Mining gene expression data for rational identification of novel drug targets and vaccine candidates against the cattle tick, Rhipicephalus microplus

Abstract

Control of complex parasites via vaccination remains challenging, with the current combination of vaccines and small drugs remaining the choice for an integrated control strategy. Studies conducted to date, are providing evidence that multicomponent vaccines will be needed for the development of protective vaccines against endo- and ectoparasites, though multicomponent vaccines require an in-depth understanding of parasite biology which remains insufficient for ticks. With the rapid development and spread of acaricide resistance in ticks, new targets for acaricide development also remains to be identified, along with novel targets that can be exploited for the design of lead compounds. In this study, we analysed the differential gene expression of Rhipicephalus microplus ticks that were fed on cattle vaccinated with a multi-component vaccine (Bm86 and 3 putative Bm86-binding proteins). The data was scrutinised for the identification of vaccine targets, small drug targets and novel pathways that can be evaluated in future studies. Limitations associated with targeting novel proteins for vaccine and/or drug design is also discussed and placed into the context of challenges arising when targeting large protein families and intracellular localised proteins. Lastly, this study provide insight into how Bm86-based vaccines may reduce successful uptake and digestion of the bloodmeal and overall tick fecundity.

Keywords: Rhipicephalus microplus; Antigen selection; Bm86; DNA microarray; Drug target identification; Multicomponent vaccine.

Fig. 1

Antigen-specific antibody responses in cattle vaccinated with a four-antigen combinatorial vaccine (A–D). Indicated is total IgG serum antibody responses raised in vaccinated cattle against recombinant Bm86, RmAg1, RmAg2 and RmAg3 over a period of 2 months. Control animals were injected with a saline buffer solution formulated in Montanide ISA 71 VG adjuvant. Antibody responses are expressed as the OD 450 nm value for using a 1:100 (i.e., RmAg1, RmAg2 and RmAg3) and 1:4000 (i.e., Bm86) dilutions of serum collected at day 0, 42 and 56. Error bars indicate standard deviation of antibody responses between animals of the same group (n = 4), performed with technical replicates. Black arrows indicate the days of vaccination at day 1, day 28 and day 42. Red arrow indicates the day of infestation at day 55 (color figure online)

Fig. 2

Hierarchical clustering of differentially expressed transcripts based on normalised log₂FC- and log₂FC-values obtained from microarray and RT-qPCR analyses, respectively. Each block represents the log₂FC values of differentially expressed transcripts obtained from microarray analysis (row 1) and RT-qPCR analysis (row 2). Different colour represents difference in expression: Red: increased expression; Blue: decreased expression. Rho: degree of correlation between expression values from DNA microarray- and RT-qPCR analyses determined by Spearman’s rank-order correlation (color figure online)

Fig. 3

Proposed impact of vaccination on the ecdysteroid pathway in the midgut tissues of female cattle ticks. Ecdysteroid metabolism starts with the uptake of cholesterol and the synthesis of 20E. The enzymes (encoded for by the Halloween genes) needed for the synthesis of 20E is downregulated, and as such the 20E-mediated activation of transcription is suppressed resulting in the lack of expression of 20E effector genes such as vitellogin that impact egg production. Receptors such as the Nieman Pick proteins type C 1a (NPC1a) and a palmitoyltransferase ZDHHC17-like protein involved in lipoprotein transport and protein palmitoylation is upregulated to allow for uptake of cholesterol under stress from vaccination. The cyotoplasmic ecdysteroid receptor (EcR) remains present and does not translocate to the nucleus to activate transcription. To assemble the tranxription machinery, a number of components have been described in the 20E activated transcription complex. We propose that the activation of cytoplasmic components via signaling is disrupted in ticks fed on vaccinated animals. An example of a possible effect on ultraspiracle (USP) is shown

Fig. 4

Proposed pathway induced by oxidative stress in the midgut tissues of female cattle ticks feeding on vaccinated cattle hosts. Since important iron transport proteins (i.e., Ferritin-1 or FER-1) are down-regulated in the midguts of ticks feeding on vaccinated hosts, an increasing hypoxic environment results as oxygen is depleted by free ferric iron. Depleting oxygen levels causes the inhibition of a hypoxia-inducible prolyl hydroxylase 2 (PHD-2) enzyme, as well as a concomitant decrease in ubiquitin-mediated proteasomal degradation of the transcriptional regulator Hypoxia-inducible factor-1 alpha (HIF-1). Stabilized by Heat shock protein 90 (HSP90), HIF-1A translocates to the nucleus where it forms a bi-functional regulator with its beta subunit HIF-1B that binds to chromosomal hypoxia-induced response elements (HRE) to initiate transcription of targets that promote cellular adaptation to oxidative stress and hypoxia that include energy metabolism (e.g. enzymes and transporters) and cell survival (e.g. growth factors such as granulin or GRN). Asterisks indicates proteins that were identified in the transcriptome, but was not significantly differentially expressed. Green and red arrows indicate transcripts that were identified to be significantly up– and down-regulated, respectively. Targets for development of novel antiparasitic chemotherapeutics and vaccine targets are indicated by black squares and triangles, respectively (color figure online)