Altered landscape of total RNA, tRNA and sncRNA modifications in the liver and spleen of mice infected by Toxoplasma gondii

Xiao-Xuan Zhang; Yu-Zhe Sun; Wei Wang; Yang Gao; Xin-Yu Wei; Hong-Chao Sun; Chun-Ren Wang; Hong-Bo Ni; Xing Yang; Hany M. Elsheikha; Huan-Ping Guo

doi:10.1371/journal.pntd.0012281

Abstract

Background

Pathogens can impact host RNA modification machinery to establish a favorable cellular environment for their replication. In the present study, we investigated the effect of Toxoplasma gondii infection on host RNA modification profiles and explored how these modifications may influence the host-parasite interaction.

Methodology/principal findings

We analyzed the modification levels of ∼ 80 nt tRNA and 17–50 nt sncRNAs in mouse liver, spleen, and serum using liquid chromatography and tandem mass spectrometry analysis. The results revealed alterations in RNA modification profiles, particularly during acute infection. The liver exhibited more differentially abundant RNA modifications than the spleen. RNA modification levels in serum were mostly downregulated during acute infection compared to control mice. Correlations were detected between different RNA modifications in the liver and spleen during infection and between several RNA modifications and many cytokines. Alterations in RNA modifications affected tRNA stability and protein translation.

Conclusions/significance

These findings provide new insight into the role of RNA modifications in mediating the murine host response to T. gondii infection.

Author summary

The dynamic landscape of host tissue RNA modifications and its possible association with the immune response to Toxoplasma gondii infection are poorly understood. Here, we analyzed the patterns of 20 RNA modification types in the liver, spleen and serum of mice infected by T. gondii. We characterized the levels of modifications of total RNA, ∼ 80 nt tRNA and 17–50 nt sncRNAs using LC-MS/MS. Differentially abundant RNA modifications were more frequent during acute infection compared to chronic infection and were more pronounced in the liver than in the spleen. Multiple tRNA modifications were co-regulated during chronic infection. Several RNA modifications showed significant correlation with serum cytokines, highlighting the role of RNA modifications in the immune response to T. gondii infection. Alterations of RNA modifications affected tRNA stability and the translation of eukaryotic initiation factor 4A/E. These findings provide new insight into the role of RNA modifications in the pathogenesis of and immune response to T. gondii infection.

Citation: Zhang X-X, Sun Y-Z, Wang W, Gao Y, Wei X-Y, Sun H-C, et al. (2024) Altered landscape of total RNA, tRNA and sncRNA modifications in the liver and spleen of mice infected by Toxoplasma gondii. PLoS Negl Trop Dis 18(6): e0012281. https://doi.org/10.1371/journal.pntd.0012281

Editor: Tiago WP Mineo, Universidade Federal de Uberlandia, BRAZIL

Received: July 2, 2023; Accepted: June 7, 2024; Published: June 21, 2024

Copyright: © 2024 Zhang 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 authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

Funding: This research has received funding from the National Natural Science Foundation of China (Grant No.32302899 to H.G.) and the National Science and Technology Program during the Twelfth Five-year Plan Period (Grant No. to 31902238 to X.Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors declare that there are no competing interests.

Introduction

Toxoplasma gondii is an obligate intracellular apicomplexan protozoan with a worldwide distribution and a substantial public health impact [1]. This parasite cycles between a felid definitive host and a wide range of vertebrate warm-blooded animals as intermediate hosts. It can also infect humans through the ingestion of raw meat containing the parasite tissue cysts or by drinking of water contaminated with the oocysts excreted in cat feces [2]. While generally harmless, T. gondii can cause serious disease in immunocompromised individuals, such as transplant patients and people living with HIV/AIDS [2] as well as in infants congenitally infected during pregnancy [3,4].

Entirely dependent on the host cell machinery for replication, T. gondii is known to co-opt host molecules and pathways for its benefit [5]. Previous transcriptomic [6–8], proteomic [9,10], and metabolomic [11,12] studies identified many host genes, proteins and metabolites whose expression is altered during infection, playing key roles in pathways (e.g. immune-metabolic) regulating various aspects of the host-parasite interaction [6,10,13,14]. These studies suggest a complex interaction between T. gondii and its host, highlighting the need for more understanding to develop better control strategies for T. gondii infection.

RNA modifications play key roles in various biological and pathological processes [15–17]. They are vital players in regulating gene expression in response to a dynamic cellular microenvironment. Over 170 different types of enzymatic RNA modifications have been identified in eukaryotes, many of which are critical for maintaining RNA stability and optimizing its activities and functions [18,19]. In a previous study, we elucidated strain-specific differences in the levels of tRNA and 17–50 nt sncRNA modifications in T. gondii, detecting significant correlations between multiple RNA modifications and mRNA expression of some virulence factors, highlighting RNA modifications’ role in parasite pathogenicity [20]. Other studies have shown that N⁶-methyladenosine (m⁶A) promotes T. gondii replication [21] and bradyzoite differentiation [22]. However, the RNA modification profiles of host tissues during T. gondii infection and their potential roles in influencing the host response are poorly understood.

The liver and spleen, as major immune organs, play key roles in mediating the host immune response to infection. Unravelling the epitranscriptomic modifications in these tissues during T. gondii infection can improve or understanding of the underlying immune-regulatory processes. Herein, we used liquid chromatography and tandem mass spectrometry (LC-MS/MS) to detect the spectrum of RNA modifications in the liver, spleen, and serum of BALB/c mice during the acute and chronic stages of T. gondii infection. We explored the possible roles of RNA modifications in the host immune response and analyzed the expression of several cytokines in the spleen. Our results provide a comprehensive profile of RNA modifications in key mouse tissues infected by T. gondii.

Methods

Ethics statement

All animal experiments were conducted under a protocol approved by the Animal Research Ethics Committee of Qingdao Agricultural University (Approval number: RECQAU201935).

Animals

Female, 8-10-week-old, BALB/c mice were purchased from Spaefer Biotechnology Company (Beijing, China). All the mice were housed under a 12-h dark/light cycle with free access to food and water at the animal facility of Qingdao Agricultural University. After one week of acclimatization to the facility’s environment, the mice (n = 12) were randomly allocated into two groups: a control group (n = 6) and an infection group (n = 6). The mice in the infection group were infected with 20 T. gondii PRU (type I) strain cysts in 1 ml phosphate buffered saline (PBS), while those in the control group were treated with an equal volume of PBS only. At 11 and 33 days post infection (dpi), three mice from each group were euthanized. These time points correspond to the time required for the development of acute and chronic T. gondii infection in mice. Following euthanasia, the liver, spleen, and blood serum were immediately collected. The spleen and liver were stored in liquid nitrogen and the serum samples were stored at -80°C. The establishment of T. gondii infection was confirmed by PCR as previously described [23].

Extraction of total RNA from liver and spleen

Total RNA was extracted from the liver and spleen using TRIzol (Life Technologies, Carlsbad, USA) according to the manufacturer’s protocol. Briefly 1 ml TRIzol was added to a 1.5 ml microtube containing pulverized tissues and vigorously vortexed. Then, 200 μl chloroform was added to the sample, which was vortexed and incubated for 10 min at room temperature, followed by centrifugation at 12,000 × g for 15 min at 4°C. The supernatant was transferred to a new 1.5 ml microtube and mixed with an equal volume of isopropanol. After gentle mixing, the mixture was incubated for 10 min at room temperature, followed by centrifugation as described above. The total RNA pellet was resuspended in RNase-free water and stored at -80°C for further use. The quantity and purity of the isolated RNA were determined using a NanoDrop spectrophotometer (ND-1000; Thermo Scientific, DE, USA).

Isolation of ∼ 80 nt and 17–50 nt RNA fragments from total RNA of liver and spleen

Total RNA isolated from the liver and spleen was separated on a 15% denaturing polyacrylamide gel electrophoresis (PAGE) gel, and different RNA fragments were isolated. Briefly, 4 μg of total RNA was loaded onto the gel and electrophoresed for 1 h in 1× Tris-Borate-EDTA buffer (TBE) buffer at 200 V. The gel was stained by SYBR GOLD (Invitrogen, Waltham, MA, USA). The ∼ 80 nt and 17–50 nt RNA fragments were excised and extracted from the gel using a single-stranded RNA marker-based gel excision approach according to the RNA marker kit (NEB, Ipswich, MA, USA) as previously described [24]. The isolated specific RNA fragments included a mixture of RNAs with a similar range of nucleotide lengths. The quantified level of RNA modifications reflects the total RNA modification levels in the different RNAs contained in each RNA group. In a previous study, ∼ 80 nt RNA isolated from mouse tissue was considered enriched in tRNAs [25]. Therefore, we considered ∼ 80 nt RNA fragments as tRNA here. In another study, PANDORA-seq (an improved sncRNA sequencing method that expands the repertoire of sncRNAs by overcoming RNA modification) was used to characterize 15–50 nt sncRNAs in mouse tissues, which were found enriched in rRNA-derived small RNAs (rsRNAs), tsRNAs, miRNAs, piRNAs, and YRNA-derived small RNAs (ysRNAs) [26]. Therefore, it is reasonable to assume that the 17–50 nt sncRNAs isolated in the present study are mainly composed of the aforementioned sncRNAs.

Extraction of total RNA from serum

Total RNA was extracted from the serum using TRIzol LS reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. Briefly, serum was added to a microtube, mixed with 3 volume of TRIzol LS reagent, and vigorously vortexed. The mixture was incubated for 5 min at room temperature. Then, 1/5 volume chloroform was added to the mixture, followed by vortexing. After incubation for 10 min at room temperature, the samples were centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was transferred to another microtube, mixed with an equal volume of isopropanol, and stored at -80°C for 30 min to precipitate the RNA. After centrifugation at 12,000 × g for 30 min at 4°C, the pellet was washed with 75% ethanol. Finally, the RNA pellet was suspended in RNase-free water and stored at -80°C until further use.

Validation of expression using quantitative (q)RT-PCR

Quantitative reverse transcription PCR (qRT-PCR) was used to validate the expression of key cytokines, including interleukin (IL)-2, IL-4, IL-5, IL-6, IL-12, tumor necrosis factor-beta (TNF-β), and interferon gamma (IFN-γ) in mouse spleen. The total RNA samples extracted from the spleen were treated with DNase I (RQ1 RNase-free DNase-Promega, USA) to remove genome DNA. RNA was then used to synthesize cDNA using the M-MuLV Reverse Transcriptase Reaction system (NEB, Ipswich, MA, USA) with a common primer. The obtained cDNA was diluted three times for quantitative PCR (qPCR). All the primers used in the study are listed in Table 1. We used SYBR Green (Promega, USA) for detection of gene expression on a LightCycler 480, according to the manufacturer’s instructions. The relative fold change was calculated using the 2^−ΔΔCT method, with actin was used as a reference gene to normalize the relative expression levels.

Download:

Table 1. Primer sequences used in RT-PCR analysis.

https://doi.org/10.1371/journal.pntd.0012281.t001

Quantitative analysis of RNA modifications by LC-MS/MS

Total RNA and various types of RNA fragments were digested into mononucleotides as described previously [27,28]. Briefly, RNAs were digested in a 30 μl reaction system containing 3 μl of 10× RNA hydrolysis buffer (2500 mM Tris-HCl, pH 8.0; 50 mM MgCl₂; and 5 mg/mL BSA), 1 IU benzonase (Sigma-Aldrich, St Louis, MO, USA), 0.2 IU alkaline phosphatase (Sigma-Aldrich, St. Louis, MO, USA), and 0.05 IU phosphodiesterase I (Thermo Fisher Scientific, Grand Island, NY, USA) at 37°C for 3 h. The enzymes in the digestion mixture were then removed using a Nanosep 3K spin filter (Pall Corporation, Ann Arbor, MI, USA) for subsequent liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis. The abundance of each RNA modification was quantified according to a standard curve and established RNA modification relative quantification methods [27,28]. The modified nucleotide standards used in this study are listed in S1A Fig. The percentage of each modified ribonucleoside was normalized to the total amount (molar concentration) of all the quantified ribonucleosides detected.

Northern blot analysis

Northern blotting was performed to evaluate tRNA expression levels in the mouse liver during T. gondii infection as previously described [29]. Total RNA was separated on a 15% urea-PAGE gel. The gel was stained, imaged, and immediately transferred onto Roche Nylon Membranes (Roche, Basel, Switzerland) and cross-linked using UV. The membrane was prehybridized using Roche DIG hybridization buffer (Roche, Basel, Switzerland). For detection of tRNA^Ala, tRNA^Gly, tRNA^Val and tRNA^Leu, the membrane was incubated overnight at 42°C with DIG labeled probes (5’-tRNA^Leu: 5’-DIG-CCTTAGACCGCTCGGCCATCCTGAC; 5’-tRNA^Ala: 5’-DIG-CGCTCTACCACTGAGCTACACCCCC; 5’-tRNA^Val: 5’-DIG-GTGATAACCACTACACTACGGAAAC; 5’-tRNA^Gly: 5’-DIG-AATTCTACCACTGAACCACCCATGC). After 24 h, the membrane was washed with a low stringency buffer (2 × SSC with 0.1% [wt/vol] SDS), followed by two washes with a high stringency buffer (0.1 × SSC with 0.1% [wt/vol] SDS) and washing buffer (1 × SSC). The membrane was then incubated with blocking buffer (Roche, Basel, Switzerland) at room temperature for 3 h, followed by incubation in a blocking buffer with anti-Digoxigenin-AP Fab fragment (Roche, Basel, Switzerland) diluted 1:10,000. After further incubation in developing buffer at room temperature, the membrane was coated with CSPD (Roche, Basel, Switzerland) for 15 min at 37°C in the dark and imaged using a Bio-Rad system (USA).

Western blot analysis

Western blotting was performed to evaluate the expression levels of eukaryotic initiation factors 4A (eIF4A) and 4E (eIF4E) in the liver of mice infected by T. gondii for 11 days. Briefly, lysates from the liver were mixed with 5× SDS gel loading buffer, denatured, and separated by SDS-PAGE. Primary antibodies used were: Beta Actin Mouse McAb (Proteintech, Chicago, USA), eIF4A (C32B4) rabbit mAb (CST, Massachusetts, USA), and eIF4E (CST, Massachusetts, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (Proteintech, Chicago, USA) and HRP-conjugated goat anti-mouse immunoglobulin G (Beyotime, Shanghai, China) were used as secondary antibodies. The blots were scanned using a Bio-Rad system (USA). The mean values of eIF4A and eIF4E amounts and standard deviations were calculated from three biological replicates.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software Inc, San Diego, CA, USA). The unpaired Student’s t-test and Pearson’s correlation analysis were performed to examine the correlations between RNA modifications and the expression levels of the RNA-modifying methyltransferase enzymes or cytokines. All results represent the means + standard error of the mean (SEM), and a p-value of < 0.05 was considered statistically significant.

Results

Acute T. gondii infection causes extensive alterations in RNA modifications

To determine the impact of T. gondii infection on the host epitranscriptome, we performed LC-MS/MS analysis on liver, spleen, and serum samples from mice infected by T. gondii for 11 or 33 days, comparing them to uninfected mice. We quantified 20 types of RNA modifications and four unmodified ribonucleosides (A, U, C and G) in the total RNA (S1B Fig). The liver and spleen showed distinct RNA modification signatures, with the liver exhibiting a higher relative abundance of most RNA modifications compared to the spleen (Fig 1A and 1B). These RNA modifications displayed different expression patterns between the liver and spleen and between acute and chronic infections. We detected 12 and 10 differentially abundant RNA modifications in the liver and spleen, respectively (Fig 1C and 1D). In acute infection, 10 types of RNA modifications (m⁵C, m²₂G, m²G, m³C, m¹G, m¹A, m¹I, m⁵U, m⁵Um, m⁷G) were significantly decreased in the liver, while two RNA modification types (m²₂7G, p < 0.01, and m³U, p < 0.0001) were increased compared to control mice (S1C Fig). Conversely, in the spleen of acutely infected mice, three RNA modification types (Um, m²₂⁷G and Cm) were downregulated and seven types (ac4C, m¹G, m³C, m⁷G, m²₂G, m⁵C, m¹A) were upregulated (S1D Fig). In chronic infection, only Um was significantly decreased in the liver and no distinct RNA modification were deregulated in the spleen (Figs 1C and 1D and S1E). Serum RNA modifications were mostly downregulated during acute infection (I, ac⁴C, Am, p < 0.05; m⁵Um, m⁵C, m⁷G, m⁶A, p < 0.01; m¹A, m²₂G, m¹I, m³C, m¹G, m²G; p < 0.001) and slightly during chronic infection (m⁵U, Psi, p < 0.05), compared to uninfected mice. However, three RNA modifications (Am, Cm, Gm, p < 0.05) were upregulated during chronic infection compared to uninfected mice (S2 Fig). Taken together, these results indicate that acute T. gondii infection induces more RNA modifications in the liver and spleen total RNA than chronic infection.

Download:

Fig 1. Toxoplasma gondii infection induces RNA modifications in the liver and spleen total RNA of mice.

(A-B) Heatmaps showing RNA modifications in the liver and spleen in the control group (NC) versus acutely infected (A) and chronically infected groups (B) (n = 3/group). Color values represent log10 (percentage of RNA modification levels). (C-D) Radar charts displaying RNA modifications in the liver (C) and spleen (D) between control and acutely infected groups. (E-F) Radar charts showing RNA modifications in the liver (E) and spleen (F) between control and chronically infected groups. The average RNA modification level in the NC group was set to 1.

https://doi.org/10.1371/journal.pntd.0012281.g001

T. gondii infection alters tRNA and sncRNA modification levels

To identify the specific RNA types responsible for the observed RNA modification changes in the liver and spleen during T. gondii infection, we compared the differential expression of 19 RNA modifications in ∼ 80 nt RNA (mainly transfer RNA (tRNA) and 17–50 nt (mostly microRNA (miRNA), some tRNA-derived small RNA (tsRNA) and ribosomal-derived small RNA (rsRNA)) during acute and chronic infection. Fig 2A–2D shows the differences in the abundance of RNA modifications between control, acute, and chronic infected groups. During acute infection, m²₂⁷G and Am exhibited high differential abundance in the liver and spleen ∼80 nt RNA, respectively (Fig 2A and 2B), while m²₂G and Am were more significantly upregulated in liver and spleen 17–50 nt RNA respectively (Fig 2C and 2D). For ∼80 nt RNA, 6 and 9 modifications were significantly changed in the liver and spleen, respectively, during acute infection, whereas 16 and 11 modifications were significantly regulated in the liver and spleen during chronic infection (Fig 2E). Regarding 17–50 nt RNA, 16 and 9 modifications were significantly altered in liver and spleen, respectively, during acute infection, while 11 and 9 types were upregulated, respectively during chronic infection (Fig 2F).

Principal component analysis (PCA) of RNA modifications during T. gondii infection showed distinct clusters by tissue type (liver and spleen) and infection stage (acute and chronic) for both 17–50 nt sncRNA (Fig 2G) and ∼80 nt tRNA (Fig 2H). This clear segregation suggests that RNA modification patterns, particularly in the tRNA fraction, retain tissue-specific characteristics even after T. gondii infection. These findings corroborate with previous studies showing distinct RNA modification profiles between different tissues and even between regions of the same tissue [30]. Additionally, others have reported tissue-specific RNA modifications in hypoxic mice [25] and correlations of RNA modification patterns with parasite strains of differing virulence [20].

Download:

Fig 2. T. gondii infection alters tRNA and sncRNA modification signatures in the liver and spleen of mice.

(A-B) Radar charts showing the relative tRNA modifications in liver (A) and spleen (B) between control, acute, and chronic infection mouse groups. The average of RNA modification in three repeats was calculated. Then, the level of each RNA modification in the NC group was considered as 1. (C-D) Radar charts displaying relative sncRNA modifications in liver (C) and spleen (D) between control, acute, and chronic infection groups. (E) Comparison of tRNA modifications between control (NC) and infection groups across tissues. (F) Comparison of sncRNA modifications between control and infection groups across tissues. (G-H) Principal component analysis (PCA) of RNA modifications in ∼80 nt RNA and 17–50 nt RNA in the liver and spleen during acute and chronic infection. Eighteen types of RNA modifications were analyzed based on their abundance (n = 3/group). Statistical analysis used unpaired student’s t-test with three independent replicates.

https://doi.org/10.1371/journal.pntd.0012281.g002

Linear correlations between different RNA modifications during T. gondii infection

Coregulation can occur between different RNA modifications. For example, the abundance of sperm tsRNA m⁵C correlates with m²G abundance in a high-fat diet-induced metabolic disorder mouse model, and knockout of the m⁵C methyltransferase Dnmt2 decreases both m⁵C and m²G levels in sperm tsRNA [24]. We systematically analyzed correlations among RNA modifications in 17–50 nt RNA and ∼80 nt RNA in the liver and spleen during acute T. gondii infection. Both positive and negative linear correlations between RNA modifications were comparable in 17–50 nt RNA during acute and chronic infection in liver and spleen (Fig 3A–3D). Interestingly, most of RNA modifications exhibited positive linear correlations in ∼80 nt tRNA in the liver and spleen during acute infection (Fig 3E–3H), especially in the spleen (Fig 3G). During chronic infection, most ∼80 nt tRNA modifications in the liver showed positive linear correlations, whereas spleen tRNA modifications showed negative correlations (Fig 3F and 3H). These results suggest that RNA modifications are involved in the pathogenesis of T. gondii infection, with tissue-specific differences in the correlations between different ∼80 nt tRNA modifications during chronic infection.

Download:

Fig 3. Correlation analysis of RNA modifications in the liver and spleen during T. gondii infection.

(A-D) Pearson correlation analysis of sncRNA modifications in liver during (A) acute and (B) chronic infection, and spleen during (C) acute and (D) chronic infection. (E-H) Pearson correlation analysis of tRNA modifications in liver during (E) acute and (F) chronic infection, and spleen during (G) acute and (H) chronic infection. Correlation coefficients were computed using GraphPad Prism 8, and Pearson R values are presented as heatmaps. Significant differences are indicated by asterisk as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

https://doi.org/10.1371/journal.pntd.0012281.g003

RNA modifications correlate with cytokine levels

RNA modifications are closely associated with cytokine expression levels, reflecting the pivotal role of spleen-secreted cytokines in the host response to T. gondii infection. We evaluated cytokine expression in the spleen using RNA-seq analysis and confirmed the results via qRT-PCR, demonstrating consistent fold changes between infected and control mice (p < 0.0001; r = 0.85). Levels of IL-2, IL-4, IL-5, IL-6, IL-12, TNF-β, and INF-γ were significantly elevated in infected mice compared to controls (Fig 4A–4G). Furthermore, cytokine levels were significantly higher in the acute infection group compared to the chronic infection group, with the exception of IL-5 (Fig 4C). To better understand the biological relevance of RNA modifications during T. gondii infection, correlations between modification abundance in total RNA, specific fragments, and cytokine levels were examined. We found positive correlations between levels of total RNA modifications (m¹A, m⁵C, m³C, ac⁴C, m⁷G, m²₂G and m¹G) and cytokine levels (Fig 4H). In the ∼ 80 nt RNA fragments, m⁵U, m³C and m²₂G exhibited significant linear correlation with multiple cytokines (Fig 4I). Additionally, levels of sncRNA modifications I, Am, m⁵C, m³C and m²₂G correlated with cytokines (Fig 4J).

Download:

Fig 4. Differential RNA modifications correlate with cytokines.

(A-G) Comparative analysis of cytokine levels in the spleen among control, acute infection, and chronic infection groups. (H-J) Hierarchical clustering and Spearman correlation heat maps showing the linear relationship between modifications levels of (H) total RNA, (I) tRNA, (J) sncRNA, and cytokine expression. RNA modifications exhibiting the strongest correlation with cytokines are shown in red. All data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with uncorrected Fisher’s LSD in panels A-G. Significance levels are denoted by asterisk as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

https://doi.org/10.1371/journal.pntd.0012281.g004

Alteration of RNA modification signature in 80 nt RNA affects the tRNA stability

To investigate the influence of RNA modifications on tRNA stability, we examined the expression of several tRNAs with high codon usage in mouse proteins. Given the more pronounced change in RNA modification signatures during acute infection compared to chronic infection (Fig 2A and 2B), we assessed the levels of tRNA^Ala, tRNA^Gly, tRNA^Val and tRNA^Leu in the liver during acute infection using northern blotting. The expression levels of these tRNAs decreased, while the corresponding tsRNA levels increased (Fig 5A), suggesting that changes in tRNA modifications during T. gondii infection in mice affect the tRNA pool, potentially influencing protein translation efficiency. To examine this assumption, we used Western blotting to measure the expression of eIF4E/A and detected a reduction in their abundance (Fig 5B), indicating an impact on translation initiation, as eIF4E/A are essential for this process.

Download:

Fig 5. T. gondii alters tRNA stability and translation initiation factors expression in the liver during acute infection.

(A) Northern blot analysis of tRNA^Ala, tRNA^Gly, tRNA^Val and tRNA^Leu in the liver of mice infected by T. gondii for 11 days. Data represent mean ± SD from 3 biological replicates, with a representative northern blot membrane shown. (B) Western blot analysis of eIF4A and eIF4E levels in the liver of mice infected by T. gondii for 11 days. Data represent mean ± SD from 3 biological replicates, with a representative Western blot membrane shown.

https://doi.org/10.1371/journal.pntd.0012281.g005

Discussion

RNA modifications play important roles in the virulence and infection processes of T. gondii [21,22]. However, the changes in RNA modifications within mouse tissues post T. gondii infection remain largely unexplored. Here, we provide new insight into the role of epitranscriptomic modifications in the host’s response to T. gondii infection. We investigated the abundance of various RNA modifications in the primary immune organs (liver and spleen) and serum of mice infected by T. gondii during acute and chronic infection phases. Our analysis revealed that RNA modifications were more prevalent during acute T. gondii infection compared to chronic infection, with the liver exhibiting more differentially abundant RNA modifications than the spleen. Additionally, RNA modifications in nt tRNA induced by T. gondii infection affected tRNA stability. These results suggest that tRNA and sncRNA modifications are involved in T. gondii infection, highlighting the potential roles of RNA modifications in the host response to T. gondii infection.

Over 170 chemical modifications have been identified in coding and various noncoding RNAs across all living organisms, including bacteria, archaea, and eukaryotes [18]. However, we quantified only 20 modified RNA nucleobases in mouse tissues due to the limited availability of RNA standards. Single-stranded RNA marker-based gel excision is used to separate different RNA fragments [20,25], and the isolated specific RNA fragments represent a mixture of RNAs with similar nucleotide length ranges. The RNA modification abundance reflects the RNAs contained in each group, potentially affecting the RNA modification profiles of specific RNA classed. For example, sncRNA shares a similar nucleotide length with mature tRNA, and the abundance of RNA modifications on sncRNA may contribute to the alteration of RNA modifications in tRNA post-T. gondii infection.

T. gondii infection progresses from an acute stage, characterized by rapid replication of tachyzoites, to a chronic stage marked by the formation of persistent dormant cysts [31,32]. A robust Th1-type immune response mediated by pro-inflammatory cytokines, including IFN-γ, TNFα, IL-1β, IL-2, IL6, and IL-12p70, is required to limit T. gondii infection during acute infection [33]. During chronic infection, anti-inflammatory cytokines, such as IL-5, restrain excessive inflammatory responses and reduce immunopathological damage to the host [34]. Our results are consistent with previous studies, showing that levels of the pro-inflammatory cytokines IL-2, IL-4, IL-5, IL-6, IL-12, TNF-β, and INF-γ increase during the acute stage, while the level of the anti-inflammatory cytokine IL-5 increases during the acute stage and decreases during the chronic stage [34].

A recent study found that deletion of Nsun2 promotes type I interferon (IFN) response, significantly reducing gene expression and replication of viruses [35]. This m⁵C-mediated antiviral innate immunity is attributed to a decrease in the host m⁵C methylome and the promotion of polymerase III–transcribed noncoding RNAs that are recognized by the cytosolic RNA sensor RIG-I, inducing effective type I IFN signaling [35]. Whether m⁵C tRNA modification plays a role in the innate immune response to T. gondii infection by regulating type I interferon remains to be investigated. Nonetheless, the role of RNA modifications in immunity is well-established [36–38]. Intriguingly, positive correlations were detected between the modification levels of total RNA (m¹A, m⁵C, m³C, ac⁴C, m⁷G, m²₂G and m¹G) and cytokines, and between tRNA (m⁵U, m³C and m²₂G) or sncRNA (I, Am, m⁵C, m³C and m²₂G), and many cytokines. Most of these RNA modifications play roles in immunity by regulating the biological processes of immune cells [39]. Given these facts, it is reasonable to assume that RNA modifications play a role in regulating the host defense mechanism against T. gondii infection during acute infection.

In the present study, levels of tRNA^Ala, tRNA^Gly, tRNA^Val and tRNA^Leu were decreased, while the corresponding levels of tsRNA were increased in the liver during acute infection. This finding agrees with previous studies showing that post-transcriptional modifications in tRNAs influence the regulatory role played by tRNA-derived fragments (tRFs) in stem cells [40] and the pathophysiology of various diseases and pathological conditions [41]. The deposition of tRNA post-transcriptional nucleoside modifications is a dynamic and effective way for cells to regulate and adapt protein translation to external cues, via tsRNAs [16]. A previous study showed that 5’tsRNA^Ala displaces the translation initiation factor eIF4E/G/A from m⁷G-capped mRNA, resulting in a reduction in translation [40]. Also, exposure to hypoxia alters the abundance of tRNA modifications in mouse tissues, affecting tRNA stability and translation control [25]. Here, we showed that T. gondii reduced the abundance of 5’tsRNA^Ala and eIF4E/A, essential for translation initiation, suggesting that altered tRNA modifications by infection can destabilize tRNA and reduce the levels of tsRNA and eIF4E/A, compromising protein translation efficiency.

Changes in tsRNAs may also play a role in modulating the host-pathogen interactions, for example, via immune regulation during infection. A previous study on Mycobacterium showed that tRNA and its derived fragments attenuate host immune responses toward Mycobacterium by modulating a caspase-8-dependent pathway and inducing apoptosis in host monocytes [42]. tRNA fragments can activate pattern-recognition receptors, such as Toll-like receptors, which play essential roles in the host immune response to infection [43]. The role of tsRNAs in the pathogenesis of T. gondii infection, whether through modulating the host immune response, altering genes at the post-transcriptional level, or interfering with intercellular and intracellular communications, remains to be investigated.

In conclusion, our results provide new insight into the epitranscriptomic modifications of the murine tissue in response to T. gondii infection, showing the parasite’s ability to cause significant modifications in the liver and spleen ∼ 80 nt tRNAs and 17–50 nt sncRNAs during acute and chronic infection. Correlations were also detected between different types of RNA modifications and between RNA modifications and cytokines. T. gondii also decreased the level of 5’tsRNAs and eIF4E/A in the liver during acute infection, influencing protein synthesis and the host cell response to infection. Whether the detected changes in RNA modifications represent an adaptative response to T. gondii infection or a passive host cell response to infection-induced stress remains to be elucidated. However, some detected modifications are involved in modulating host immune response mechanisms and cell cycle regulation–mechanisms relevant to the pathogenesis of T. gondii infection. This work paves the way for genetic manipulation and more mechanistic insight into the cellular pathways affected by the RNA modifications identified here, such as m²₂⁷G in the liver and Am in the spleen, and their roles in influencing the outcome of T. gondii infection.

Supporting information

S1 Fig. Detection of RNA modifications in the RNA of mouse tissues.

(A) A schematic illustration depicting the experimental procedures used for detecting and quantifying RNA modifications in the spleen, liver, and serum of mice. (B) List of the used nucleobase standards. (C) Comparison of RNA modifications in total RNA of the liver between the control group and the acute infection group. (D) Comparison of RNA modifications in total RNA of the spleen between the control group and the acute infection group. (E) Comparison of RNA modifications in the total RNA of liver between the control group and the chronic infection group. All results are shown as mean ± SEM based on three biological replicates. Statistical analysis was conducted using unpaired Student’s t-test. Significant differences are indicated by asterisk as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

https://doi.org/10.1371/journal.pntd.0012281.s001

(TIF)

S2 Fig. The abundance of RNA modifications in the total RNA of the mouse serum.

(A) Comparison of RNA modifications in the serum total RNA between the control group and the acute infection group. (B) Comparison of RNA modifications in the serum total RNA between the control group and the chronic infection group. All results are shown as mean ± SEM based on biological triplicate. Statistical analysis was conducted using unpaired Student’s t-test. Significant differences are indicated by asterisk as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

https://doi.org/10.1371/journal.pntd.0012281.s002

(TIF)

References

1. Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet. 2004;363(9425):1965–76. pmid:15194258
- View Article
- PubMed/NCBI
- Google Scholar
2. Elsheikha HM, Marra CM, Zhu XQ. Epidemiology, pathophysiology, diagnosis, and management of cerebral toxoplasmosis. Clin Microbiol Rev. 2021;34(1). pmid:33239310
- View Article
- PubMed/NCBI
- Google Scholar
3. Elsheikha HM. Congenital toxoplasmosis: priorities for further health promotion action. Public Health. 2008;122(4):335–53. pmid:17964621
- View Article
- PubMed/NCBI
- Google Scholar
4. Torgerson PR, Mastroiacovo P. The global burden of congenital toxoplasmosis: a systematic review. Bull World Health Organ. 2013;91(7):501–8. pmid:23825877
- View Article
- PubMed/NCBI
- Google Scholar
5. Rosenberg A, Sibley LD. Toxoplasma gondii secreted effectors co-opt host repressor complexes to inhibit necroptosis. Cell Host Microbe. 2021;29(7):1186–98 e8. pmid:34043960
- View Article
- PubMed/NCBI
- Google Scholar
6. Wang M, Zhang FK, Elsheikha HM, Zhang NZ, He JJ, Luo JX, et al. Transcriptomic insights into the early host-pathogen interaction of cat intestine with Toxoplasma gondii. Parasit Vectors. 2018;11(1):592. pmid:30428922
- View Article
- PubMed/NCBI
- Google Scholar
7. Wang SS, Zhou CX, Elsheikha HM, He JJ, Zou FC, Zheng WB, et al. Temporal transcriptomic changes in long non-coding RNAs and messenger RNAs involved in the host immune and metabolic response during Toxoplasma gondii lytic cycle. Parasit Vectors. 2022;15(1):22. pmid:35012632
- View Article
- PubMed/NCBI
- Google Scholar
8. Hu RS, He JJ, Elsheikha HM, Zou Y, Ehsan M, Ma QN, et al. Transcriptomic Profiling of mouse brain during acute and chronic infections by Toxoplasma gondii oocysts. Front Microbiol. 2020;11:570903. pmid:33193165
- View Article
- PubMed/NCBI
- Google Scholar
9. He JJ, Ma J, Elsheikha HM, Song HQ, Zhou DH, Zhu XQ. Proteomic profiling of mouse liver following acute Toxoplasma gondii infection. PLoS One. 2016;11(3):e0152022. pmid:27003162
- View Article
- PubMed/NCBI
- Google Scholar
10. He JJ, Ma J, Wang JL, Zhang FK, Li JX, Zhai BT, et al. iTRAQ-based quantitative proteomics analysis identifies host pathways modulated during Toxoplasma gondii infection in swine. Microorganisms. 2020;8(4). pmid:32260483
- View Article
- PubMed/NCBI
- Google Scholar
11. Zhou CX, Zhou DH, Elsheikha HM, Liu GX, Suo X, Zhu XQ. Global metabolomic profiling of mice brains following experimental infection with the cyst-forming Toxoplasma gondii. PLoS One. 2015;10(10):e0139635. pmid:26431205
- View Article
- PubMed/NCBI
- Google Scholar
12. Ma J, He JJ, Hou JL, Zhou CX, Elsheikha HM, Zhu XQ. Ultra performance liquid chromatography-tandem mass spectrometry-based metabolomics reveals metabolic alterations in the mouse cerebellum during Toxoplasma gondii infection. Front Microbiol. 2020;11:1555. pmid:32765450
- View Article
- PubMed/NCBI
- Google Scholar
13. Chen XQ, Elsheikha HM, Hu RS, Hu GX, Guo SL, Zhou CX, et al. Hepatic metabolomics investigation in acute and chronic murine toxoplasmosis. Front Cell Infect Microbiol. 2018;8:189. pmid:29922602
- View Article
- PubMed/NCBI
- Google Scholar
14. Ma J, He JJ, Hou JL, Zhou CX, Zhang FK, Elsheikha HM, et al. Metabolomic signature of mouse cerebral cortex following Toxoplasma gondii infection. Parasit Vectors. 2019;12(1):373. pmid:31358041
- View Article
- PubMed/NCBI
- Google Scholar
15. Roundtree IA, Evans ME, Pan T, He C. Dynamic RNA modifications in gene expression regulation. Cell. 2017;169(7):1187–200. pmid:28622506
- View Article
- PubMed/NCBI
- Google Scholar
16. Frye M, Harada BT, Behm M, He C. RNA modifications modulate gene expression during development. Science. 2018;361(6409):1346–9. pmid:30262497
- View Article
- PubMed/NCBI
- Google Scholar
17. Wiener D, Schwartz S. The epitranscriptome beyond m(6)A. Nat Rev Genet. 2021;22(2):119–31. pmid:33188361
- View Article
- PubMed/NCBI
- Google Scholar
18. Boccaletto P, Machnicka MA, Purta E, Piatkowski P, Baginski B, Wirecki TK, et al. MODOMICS: a database of RNA modification pathways. 2017 update. Nucleic Acids Res. 2018;46(D1):D303–D7. pmid:29106616
- View Article
- PubMed/NCBI
- Google Scholar
19. Nachtergaele S, He C. Chemical modifications in the life of an mRNA transcript. Annu Rev Genet. 2018;52:349–72. pmid:30230927
- View Article
- PubMed/NCBI
- Google Scholar
20. Wang W, Yang Y, Guo H, Li MH, Chen XQ, Wei XY, et al. Unravelling strain-specific modifications of Toxoplasma gondii tRNA and sncRNA using LC-MS/MS. Microbiol Spectr. 2023;11(3):e0356422. pmid:37036375
- View Article
- PubMed/NCBI
- Google Scholar
21. Holmes MJ, Padgett LR, Bastos MS, Sullivan WJ, Jr. m6A RNA methylation facilitates pre-mRNA 3’-end formation and is essential for viability of Toxoplasma gondii. PLoS Pathog. 2021;17(7):e1009335. pmid:34324585
- View Article
- PubMed/NCBI
- Google Scholar
22. Nakamoto MA, Lovejoy AF, Cygan AM, Boothroyd JC. mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii. RNA. 2017;23(12):1834–49. pmid:28851751
- View Article
- PubMed/NCBI
- Google Scholar
23. Hu RS, He JJ, Elsheikha HM, Zhang FK, Zou Y, Zhao GH, et al. Differential brain MicroRNA expression profiles after acute and chronic infection of mice with Toxoplasma gondii oocysts. Front Microbiol. 2018;9:2316. pmid:30333806
- View Article
- PubMed/NCBI
- Google Scholar
24. Zhang Y, Zhang X, Shi J, Tuorto F, Li X, Liu Y, et al. Dnmt2 mediates intergenerational transmission of paternally acquired metabolic disorders through sperm small non-coding RNAs. Nat Cell Biol. 2018;20(5):535–40. pmid:29695786
- View Article
- PubMed/NCBI
- Google Scholar
25. Guo H, Xia L, Wang W, Xu W, Shen X, Wu X, et al. Hypoxia induces alterations in tRNA modifications involved in translational control. BMC Biol. 2023;21(1):39. pmid:36803965
- View Article
- PubMed/NCBI
- Google Scholar
26. Shi J, Zhang Y, Tan D, Zhang X, Yan M, Zhang Y, et al. PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications. Nat Cell Biol. 2021;23(4):424–36. pmid:33820973
- View Article
- PubMed/NCBI
- Google Scholar
27. He T, Guo H, Shen X, Wu X, Xia L, Jiang X, et al. Hypoxia-induced alteration of RNA modifications in the mouse testis and spermdagger. Biol Reprod. 2021;105(5):1171–8.
- View Article
- Google Scholar
28. Guo H, Shen X, Hu H, Zhou P, He T, Xia L, et al. Alteration of RNA modification signature in human sperm correlates with sperm motility. Mol Hum Reprod. 2022;28(9). pmid:35959987
- View Article
- PubMed/NCBI
- Google Scholar
29. He T, Guo H, Xia L, Shen X, Huang Y, Wu X, et al. Alterations of RNA modification in mouse germ cell-2 spermatids under hypoxic stress. Front Mol Biosci. 2022;9:871737. pmid:35775084
- View Article
- PubMed/NCBI
- Google Scholar
30. Richter F, Plehn JE, Bessler L, Hertler J, Jorg M, Cirzi C, et al. RNA marker modifications reveal the necessity for rigorous preparation protocols to avoid artifacts in epitranscriptomic analysis. Nucleic Acids Res. 2022;50(8):4201–15. pmid:34850949
- View Article
- PubMed/NCBI
- Google Scholar
31. Nguyen TD, Bigaignon G, Van Broeck J, Vercammen M, Nguyen TN, Delmee M, et al. Acute and chronic phases of Toxoplasma gondii infection in mice modulate the host immune responses. Infect Immun. 1998;66(6):2991–5. pmid:9596779
- View Article
- PubMed/NCBI
- Google Scholar
32. Hester J, Mullins J, Sa Q, Payne L, Mercier C, Cesbron-Delauw MF, et al. Toxoplasma gondii antigens recognized by IgG antibodies differ between mice with and without active proliferation of tachyzoites in the brain during the chronic stage of infection. Infect Immun. 2012;80(10):3611–20. pmid:22851753
- View Article
- PubMed/NCBI
- Google Scholar
33. Sana M, Rashid M, Rashid I, Akbar H, Gomez-Marin JE, Dimier-Poisson I. Immune response against toxoplasmosis-some recent updates RH: Toxoplasma gondii immune response. Int J Immunopathol Pharmacol. 2022;36:3946320221078436. pmid:35227108
- View Article
- PubMed/NCBI
- Google Scholar
34. Hwang YS, Shin JH, Yang JP, Jung BK, Lee SH, Shin EH. Characteristics of infection immunity regulated by Toxoplasma gondii to maintain chronic infection in the brain. Front Immunol. 2018;9:158. pmid:29459868
- View Article
- PubMed/NCBI
- Google Scholar
35. Zhang Y, Zhang LS, Dai Q, Chen P, Lu M, Kairis EL, et al. 5-methylcytosine (m(5)C) RNA modification controls the innate immune response to virus infection by regulating type I interferons. Proc Natl Acad Sci U S A. 2022;119(42):e2123338119. pmid:36240321
- View Article
- PubMed/NCBI
- Google Scholar
36. Wang H, Hu X, Huang M, Liu J, Gu Y, Ma L, et al. Mettl3-mediated mRNA m(6)A methylation promotes dendritic cell activation. Nat Commun. 2019;10(1):1898. pmid:31015515
- View Article
- PubMed/NCBI
- Google Scholar
37. Song H, Song J, Cheng M, Zheng M, Wang T, Tian S, et al. METTL3-mediated m(6)A RNA methylation promotes the anti-tumour immunity of natural killer cells. Nat Commun. 2021;12(1):5522. pmid:34535671
- View Article
- PubMed/NCBI
- Google Scholar
38. Elsabbagh RA, Rady M, Watzl C, Abou-Aisha K, Gad MZ. Impact of N6-methyladenosine (m(6)A) modification on immunity. Cell Commun Signal. 2022;20(1):140. pmid:36085064
- View Article
- PubMed/NCBI
- Google Scholar
39. Cui L, Ma R, Cai J, Guo C, Chen Z, Yao L, et al. RNA modifications: importance in immune cell biology and related diseases. Signal Transduct Target Ther. 2022;7(1):334. pmid:36138023
- View Article
- PubMed/NCBI
- Google Scholar
40. Guzzi N, Ciesla M, Ngoc PCT, Lang S, Arora S, Dimitriou M, et al. Pseudouridylation of tRNA-derived fragments steers translational control in stem cells. Cell. 2018;173(5):1204–16 e26. pmid:29628141
- View Article
- PubMed/NCBI
- Google Scholar
41. Pandey KK, Madhry D, Ravi Kumar YS, Malvankar S, Sapra L, Srivastava RK, et al. Regulatory roles of tRNA-derived RNA fragments in human pathophysiology. Mol Ther Nucleic Acids. 2021;26:161–73. pmid:34513302
- View Article
- PubMed/NCBI
- Google Scholar
42. Obregon-Henao A, Duque-Correa MA, Rojas M, Garcia LF, Brennan PJ, Ortiz BL, et al. Stable extracellular RNA fragments of Mycobacterium tuberculosis induce early apoptosis in human monocytes via a caspase-8 dependent mechanism. PLoS One. 2012;7(1):e29970. pmid:22253841
- View Article
- PubMed/NCBI
- Google Scholar
43. Keegan C, Krutzik S, Schenk M, Scumpia PO, Lu J, Pang YLJ, et al. Mycobacterium tuberculosis transfer RNA Induces IL-12p70 via synergistic activation of pattern recognition receptors within a cell network. J Immunol. 2018;200(9):3244–58. pmid:29610140
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet. 2004;363(9425):1965–76. pmid:15194258
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Elsheikha HM, Marra CM, Zhu XQ. Epidemiology, pathophysiology, diagnosis, and management of cerebral toxoplasmosis. Clin Microbiol Rev. 2021;34(1). pmid:33239310
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Elsheikha HM. Congenital toxoplasmosis: priorities for further health promotion action. Public Health. 2008;122(4):335–53. pmid:17964621
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Torgerson PR, Mastroiacovo P. The global burden of congenital toxoplasmosis: a systematic review. Bull World Health Organ. 2013;91(7):501–8. pmid:23825877
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Rosenberg A, Sibley LD. Toxoplasma gondii secreted effectors co-opt host repressor complexes to inhibit necroptosis. Cell Host Microbe. 2021;29(7):1186–98 e8. pmid:34043960
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Wang M, Zhang FK, Elsheikha HM, Zhang NZ, He JJ, Luo JX, et al. Transcriptomic insights into the early host-pathogen interaction of cat intestine with Toxoplasma gondii. Parasit Vectors. 2018;11(1):592. pmid:30428922
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Wang SS, Zhou CX, Elsheikha HM, He JJ, Zou FC, Zheng WB, et al. Temporal transcriptomic changes in long non-coding RNAs and messenger RNAs involved in the host immune and metabolic response during Toxoplasma gondii lytic cycle. Parasit Vectors. 2022;15(1):22. pmid:35012632
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Hu RS, He JJ, Elsheikha HM, Zou Y, Ehsan M, Ma QN, et al. Transcriptomic Profiling of mouse brain during acute and chronic infections by Toxoplasma gondii oocysts. Front Microbiol. 2020;11:570903. pmid:33193165
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. He JJ, Ma J, Elsheikha HM, Song HQ, Zhou DH, Zhu XQ. Proteomic profiling of mouse liver following acute Toxoplasma gondii infection. PLoS One. 2016;11(3):e0152022. pmid:27003162
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. He JJ, Ma J, Wang JL, Zhang FK, Li JX, Zhai BT, et al. iTRAQ-based quantitative proteomics analysis identifies host pathways modulated during Toxoplasma gondii infection in swine. Microorganisms. 2020;8(4). pmid:32260483
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Zhou CX, Zhou DH, Elsheikha HM, Liu GX, Suo X, Zhu XQ. Global metabolomic profiling of mice brains following experimental infection with the cyst-forming Toxoplasma gondii. PLoS One. 2015;10(10):e0139635. pmid:26431205
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Ma J, He JJ, Hou JL, Zhou CX, Elsheikha HM, Zhu XQ. Ultra performance liquid chromatography-tandem mass spectrometry-based metabolomics reveals metabolic alterations in the mouse cerebellum during Toxoplasma gondii infection. Front Microbiol. 2020;11:1555. pmid:32765450
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Chen XQ, Elsheikha HM, Hu RS, Hu GX, Guo SL, Zhou CX, et al. Hepatic metabolomics investigation in acute and chronic murine toxoplasmosis. Front Cell Infect Microbiol. 2018;8:189. pmid:29922602
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Ma J, He JJ, Hou JL, Zhou CX, Zhang FK, Elsheikha HM, et al. Metabolomic signature of mouse cerebral cortex following Toxoplasma gondii infection. Parasit Vectors. 2019;12(1):373. pmid:31358041
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Roundtree IA, Evans ME, Pan T, He C. Dynamic RNA modifications in gene expression regulation. Cell. 2017;169(7):1187–200. pmid:28622506
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Frye M, Harada BT, Behm M, He C. RNA modifications modulate gene expression during development. Science. 2018;361(6409):1346–9. pmid:30262497
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Wiener D, Schwartz S. The epitranscriptome beyond m(6)A. Nat Rev Genet. 2021;22(2):119–31. pmid:33188361
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Boccaletto P, Machnicka MA, Purta E, Piatkowski P, Baginski B, Wirecki TK, et al. MODOMICS: a database of RNA modification pathways. 2017 update. Nucleic Acids Res. 2018;46(D1):D303–D7. pmid:29106616
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Nachtergaele S, He C. Chemical modifications in the life of an mRNA transcript. Annu Rev Genet. 2018;52:349–72. pmid:30230927
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Wang W, Yang Y, Guo H, Li MH, Chen XQ, Wei XY, et al. Unravelling strain-specific modifications of Toxoplasma gondii tRNA and sncRNA using LC-MS/MS. Microbiol Spectr. 2023;11(3):e0356422. pmid:37036375
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Holmes MJ, Padgett LR, Bastos MS, Sullivan WJ, Jr. m6A RNA methylation facilitates pre-mRNA 3’-end formation and is essential for viability of Toxoplasma gondii. PLoS Pathog. 2021;17(7):e1009335. pmid:34324585
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Nakamoto MA, Lovejoy AF, Cygan AM, Boothroyd JC. mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii. RNA. 2017;23(12):1834–49. pmid:28851751
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Hu RS, He JJ, Elsheikha HM, Zhang FK, Zou Y, Zhao GH, et al. Differential brain MicroRNA expression profiles after acute and chronic infection of mice with Toxoplasma gondii oocysts. Front Microbiol. 2018;9:2316. pmid:30333806
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Zhang Y, Zhang X, Shi J, Tuorto F, Li X, Liu Y, et al. Dnmt2 mediates intergenerational transmission of paternally acquired metabolic disorders through sperm small non-coding RNAs. Nat Cell Biol. 2018;20(5):535–40. pmid:29695786
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref25] 25. Guo H, Xia L, Wang W, Xu W, Shen X, Wu X, et al. Hypoxia induces alterations in tRNA modifications involved in translational control. BMC Biol. 2023;21(1):39. pmid:36803965
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref26] 26. Shi J, Zhang Y, Tan D, Zhang X, Yan M, Zhang Y, et al. PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications. Nat Cell Biol. 2021;23(4):424–36. pmid:33820973
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref27] 27. He T, Guo H, Shen X, Wu X, Xia L, Jiang X, et al. Hypoxia-induced alteration of RNA modifications in the mouse testis and spermdagger. Biol Reprod. 2021;105(5):1171–8.
View Article
Google Scholar

[106] View Article

[107] Google Scholar

[ref28] 28. Guo H, Shen X, Hu H, Zhou P, He T, Xia L, et al. Alteration of RNA modification signature in human sperm correlates with sperm motility. Mol Hum Reprod. 2022;28(9). pmid:35959987
View Article
PubMed/NCBI
Google Scholar

[109] View Article

[110] PubMed/NCBI

[111] Google Scholar

[ref29] 29. He T, Guo H, Xia L, Shen X, Huang Y, Wu X, et al. Alterations of RNA modification in mouse germ cell-2 spermatids under hypoxic stress. Front Mol Biosci. 2022;9:871737. pmid:35775084
View Article
PubMed/NCBI
Google Scholar

[113] View Article

[114] PubMed/NCBI

[115] Google Scholar

[ref30] 30. Richter F, Plehn JE, Bessler L, Hertler J, Jorg M, Cirzi C, et al. RNA marker modifications reveal the necessity for rigorous preparation protocols to avoid artifacts in epitranscriptomic analysis. Nucleic Acids Res. 2022;50(8):4201–15. pmid:34850949
View Article
PubMed/NCBI
Google Scholar

[117] View Article

[118] PubMed/NCBI

[119] Google Scholar

[ref31] 31. Nguyen TD, Bigaignon G, Van Broeck J, Vercammen M, Nguyen TN, Delmee M, et al. Acute and chronic phases of Toxoplasma gondii infection in mice modulate the host immune responses. Infect Immun. 1998;66(6):2991–5. pmid:9596779
View Article
PubMed/NCBI
Google Scholar

[121] View Article

[122] PubMed/NCBI

[123] Google Scholar

[ref32] 32. Hester J, Mullins J, Sa Q, Payne L, Mercier C, Cesbron-Delauw MF, et al. Toxoplasma gondii antigens recognized by IgG antibodies differ between mice with and without active proliferation of tachyzoites in the brain during the chronic stage of infection. Infect Immun. 2012;80(10):3611–20. pmid:22851753
View Article
PubMed/NCBI
Google Scholar

[125] View Article

[126] PubMed/NCBI

[127] Google Scholar

[ref33] 33. Sana M, Rashid M, Rashid I, Akbar H, Gomez-Marin JE, Dimier-Poisson I. Immune response against toxoplasmosis-some recent updates RH: Toxoplasma gondii immune response. Int J Immunopathol Pharmacol. 2022;36:3946320221078436. pmid:35227108
View Article
PubMed/NCBI
Google Scholar

[129] View Article

[130] PubMed/NCBI

[131] Google Scholar

[ref34] 34. Hwang YS, Shin JH, Yang JP, Jung BK, Lee SH, Shin EH. Characteristics of infection immunity regulated by Toxoplasma gondii to maintain chronic infection in the brain. Front Immunol. 2018;9:158. pmid:29459868
View Article
PubMed/NCBI
Google Scholar

[133] View Article

[134] PubMed/NCBI

[135] Google Scholar

[ref35] 35. Zhang Y, Zhang LS, Dai Q, Chen P, Lu M, Kairis EL, et al. 5-methylcytosine (m(5)C) RNA modification controls the innate immune response to virus infection by regulating type I interferons. Proc Natl Acad Sci U S A. 2022;119(42):e2123338119. pmid:36240321
View Article
PubMed/NCBI
Google Scholar

[137] View Article

[138] PubMed/NCBI

[139] Google Scholar

[ref36] 36. Wang H, Hu X, Huang M, Liu J, Gu Y, Ma L, et al. Mettl3-mediated mRNA m(6)A methylation promotes dendritic cell activation. Nat Commun. 2019;10(1):1898. pmid:31015515
View Article
PubMed/NCBI
Google Scholar

[141] View Article

[142] PubMed/NCBI

[143] Google Scholar

[ref37] 37. Song H, Song J, Cheng M, Zheng M, Wang T, Tian S, et al. METTL3-mediated m(6)A RNA methylation promotes the anti-tumour immunity of natural killer cells. Nat Commun. 2021;12(1):5522. pmid:34535671
View Article
PubMed/NCBI
Google Scholar

[145] View Article

[146] PubMed/NCBI

[147] Google Scholar

[ref38] 38. Elsabbagh RA, Rady M, Watzl C, Abou-Aisha K, Gad MZ. Impact of N6-methyladenosine (m(6)A) modification on immunity. Cell Commun Signal. 2022;20(1):140. pmid:36085064
View Article
PubMed/NCBI
Google Scholar

[149] View Article

[150] PubMed/NCBI

[151] Google Scholar

[ref39] 39. Cui L, Ma R, Cai J, Guo C, Chen Z, Yao L, et al. RNA modifications: importance in immune cell biology and related diseases. Signal Transduct Target Ther. 2022;7(1):334. pmid:36138023
View Article
PubMed/NCBI
Google Scholar

[153] View Article

[154] PubMed/NCBI

[155] Google Scholar

[ref40] 40. Guzzi N, Ciesla M, Ngoc PCT, Lang S, Arora S, Dimitriou M, et al. Pseudouridylation of tRNA-derived fragments steers translational control in stem cells. Cell. 2018;173(5):1204–16 e26. pmid:29628141
View Article
PubMed/NCBI
Google Scholar

[157] View Article

[158] PubMed/NCBI

[159] Google Scholar

[ref41] 41. Pandey KK, Madhry D, Ravi Kumar YS, Malvankar S, Sapra L, Srivastava RK, et al. Regulatory roles of tRNA-derived RNA fragments in human pathophysiology. Mol Ther Nucleic Acids. 2021;26:161–73. pmid:34513302
View Article
PubMed/NCBI
Google Scholar

[161] View Article

[162] PubMed/NCBI

[163] Google Scholar

[ref42] 42. Obregon-Henao A, Duque-Correa MA, Rojas M, Garcia LF, Brennan PJ, Ortiz BL, et al. Stable extracellular RNA fragments of Mycobacterium tuberculosis induce early apoptosis in human monocytes via a caspase-8 dependent mechanism. PLoS One. 2012;7(1):e29970. pmid:22253841
View Article
PubMed/NCBI
Google Scholar

[165] View Article

[166] PubMed/NCBI

[167] Google Scholar

[ref43] 43. Keegan C, Krutzik S, Schenk M, Scumpia PO, Lu J, Pang YLJ, et al. Mycobacterium tuberculosis transfer RNA Induces IL-12p70 via synergistic activation of pattern recognition receptors within a cell network. J Immunol. 2018;200(9):3244–58. pmid:29610140
View Article
PubMed/NCBI
Google Scholar

[169] View Article

[170] PubMed/NCBI

[171] Google Scholar

Figures

Abstract

Background

Methodology/principal findings

Conclusions/significance

Author summary

Introduction

Methods

Ethics statement

Animals

Extraction of total RNA from liver and spleen

Isolation of ∼ 80 nt and 17–50 nt RNA fragments from total RNA of liver and spleen

Extraction of total RNA from serum

Validation of expression using quantitative (q)RT-PCR

Quantitative analysis of RNA modifications by LC-MS/MS

Northern blot analysis

Western blot analysis

Statistical analysis

Results

Acute T. gondii infection causes extensive alterations in RNA modifications

T. gondii infection alters tRNA and sncRNA modification levels

Linear correlations between different RNA modifications during T. gondii infection

RNA modifications correlate with cytokine levels

Alteration of RNA modification signature in 80 nt RNA affects the tRNA stability

Discussion

Supporting information

S1 Fig. Detection of RNA modifications in the RNA of mouse tissues.

S2 Fig. The abundance of RNA modifications in the total RNA of the mouse serum.

References