© M. Carreno et al., published by EDP Sciences, 2025

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 revelation 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 activated 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 containing 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 duplicate 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 consistently 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 carbonate (NH₄HCO₃), the gel plugs were dehydrated with acetonitrile. 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 evaporated under vacuum prior to MS analysis.

Mass spectrometry analysis

NanoLC-MS/MS analysis was performed using a nanoACQUITY 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^TM M/Z Symmetry C18 precolumn (100 Å, 5 μm, 180 μm × 20 mm, Waters Corporation) using 99% solvent A (0.1% formic acid in water) and 1% solvent B (0.1% formic acid in acetonitrile) 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^TM 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 isolation using a 2 m/z window, fragmentation using higher-energy collisional dissociation (HCD, normalized collision energy of 27), dynamic exclusion of already fragmented precursors 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://proteowizard.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/ (ToxoDB-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.profiproteomics.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 optimized 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.

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.

IgM triplet revelation using NNTPos and NNTNeg sera

A total of 32 transfers were performed for IgM triplet revelation 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).

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).

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 software (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 the 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 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, Rhoptry Neck proteins (RON), Apical Membrane Antigen 1 (AMA1), and Rhoptry 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_TgondiiME49_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 rhoptry 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 futures 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.

Acknowledgments

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]. Access here

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. Access here

References

All Tables

All Figures

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.

In the text

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).

In the text