Background

Toxoplasma gondii can infect humans and many animals worldwide [1]. In humans, infection is usually asymptomatic or causes mild flu-like symptoms [1]. However, toxoplasmosis can have serious health effects in immunocompromised patients and cause miscarriage or birth defects in pregnant women [2]. Toxoplasmosis can also have an impact on the animal health and fertility, especially in pigs, resulting in serious economic losses [3, 4].

The cat and other felids serve as the definitive host where they support the formation of T. gondii oocysts, which are excreted with feces. T. gondii oocysts can contaminate the soil in areas where cats are present, and the oocysts can survive for over a year in non-extreme environments [5,6,7]. Humans as well as domesticated and wild animals can be infected by consuming water and vegetables containing T. gondii oocysts or by ingesting meat containing the parasite tissue cysts [8, 9]. Given the critical role that the cats play in the transmission of T. gondii, it is important to enhance our understanding of the pathogenesis of toxoplasmosis in cats. However, many of the mechanisms underlying T. gondii-induced molecular and pathological changes in domestic cats remain unknown.

Different technologies have been used to study the molecular attributes of T. gondii, and the molecular mechanisms associated with T. gondii infection in different hosts, including proteomics [10,11,12], transcriptomics [13,14,15], and metabolomics [16,17,18]. Over the past decade, proteomic approaches have been widely used to improve the understanding of host–pathogen interactions [19, 20]. Proteomics enables a comprehensive high-throughput analysis of proteins in organisms and can reveal the proteomic responses of organisms or tissues to perturbations caused by drug treatments or diseases [21]. A proteomic technique called isobaric tags for relative and absolute quantification (iTRAQ) can provide more reliable quantitative measurements and enable the comparison between samples, compared to proteomic analysis based on two-dimensional differential in-gel electrophoresis [22]. The iTRAQ-based quantitative proteomic technique has been used to study T. gondii-host interactions [23, 24]. However, the proteomic response of the different body organs of cats during T. gondii infection has yet to be investigated.

In this study, the iTRAQ was used to study the global proteomic alterations in the major body organs of cats during acute T. gondii infection. The results showed that T. gondii infection affected protein expression and pathways in all examined organs. Additionally, we correlated the proteome data with a transcriptome data of the same organs obtained from the same cats in a previous study [25], to expand our understanding of the differentially expressed transcripts and proteins which play a role in cat–T. gondii interactions.

Methods

Ethics statement

This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Permit number: LVRIAEC-2014–001). The animals used in the experiments were handled in strict accordance with the Animal Ethics Procedures and Guidelines of the People's Republic of China. Every effort was made to minimize animal distress during the experiment.

Parasite infection and sample collection

Two litters (n = 12) of domestic cats (Felis catus) of the Chinese Lihua breed were purchased from a local breeder. Cats were between 2–3 months of age. All cats were tested serologically and found free of T. gondii and the viruses, including feline calicivirus, coronavirus, feline immunodeficiency virus, feline leukemia virus, and feline parvovirus as previously descripted [25]. These cats were divided into four groups, three cats each. Two groups were infected and two groups were used as a control. Cats were housed in a controlled environment on a commercial cat food (Royal Canine Inc., St. Charles, MO, USA). Based on the daily energy needs of individual cats, they were fed individually once per day and water was provided ad libitum.

T. gondii PRU strain (Genotype II) maintained in Kunming mice in our laboratory was used in this study [26]. This T. gondii strain was used because most cases of human toxoplasmosis are associated with T. gondii strains of genotype II [27]. The number of T. gondii cysts was counted using a light microscope and adjusted to 100 cysts/ml in PBS. Each cat in the infected group was orally infected with 100 cysts. Cats in the control group were given PBS orally only. On day 7 post-infection, the cats were euthanized by overdose of isoflurane. This procedure was performed by a qualified veterinarian with knowledge of anesthetic techniques. After confirmation of complete unresponsiveness to stimuli and permanent cessation of chest movement and heart rate, the tissue samples were harvested and immediately stored at –80 °C until processing.

Confirmation of T. gondii infection

Total genomic DNA of each tissue sample was isolated by using TIANamp Genomic DNA kit (TianGen™, Beijing, China) according to manufacturer’s recommendations. The extracted DNA was used to detect the T. gondii infection by using a PCR assay that targets T. gondii B1 gene [28], The genotype of the detected T. gondii was determined by using PCR–RFLP as described previously [29].

Protein extraction

Tissue samples were ground into powder in liquid nitrogen and were treated with Lysis buffer (7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 40 mmol/L tris–HCl, pH 8.5) containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF) and 2 mmol/L ethylenediaminetetraacetate (EDTA) for 5 min. Then, 10 mmol/L dithiothreitol (DTT) was added to the samples. The suspension was sonicated at 200 W for 15 min and centrifuged at 4 °C, 30,000 × g for 15 min. After mixing the supernatant with chilled acetone containing 10% (v/v) trichloroacetic acid, the mixture was stored at − 20 °C for overnight. The mixture was centrifuged at 4000 × g for 10 min at 4 °C. The pellet of the protein was washed three times with pre-cold acetone. The air-dried pellet was dissolved into Lysis buffer (7 mol/L urea, 2 mol/L thiourea, 4% NP40, 20 mmol/L Tris–HCl, pH 8.0 − 8.5) and was sonicated at 200 W for 15 min. The supernatant was collected into a new tube after centrifuging at 50,000 × g for 10 min at 4 °C. Then, 10 mmol/L DTT was added into the supernatant and the mixture was incubated for 1 h at 56 °C. The above mixture was placed in the dark for 1 h after adding 55 mmol/L iodoacetamide (IAM) to block free thiols and preserve cysteines. Then the mixture was added with chilled acetone again and was centrifuged at 4000 × g for 10 min at 4 °C to collect the pellet. After dissolving the pellet in 500 μl 0.5 mol/L triethylammonium bicarbonate (TEAB) (Applied Biosystems, Milan, Italy), the pellet was transferred to a new tube after discarding the supernatant. Finally, Bradford Protein Assay (Bio-Rad, Hercules, USA) was used to determine the protein concentration.

iTRAQ labeling and SCX fractionation

Proteins (100 μg) per sample were digested using Trypsin Gold (Promega, Madison, WI, USA) at a 30:1 protein: trypsin ratio for 16 h at 37 ℃. After digestion, peptides were dried by vacuum centrifugation and were reconstituted in 0.5 mol/L TEAB and processed according to the manufacturer’s instructions for 8-plex iTRAQ reagent (Applied Biosystems, Calif, USA). Strong cation exchange (SCX) chromatography was conducted using LC-20AB HPLC Pump system (Shimadzu, Kyoto, Japan). The iTRAQ-labeled peptide mixtures were reconstituted with 4 ml buffer A (25 mm NaH2PO4 in 25% ACN, pH 2.7) and loaded onto a 4.6 × 250 mm Ultremex SCX column containing 5 μmol/L particles (Phenomenex, Calif, USA). The peptides were eluted at a flow rate of 1 ml/min with a gradient of buffer A for 10 min, 5–60% buffer B (25 mm NaH2PO4, 1 mol/L KCl in 25% ACN, pH 2.7) for 27 min, 60–100% buffer B for 1 min. The system was then maintained at 100% buffer B for 1 min before equilibrating with buffer A for 10 min prior to the next injection. Elution was monitored by measuring the absorbance at 214 nm, and fractions were collected every 1 min. The eluted peptides were pooled into 20 fractions, desalted with a Strata X C18 column (Phenomenex, Calif, USA) and vacuum dried.

Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis

Each fraction was re-suspended in buffer A (5% ACN, 0.1% FA) and centrifuged at 20,000 × g for 10 min, and the final concentration of peptide was 0.5 μg/μl. Approximately 10 μl supernatant was loaded on a LC-20AD nanoHPLC (Shimadzu, Kyoto, Japan) by an auto sampler onto a 2 cm C18 trap column. The peptides were then eluted onto a 10 cm analytical C18 column (inner diameter 75 μm) packed in-house. The samples were loaded at 8 μl/min for 4 min; then the 35 min gradient was run at 300 nl/min starting from 2 and increasing to 35% buffer B (95% ACN, 0.1% FA), followed by 5 min linear gradient to 60% and 2 min linear gradient to 80%, and maintenance at 80% buffer B for 4 min, and finally a return to 5% for 1 min.

Data acquisition was conducted with a TripleTOF 5600 System (AB SCIEX, Concord, ON) fitted with a Nanospray III source (AB SCIEX, Concord, ON) and a pulled quartz tip as the emitter (New Objectives, Woburn, MA). Data was acquired using an ion spray voltage of 2.5 kV, curtain gas of 30 psi, nebulizer gas of 15 psi, and an interface heater temperature of 150 °C. The MS was operated with a resolution power (RP) of ≥ 30,000 (full width at half maxima; FWHM) for TOF MS scans. For information dependent acquisition (IDA), the survey scans were acquired in 250 MS and as many as 30 product ion scans were collected if exceeding a threshold of 120 counts per second (counts/s) and with a 2 + to 5 + charge-state. Total cycle time was fixed to 3.3 s. Q2 transmission window was 100 Da for 100%. Four-time bins were summed for each scan at a pulser frequency value of 11 kHz via monitoring of the 40 GHz multichannel TDC detector with four-anode channel detect ion. A sweeping collision energy setting of 35 ± 5 eV coupled with iTRAQ adjust rolling collision energy was applied to all precursor ions for collision-induced dissociation. Dynamic exclusion was set for 1/2 of peak width (15 s), and then the precursor was refreshed off the exclusion list.

Protein identification and data analysis

Mascot search engine (version 2.3.02, Matrix Science, London, UK) against the Uniprot database was used for protein identification. Mascot software was used to conduct the student’s t-test. The resulting dataset was auto bias-corrected to the biological replicates. Proteins with P values < 0.05 and fold change > 1.2 or < 0.83 were considered as differentially expressed proteins (DEPs). Web-based software (http://www.geneontology.org) was used to conduct gene ontology (GO) annotation analysis of the potential biological functions of significantly dysregulated proteins [30]. Kyoto Encyclopedia of Genes and Genomes database (KEGG, http://www.genome.p/kegg) was used to perform pathway analysis [31]. EuKaryotic Orthologous Groups (KOG, https://ftp.ncbi.nih.gov/pub/COG/KOG/) was used to detect the functional annotation of the DEPs. STRING 10.0 (http://string-db.org) was employed to explore the interaction network and functional relations of the DEPs.

Correlation analysis between the proteome and transcriptome

In a previous study, parallel tissue samples were harvested from the same cat organs (brain, heart, liver, lung, small intestine and spleen), on day 7 after initial T. gondii infection. RNAs isolated from those samples were used in RNA sequencing to profile the transcriptomes of multiple cat organs [25]. The basic principle of the association analysis between proteome and transcriptome is the central dogma. The data screening and difference definitions of this association analysis were as follow: protein-fold change > 1.2, gene fold change > 2, gene significant < 0.001, GO and pathway significant < 0.05. Correlation was considered if a gene/protein was differentially expressed at both the proteome and transcriptome level after T. gondii infection. We also analyzed the level of concordance between transcripts and proteins of genes that belong to the same pathway. Pathways with good congruence between changes of the protein and transcript levels suggest that changes in the expression of transcripts may have changed the expression of the respective proteins; hence, modest alterations occur at the post-transcriptional regulation level.

Results

Confirmation of T. gondii infection

Positive PCR results were detected for the six organs obtained from infected cats (Additional file 1: Fig. S1A), and the positive PCR products were identified as T. gondii genotype II by RFLP analysis. As anticipated, all samples from control cats were negative (Additional file 1: Fig. S1A). T. gondii oocysts were observed in the feces of infected cats (Additional file 1: Fig. S1B).

The identification and quantification of proteins

We used iTRAQ-based quantitative proteomic approach to identify the protein species and their expression levels in different tissue samples of acutely infected cats. A total of 2,118,641 spectra were obtained, with an average of 353,107 spectra per organ. According to the Mascot program searching, an average of 78,698 spectra were matched and an average of 68,794 high scoring unique peptides were obtained. 32,657 proteins were identified in the six organs, with an average of 5443 proteins per organ (Table 1). For differential expression analysis, 855, 575, 463, 283, 255, and 125 proteins were identified as DEPs in the heart, small intestine, liver, spleen, lung, and brain, respectively, compared to non-infected groups (Table 2). Of the 2556 DEPs, 1325 and 1231 were identified as up- and down-regulated proteins in infected vs uninfected cats. Liver had the largest number of up-regulated proteins (319), followed by small intestine (309), heart (298), spleen (185), lung (155), while the lowest number was detected in the brain (59). Interestingly, the largest number of down-regulated proteins was detected in the heart (557), followed by small intestine (266), liver (144), lung (100), spleen, while the lowest number was detected in the brain (66). The numbers of the DEPs of each organ are listed in Table 2.

Table 1 Summary of the proteomics results
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Table 2 Summary of the identified proteins in the cat organs
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Functional enrichment analysis of all proteins

GO and KEGG pathway enrichment analyses were performed on all proteins and the detailed results are shown in Additional file 2: Table S1 and Additional file 3: Table S2. Based on the GO enrichment results of the biological process (BP) category, 5351 and 3274 proteins were significantly enriched in binding and catalytic activity. Proteins were annotated into different cellular components (CC) category, including cell body (13,341), organelle (9373), membrane (6302), and macromolecular complex (2603). For the molecular function (MF) category, 6051, 4395, 4198 and 3923 proteins were associated with cellular process, metabolic process, biological process and regulation of biological process, respectively (Fig. 1). KEGG enrichment identified six functional categories, including cellular processes, environmental information processing, genetic information process, human diseases, metabolism, and organismal system. Most proteins were enriched in transport and catabolism (938), signal transduction (1614), translation (561), infectious diseases: viral (1013), global and overview maps (1422), and immune system (1039) (Fig. 2).

Fig. 1
figure 1

Horizontal bar graph of gene ontology (GO) classification of proteins identified in the brain, liver, lung, spleen, heart and small intestine of cats infected by Toxoplasma gondii on day 7 post-infection. X-axis shows the number of proteins and y-axis shows the GO terms

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Fig. 2
figure 2

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of the proteins identified in the brain, liver, lung, spleen, heart and small intestine of cats infected by Toxoplasma gondii on day 7 post-infection. X-axis shows the number of proteins and y-axis shows the KEGG pathways

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Enrichment analysis of differentially expressed proteins

In the MF category, most DEPs were enriched in binding, catalytic activity, molecular function regulator, structural molecule activity and transporter activity (Table 3). For CC category, most DEPs were enriched in cell part, cell, organelle, extracellular region, membrane, and macromolecular complex (Table 3). In the BP category, most DEPs were involved in cellular process, metabolic process, biological regulation, regulation of biological process, and response to stimulus. The DEPs in brain were enriched in multicellular organismal process (Table 3).

Table 3 Summary of GO enrichment analysis of the cat organs
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KOG enrichment analysis showed that most DEPs were enriched in amino acid transport and metabolism (small intestine, lung), energy production and conversion (spleen, heart), lipid transport and metabolism (liver, brain) in metabolism. In information storage and processing, most DEPs were enriched in translation, ribosomal structure and biogenesis (small intestine, lung, spleen), RNA processing and modification (liver), chromatin structure and dynamics replication (heart), replication, recombination and repair (brain). In cellular processes and signaling, the DEPs were mainly associated with signal transduction mechanisms (brain, heart, spleen, liver, lung) and posttranslational modification, protein turnover, chaperones (small intestine) (Table 4).

Table 4 Summary of KOG enrichment analysis of the cat organs
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As shown in Table 5, KEGG pathway enrichment revealed that in cellular processes, most DEPs were associated with cell growth and death (small intestine, spleen, brain) or transport and catabolism (lung, liver, heart). In environmental information processing, most DEPs were associated with signal transduction and translation (small intestine, liver,spleen, lung) or folding, sorting and degradation (brain, heart). In genetic information processing, most DEPs were associated with folding, sorting and degradation (brain, heart) or translation (spleen, liver, lung, small intestine). In human diseases, most DEPs were associated with cancers: overview (small intestine) or infectious diseases (lung, liver): viral or cardiovascular diseases (spleen) or neurodegenerative diseases (heart) or cardiovascular disease (brain). In metabolism, most DEPs were associated with global and overview maps. In organismal systems, most DEPs were associated with immune system (brain, spleen, liver, lung, small intestine) or environmental adaption (heart).

Table 5 Summary of the pathway enrichment analysis of the cat organs showing enriched pathways annotated according to the six main KEGG categories
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Correlations between the proteome and transcriptome data

A low correlation between the level of transcript and protein changes was observed for all quantified proteins (R = 0.027) and differentially expressed proteins (R = 0.487). In all proteins, 41, 56, 64, 44, 46, 28 proteins were commonly identified at the protein and transcript levels in the brain, heart, liver, lung, small intestine, and spleen, respectively (Fig. 3). While for the DEPs, only 19, 17, 9, 9, 3 proteins were common at the transcript and protein levels in the liver, small intestine, heart, lung, and spleen (Fig. 4; Additional file 4: Table S3). Among those, 3 proteins (indoleamine 2, 3-dioxygenase 1 and guanylate binding protein 1), 12 proteins (e.g. indoleamine 2, 3-dioxygenase 1, caveolae associated protein, parvin beta), 8 proteins (e.g. indoleamine 2, 3-dioxygenase 1, synapsin I, matrix metallopeptidase), 8 proteins (e.g. indoleamine 2, 3-dioxygenase 1, tryptophanyl-tRNA synthetase, apolipoprotein A1) and 3 proteins (transmembrane p24 trafficking protein 1, CDP-diacylglycerol–inositol 3-phosphatidyltransferase, importin subunit alpha) were upregulated in the heart, liver, lung, small intestine and spleen, respectively. Seven proteins (e.g. collagen type XV alpha 1, collagen type I alpha 1, FTO alpha-ketoglutarate dependent dioxygenase), 7 proteins (e.g. glucosylceramidase beta 3, NDRG family member 2, carboxylic ester hydrolase), 1 protein (reticulon) and 9 proteins (e.g. UDP-glucuronosyltransferase, ectonucleotide pyrophosphatase/phosphodiesterase 6, 3-hydroxyanthranilate 3, 4-dioxygenase) were downregulated in the heart, liver, lung and small intestine, respectively (Additional file 4: Table S3). Interestingly, there were no common DEPs between the proteome and transcriptome in the brain (Fig. 4).

Fig. 3
figure 3

Venn diagrams showing overlap between all proteins identified in the proteome and differentially expressed genes identified in the transcriptome of the brain, heart, liver, lung, small intestine and spleen of cats infected by Toxoplasma gondii on day 7 post-infection

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Fig. 4
figure 4

Venn diagrams showing overlap between differentially expressed proteins (DEPs) identified in the proteome and differentially expressed genes (DEGs) identified in the transcriptome of the brain, heart, liver, lung, small intestine and spleen of cats infected by Toxoplasma gondii on day 7 post-infection

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For the GO-correlation analysis, the DEPs were mainly enriched in intracellular membrane-bound, extracellular region, membrane part, and cytoplasmic part in the CC category (Fig. 5). In the MF category, most DEPs were enriched in enzyme binding, ion binding, organic cyclic compound bind, enzyme regulator activity, oxidoreductase activity, and protein complex binding (Fig. 6). In the BP category, most DEPs were enriched in the regulation of molecular function, small molecule metabolic process, negative regulation of response, developmental process, and response to external stimulus (Fig. 7). KEGG pathway enrichment correlation revealed that DEPs are mainly enriched in herpes simplex infection, metabolic pathway, ECM-receptor interaction, Toll-like receptor signaling, and pentose phosphate pathway (Fig. 8).

Fig. 5
figure 5

Gene Ontology (GO)-correlation enrichment of the differentially expressed proteins (DEPs) in the cellular component showing the proportion of changes at the protein and transcript levels in the brain, heart, liver, lung, small intestine and spleen of cats infected by Toxoplasma gondii on day 7 post-infection

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Fig. 6
figure 6

Gene Ontology (GO)-correlation enrichment of the differentially expressed proteins (DEPs) in the molecular function showing the proportion of changes at the protein and transcript levels in the brain, heart, liver, lung, small intestine and spleen of cats infected by Toxoplasma gondii on day 7 post-infection

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Fig. 7
figure 7

Gene Ontology (GO)-correlation enrichment of the differentially expressed proteins (DEPs) in the biological process showing the proportion of changes at the protein and transcript levels in the brain, heart, liver, lung, small intestine and spleen of cats infected by Toxoplasma gondii on day 7 post-infection

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Fig. 8
figure 8

Kyoto Encyclopedia of Genes and Genomes (KEGG)-correlation enrichment of the differentially expressed proteins (DEPs) showing the proportion of changes at the protein and transcript levels in the brain, heart, liver, lung, small intestine and spleen of cats infected by Toxoplasma gondii on day 7 post-infection

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Discussion

Cats play a fundamental role in the biology and epidemiology of T. gondii because they are the only definitive hosts that can produce and excrete T. gondii oocysts. In a previous study, we determined the transcriptomic profiles of the main cat organs (brain, heart, spleen, liver, lung and small intestine) at 7 days post infection by T. gondii [25]. Aiming for a more comprehensive picture of the molecular response of cat tissues to T. gondii infection, we studied the global differential protein expression profile of the same cat tissues and at the same time point post infection using iTRAQ-based quantitative proteomics.

A total of 2556 DEPs were identified in all cat organs; of which 1325 and 1231 were identified as up-regulated and down-regulated proteins, respectively. The proteomic profiling also showed that 319, 309 and 298 DEPs were up-regulated in the liver, small intestine and heart, while 557 and 266 DEPs were down-regulated in the heart and small intestine, respectively. These results show that liver had the largest up-regulated proteins (n = 319), while the heart had the largest down-regulated proteins (n = 557). This corroborates part of the results in a previous transcriptomic study where the liver had the highest number of differentially expressed transcripts, suggesting that T. gondii causes significant changes in the liver [25]. The results also suggest that heart had the highest level of DEPs (298 up-regulated + 557 down-regulated = 855 DEPs), indicating a significant impact of T. gondii on protein regulation in the heart compared to other organs. One would expect the heart to exhibit significant changes in proteins owing to the contractile function of the organ which requires a lot of metabolism and bioenergetics as was evident by the KOG enrichment of DEPs in energy production and conversion (Table 4). The difference in the differentially regulated transcripts and proteins between different tissues may suggest tissue-specific variations in the induction and turnover rates of transcripts and proteins.

The functional enrichment of the DEPs in the six cat tissues identified some important up-regulated proteins in heart, such as MHC 1, MHC II, heat shock protein, phosphoinositide phospholipase C (TgPI-PLC). MHC 1 plays a key role in processing and presenting T. gondii proteins to CD8+ T-cells [32]. Expression of MHC II by microglia and astrocytes is crucial for CD4+-mediated immune responses in the central nervous system [33]. These proteomic results concur with previous transcriptomic analysis showing T. gondii’s ability to induce significant expression of immune related genes in all infected organs in cats [25]. Heat shock protein can play key roles in the pathogenesis of T. gondii [34]. TgPI-PLC is localized in the internal leaflet of the plasma membrane and encodes a functional PI-PLC in T. gondii genome [35]. Some of the up-regulated proteins remain uncharacterized, and further studies should be carried out to elucidate their biological functions in the context of T. gondii infection.

Enrichment analysis of the identified proteins suggest that T. gondii alters the xenobiotic metabolism. Oxidoreductase activity was enriched and up-regulated in the heart, liver and small intestine, suggesting that potential involvement in drug metabolism in the liver and that targeting oxidoreductase may be part of a mechanism by which T. gondii interferes with drug pharmacokinetic [36]. This result disagrees with a previous study showing downregulation of oxidoreductase activity protein in mouse liver [37]. However, this difference may be attributed to different experimental methods or host specificity. In the present study, feline aminopeptidase was downregulated in small intestine. A previous study showed that feline aminopeptidase N serves as an entry receptor for viruses [38]. Whether feline aminopeptidase N serves as a functional receptor for T. gondii entry into the small intestine remains to be investigated.

We further examined the transcriptome and proteome data sets to identify differences in the correlation between proteins and transcripts with differential expression patterns in different tissues in response to T. gondii infection. Correlations were detected between proteins and the corresponding transcripts in the liver (n = 19), small intestine (n = 17), heart (n = 9), lung (n = 9), with spleen showing the least transcript–protein correlation (n = 3). Detailed information is available as supplemental material (see Additional file 4: Table S3). Overall, this result shows a limited accordance between alterations in proteins and transcripts, suggesting that protein synthesis, protein stability, post-transcriptional modifications may have played a role in determining the protein levels beyond transcript levels between different tissues [39].

It is noteworthy that the correlation between the differential expression pattens of indoleamine 2,3-dioxygenase at both protein and transcript levels was detected in all organs, with the exception of spleen. IDO was co-expressed in 5 feline tissues (brain, heart, liver, small intestine, and spleen) in the previous transcriptomic analysis [25]. Induction of IDO, mediated by interferon gamma (IFN-γ), plays a key role in degradation of the essential amino acid tryptophan, inhibiting the growth of T. gondii [40], and limiting neuroinflammation [41,42,43]. These findings largely corroborate previous studies linking IDO to various mechanisms that mediate the pathophysiology of T. gondii infection [36, 37, 44,45,46,47,48].

Matrix metallopeptidases (MMPs) are proteolytic enzymes that degrade extracellular matrix proteins such as collagen, fibronectin and laminin. In the present study, matrix metallopeptidase was upregulated at both the transcript and protein levels in T. gondii infected lungs compared with the lungs of uninfected cats (Additional file 4: Table S3). Interestingly, downregulation of protein and transcript of collagen type I alpha 1 and collagen type XV alpha 1 was detected in the heart. A previous study showed that matrix metalloproteinase (MMP)-2 and MMP-9 cleave fibronectin and induce astroglia reaction and leukocyte migration to the sites of T. gondii infection in the brain [49]. Several molecules involved in the immune and inflammatory responses during toxoplasmosis, such as interleukin (IL)-1, IL-23 and tumor necrosis factor alpha (TNFα), can increase MMP production in the brain [50]. Additionally, MMP-8, MMP-10 and tissue inhibitor of metalloproteinases-1 (TIMP-1) have important functions in regulating the perivascular accumulation and influx of lymphocytes into the brain to prevent the reactivation of dormant T. gondii cysts [51]. These results suggest that regulation of extracellular matrix and collagen may be involved in the immune response of cats during early T. gondii infection.

Understanding the pathogenic mechanisms of T. gondii infection in cats is critical for the development of intervention technologies against T. gondii infection because cats play a key role in the transmission of T. gondii. The identification of 2556 DEPs in six cat tissues responsive to T. gondii infection provides new and valuable resources for further exploration of the intricate mechanism of toxoplasmosis pathogenesis in cats.

Conclusions

iTRAQ-based quantitative proteomic analysis identified 32,657 proteins; of those 2556 were DEPs in six cat tissues at day 7 after infection by T. gondii PRU strain. The proteins were not expressed at similar levels across cat tissues and the correlation of transcript/protein ratios across tissues was very low. Functional annotation and enrichment analyses revealed that the DEPs were localized in different cellular compartments and were associated with various cellular and metabolic processes, such as immune response, drug metabolism, tryptophan catabolism, extracellular matrix remodeling, and information storage and processing. These proteomic profiles serve as a rich foundation for future investigations of the intricate mechanisms that allow T. gondii to adapt and survive in different feline tissues.