALV1)</td></tr><tr><td>ND</td><td>ND</td><td>Chaperonin cpn60, putative</td><td>Chaperonin cpn60, putative</td></tr><tr><td>Heat Shock Protein 70 (HSP70)</td><td>Heat Shock Protein 70 (HSP70)</td><td>Heat Shock Protein 70 (HSP70)</td><td>Heat Shock Protein 70 (HSP70)</td></tr><tr><td>ND</td><td>ND</td><td>ND</td><td>RNA recognition motif-containing protein</td></tr><tr><td>Chaperonin protein BiP</td><td>Chaperonin protein BiP</td><td>Chaperonin protein BiP</td><td>Chaperonin protein BiP</td></tr><tr><td>Hypothetical protein</td><td>Hypothetical protein</td><td>Hypothetical protein</td><td>Hypothetical protein</td></tr><tr><td>Tryptophanyl-tRNA synthetase (TrpRS2)</td><td>Tryptophanyl-tRNA synthetase (TrpRS2)</td><td>Tryptophanyl-tRNA synthetase (TrpRS2)</td><td>Tryptophanyl-tRNA synthetase (TrpRS2)</td></tr><tr><td>Heat Shock Protein (HSP)</td><td>Heat Shock Protein (HSP)</td><td>Heat Shock Protein (HSP)</td><td>Heat Shock Protein (HSP)</td></tr><tr><td rowspan="6">90 kDa band</td><td>Heat Shock Protein 90 (HSP90)</td><td>Heat Shock Protein 90 (HSP90)</td><td>Heat Shock Protein 90 (HSP90)</td><td>Heat Shock Protein 90 (HSP90)</td></tr><tr><td>Hypothetical protein</td><td>Hypothetical protein</td><td>Hypothetical protein</td><td>ND</td></tr><tr><td>Tetratricopeptide repeat-containing protein</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>ND</td><td>ND</td><td>Cell Division protein C48CY (CDC48CY)</td><td>Cell Division protein C48CY (CDC48CY)</td></tr><tr><td>ND</td><td>ND</td><td>Dynamin-Related Protein B (DRPB)</td><td>Dynamin-Related Protein B (DRPB)</td></tr><tr><td>Heat Shock Protein 90, putative</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td rowspan="7">100 kDa band</td><td>ND</td><td>ND</td><td>ND</td><td>FUSE-Binding Protein 2 / KH-type splicing regulatory protein (FUBP2)</td></tr><tr><td>DnaK family protein</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>Aconitate hydratase ACN/IRP</td><td>ND</td><td>Aconitate hydratase ACN/IRP</td><td>Aconitate hydratase ACN/IRP</td></tr><tr><td>Cytosolic tRNA-Ala synthetase</td><td>Cytosolic tRNA-Ala synthetase</td><td>Cytosolic tRNA-Ala synthetase</td><td>Cytosolic tRNA-Ala synthetase</td></tr><tr><td>Peptidase M16 inactive domain-containing protein</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>ND</td><td>Inner Membrane Complex protein 2A (IMC2A)</td><td>ND</td><td>ND</td></tr><tr><td>Toxolysin 4 (TLN4)</td><td>Toxolysin 4 (TLN4)</td><td>Toxolysin 4 (TLN4)</td><td>ND</td></tr><tr><td></td><td>ND</td><td>Ulp1 protease family, C-terminal catalytic domain-containing protein</td><td>ND</td></tr></tbody></table>
invades host cells through a process known as gliding motility, which involves the coordinated action of proteins such as Myosin A, Actin filaments, Gliding-Associated Proteins (GAP), Microneme proteins, Rhotry Neck proteins (RON), Apical Membrane Antigen 1 (AMA1), and Rhotry proteins (ROPs).

These proteins work together to facilitate the attachment of toxoplasma to host cells, form a mobile junction, and ultimately invade and exit these host cells.

In our study, we sought to identify the proteins involved in the IgM triplet by 2-DE followed by LC-MS analysis.

Around the area of 75–100 kDa four spots of interest were found.

Several proteins are good candidates for the IgM triplet. These include eight proteins (EMP24/GP25L/p24 family protein; Vacuolar ATP synthase subunit A, putative; ALV1; HSP70; Chaperonin Protein BiP; hypothetical protein; TrpRS2, and HSP) for the 75 kDa band, two proteins (HSP90 and hypothetical protein) for the 90 kDa band, and three proteins (Aconitate Hydratase ACN/IRP; Cytosolic tRNA-Ala synthetase; and TLN4) for the 100 kDa band. It is unlikely that all thirteen identified proteins contribute to the immunological response underlying the IgM triplet. The IgM triplet is composed of three narrow and well-defined high MW bands, suggesting that only a few of the thirteen proteins are truly implicated, perhaps one or two proteins per band. The remaining proteins are likely co-migrating entities, either due to similar native molecular weights or the presence of post-translational modifications. The higher the abundance of protein within the parasite, the more likely it is to be recognized by the host immune system. According to the ToxoDB database (ToxoDB-10.0_Tgondii-ME49_AnnotatedProteins.fasta, 8,322 sequences), transcript levels exceed 1,000 transcripts per million (TPM) for proteins such as HSP70, HSP90, and ALV1, suggesting that these proteins are strong candidates as true targets of the IgM triplet. In contrast, other proteins identified, such as TLN4, exhibit TPM values well below 1,000, which argues against their significant involvement in IgM triplet formation. Additional studies will be required to identify which of these proteins represent the real targets of the IgM triplet.

It turns out that each of these proteins is involved in some way in the cell invasion process, and therefore probably in the transplacental invasion process of *T. gondii*.

For the 75 kDa and 90 kDa IgM triplet bands, HSP family predominate. More specifically, HSP70 and HSP90 were found, with respective MWs of 73 and 82 kDa. In a previous work, we showed that these proteins are known to be essential for *T. gondii* host cell invasion and virulence [2]. For the 75 kDa band, HSP70 is particularly well characterized in protozoa, and is also capable of interfering with its host's immune system by modulating nitric oxide production by macrophages and reducing their phagocytosis capacity [2]. Lyons and Johnson have shown that the expression of HSP70 induced by immunological stress in *T. gondii* can protect it from the cellular damage associated with host invasion [16].

Another major protein for the 75 kDa band of the IgM triplet is the Chaperonin Protein BiP with a recorded MW of 73 kDa. It is a protein present in the endoplasmic reticulum (ER) of toxoplasma that may play a role in parasite invasion

via its action on the secretory pathway. It is currently known to act as a molecular chaperone in protein folding and exit from the ER [2, 9]. For the 75 kDa band, there are four other proteins: Vacuolar ATP synthase subunit A, putative, 68 kDa; ALV1, 70 kDa; TrpRS2, 77 kDa; and EMP24/GP25L/p24 family protein, 65 kDa. V-ATPase subunit A is a catalytic subunit of the V-ATPase complex capable of pumping protons across membranes using the energy released by ATP hydrolysis. Like HSP proteins, this complex is synthesized in response to ionic stress to protect the toxoplasma from the host immune response. This pump also plays a role in the maturation of secretory proteins in endosomal compartments, such as microneme and rhotry proteins [24].

ALV1 is a protein of the *T. gondii* inner membrane complex that has been little studied to date. It is associated with the membrane cytoskeleton of toxoplasma and therefore plays a role in the parasite's motility and virulence [6, 7]. TrpRS2 is an enzyme located in the apicoplast of *T. gondii*, whose main function is the aminoacylation of tRNA during proteins synthesis [11]. The EMP24/GP25L/p24 protein family is a family of proteins that plays an important role in protein transport between the ER and the Golgi apparatus and specialized organelles such as micronemes or rhoptries in *T. gondii* [5].

To finish with the 75 kDa band, another 74 kDa protein could also be involved, although it is less representative. It has been named "hypothetical protein" because it has not yet been characterized.

For the 90 kDa band, HSP90 plays a major role. Sun *et al.* showed that HSP90 knockout strains of *T. gondii* had a 57.5% decreased invasion capacity 1 hour after infection of Vero cells, and a 58.1% decreased intracellular growth at 24 h [25]. These are highly conserved chaperone proteins present in the mitochondria of toxoplasma and produced in response to the stress generated in the host by parasitic infection. They are involved in the correct folding of proteins, the degradation of damaged proteins and their transport across the mitochondrial membrane [2, 12, 25]. Another 85 kDa protein may also be involved, although it is less representative than HSP90. As with the 75 kDa band, it has been named "hypothetical protein", as it has not yet been characterized.

For the 100 kDa band, three proteins stand out: Aconitate hydratase ACN/IRP (aconitase) with a MW of 115 kDa; Cytosolic tRNA-Ala synthetase with a MW of 140 kDa; and TLN4 with a MW of 247 kDa. The first protein mentioned is aconitase, a mitochondrial enzyme also presents in the apicoplast of toxoplasma. It plays an important role in the energy metabolism of *T. gondii*, essential to the parasite invasion process, thanks to its role in the Krebs cycle. In the study by MacRae *et al.*, inhibition of aconitase considerably reduced the sliding motility of toxoplasma [17].

It also plays a role in regulating the parasite's iron metabolism. Aconitase thus plays a key role in maintaining cellular homeostasis in *T. gondii*. Like HSP70, it is capable of interfering with the host immune system by regulating the production of reactive oxygen species that are harmful to the parasite [22]. The second most widely represented protein is Cytosolic tRNA-Ala synthetase. Like TrpRS2, the primary function of Cytosolic tRNA-Ala synthetase is aminoacylation of tRNA, an essential step in protein synthesis. It also helps regulate protein synthesis
by hydrolyzing misacylated amino acids [8]. Finally, the third protein is TLN4, a member of the M16A metalloprotease family. It plays a crucial role in the exit of the parasite from the host cell, allowing it to continue its lytic cycle by infecting other cells [2, 10]. Proteins secreted by apical micronemes are central components for host cell recognition, invasion, exit, and virulence. TLN4 may act as a maturase or regulator for these microneme proteins. TLN4 is first synthesized as a large precursor (~260 kDa) and then cleaved into several proteolytic fragments within the parasite's secretory system [15].

These proteins are closely linked and work together to promote parasite invasion, either by acting as transporters for certain proteins in the secretory pathway, by regulating their maturation, or by modulating the host immune system to promote toxoplasma virulence. These processes are necessary prerequisites for the parasite to cross epithelial and immunological barriers, including those of the placenta [2, 23]. While our study does not establish a direct mechanistic role for these proteins in vertical transmission, these proteins represent strong candidates for future studies about the placental invasion process with a new therapeutic perspective.

Importantly, there is a clear need to develop treatments aimed at blocking transplacental passage of *T. gondii*, thus preventing exposure of the fetus [18].

In terms of diagnostic prospects, IgM triplet immunoblot testing constitutes a pathognomonic marker of congenital toxoplasmosis, conferring high specificity for the diagnosis of congenital infection in neonates. However, the IgM triplet may be absent in an infected newborn who lacks IgM but shows IgG neosynthesis. So, developing a diagnostic test only based on IgM triplet detection isn't essential. Nonetheless, it is important to ensure that this triplet is present in all diagnostic tests for congenital toxoplasmosis.

## Conclusion

The mechanisms underlying transplacental transmission of *Toxoplasma gondii* from an infected mother to her fetus are complex and not yet fully elucidated. Many proteins, secreted or not by toxoplasma for cellular invasion, could be involved in this process. It turns out that the IgM triplet, a pathognomonic marker of CT identified by L'Ollivier *et al.* and Peyclit *et al.* in mother-infant IbPP, appears to specifically target several of these proteins. This study allowed us to identify 13 proteins potentially targeted by the IgM triplet, all parts of the effector arsenal deployed by *T. gondii*. Further research is needed to confirm the precise roles of these proteins, with the ultimate goal of improving neonatal diagnosis and the clinical management of CT.

## Acknowledgements

The authors are grateful to: Said Azza (Microbes, Evolution, Phylogénie et Infection (MEPHI)-Méditerranée Infection, 13005 Marseille, France) for his contributions to the two-dimensional electrophoresis study and assistance; Members of National Reference Centre on Toxoplasmosis: Dr. AS. Deleplanque (CHU Lille), Dr. C. Garnaud (CHU Grenoble), Prof. S. Houze (AP-HP, Paris), Dr. RA. Lavergne (CHU Nantes), Dr. M. Leveque (CHU Montpellier), Prof. C. Pomares (CHU Nice), and Dr. F. Touafek (AP-HP, Paris) for their contribution to

interpretation of the IgM triplet in neonate sera. The mass spectrometry analysis was supported by the French Proteomic Infrastructure (ProFI; ANR-10-INBS-08-03).

## Funding

This work was supported by a grant from the French Government managed by the National Research Agency under the "Investissements d'avenir (Investments for the Future)" program, with the reference ANR-10-IAHU-03 (Méditerranée Infection), by the Contrat Plan Etat-Région and European funding FEDER IHUPERF.

## Conflicts of interest

The authors declare that they have no conflict of interest.

## Data availability statement

All data are available within the article.

## Author contribution statement

Conceptualization, C.L.; methodology, C.L., M.C.; resources, M. C., S.A., O.V., L.P., H.D., C.S.R., C.L.; writing – original draft preparation, C.L., M.C.; writing – review and editing, C.L.; supervision, C. L. All authors have read and agreed to the published version of the manuscript.

## Supplementary materials

*Supplementary data S1:* Example of an IgM and IgG mother and child immunoblot pair profile with identical profiles and the infant's IgM triplet (pink box). The child was proved to be infected subsequently during follow-up. m, mother; i, infant. Reproduced from Peyclit *et al.* 2023 [21].

*Supplementary data S2:* Immunoblot obtained using sera positive for the IgM triplet (NNTPos; Patients 1BB to 16BB) or negative for this same IgM triplet (NNTNeg; Patients 17BB to 19BB). The black box indicates the location of spots 21 to 24 only found on NNTPos sera and marked by arrows when present. The immunoblot obtained with the NNTNeg sera from the patient 19BB shows spots 25 and 26, which were excluded because they were not specific to the IgM triplet.

The supplementary material of this article is available at https://www.parasite-journal.org/10.1051/parasite/2025065/olm.

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**Cite this article as:** Carreno M, Villard O, Villena I, Peyclit L, Diemer H, Schaeffer-Reiss C & L'Ollivier C. 2025. Protein characterization of the IgM triplet involved in the diagnosis of congenital toxoplasmosis. *Parasite* **32**, 72. https://doi.org/10.1051/parasite/2025065.
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## PARASITE

An international open-access, peer-reviewed, online journal publishing high quality papers on all aspects of human and animal parasitology

Reviews, articles and short notes may be submitted. Fields include, but are not limited to: general, medical and veterinary parasitology; morphology, including ultrastructure; parasite systematics, including entomology, acarology, helminthology and protistology, and molecular analyses; molecular biology and biochemistry; immunology of parasitic diseases; host-parasite relationships; ecology and life history of parasites; epidemiology; therapeutics; new diagnostic tools.

All papers in *Parasite* are published in English. Manuscripts should have a broad interest and must not have been published or submitted elsewhere. No limit is imposed on the length of manuscripts.

*Parasite* (open-access) continues *Parasite* (print and online editions, 1994-2012) and *Annales de Parasitologie Humaine et Comparée* (1923-1993) and is the official journal of the Société Française de Parasitologie.

Editor-in-Chief:
Jean-Lou Justine, Paris

Submit your manuscript at
https://www.editorialmanager.com/parasite