Transcriptomics reveal potential vaccine antigens and a drastic increase of upregulated genes during Theileria parva development from arthropod to bovine infective stages

Abstract

Theileria parva is a protozoan parasite transmitted by the brown ear tick Rhipicephalus appendiculatus that causes East Coast fever (ECF) in cattle, resulting in substantial economic losses in the regions of southern, eastern and central Africa. The schizont form of the parasite transforms the bovine host lymphocytes into actively proliferating cancer-like cells. However, how T. parva causes bovine host cells to proliferate and maintain a cancerous phenotype following infection is still poorly understood. On the other hand, current efforts to develop improved vaccines have identified only a few candidate antigens. In the present paper, we report the first comparative transcriptomic analysis throughout the course of T. parva infection. We observed that the development of sporoblast into sporozoite and then the establishment in the host cells as schizont is accompanied by a drastic increase of upregulated genes in the schizont stage of the parasite. In contrast, the ten highest gene expression values occurred in the arthropod vector stages. A comparative analysis showed that 2845 genes were upregulated in both sporozoite and schizont stages compared to the sporoblast. In addition, 647 were upregulated only in the sporozoite whereas 310 were only upregulated in the schizont. We detected low p67 expression in the schizont stage, an unexpected finding considering that p67 has been reported as a sporozoite stage-specific gene. In contrast, we found that transcription of p67 was 20 times higher in the sporoblast than in the sporozoite. Using the expression profiles of recently identified candidate vaccine antigens as a benchmark for selection for novel potential vaccine candidates, we identified three genes with expression similar to p67 and several other genes similar to Tp1-Tp10 schizont vaccine antigens. We propose that the antigenicity or chemotherapeutic potential of this panel of new candidate antigens be further investigated. Structural comparisons of the transcripts generated here with the existing gene models for the respective loci revealed indels. Our findings can be used to improve the structural annotation of the T. parva genome, and the identification of alternatively spliced transcripts.

Fig 1

A) Theileria parva life cycle stages (not drawn to scale). The tick and cattle stages (sporoblast, sporozoite and schizont) shadowed are the ones reported in the present study. The red line separates the bovine from the tick vector stages. B) Flow chart of the origin of the stabilate T. parva Muguga 3087 (stabilate 3087) used in this study (shadowed box). The cow F100 was infected with TpM 3087 to generate F100 TpM. The stabilate 4138 is the Muguga component of three stock components of the Muguga Cocktail ITM live vaccine. The date (day, month and year) and place of preparation are indicated where known. NVRC; National Veterinary Research Centre, Muguga, Kenya. CVL; Central Veterinary Laboratory, Lilongwe, Malawi.

Fig 2. Kallisto and TopHat2 comparison and similarity between samples.

A) Percentage of paired-end reads mapping to the reference transcriptome using Kallisto (blue line) and to the reference genome using TopHat2/Bowtie2 (orange line). B) Jensen-Shannon divergence (JSD) heatmap between each pair of samples.

Fig 3. Analysis of unmapped reads.

A) Count (>1000 showed only) of the Blast hits from unmapped reads after using Kallisto for the three infection stages. B) Count of Blast hits to T. parva genome (only targets with >1000 showed only). C) Heatmap showing differentially expressed genes (p-adj < 0.05) obtained from the unmapped reads for each infection stage.

Fig 4. A pairwise comparison of sporoblast against sporozoite and schizont stages.

2845 genes differentially expressed in sporozoite and schizont stages in comparison to sporoblast, plus 647 and 310 only differentially expressed relative to the sporozoite or the schizont stages, respectively.

Fig 5. Distribution of differentially expressed genes (p-adj < 0.01).

A). Total number of genes upregulated (coral) and downregulated (green) in the three life cycle stages of the parasite. B) Venn diagram comparing the upregulated genes in sporozoite and schizont to the downregulated genes in sporozoite. C) Venn diagram comparing the upregulated genes in sporoblast and sporozoite to the downregulated genes in schizont. D) List of four downregulated genes in the schizont stage of the parasite. TMD, transmembrane domain; SP, signal peptide; (-), none.

Fig 6. Heatmap showing the log₁₀ (TPM values) for enriched GO terms enriched in the upregulated genes in the different infection stages obtained from DAVID.

Fig 7. Indels identified in the transcripts.

Multiple nucleotide and amino acid sequences alignments of T. parva gene regions with indels using the GT-AG dinucleotides DNA sequence requirement at the first two (GT) and last two (AG) positions of introns in pre-mRNAs (highlighted in bold). (^a) indicates difference in both original genome annotation and re-annotation; # indicates introns not in-frame.

Fig 8. PCR verification of deletion (TP04_0272) and insertion (TP01_0193) identified by MiSeq.

Samples are as follows: Lanes 1–4, genomic DNA from T. parva strain Muguga 3087 (lane 1); T. parva strain Marikebuni 3292 (lane 2); T. parva strain Kiambu5 (lane 3); T. parva F100 TpM (lane 4); Lanes 5–8, mRNA from sporozoite of T. parva Muguga 3087 (lanes 5, 5a and 5b); mRNA schizont T. parva Muguga 3087 (lane 6); schizont of T. parva Muguga 3087 (lane 7); piroplasm of T. parva Muguga 3087 (lane 8). Also shown are DNA size markers (lanes M), with length in base pair (pb).

Fig 9. Heatmap showing the log₁₀ of normalized TPM values for known Theileria parva antigens in samples from three parasite life cycle stages.

Yellowish is high expression and red is low expression. Selected known candidate vaccine antigens are listed.

Fig 10. Clusters of expression (TPM) for known Theileria parva antigens.

The genes with similar expression to the known antigens are shown in each plot legend.