Label-free quantitative proteomic analyses of mouse astrocytes provides insight into the host response mechanism at different developmental stages of Toxoplasma gondii

Toxoplasma gondii (T. gondii) is an opportunistic parasite that can infect the central nervous system (CNS), causing severe toxoplasmosis and behavioral cognitive impairment. Mortality is high in immunocompromised individuals with toxoplasmosis, most commonly due to reactivation of infection in the CNS. There are still no effective vaccines and drugs for the prevention and treatment of toxoplasmosis. There are five developmental stages for T. gondii to complete life cycle, of which the tachyzoite and bradyzoite stages are the key to the acute and chronic infection. In this study, to better understanding of how T. gondii interacts with the host CNS at different stages of infection, we constructed acute and chronic infection models of T. gondii in astrocytes, and used label-free proteomics to detect the proteome changes before and after infection, respectively. A total of 4676 proteins were identified, among which 163 differentially expressed proteins (fold change ≥ 1.5 or ≤ 0.67 and p-value ≤ 0.05) including 109 up-regulated proteins and 54 down-regulated proteins in C8-TA vs C8 group, and 719 differentially expressed proteins including 495 up-regulated proteins and 224 down-regulated proteins in C8-BR vs C8-TA group. After T. gondii tachyzoites infected astrocytes, differentially expressed proteins were enriched in immune-related biological processes to promote the formation of bradyzoites and maintain the balance of T. gondii, CNS and brain. After T. gondii bradyzoites infected astrocytes, the differentially expressed proteins up-regulated the host’s glucose metabolism, and some up-regulated proteins were strongly associated with neurodegenerative diseases. These findings not only provide new insights into the psychiatric pathogenesis of T. gondii, but also provide potential targets for the treatment of acute and chronic Toxoplasmosis.


Introduction
Toxoplasma gondii (T.gondii) is an obligate intracellular parasite that can infect almost all warm-blooded animals, including humans and livestock, and causes zoonotic parasitic diseases [1][2][3][4].It is estimated that 30% of the global population was infected by T. gondii [5].In China, approximately 8.2% of Chinese population individuals have been affected by this parasite [6].T. gondii has a complex life cycle, and almost all warm-blooded animals can become intermediate hosts.During the intracellular life, T. gondii undergoes five distinct developmental stages including tachyzoite, bradyzoite, sporozoite, schizont, and gametocyte.Tachyzoites reside within pseudocyst, while bradyzoites are present in the tissue cyst.The schizont and gametophyte are the sexual reproductive stages, ultimately formed into oocyst.In the intermediate hosts, there are two infectious stages: tachyzoite and bradyzoite.Due to the minimal morphological disparity between tachyzoites and bradyzoites, the bradyzoite-specific expression protein BAG1 is usually used for the identification of bradyzoites [7].
The mutual transformation of tachyzoites and bradyzoites is the central link in the pathogenesis of T. gondii.Tachyzoites can cause acute infection, while bradyzoites are the main cause of chronic infection.Acute toxoplasmosis is more prevalent in immunodeficiency patients [8][9][10].However, when the tachyzoites are transformed into bradyzoites and form cysts in normal immune responses, they will latently parasitize in the host's brain and ocular chorioretinal regions.When the hosts immune system is impaired, the latent bradyzoites (in the CNS) could burst out of the cysts, reconvert to replicative tachyzoites and triggering a new round of infection [11].In the chronic stages of infection, the host may have alterations in behavior and cognition [12][13][14][15].For example, the chronic infection in rodent hosts, can lead to excessive exercise, decreased anxiety, reduced new phobias, and predator vigilance.In particular, T. gondii could induce changes in rodent olfactory preferences, converting an innate aversion for cat odor into attraction, in order to enhance their own transmission [16].Nevertheless, Boillat et al found that T. gondii infection could commonly shift the host's aversion to predators, which is not unique to cats.And this alternation may be related to cysts in the host brain [17].As far as humans are concerned, a large amount of seroepidemiological data have indicated that T. gondii infection has increased the incidence of mental illness, especially focusing on the relationship among schizophrenia, suicide and traffic accidents [18][19][20][21].
T. gondii can infect a variety of cells in the CNS, including neuronal cells, astrocytes, microglia and Purkinje cells.Previous studies have reported that astrocytes in CNS were more susceptible to tachyzoites [22].Astrocytes are the most abundant glial cell type in CNS, constitute a heterogeneous cell population and could maintain neural homeostasis [23].Astrocytes play an important role in regulation of energy metabolism, brain barrier, synaptic structure and plasticity [24][25][26][27].They can also interact with CD4+ and CD8+ T cells to prevent neuronal damage [28].However, the regulation details of astrocytes infected with tachyzoites or / and bradyzoites are still unknown.Therefore, exploring the response of astrocytes infected with different stages of T. gondii will help to understand the mechanism of interaction between T. gondii and host CNS, and lay a solid foundation for the study of acute and chronic Toxoplasmosis.
The aim of the present study was to figure out the changes of host protein expression before and after infection with tachyzoite and bradyzoite in C8-D1A cells, respectively by using the label-free proteome detection.Exploring the function of differentially expressed proteins to reveal the molecular mechanism of astrocytes to parasites at different stages of T. gondii infection, which can provide new insights into the pathogenic mechanism of acute and chronic toxoplasmosis, and show new targets for the development of anti-T.gondii therapeutic drugs.

T. gondii and cell culture
T. gondii tachyzoites (RH strain) were maintained using human foreskin fibroblast (HFF) cells.Cells were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 100 IU/mL penicillin, and 100 μg/mL streptomycin (Beyotime, China) at 37˚C in a humidified atmosphere of 5% CO 2 .After the infected HFF monolayer was lysed, cell mixture was collected and filtered through a 5 μm filter to obtain T. gondii tachyzoites, counted and stored at -80˚C.C8-D1A mouse astrocytes were cultured under the identical conditions as HFF cells.

Sample preparation and collection
C8-D1A (5×10 5 cells) were seeded on 8 mm coverslips in 6-well plates (Costar, USA), and cultured in 37˚C with 5% CO 2 .When cells formed confluent monolayers, replace medium with 3% FBS.RH strain of T. gondii were added to C8-D1A (The ratio of cells to tachyzoites was 10:1) for 24 h.PBS washed two times and collected samples, centrifuged at 1000 g for 5 minutes, stored at -80˚C, and the group was named C8-TA.Alkaline-induced transformation of tachyzoites in vitro were performed.The T. gondii tachyzoites were added to cells at a ratio of 1:10 and cultured for 8 h at 37˚C with 5% CO 2 , then continued to culture for 96 h in alkaline media (pH 8.2) and changed the medium every 1-2 days.PBS washed two times and collected samples, centrifuged at 1000 g for 5 minutes, stored at -80˚C, the group was named C8-BR.The uninfected group was named C8.Because of three replicates for each group, nine samples were used for the following experiment.

RT-PCR
The expression of bradyzoite antigen 1 (BAG1) was detected using RT-PCR.Total RNA was extracted from the C8-BR group and the C8-TA group with TRIzol (Sigma-Aldrich, USA).The isolated RNA was converted to cDNA using Takara PrimerScript RT reagent Kit (Takara, Japan) according to the instructions.BAG1 primers were as follows: Forward primer: 5'-TCGCCTCTCAACAGCTAGAC-3'; Reverse primer: 5'-CCCTGAATCCTCGACCTTGAT-3'; The PCR reaction conditions were 94˚C for 5 min, 94˚C for 40 s, 56˚C for 40 s, and 72˚C for 1 min.PCR products were analyzed by 1% agarose gel electrophoresis.

Protein extraction and trypsin digestion
All of the samples were sonicated three times on ice using a high intensity ultrasonic processor (Scientz, China) in lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail).Centrifuged at 12,000 g at 4˚C for 10 min to remove remaining debris.BCA kit was used to determine the protein concentration according to the manufacturer's instructions.For digestion, the protein solution of each sample was reduced with 5 mM dithiothreitol for 30 min at 56˚C and then alkylated with 11 mM iodoacetamide in the dark for 15 min at room temperature.100 mM TEAB was added to dilute the sample.Finally, trypsin was added for the first digestion overnight (trypsin-to-protein mass ratio was 1:50) and performed a subsequent 4 h-digestion (trypsin-to-protein mass ratio was 1:100).

LC-MS/MS analysis
The tryptic peptides were dissolved in 0.1% formic acid (solvent A), and directly loaded onto a home-made reversed-phase analytical column (15-cm length, 75 μm i.d.).The gradient was comprised of an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system.The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive Plus (Thermo, USA) coupled online to the UPLC.The electrospray voltage applied was 2.0 kV.The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000.Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500.A datadependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion.Automatic gain control (AGC) was set at 5E4.

Database search
The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8).Tandem mass spectra were searched against Mus musculus data in the Uniprot database concatenated with reverse decoy database.Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages.Mass tolerance for precursor ions was set as 20 ppm in First search and 5 ppm in Main search, and the mass tolerance for fragment ions was set as 0.02 Da.Carbamidomethyl on Cys was specified as fixed modification, oxidation on Met was specified as variable modifications.Label-free quantification method was LFQ, FDR was adjusted to < 1% and the minimum score for peptides was set > 40.

Bioinformatic analysis
Wolfpsort (http://www.genscript.com/psort/wolf_psort.html)was used to predicate subcellular localization of differentially expressed proteins.Eukaryotic orthologous group (http:// genome.jgi.doe.gov/help/kogbrowser.jsf) was performed on all differentially expressed proteins for further functional classification by aligning their sequences with the KOG database.The UniProt-GOA database (http://www.ebi.ac.uk/GOA/) together with InterProScan soft (http://www.ebi.ac.uk/InterProScan/) were used to analyze biological process, cellular component and molecular function of differentially expressed proteins.The Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg) was used to analyze signaling pathways involved in differentially expressed proteins.A two-tailed Fisher's exact test was employed to test the enrichment of the differentially expressed protein against all identified proteins.A corrected p-value < 0.05 was considered significant in database analysis.
Cluster membership was visualized by a heat map using the "heatmap.2"function from the "gplots" R-package.

Parallel Reaction Monitoring (PRM) Validation
To verify the accuracy of label-free proteome quantification analysis, we selected 18 differentially expressed proteins for PRM assay.The methods of protein extraction and trypsin digestion were as described above.In LC-MS/MS Analysis, the gradient was comprised of an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 38 min, 23% to 35% in 14 min and climbing to 80% in 4 min then holding at 80% for the last 4 min, all at a constant flow rate of 700 nL/min on an EASY-nLC 1000 UPLC system.Automatic gain control (AGC) was set at 3E6 for full MS and 1E5 for MS/MS.The maximum IT was set at 20 ms for full MS and auto for MS/MS.The isolation window for MS/MS was set at 2.0 m/z.Peptide parameters were as follows: enzyme was set as Trypsin [KR/P], Max missed cleavage set as 2. The peptide length was set as 8-25.precursor charges were set as 2, 3, ion charges were set as 1, 2, ion types were set as b, y, p.The product ions were set as from ion 3 to last ion, the ion match tolerance was set as 0.02 Da.

Identification of differentially expressed proteins between C8-TA, C8-BR and C8 groups
We used T. gondii tachyzoites (RH strain) infected C8-D1A cells.After 24 h, the pseudocysts were observed in the cells (S1 Fig) .Next, we performed Alkaline-induced transformation of tachyzoites in vitro.After 96 h, the bradyzoite-specific gene BAG1 was detectable (S2 Fig) .These results indicated that we have successfully constructed in vitro models of tachyzoite and bradyzoite infection in C8-D1A astrocytes.We used label-free proteome to quantitatively analyze the host proteins and in tachyzoite and tachyzoite to bradyzoite transformation stages of T. gondii infection.Among the 9 samples, a total of 32906 peptides and 30808 unique peptides were identified.We identified 4676 proteins as host protein, of which 3415 proteins were quantified (S1 Table ).We defined fold change � 1.5 or � 0.67 and p-value � 0.05 as the criteria to analyze the differentially expressed proteins of C8-TA, C8-BR and C8 group (Fig 1A).There were a total of 163 differentially expressed proteins, of which 109 were up-regulated and 54 were down-regulated in C8-TA group compared with C8 group (S2A

Tachyzoite infection altered multiple immunoregulatory processes in astrocytes
To investigate the biological functions of the differentially expressed proteins between the C8-TA group and the C8 group, we firstly analyzed the subcellular localization of the differentially expressed proteins.As shown in Fig 2A , 38.27% differentially expressed proteins were located in cytoplasma, 14.81% differentially expressed proteins were located in plasma membrane, and 17.28% differentially expressed proteins were located in nucleus in C8-TA vs C8    immune-related biological processes, such as defense response to other organism, defense response to virus, response to interferon-beta, innate immune response, detection of virus, which indicated that the differentially expressed proteins were primarily involved in immune regulations.
Next, we comprehensively analyzed the biological functions of up-regulated and down-regulated proteins in C8-TA vs C8 group.The GO-BP results showed that, the up-regulated proteins in C8-TA vs C8 group were mainly involved in the following biological processes: cellular hypotonic response, response to light intensity, learning, ribosomal small subunit biogenesis, negative regulation of transporter activity (S4B Table and Fig 3A).The down-regulated proteins were mainly related to immune responses, such as defense response to other organisms, defense response to virus, response to interferon-beta, response to virus, and innate immune response (S4C Table and Fig 3B).KEGG enrichment analysis of up-regulated and down-regulated proteins revealed the signaling pathways during the infection of T. gondii tachyzoites (S5A and S5B Table ).As shown in Fig 3C and 3D, the up-regulated proteins were mapped to 18 signaling pathways, and the down-regulated proteins were mapped to 10 signaling pathways in C8-TA vs C8 group.As expected, the down-regulated proteins were enriched in inflammatory signaling pathways, such as Herpes simplex virus 1 infection, RIG-I-like receptor signaling pathway, Amoebiasis, Systemic lupus erythematosus.The above findings indicated that the host immune response was activated during T. gondii invasion.Interestingly, our results showed that the down-regulated proteins may play a major role in immune regulation.This was different from the biological traits of T. gondii invaded epithelial cells, which indicated that the immune response of the host was gradually weakened when tachyzoites invaded astrocytes for 24 h.This might be a protective measurement for T. gondii to avoid host astrocytes generating strong neuro-immune response.

The conversion of the tachyzoites into bradyzoites upregulated glucose metabolism of astrocytes
Tachyzoite to bradyzoite differentiation is a key aspect of T. gondii biology and pathogenesis.To better understand the mechanism of interaction between T. gondii and the host during differentiation of T. gondii, we performed a comprehensive bioinformatics analysis in C8-BR vs C8-TA group.First, we analyzed the sublocalization of all differentially expressed proteins by WoLF PSORT software (Fig 4A).The results showed that 33% of the differentially expressed proteins were localized in cytoplasm, 26% in nucleus, and 17% in mitochondria.In up-regulated proteins, most of them were located in the cytoplasm (36%), followed by nucleus (21.41%) and mitochondria (20.61%) (Fig 4B).In down-regulated proteins, most of them were located in the nucleus (37%), followed by cytoplasm (26%) and plasma membrane (11%) (Fig 4C ).
We used KOG database to predict the function and classification of up and down-regulated proteins (S6A and 6B Table and Fig 4D and 4E).The results showed that the functions of upregulated proteins were consistent with down-regulated proteins, and the differentially expressed proteins were mainly enriched in the following categories:

[O] Posttranslational modification, protein turnover, chaperones, [T] Signal transduction mechanisms, [C] Energy production and conversion, [U] Intracellular trafficking, secretion, and vesicular transport,
[A] RNA processing and modification pathway, suggested that during the chronic infection stage, epigenetic mechanisms may mediate the effects on host nutrient metabolism in the coexistence of host and bradyzoites.GO enrichment analysis showed that the differentially expressed proteins were mainly involved in metabolism-related biological processes including dicarboxylic acid metabolic process, oxidoreduction coenzyme metabolic process, hexose biosynthetic process, monocarboxylic acid metabolic process, tricarboxylic acid metabolic process (S7A Table and S3A Fig).Among them, glucose metabolism related biological processes were up-regulated, such as dicarboxylic acid metabolic process, oxidoreduction coenzyme metabolic process, carboxylic acid metabolic process, tricarboxylic acid metabolic process, nucleoside phosphate metabolic process (S7B Table and Fig 5A).However, there were no significant biological processes enrichment for down-regulated proteins.The top five categories of downregulated proteins were protein localization to endoplasmic reticulum, ribosomal large subunit biogenesis, peptide biosynthetic process, DNA conformation change, peptide metabolic process (S7C Table and Fig 5B).
KEGG pathway enrichment was used to identify pathways of differentially expressed proteins (S8A Table and S3B Fig), the results showed that differentially expressed proteins significantly enriched in metabolism-related pathways, the top five were Glycolysis/ Gluconeogenesis, Citrate cycle (TCA cycle), Phenylalanine metabolism, Phagosome, Pyruvate metabolism.We further investigated the function of up and down-regulated proteins and found that up-regulated proteins were mainly involved in metabolism-related signaling pathways including Citrate cycle (TCA cycle), Glycolysis/Gluconeogenesis, Proteasome, Phagosome, Pyruvate metabolism, which was consistent with the GO-BP (S8B Table and Fig 5C).The part of down-regulated proteins involved in amino acid metabolism, such as Tyrosine metabolism, Phenylalanine metabolism (S8C Table and Fig 5D).During the transformation from tachyzoite infection stage to bradyzoite infection stage, the function of host proteins may have undergone a gradual transition from immune emergency mode to up-regulation metabolic pathways.The effects on host metabolism during transformation suggested that the increased host's metabolism could accelerate the decomposition of glucose, so the metabolism of T. gondii was decreased, which was more conducive to its long-lived in their hosts.

Verification of differentially expressed proteins
To evaluate the accuracy of label-free proteome quantification techniques, a total of 18 differentially expressed proteins were selected for PRM analysis.Based on GO and KEGG annotations, we chose Bystin (BYSL), Fibronectin (FN1), Signal transducer and activator of   free proteome quantification.Next, we used western blotting to verify the expression of DDX58, ECHS1, DLD, SIRT2, GOT1(Fig 7).The results were also consistent with label-free proteome quantification.The above results suggested that label-free proteome quantification outcomes were relatively reproducible and reliable.

Discussion
The invasion of T. gondii tachyzoites and bradyzoites in the host causes acute infection and chronic infection respectively, resulting in clinical manifestations of toxoplasma encephalopathy, chorioretinitis, miscarriage, stillbirth and schizophrenia [18,[29][30][31].Since there is no effective method to eliminate tissue cysts up to now, the damage of host CNS caused by T. gondii accompanied with mental and behavioral disorders has been brought into focus.Therefore, understanding the proteomics changes in host neurogliocytes after tachyzoites/bradyzoites infection would identify the negative effects of different stages on the host, and provide targets for developing new vaccines and drugs to treat brain damages caused by T. gondii.
In the present study, our results showed that the differentially expressed proteins in the acute infection stage were mainly enriched in immune-related biological processes, while the differentially expressed proteins in the chronic infection stage were mainly enriched in metabolic-related biological processes.Therefore our results suggested that T. gondii would regulate host neurogliocytes by distinct modes in each of the two stages.Similarly, previous research on tachyzoite infection stage indicated that the up-regulated proteins were also involved in immune inflammation-related pathways to prevent acute infection [32], while those similar studies were mainly based on epithelial cells such as HFF cells.
As we know, invasion of T. gondii can cause inflammation in the host, and astrocytes have been proven to be a pivotal regulator of CNS inflammatory responses [33].The borders and scars of astrocytes could serve as functional barriers that restrict the entry of inflammatory cells into CNS parenchyma in health and disease, simultaneously, it also has powerful proinflammatory potential.Therefore, we have established acute/chronic infection models with mouse astrocytes.Interestedly, although our differentially expressed proteins results in tachyzoites infection models were also involved in immune inflammation-related pathways, changes in protein expression were very different to the results of previous studies in epithelial cells.
In our study, the down-regulation proteins rather than up-regulation proteins were involved in immune regulation-related processes, especially in defense response pathways and innate immune response pathways.Consistently, Cekanaviciute reported that after T. gondii infection in the host CNS, the immune function must be restricted to prevent excessive neuronal damage, in order to keep a balance among T. gondii, brain and the immune system [34].As a result, we have focused on four down-regulated and immune-related proteins including STAT1, GBP4, IFIT3 and DDX58 after tachyzoite infection.STAT1 is an important immune inflammatory factor in CNS and is involved in the immune regulation of various cells.Studies have shown that inhibition of STAT1 can promote bradyzoite formation [35].In this study, the expression of STAT1 was significantly decreased, suggesting that after tachyzoite infection in astrocytes, the down-regulated STAT1 might participate in promoting the transformation of tachyzoites to bradyzoites, as well as inhibiting the persistent infection of tachyzoites.GBP4 and IFIT3 are IFN-γ-inducing proteins.Hu et al found that GBP4 could negatively regulate virus-induced type I IFN and antiviral responses by interacting with IFN regulatory factor (IRF) 7 during viral infection, thus the following researches on their roles in astrocytes during T. gondii infection may reveal a new mechanism by which astrocytes against parasites [36].DDX58, also known as RIG-I, can function as an innate antiviral immune response receptor and play an important role in antiviral innate immunity [37][38][39].In vesicular stomatitis virus (VSV)-infected astrocytes, RIG-I knockdown significantly reduced inflammatory cytokine production [40], so the down regulation of DDX58 may be also involved in the transformation of tachyzoites to bradyzoites in host CNS.
The conversation from tachyzoites to bradyzoites of T. gondii is a key to establish chronic infection and an important link in the pathogenesis of T. gondii.We comprehensively analyzed the differentially expressed proteins between the bradyzoites infection group and the tachyzoite infection group.The results showed that the differentially expressed proteins were mainly involved in metabolism-related biological processes and signaling pathways, and the detailed GO and KEGG analyses indicated that the host's glucose metabolism and a part of the amino acid metabolism process have been changed significantly during chronic infection.Further, we found that the up-regulated proteins were mainly involved in glucose metabolism and entered the TCA process, especially for ECHS1.
ECHS1 is a key enzyme involved in the metabolism of fatty acyl-CoA esters [41].In fatty acid β-oxidation, it could increase the synthesis of acetyl-CoA and promote the TCA cycle, thereby increasing the process of glucose metabolism.It has been reported that ECHS1 deficiency (ECHS1D) led to the impaired ATP production and metabolic acidosis in patients [42].As an obligate intracellular parasite, T. gondii obtains all nutrients from host cells to support its intracellular growth and proliferation.Glucose and glutamine are raw materials for tachyzoites to complete the classic TCA and then synthesize ATP [43,44].Therefore the increase of host glucose metabolism would accelerate the consumption of glucose, resulting in the decreasing uptake of glucose by T. gondii.In the present study, the up-regulated ECHS1 may be an important host receptor of invading parasites, and associated with the inhibition of parasites self-glucose metabolism, thereby promoting the formation of intracellular bradyzoites and establish long-term latent infection in the host.
Neuronal degeneration caused by chronic infection of T. gondii is an important pathogenesis of neurodegenerative diseases, but the mechanism has not been fully elucidated.Our results also found important clues and potential targets for this process, such as DLD, SIRT2 and GOT1.
DLD, also known as dihydrolipoyl dehydrogenase, is a mitochondrial enzyme that is essential for eukaryotic cell metabolism [45].Ahmad reported that DLD was related to Alzheimer's disease (AD), and inhibition of DLD expression would lead to a significant recovery of Aβ pathological degradation.This could be explained that the inhibition of DLD would down-regulate metabolism-related signaling pathways and reduce the host's energy metabolism, which would be beneficial to alleviate the symptoms of AD [46].In this study, we found that DLD was significantly up-regulated in the bradyzoites infection group, which may provide a new insight for explaining the mechanism of T. gondii infection-induced Alzheimer's disease.In addition, DLD is also associated with severe diseases in infants, causing developmental delay, hypotonia and metabolic acidosis [47].Recent studies have shown that Leishmania's self-encoded DLD induced a protective cellular immune response in L. major-infected mice, which could serve as a design site for Duchenne vaccines for kala-azar prevention [25,48].
SIRT2 is an NAD+-dependent deacetylase that is widely involved in cell division, angiogenesis [49][50][51], energy metabolism [52] and neurodegenerative [53,54], cardiovascular disease [55,56], oxidative stress [57] and many cancers [58,59], etc.Studies have found that SIRT2 was the most abundant sirtuin expressed in mammalian CNS, especially in cortex, striatum, hippocampus, and spinal cord, suggesting that it might have a role in CNS [60].Dopaminergic (DA) neurons plays a vital role in CNS, and DA neuron hyperfunction was involved in several neurological disorders including schizophrenia and Parkinson's Disease.SIRT2 expression was dramatically increased during the differentiation of human embryonic stem cells (hESCs) into midbrain DA neurons [61].In addition, SIRT2 knockout (SIRT2−/−) mice displayed fewer DA neurons and less dense striatal fibers in the substantia nigra [62].SIRT2 inhibition also improves cognitive impairment in different AD animal models and promotes neuronal survival [63,64].This suggested that SIRT2 can be a potential therapeutic target for neurodegenerative disease.Moreover, SIRT2 is also involved in ATP synthesis.Recent studies have shown that SIRT2 localized to the inner mitochondrial membrane of the mouse brain, and mice lacking SIRT2 showed decreased ATP levels in the striatum [65].In our study, the expression of SIRT2 was up-regulated after chronic infection, which is consistent with Mcconkey's report, they found that dopaminergic cells and brain tissues encysted with cerebral cysts have increased levels of dopamine synthesis and release [66].Moreover, treatment of rats and mice with dopamine receptor antagonists could inhibit the behavioral changes induced by T. gondii infection [67,68].In addition, some parasites such as Leishmania, T. brucei, and Schistosoma could encode SIRT2 homologous protein, and more importantly, it was essential for parasite growth [69][70][71].Thus SIRT2 might serve as a potential treatment target for psychiatric disorders induced by manipulative parasites.
Neuroendocrine programs and neurotransmitter imbalance may act as the physiological basis for T. gondii induced psychiatric and behavioral disorders [72].Glutamate (GLU) is the most abundant neurotransmitter in the brain, and its excitability plays a crucial role in brain structure and function.GOT1 aspartate aminotransferase is a type of aminotransferase that catalyzes the transamination of aspartate and α-ketoglutarate to form glutamate and oxaloacetate.Glutamate dehydrogenase (GLUD1) is a mitochondrial enzyme that catalyzes the reductive fixation of ammonia to α-ketoglutarate to form glutamate.Both GOT1 and GLUD1 expression were found to be up-regulated in our study, suggesting that the glutamate synthesis might be increased in the chronic infection group.Our finding is also consistent with a recent study, which showed that chronic infection with T. gondii could cause an increase in extracellular glutamate and a two-fold decrease in glutamate transporter expression in glial cells [73].As hyperexcitability of GLU is neurotoxic and leading to brain damage, neurological disorders (eg, ALS, multiple sclerosis, AD, Huntington's disease, Parkinson's disease) and psychiatric disorders (eg, schizophrenia, depression, bipolar disorder).Our finding provides new evidence to explain the host mental behavioral disorders induced by chronic T. gondii infection.
In conclusion, our results systematically analyzed the proteomic changes in astrocytes infected with not only tachyzoites, but also bradyzoites, respectively.We surprisingly discovered that T. gondii tachyzoites can cause down-regulation of immune-related pathways in astrocytes, and the inhibited expression of DDX58 might be associated with the transformation of tachyzoites to bradyzoites.However, during the bradyzoites infection stage, metabolism rather than immune pathways of the host was changed significantly.Both the glucose metabolism pathways and the expression of some metabolism-related enzymes in astrocytes were significantly up-regulated, such as ECHS1, DLD, SIRT2, and GOT1.Since their expression was closely related to chronic degenerative diseases and psychiatric diseases, which could provide a new explanation for host mental and behavioral disorders induced by chronic infection of T. gondii.

Fig 1 .
Fig 1. Proteome analysis of differentially expressed proteins in tachyzoite and tachyzoite to bradyzoite transformation stages of T. gondii infection.(A) Heat map shows the differentially expressed proteins in the three groups.(B) Volcano plot shows differentially expressed proteins between the C8-TA group and the C8 group.(C) Volcano plot shows differentially expressed proteins between the C8-BR group and the C8-TA group.https://doi.org/10.1371/journal.pntd.0011102.g001 Fig 2B).The results showed that the top five KOG classifications were [U] Intracellular trafficking, secretion, and vesicular transport, [J] Translation, ribosomal structure and biogenesis, [O] Posttranslational modification, protein turnover, chaperones, [Z] Cytoskeleton, [A] RNA processing and modification.GO enrichment analysis was performed for the functional annotation of differentially expressed proteins including three categories: biological process (GO-BP), cellular compartment (GO-CC) and molecular function (GO-MF) (S4A Table and Fig 2C).We focused on the biological processes and found that the differentially expressed proteins mainly enriched in

Fig 2 .
Fig 2. Location and functional classification of differentially expressed proteins between the C8-TA group and the C8 group.(A) Location of subcellular structures of differentially expressed proteins between the C8-TA group and the C8 group.(B) KOG classification of differentially expressed proteins between the C8-TA group and the C8 group.(C) GO enrichment analysis of differentially expressed proteins between C8-TA vs C8 group included biological process, cellular composition, and biological function.https://doi.org/10.1371/journal.pntd.0011102.g002

Fig 3 .
Fig 3. GO and KEGG pathway enrichment analysis of up-regulated and down-regulated proteins between C8-TA group and C8 group.(A) GO enrichment analysis of up-regulated proteins.(B) GO enrichment analysis of down-regulated proteins.(C) KEGG pathway enrichment analysis of up-regulated proteins.(D) KEGG pathway enrichment analysis of down-regulated proteins.https://doi.org/10.1371/journal.pntd.0011102.g003

Fig 4 .
Fig 4. Subcellular localization and functional classification of differentially expressed proteins between C8-BR group and C8-TA group.(A) The location of subcellular structures of all differentially expressed proteins.(B) The location of subcellular structures of up-regulated proteins.(C) The location of subcellular structures of down-regulated proteins.(D) The KOG classification of up-regulated proteins.(E) The KOG classification of downregulated proteins.https://doi.org/10.1371/journal.pntd.0011102.g004 Fig 6).The results showed that the changing trends of 18 differentially expressed proteins in PRM were consistent with label-

Fig 5 .
Fig 5. GO and KEGG pathway enrichment analysis of up-regulated and down-regulated differentially expressed proteins between C8-BR group and C8-TA group.(A) GO enrichment analysis of up-regulated proteins.(B) GO enrichment analysis of down-regulated proteins.(C) KEGG pathway enrichment analysis of up-regulated proteins.(D) KEGG pathway enrichment analysis of down-regulated proteins.https://doi.org/10.1371/journal.pntd.0011102.g005

Fig 7 .
Fig 7. Verification of the differentially expressed proteins in different stages (tachyzoite and bradyzoite) of T. gondii infection.(A) Western blotting analysis was used to detect the protein expression level of differentially expressed proteins in C8 group and C8-TA group.(B) Histogram analysis of the western blotting results in C8 group and C8-TA group.Statistical significance was analyzed using an unpaired Student's t-test (***p < 0.001).(C) Western blotting analysis was used to detect the protein expression level of differentially expressed proteins in C8-TA group and C8-BR group.(D) Histogram analysis of the western blotting results in C8-TA group and C8-BR group.Statistical significance was analyzed using an unpaired Student's t-test (***p < 0.001).https://doi.org/10.1371/journal.pntd.0011102.g007 Table and Fig 1B).In addition, there were 719 differentially expressed proteins, of which 495 host proteins were up-regulated and 224 host proteins were down-regulated in C8-BR group compared with C8-TA group (S2B Table and Fig 1C).