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Global profiling of protein lysine malonylation in Toxoplasma gondii strains of different virulence and genetic backgrounds

['Lan-Bi Nie', 'State Key Laboratory Of Veterinary Etiological Biology', 'Key Laboratory Of Veterinary Parasitology Of Gansu Province', 'Lanzhou Veterinary Research Institute', 'Chinese Academy Of Agricultural Sciences', 'Lanzhou', 'People S Republic Of China', 'College Of Animal Science', 'Technology', 'Jilin Agricultural University']

Date: 2022-05

Abstract Lysine malonylation is a post-translational modification (PTM), which regulates many cellular processes. Limited information is available about the level of lysine malonylation variations between Toxoplasma gondii strains of distinct genetic lineages. Yet, insights in such variations are needed to understand the extent to which lysine malonylation contributes to the differences in the virulence and repertoire of virulence factors between T. gondii genotypes. In this study, we profiled lysine malonylation in T. gondii using quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immuno-affinity purification. This analysis was performed on three T. gondii strains with distinctive pathogenicity in mice, including RH strain (type I), PRU strain (type II), and VEG strain (type III). In total, 111 differentially malonylated proteins and 152 sites were upregulated, and 17 proteins and 17 sites were downregulated in RH strain versus PRU strain; 50 proteins and 59 sites were upregulated, 50 proteins and 53 sites were downregulated in RH strain versus VEG strain; and 72 proteins and 90 sites were upregulated, and 7 proteins and 8 sites were downregulated in VEG strain versus PRU strain. Differentially malonylated proteins were involved in key processes, such as those mediating the regulation of protein metabolism, stress response, glycolysis, and actin cytoskeleton. These results reveal an association between lysine malonylation and intra-species virulence differences in T. gondii and offer a new resource for elucidating the contribution of lysine malonylation to energy metabolism and virulence in T. gondii.

Author summary Lysine malonylation has been shown to play important roles in various biological processes in Toxoplasma gondii. Here, we used quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immuno-affinity purification to test the hypothesis that lysine malonylation underpins the inter-genotype differences in the virulence of T. gondii. Several up-regulated and down- regulated malonylated proteins were identified in the tachyzoites of RH (type I) strain, PRU (type II) strain, and VEG (type III) strain. Differentially regulated malonylated proteins were enriched in many biological and metabolic pathways, and were found to contribute T. gondii energy metabolism, stress response, and infectivity, suggesting the role of lysine malonylation in the regulation of T. gondii virulence. These findings expand our knowledge of lysine malonylation in T. gondii and provide more insight into the mechanisms mediating the virulence differences between T. gondii strains of different genotypes.

Citation: Nie L-B, Liang Q-L, Wang M, Du R, Zhang M-Y, Elsheikha HM, et al. (2022) Global profiling of protein lysine malonylation in Toxoplasma gondii strains of different virulence and genetic backgrounds. PLoS Negl Trop Dis 16(5): e0010431. https://doi.org/10.1371/journal.pntd.0010431 Editor: Javier Sotillo, Instituto de Salud Carlos III, SPAIN Received: March 5, 2022; Accepted: April 18, 2022; Published: May 16, 2022 Copyright: © 2022 Nie et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The datasets supporting the findings of this article are included within the article. The mass spectrometry data has been submitted to the ProteomeXchange Consortium with the identifier PXD029366. Funding: This work was supported by the National Key Research and Development Program of China (Grant Nos. 2021YFC2300800 and 2021YFC2300802) to XQZ, the Fund for Shanxi “1331 Project” (Grant No. 20211331-13) to XQZ, the Yunnan Expert Workstation (Grant No. 202005AF150041) to XQZ and the Agricultural Science and Technology Innovation Program (ASTIP) (Grant No. CAAS-ASTIP-2016-LVRI-03) to XQZ. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction The obligate intracellular parasite Toxoplasma gondii infects a wide range of warm-blooded animals and is highly prevalent in humans [1,2]. This parasite imposes a significant risk to patients with a compromised immune system and to pregnant woman [3–5]. T. gondii strains are grouped into three genetically distinct lineages, known as types I, II, and III, which vary significantly in virulence in mice [6,7]. Besides these three established genotypes, atypical T. gondii genotypes have been reported, such as Chinese I (ToxoDB 9) in China [8–10], Africa 1 and Africa 3 in Africa, and type 12 in North American wild animals [11]. The ability of T. gondii to establish an infection relates to the expression of a wide range of virulence factors. These effector proteins play important roles in promoting the parasite invasion and colonization of host cells, and evasion of innate and adaptive immune responses. Early transcriptomic and proteomics studies have shown that virulence factors produced by T. gondii vary between different clonal lineages [12,13], possibly attributed to the plasticity in proteins required for parasite invasion [14]. Lysine malonylation is a type of protein post-translational modification (PTM) reported in eukaryotes and bacteria [15,16]. The regulatory role of lysine malonylation in many biological processes has been established in various organisms [17–19]. Lysine malonylation affects energy metabolism, mitochondrial function, and fatty acid synthesis [20]. However, this type of PTM remains poorly understood in T. gondii, with only one study having investigated its expression pattern in one T. gondii strain [21]. It is unclear what regulatory mechanism lysine malonylation mediates in T. gondii and whether malonylation contributes to the genotype-related differences in the virulence of T. gondii. Recent studies have suggested a role for lysine acetylation in the virulence T. gondii strains of different genotypes [22,23]. Therefore, it is reasonable to hypothesize that lysine malonylation contributes to the proteomic differences between T. gondii strains of different genotypes, especially in proteins related to virulence and pathogenicity. Here, we used liquid chromatography-tandem mass spectrometry with immuno-affinity purification to investigate the differences in lysine malonylation between T. gondii strains of different genotypes, including RH strain (type I), PRU strain (type II), and VEG strain (type III). Our data provide new insight into the role of lysine malonylation in the genotypic differences in T. gondii virulence.

Materials and methods Cell and parasite culture Tachyzoites of three T. gondii strains, RH strain (type I), PRU strain (type II) and VEG strain (type III), were used in this study. All strains were maintained by serial passage in human foreskin fibroblast (HFF) cells originally obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). HFFs were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FBS, Gibco, USA), 100 U/ml antibiotics (penicillin-streptomycin solution). The infected cell cultures were incubated at 37°C with 5% CO 2 . Tachyzoites were separated from the feeder host cells by passed through 25-gauge syringe needles. A 3 μm membrane filter (Millipore) was used to remove the cell debris, and the tachyzoites were washed with phosphate-buffered saline (PBS) and centrifuged at 2,000 × g twice. Purified tachyzoite pellets were stored at –80°C until use. Protein extraction To extract total protein from RH, PRU and VEG strain, ~ 3 × 109 tachyzoites were transferred from –80°C freezer and thawed at room temperature. Lysis buffer (1% dodecyl sulfate, sodium salt [SDS], 1% protease inhibitor cocktail, 5 mM dithiothreitol [DTT], 3 μM trichostatin A [TSA] and 50 mM nicotinamide [NAM]) was added to tachyzoites and the crude lysate was sonicated three times on ice (220 W, sonicated 3 seconds, stop for 5 seconds, and repeat three times). The samples were centrifuged at 2,000 ×g for 10 min at 4°C to remove the cell debris. The clear supernatant was collected and transferred to a new centrifuge tube and stored at –80°C. Bradford protein assay (Bio-Rad Laboratories, Hercules, CA) was used to determine the protein concentration in the supernatant. Trypsin digestion An equal quantity of 200 μg of protein from each sample was prepared. Each biological replicate was analyzed in three technical triplicates. Trichloroacetic acid was slowly added to each sample to a final concentration of 20%, and the sample was mixed by vortexing and precipitated at 4°C for 2 h. The protein pellet was obtained by centrifugation at 4, 500 ×g for 5 min, the supernatant was discarded, and the precipitate was washed with chilled acetone for 2–3 times. After the protein pellets were air-dried, 200 mM triethylammonium bicarbonate (TEAB) was added to each sample to resuspend the protein pellet by ultrasound sonication, and then the trypsin was added at a ratio of 1:50 (protease: protein, M/M) overnight. DTT was added to a final concentration of 5 mM. Protein reduction was performed at 56°C for 30 min followed by alkylation by adding iodoacetamide (IAA) at a final concentration of 11 mM and incubation for 15 min in the dark at room temperature. Modification enrichment After trypsin digestion, resulting peptides were dissolved in IP buffer solution (100 mM NaCl, 1 mm EDTA, 50 mM Tris HCl, 0.5% NP-40, pH 8.0). The supernatant was transferred to a pre-washed pan anti-malonyllysine antibody resin (No. PTM-904, PTM Bio, Hangzhou). The peptide solution and antibody bead mixture were placed overnight on a shaker at 4°C. After incubation, the resin was washed with IP buffer solution four times and twice with deionized water. Finally, the resin bound peptides were eluted with 0.1% trifluoroacetic acid for three times. The eluent was collected and vacuum dried. After drying, peptides were desalted using C18 ziptips, and the clean peptides were vacuum dried for LC-MS/MS analysis. LC-MS/MS analysis Enriched peptides were dissolved in liquid chromatography mobile phase A and separated by ultra-performance liquid chromatography (UPLC). The mobile phase A was aqueous solution containing 0.1% formic acid and 2% acetonitrile, the mobile phase B was aqueous solution containing 0.1% formic acid and 100% acetonitrile. The gradient involved 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 and then holding at 80% for the last 3 min. The peptides were separated by UPLC system, then ionized by capillary ion source and analyzed by tims-TOF Pro mass spectrometer. The ion source voltage was set at 2.0 kV, peptide parent ion and its secondary fragments were detected and analyzed by high-resolution TOF mass analyzer. The scanning range of secondary mass spectrometry was set at 100–1700. The data acquisition mode was PASEF. After a first-order mass spectrometer collected, 10 times of the PASEF mode was used to collect the second-order spectrum with the charge number of parent ions in the range of 0–5. The dynamic exclusion time of tandem mass spectrometry scanning was set to 30 seconds to avoid repeated scanning of parent ions. Database search Raw mass spectrometry data was searched against T. gondii database ToxoDB 48 (8,322 sequences) using MaxQuant (1.6.15.0) software. A reverse library was added to calculate the false discovery rate (FDR) caused by random matching, and common contamination library was added to the database to eliminate contaminated protein in the identification results. Enzyme digestion method was set to trypsin/P, number of missing cut sites of 4, and minimum length of peptide segment of 7 amino acid residues. Maximum modification number of peptide segment was 5. Mass error was set at 0 ppm and 20 ppm for the primary parent ion of search and main search, and 20.0 ppm for the secondary fragment ion. Peptide quantification was performed using label free quantification (LFQ) model in MaxQuant, FDR of protein identification and PSM identification was set at 1%. Bioinformatics analysis Gene Ontology (GO) annotation of the proteome was based on the UniProt-GOA (http://www.ebi.ac.uk/GOA/) and ToxoDB 48 database. Briefly, protein ID was converted to UniProt ID and UniProt ID was matched to GO ID. Then, the corresponding information was extracted from UniProt-GOA database based on GO ID. In the case of absence of protein information in UniProt GOA database, InterProScan was used to predict the GO function of the protein using an algorithm based on protein sequence. The identified proteins were classified according to cell composition, molecular function, and physiological process. InterProScan based on protein sequence and the corresponding InterPro (http://www.ebi.ac.uk/interpro/) were used to annotate the protein domain. Online service tool KEGG Automatic Annotation Server (KAAS) of Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to annotate the submitted proteins, and then KEGG mapper was used to match the annotated proteins to the corresponding pathways in the database. The wolfpsort (https://wolfpsort.hgc.jp/) was used to annotate the subcellular localization of the eukaryotic proteins. Fisher’s exact test was used to detect differentially expressed malonylated proteins of GO and KEGG annotation. P-value < 0.05 was considered significant. The differential expression > 1.5 and < 1/1.5 were considered the threshold of significantly upregulated and downregulated proteins, respectively. InterPro (http://www.ebi.ac.uk/interpro/) was used to analyze the enrichment of the functional domains of differentially expressed proteins. The selected P-value matrix was transformed by − log10, the hierarchical clustering (Euclidean distance, average connection clustering) method was used for one-sided clustering analysis. The clustering relationship was visualized by heat map constructed using the function Heatmap. 2 in R language package gplots. MoMo (http://meme-suite.org/tools/momo) was used to analyze the motif characteristics of the modification sites. When the number of peptides in a specific sequence is more than 20 and P-value < 0.000001, the specific sequence was considered a motif of a modified peptide. Differentially expressed malonylated proteins screened from different comparison groups were mapped into protein-protein interaction (PPI) network database of STRING (v.10.5) (http://string-db.org/), the protein interaction relationship was extracted according to a confidence score > 0.7. R package "network D3" was used to visualize the PPI network.

Discussion We investigated a possible basis for lysine malonylation in the differences in the virulence between T. gondii strains of different genetic backgrounds. We examined three genetically distinct strains of T. gondii that differ in their virulence for mice: RH virulent strain (type I), PRU less virulent strain (type II), and VEG strain (type III), which is avirulent. By comparing the virulent RH strain with the less virulent PRU strain, we identified 17 down-regulated malonylated proteins, such as calcium-dependent protein kinase 1 (CDPK1) and ribosomal-ubiquitin protein (RPL40). The CDPK1 belongs to the serine/threonine kinase family which plays roles in the motility, organelle secretion, cell invasion, and egress of T. gondii [24]. Chemical inhibition of CDPK1 reduces T. gondii growth in vitro, reduces parasite dissemination to the central nervous system in mice, and inhibits reactivation of latent infection in immunocompromised mice [25]. RPL40 is an essential virulence factor and may play a role in the pathogenesis of acute T. gondii infection [26]. Compared with the less-virulent PRU strain and the avirulent VEG strains, several malonylated proteins were down-regulated in the virulent RH strain. These included acetyltransferase, glyceraldehyde-3-phosphate dehydrogenase 2 (GAPDH2), arginyl-tRNA synthetase family protein, 3-ketoacyl-(acyl-carrier-protein) reductase, ATPase (DUF699) protein, cytochrome C, putative, ribosomal-ubiquitin protein, RNA recognition motif-containing protein, GNAT family protein, matrix antigen 1MAG1, and CDPK1 (Fig 10). Lysine acetyltransferase is involved in stage-specific gene expression and plays a role in T. gondii response to high pH (alkaline) [23]. Compared to the wild-type strains, RH strain lacking histone acetyltransferase is less sensitive to alkaline pH and exhibits low expression of stress-related genes [27]. PPT PowerPoint slide

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TIFF original image Download: Fig 10. Venn diagram showing the unique and common differentially malonylated proteins in RH vs. PRU, RH vs. VEG and VEG vs. PRU. https://doi.org/10.1371/journal.pntd.0010431.g010 The glycolytic enzyme GAPDH has two isozymes, with diverse activities and different subcellular localization in T. gondii. GAPDH2, located in the apicoplast of T. gondii, produces nicotinamide adenine dinucleotide phosphate (NADPH), which together with thioredoxin reductase and its substrate thioredoxin forms thioredoxin system [28], which plays a key role in the parasite intracellular survival [29]. T. gondii relies on thiol-reduction systems such as thioredoxin to counter oxidative stress and maintain parasite redox status [30]. Virulence factors, such as thioredoxin reductase protects T. gondii against oxidative-burst damage from host immune cells by catalysing the conversion of oxidized thioredoxin into its reduced redox state with the consumption of NADPH [31]. Maintaining this thioredoxin-reduction state enables the parasite to resist free radical injury in host immune cells. This result corroborates previous finding showing that control of intracellular T. gondii infection of naïve macrophages by type III, but not type I, depends on NADPH activity and elevated reactive oxygen species level, independent of interferon activation, indicating that the improved survivability and infectivity of the virulent T. gondii strains may be related to their ability to block reactive oxygen species production [32]. Aldehyde dehydrogenase, chaperonin, putative, glycosyl transferase, putative (predict) were the three most common down-regulated malonylated proteins in RH vs. PRU and VEG vs. PRU (Fig 10). Also, two common down-regulated malonylated proteins (glyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH1) and phosphoglycerate kinase 1 (PGK1) were identified in RH vs. VEG and VEG vs. PRU (Fig 10). Both GAPDH1 and PGK1 are glycolytic enzymes located in cytosol and synthesize pyruvate from D-glyceraldehyde 3-phosphate. The pyruvate serves as a substrate for the pyrvuvate dehydrogenase to produce acetyl-CoA, required for the synthesis of fatty acids, which are critical for the parasite growth and proliferation [33]. The deletion of GAPDH1 causes sharp reduction in ATP levels in T. gondii, which was not compensated by GAPDH2 [34]. The enzymatic activities of GAPDH1 seems to be affected by other PTMs, with phosphorylation of the regulatory S-loop modulating glycolysis and palmitoylation regulating the association of GAPDH1 with the cortical membrane skeleton of T. gondii via Cys3 at the N-terminus [34]. To what extent malonylation of GAPDH1 contributes to the virulence and energy metabolism in T. gondii remains to be investigated. Some of the differentially malonylated proteins between RH vs. PRU strains were mainly enriched in the regulation of actin cytoskeleton and actin binding. Likewise, in PRU vs. VEG strains the differentially malonylated proteins were enriched in actin binding. Furthermore, the upregulated proteins in VEG vs. PRU strains were enriched in actin cytoskeleton organization. Actin cytoskeleton plays a key role in the motility and invasion processes of T. gondii [35]. Toxofilin, an actin-binding protein secreted by T. gondii, facilitates the parasite invasion by dismantling the actin structure of the host cells; in the absence of toxifilin, an intact cellular actin cytoskeleton impedes T. gondii invasion [36]. In conclusion, differences in lysine malonylation between T. gondii strains representative of three main genotypes were determined using mass spectrometry and immuno-affinity purification. This analysis revealed many differentially regulated malonylated proteins in RH strain (type I), PRU strain (type II) and VEG strain (type III). Malonylated proteins were enriched in diverse enzymatic, biosynthetic, and metabolic processes. While our data indicate that lysine malonylation plays a role in the regulation of T. gondii virulence, the molecular details of the function and biological significance of the identified virulence related malonylated proteins remain to be unveiled. Nevertheless, the data obtained shed new light on the molecular mechanisms underpinning virulence differences between T. gondii strains with different genetic backgrounds and emphasize the impact of post-translational modification on the virulence of T. gondii.

Acknowledgments The authors would like to thank PTM Biolabs (Hangzhou, China) for assistance with the LC-MS/MS analysis.

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