Dataset Information

Theileria parva Muguga (TpM) schizonts LC-MS/MS

ABSTRACT: The apicomplexan parasite Theileria parva (Tp) causes East Coast Fever (ECF), an often fatal tick-borne disease especially for Bos taurus cattle breeds that causes substantial economic losses for cattle farmers in sub-Saharan Africa. Replacement of the currently employed live vaccine with a more standardised, stable, affordable, and cross-protective subunit vaccine has not yet been successful and requires the identification of additional suitable parasite antigens. Carbohydrate-based molecules play an important role in the survival and establishment of pathogens including protozoan parasites and can represent strong virulence factors. The current knowledge of the glycobiology of Tp, however, is very limited which led us to investigate the glycosylation potential of Tp in this study. Screening of isolated Tp Muguga (TpM) schizonts with C-type lectin (-like) receptors identified recognition of the parasites by ruminant but not murine Macrophage C-type lectin (MCL). This suggested the presence of carbohydrate-associated schizont surface molecules. Specific binding of isolated schizonts by WGA but not ConA further suggested the presence of terminal GlcNAc residues on the surface of the parasites which is in alignment with the identification of a minimal N-glycosylation machinery in TpM schizonts. LC-MS/MS analyses of either HILIC-HPLC or WGA enriched peptides from parasite protein fractions identified eight predicted N-glycopeptides and two predicted O-glycopeptides. While putative parasite O-glycans were assigned with high confidence, assignments of putative N-glycans to parasite proteins were less confident. Confirmation of both the assignment and the composition of predicted parasite glycans requires further analyses.

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ORGANISM(S): Bos Taurus (bovine) Theileria Parva

SUBMITTER: Shi Yan

LAB HEAD: Shi Yan

PROVIDER: PXD070746 | Pride | 2026-06-29

REPOSITORIES: Pride

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Investigation of carbohydrate-based molecules of Theileria parva parasites.

Kolakowski Jeannine J Yan Shi S Stadlmann Johannes J Lepenies Bernd B Ngugi Daniel D Harris Heather H Werling Dirk D

Frontiers in veterinary science 20260608

Theileria parva (Tp), the causative agent of East Coast Fever, poses a major threat to Bos taurus cattle in sub-Saharan Africa. Progress towards a stable, affordable, cross-protective subunit vaccine covering more than one major histocompatibility complex (MHC)-presented Tp antigen has been hindered by the limited knowledge of antigens. Given the importance of glycoconjugates in protozoan pathogen biology, especially during the invasion process, we investigated the glycosylation po ...[more]

PMID: 42339100

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Project description:Plasmodium falciparum merozoite surface protein MSPDBL2 is a highly polymorphic target of naturally acquired immune responses encoded by a single copy gene under strong balancing selection in natural populations. The MSPDBL2 protein is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene expression in culture is increased in response to overexpression of the gametocyte development inducer GDV1 in an engineered parasite clone, so there is a need to characterise its natural expression variation. In this study, MSPDBL2 in mature schizonts was analyzed in the first ex vivo culture cycle of 102 clinical isolates from four endemic populations in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2 (median of 0.6% overall), but the frequency distribution was highly skewed as eleven isolates had more than 3% schizonts positive and one had 73% positive. The expression frequencies were similar across the four endemic populations, and there was no significant difference between the clinical isolates overall and frequencies of positive schizonts previously seen in cultured P. falciparum laboratory lines. To explore whether expression of other gene loci correlated with MSPDBL2 expression, whole transcriptome sequencing was performed on schizont-enriched material from 17 of the clinical isolates with a wide range of proportions of schizonts positive. Transcripts of particular parasite genes were highly significantly positively correlated with MSPDBP2 positivity in schizonts as well as with mspdbl2 gene transcript levels, with overrepresentation of many genes previously implicated as likely to be involved in gametocytogenesis, but not including other genes including the gametocytogenesis master regulator ap2g. Although MSPDBL2 expression occurs in a highly variable proportion of schizonts in clinical isolates, and correlation with expression of some gametocytogenesis-related genes is consistent with regulation by GDV1, it is not apparently a direct marker of sexual commitment and its function in the parasite remains to be determined.

Project description:Purpose: In malaria parasites, the regulation of mRNA translation, storage and degradation is a critical aspect of gene expression, especially during the life stage transition. Since the DEAD-box helicases are involved in RNA metabolism, we wanted to determine whether pfdozi disruption led to altered mRNA abundance in P. falciparum. Methods: In this study, we functionally characterized the DEAD-box RNA helicase PfDOZI in the human malaria parasite Plasmodium falciparum and discovered its functions on mRNA metabolism during development. We generate a pfdozi gene knockout (KO) parasite line. 1). The transcriptomic analysis via RNAseq to the asexual parasites at four stages (ring, early trophozoite, late trophozoite, and schizont)(10h, 20h, 30h, and 40h post-invasion) and the sexual stage (gametocytes) between KO and wildtype (WT) 3D7 parasite line were performed in triplicate. Total RNA was isolated from the purified parasites using the Quick-RNA MiniPrep kit (Zymo Research). RNA sequencing libraries were prepared using the KAPA stranded RNA-seq library preparation kit (Roche) with 500 ng RNA from each sample. Illumina adapter sequence removal and quality trimming of reads were performed using Trimmomatic. Only reads that had a minimum length of 50 base pairs were retained. Reads were then mapped to the P. falciparum 3D7 strain reference genome with HISAT2. Differentially expressed genes are determined by DESeq2 with Padj < 0.01 and absolute log2 fold change higher than 1. 2). To investigate the function of pfdozi in stress response, both KO and WT parasites at the schizont stage were cultured under stress conditions. RNAs were harvested for the transcriptomic analysis via RNAseq. 3). To identify the mRNA targets of PfDOZI in schizonts and gametocytes, we performed RNA-immunoprecipitation (RIP) coupled with next-generation sequencing from the PfDOZI::GFP parasites. A gaussian mixture model with a set of stringent criteria, including a read-depth coverage greater than 10, a fold enrichment ratio greater than 2, and posterior probabilities higher than 0.95 in both replicates. Results: 1). RNA-seq analysis identified up-regulation of 18, 4, 7 and 130 genes and down-regulation of 104, 44, 28 and 15 genes in the Δpfdozi parasite compared to WT 3D7 (fold change ≥ 2 and adjusted P (Padj) < 0.01) at ring (10h), early trophozoite (20h), late trophozoite (30h) and schizont (40h) stage, respectively. Our results showed that pfdozi disruption led to the upregulation of invasion-related genes in schizonts; 2). RNA-seq analysis revealed that the effect of pfdozi disruption on gametocytes was substantially more profound than in asexual stages, with 842 downregulated and 752 upregulated transcripts (fold change ≥2, Padj < 0.01) in the Δpfdozi gametocyte. About 80% of these markedly reduced transcripts are gametocyte/ookinete-specific transcripts. Genes related to microtubule/cytoskeleton, cellular respiration, and crystalloid were significantly enriched among the downregulated genes, which agrees with the defective morphogenesis of the Δpfdozi gametocytes. 3). RNAseq analysis showed that under the stress conditions, the WT parasites showed three-fold more upregulated transcripts (591) than downregulated (149), suggesting that significantly more transcripts were protected from degradation under stress conditions. In contrast, the Δpfdozi parasites had a much higher proportion (57%, 463) of downregulated than that of upregulated transcripts (43%, 346) under such stress conditions. 4). The RIP-seq analysis collectively identified the potential target mRNAs of the PfDOZI complex(es) in P. falciparum schizonts and gametocytes, identified 823 and 1377 transcripts as potential target mRNAs of the PfDOZI complexes in schizonts and gametocytes, respectively. Our data also showed significant overlaps with genes whose expression was disturbed upon pfdozi disruption. Conclusions: Collectively, we have demonstrated that PfDOZI, likes its metazoan orthologs, is a versatile regulator of mRNA metabolism, and these functions are dynamically regulated during development with stage-specific characteristics.

			Action	DRS
	20231107_JST_AP.raw	Raw
	20231107_JST_DP.raw	Raw
	20240131_JST_Shi_WGA_1.raw	Raw
	20240131_JST_Shi_WGA_2.raw	Raw
	20240131_JST_Shi_WGA_3.raw	Raw

Dataset Information

Theileria parva Muguga (TpM) schizonts LC-MS/MS

Dataset's files

Publications

Investigation of carbohydrate-based molecules of <i>Theileria parva</i> parasites.

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Dataset Information

Theileria parva Muguga (TpM) schizonts LC-MS/MS

Dataset's files

Publications

Investigation of carbohydrate-based molecules of &lt;i&gt;Theileria parva&lt;/i&gt; parasites.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Investigation of carbohydrate-based molecules of <i>Theileria parva</i> parasites.