Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites

Abstract

Background: Vector-borne apicomplexan parasites are a major cause of mortality and morbidity to humans and livestock globally. The most important disease syndromes caused by these parasites are malaria, babesiosis and theileriosis. Strategies for control often target parasite stages in the mammalian host that cause disease, but this can result in reservoir infections that promote pathogen transmission and generate economic loss. Optimal control strategies should protect against clinical disease, block transmission and be applicable across related genera of parasites. We have used bioinformatics and transcriptomics to screen for transmission-blocking candidate antigens in the tick-borne apicomplexan parasite, Theileria annulata.

Results: A number of candidate antigen genes were identified which encoded amino acid domains that are conserved across vector-borne Apicomplexa (Babesia, Plasmodium and Theileria), including the Pfs48/45 6-cys domain and a novel cysteine-rich domain. Expression profiling confirmed that selected candidate genes are expressed by life cycle stages within infected ticks. Additionally, putative B cell epitopes were identified in the T. annulata gene sequences encoding the 6-cys and cysteine rich domains, in a gene encoding a putative papain-family cysteine peptidase, with similarity to the Plasmodium SERA family, and the gene encoding the T. annulata major merozoite/piroplasm surface antigen, Tams1.

Conclusions: Candidate genes were identified that encode proteins with similarity to known transmission blocking candidates in related parasites, while one is a novel candidate conserved across vector-borne apicomplexans and has a potential role in the sexual phase of the life cycle. The results indicate that a 'One Health' approach could be utilised to develop a transmission-blocking strategy effective against vector-borne apicomplexan parasites of animals and humans.

Keywords: 6-Cys domain; Babesia; Bioinformatic screen; Plasmodium; Theileria annulata; Transmission-blocking vaccine.

Fig. 1

Gene expression profile of 12 transmission-blocking candidates. Microarray expression profiles (a), (b) and (c) of T. annulata candidate genes in sporozoite, schizont, merozoite Day 4, Day 7, Day 9 and piroplasm. Expression is depicted on a log₂ scale

Fig. 2

Protein alignments with related Apicomplexan genera. (a) Alignment of the conserved papain family cysteine protease domain of Serine Repeat Antigen (SERA)-like Proteins from : P. falciparum (PF3D7_0207600), P. reichenowi (PRCDC_0206900), T. orientalis (TOT_040000333), T. annulata (TA10995), T. parva (TP04_0598). (b) Alignment of the highly conserved s48-45 superfamily 6-cysteine domain from sequences of TA03640 homologues with conserved cysteine residues in green: P. vivax (PVP01_113600), P. yoellii (PYO3100), P. chabaudi (PCHAS_0111600), T. annulata (TA03640), T. parva (TP03_0268), T. orientalis (TOT_030000578), B. bigemina (BBBOND_0402900). (c) Alignment of the highly conserved 8-cysteine domain region of (revised) TA20855 homologues with predicted signal peptides (blue), transmembrane helices (red) and cysteine residues (green) highlighted: V. brassicaformis (VBRA_17621), T. gondii (TGME49_321580), P. falciparum (PF3D7_1322900) T. annulata_revised (TA20855), B. bovis (BBOV_IV006060), B. bigemina (BBBOND_0208520)

Fig. 3

qRT-PCR of candidate genes in tick stages. Quantitative RT-PCR expression analysis of RNA from T. annulata infected ticks generated at Day 2, Day 6, Day 10 and Day 15 post-detachment, relative to T. annulata merozoite Day 8 (calibrator) for: Tams1 (TA17050); putative papain-family cysteine protease (TA10955); Pfs 48/45 6-cys domain encoding gene TA03640 and 6-Cys like gene TA20855. * above (positive) or below (negative) error bars denote degrees of significant difference (* p < 0.05, ** p < 0.01, *** p < 0.0001) between fold-change at a time-point relative to merozoite calibrator RNA. Expression is depicted on a log₂ scale

Fig. 4

Diversifying selection and B cell epitopes in candidate genes. dN/dS computed from allelic sequences of (a) TA17050; (b) TA10955; (c) TA03640 and (d) TA20855, plotted against predicted B cell epitope regions (>1 amino acid, shaded areas) from GeneDB reference sequences for each protein. Epitopes with evidence of positive selection (peaks above 0) are shaded yellow, epitopes with no evidence of positive selection are shaded grey. For TA20855 and TA10955, but not TA17050 or TA03640, there was a statistically significant association between predicted epitopes and positive dN/dS, reflected in the figure by more yellow shaded areas, relative to grey