Comparative Transcriptomics of the Bovine Apicomplexan Parasite Theileria parva Developmental Stages Reveals Massive Gene Expression Variation and Potential Vaccine Antigens

Abstract

Theileria parva is a protozoan parasite that causes East Coast fever (ECF), an economically important disease of cattle in Africa. It is transmitted mainly by the tick Rhipicephalus appendiculatus. Research efforts to develop a subunit vaccine based on parasite neutralizing antibodies and cytotoxic T-lymphocytes have met with limited success. The molecular mechanisms underlying T. parva life cycle stages in the tick vector and bovine host are poorly understood, thus limiting progress toward an effective and efficient control of ECF. Transcriptomics has been used to identify candidate vaccine antigens or markers associated with virulence and disease pathology. Therefore, characterization of gene expression throughout the parasite's life cycle should shed light on host-pathogen interactions in ECF and identify genes underlying differences in parasite stages as well as potential, novel therapeutic targets. Recently, the first gene expression profiling of T. parva was conducted for the sporoblast, sporozoite, and schizont stages. The sporozoite is infective to cattle, whereas the schizont is the major pathogenic form of the parasite. The schizont can differentiate into piroplasm, which is infective to the tick vector. The present study was designed to extend the T. parva gene expression profiling to the piroplasm stage with reference to the schizont. Pairwise comparison revealed that 3,279 of a possible 4,084 protein coding genes were differentially expressed, with 1,623 (49%) genes upregulated and 1,656 (51%) downregulated in the piroplasm relative to the schizont. In addition, over 200 genes were stage-specific. In general, there were more molecular functions, biological processes, subcellular localizations, and pathways significantly enriched in the piroplasm than in the schizont. Using known antigens as benchmarks, we identified several new potential vaccine antigens, including TP04_0076 and TP04_0640, which were highly immunogenic in naturally T. parva-infected cattle. All the candidate vaccine antigens identified have yet to be investigated for their capacity to induce protective immune response against ECF.

Keywords: Theileria parva; piroplasm; schizont; transcriptome; vaccine antigens.

Figure 1

Clustering analysis of samples. (A) Samples' distance matrix of replicates clustering schizont and piroplasm replicates according to the life cycle stages. (B) Principal component analysis (PCA) of the replicates showing a variance of 99% between the schizont (blue) and piroplasm replicates (red).

Figure 2

Differential gene expression between schizont and piroplasm stages using Bioconductor EnhancedVolcano package in R. The graph shows the distribution of the upregulated genes between schizont (blue dots) and piroplasm (red dots) stages with the exception of the non-differentially expressed or unchanged genes (gray dots). Each dot in the EnhancedVolcano diagram represents one differentially expressed gene. The x-axis represents the Log2 fold change, and the y-axis shows the –Log10 adjusted P-value.

Figure 3

Gene expression profile in the schizont and piroplasm life cycle stages. (A) Heatmap of all the differentially expressed genes. (B) Heatmap of the top 20 most variable genes between schizont replicates and piroplasm replicates. The genes were clustered into two groups: from genes XM_760801.1 to XM_757627.1 where genes were upregulated in piroplasm and downregulated in schizont, and from XM_757604.1 to XM_758863.1 where genes where upregulated in schizont and downregulated in piroplasm. The genes were clustered by hierarchical clustering within each horizontal partition and replicates for the infection stages within each vertical partition by similarity. Color red denotes high expression, yellow denotes stable expression, and blue denotes low expression. The colors are scaled per row.

Figure 4

Gene Ontology (GO) functional classification for differentially expressed genes in the two developmental stages studied. (A) Molecular function category. (B) Biological process category. (C) Subcellular localization. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) database of the categorized genes downregulated in schizont and upregulated piroplasm. Genes without ontology information are not included. The proportion number of each GO and KEGG term of schizont and piroplasm is, respectively, presented in the parentheses in front of the GO and KEGG terms. Red color indicates most enriched (2), and blue, no enrichment (0). The expression level is color coded: red for over-expressed, white for unchanged. (E) Pathway map of SNARE interactions in vesicular transport. This pathway is annotated with most of the hypothetical proteins among all the referred pathways. The arrows in the pathway indicate the molecular interaction or reaction. The green boxes are hyperlinked to identified genes (encoded proteins) involved in the reaction, and the green box with a red star is hyperlinked to the hypothetical proteins involved in the pathway. Schi represents schizont stage, and Piro, piroplasm.

Figure 5

Genes with expression profiles similar to known Theileria parva antigens' genes. Sporoblast, sporozoite, and schizont_a are transcriptome data collected from our previous work (16), whereas schizont_b and piroplasm are transcriptome data generated from this study. Each line in a unique color represents a specific gene with its GenBank accession number. Each plot represents the profiling of genes (GenBank accession number listed) having a similar expression profile to the known antigen (top of the plot).

Figure 6

Analysis of unmapped reads. (A) Count of BLAST hits to unmapped reads. (B) Counts of BLAST hits of unmapped reads to T. parva genome.

Figure 7

Amino acid sequences of the recombinant fusion protein TP04_0076F. The fusion protein contains 260 aa (single letter peptide) with the vector portion underlined and the 6x His tag used for affinity purification shadowed. The TP04_0076 portion is bolded, and the methionine (M) start codon was not included in the fusion protein.

Figure 8

Enzyme-linked immunosorbent assay (ELISA) evaluation of the antigenicity of (A) TP04_0076 gene product (recombinant protein TP04_0076F) and predicted antibody target epitopes of TP04_0640 and TP04_0076 gene products (synthetic peptides (B) TP04_0076ep2, (C) TP04_0640ep1, and (D) TP04_0640ep2). A panel of 34 predetermined bovine positive controls with strong percent positivity (PP ≥ 80) to PIM antigen (red line) were included in the analysis. The PP results of each of the four recombinant proteins and synthetic peptides tested (blue line) are shown in separated panels. The green line represents the cutoff, and an assay with a PP ≥ 20 (PP ≥ 20) is considered positive.