Defining epitranscriptomic hallmarks at the host-parasite interface and their roles in virulence and disease progression in Theileria annulata-infected leukocytes

Abstract

Theileria parasites are known to induce the transformation of host bovine leukocytes, involved in rapid proliferation, evasion from apoptotic mechanisms, and increased dissemination. In this study, we reveal the involvement of m⁶A RNA modification in T. annulata infection-induced transformation of bovine leukocytes. We conducted m⁶A sequencing and bioinformatics analysis to map the mRNA methylation patterns of T. annulata-infected host leukocytes. We observe specific mRNA modifications for T. annulata-infected leukocytes and a strong correlation between the proliferation rate of the infected Leukocytes with m⁶A modifications We observe that the increased amounts of m⁶A seem to impact some cell cycle dynamics, potentially via modifications of E2F4 mRNA. Moreover, we further identify HIF-1α as a possible driver of these m⁶A RNA modifications that have clear relevance to cellular proliferation dynamics. Overall, our results provide insights into the role of m6A mRNA methylation in the molecular crosstalk between Theileria and their host leukocytes, emphasizing the critical role of mRNA methylation in host-parasite interaction.

Keywords: HIF1α; Proliferation; Theileria; m(6)A.

Fig. 1

Variation in m6A RNA methylation levels across T. annulata-infected leukocytes. A. Left and Right: RNA, extracted and treated with DNAse, was isolated from T. annulata-infected bovine macrophages (V), merozoite-producing bovine macrophages (Vm), infected bovine B cells (TBL20), and non-infected bovine B cells (BL20). m6A levels were assessed using an ELISA-like assay. B. Left and Right The proportion of m6A peaks in genes was determined from Me-RIP-seq data across various T. annulata-infected cell lines. ∗p < 0.05, ∗∗p < 0.005.

Fig. 2

Alteration in expression of m6A writers, readers, and erasers in T. annulata infected leukocytes. A. mRNA levels of m6A writers (mettl3, wtap, rbm15), readers (ythdf1 & 3, hnrnpa2b1), and erasers (alkbh5) were measured by qPCR in Theileria-infected macrophages (A) and TBL20 and BL20 (B). ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001.

Fig. 3

Link between m6A methylation and gene expression variations. A. The proportion of m6A peaks in differentially expressed (DE) genes was calculated in the various RNAseq datasets (V vs. A; V vs. Vm, A vs. Vm) of Theileria-infected macrophages. The graph represents genes that have m6A peaks in both cell lines studied but are DE in only one of them. It also exclusively represents genes that are DE in one cell line and have m6A peaks in it, as well as genes that are not DE (indicated in the graph as “none”) but have m6A peaks. B. Without specific peak calling, the level of m6A modifications were assessed by simply recording the ratio between normalized read counts from immunoprecipitated and control samples. For BL20 and TBL20 samples, the genes with the 5% highest ratios were collected. This corresponded to 1006 genes (genes with no mapped reads were omitted from the analysis) from each sample. 806 genes were shared between the top 5% genes in BL20 and TBL20 samples, leaving 200 genes that were top 5% in TBL20 but not in BL20 (‘high m6A in TBL20 only’) and 200 genes that were top-5% in BL20 but not in TBL20 (‘high m6A in BL20 only’). These two gene groups were then tested for differences in expression levels between TBL20 and BL20 control samples. C. The same analysis was done for BL3 and TBL3 samples and a similar trend was observed.

Fig. 4

The role of m6A in controlling proliferation of Theileria-infected leukocytes. A. Left and middle. qPCR and western blot validation of METTL3 upregulation in Theileria-infected leukocytes via CRISPR. Right. Confirmation of increased m6A levels following METTL3 overexpression in Theileria-infected leukocytes. B. Left: The growth rate of both virulent infected macrophages (V) and infected TBL20 B cells declined post METTL3 upregulation. The viability of both V macrophages and TBL20 B cells transfected with the METTL3 CRISPRa plasmid was evaluated by an ATP bioluminescence assay, and remains unchanged when matched with their respective controls. C. Left: FACS assessment revealed that METTL3 overexpression results in a higher fraction of cells in G2, signifying that an upsurge in m6A accelerates the host cell cycle. Cell cycle distribution was inspected using Propidium iodide on GFP-positive cells through flow cytometry. Right, METTL3 upregulation in virulent macrophages boosts the mRNA count of e2f4. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001.

Fig. 5

HIF-1α augments proliferation in Theileria-infected leukocytes Via m6A-RNA Methylation. A. Left: Western blot s comparison of HIF1A expression between virulent (V) and merozoite-producing macrophages (Vm). Right: m6A measurements of V and TBL20 B cells post-HIF1A inhibition using PX-478. B. Left & Right: HIF-1A suppression elevates expression of m6A writers, readers, and erasers in Theileria-infected leukocytes. C. Left & Right: Proliferation of Theileria-infected macrophages and B20 B cells stopped 24 h post PX-478 treatment ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 6

m6A modifications influence the dissemination potential of Theileria-infected macrophages. A. Transcription levels of adam19 increase in virulent macrophages post-transfection with METTL3 CRISPRa plasmid. B. Matrigel chamber assay showcases heightened matrigel migration following METTL3 overexpression. ∗p < 0.05.

Fig. S1

Cell cycle profiles obtained by flow cytometry (propidium iodide staining). (A) Control (control cells transfected with scrambled CRISPRa plasmid). (B) Cells transfected with METLL3 CRISPRa plasmid. Notably, METTL3 significantly influenced cell cycle arrest in the G2/M phase.

Fig. S2

Analysis of E2F4-regulated genes in Theileria-infected macrophages. A Volcano plot showing genes identified in bovine studies as potentially regulated by E2F4, specifically SIRT5, mTOR, and SREBP-1c (gene is SREBF1), based on the limited available bovine data. None of these genes display significant differential gene expression (DGE) but are all downregulated in Vm compared to V. B Analysis of human genes with putative E2F4 binding sites within 250 bp upstream regions, based on a ChIP-Seq study. Of the 4739 human genes identified, 3960 corresponding bovine genes were located in our RNA DESeq output. Among these genes, 43 are upregulated and 114 are downregulated in Vm.