Mining the secreted and membrane transcriptome of Hyalomma dromedarii ticks for identification of potential protective antigens.

Background: Members belonging to the tick genus Hyalomma function as a multi-host reservoir for several pathogens and important parasites infesting large animals, such as camels, goats, cattle and sheep. In Egypt, there is a high risk of pathogen transmission as camels and cattle are imported from Sudan and Ethiopia and shipped to slaughterhouses and animal markets located in populated areas. Hyalomma dromedarii ticks are semi-desert vectors and, similar to other members of the genus Hyalomma, characterized by long-term feeding. During this process, different physiological, biochemical and immunological interactions occur within both the feeding ticks and their hosts. These biological changes affect the different tick developmental phases. The aim of this study was to explore the transcriptome of mixed messenger RNAs (mRNAs) collected from H. dromedarii eggs, larvae, nymphs and fed and unfed adults, using the Gateway cDNA library prepared in pCMV sport6.1 vector METHODS: The clones were sequenced and searched for potential secreted, membrane-associated or transmembrane (SMaT) sequences. The identified SMaT sequences were compared to the National Center for Biotechnology Information (NCBI) non-redundant protein sequence database using Blastx. Annotation and functional classification were achieved by comparison to sequences in the UniProtKB/Swiss-Prot and VectorBase databases and to the publicly available annotated proteomes of six hard tick species (H. asiaticum, Rhipicephalus sanguineus sensu lato, Dermacentor silvarum, Rhipicephalus microplus, Ixodes scapularis and Haemaphysalis longicornis) in addition to the published H. dromedarii sialotranscriptome. For the common sequences, we predicted the physicochemical properties, secondary structures and antigenicity of the fragments similar to matched sequences in the UniProtKB/Swiss-Prot database using three different methods.

Results: The quality-trimmed sequences from the cDNA library revealed 319 SMaT transcripts among 1248 sequenced clones. Annotation of the SMaT sequences using the UniProtKB/Swiss-Prot database revealed only 232 non-redundant sequences with at least one match. According to the UniProtKB/Swiss-Prot and Vectorbase databases, the SMaT sequences were either secreted (extracellular) (29 sequences) or cellular (transmembrane and membrane-associated) (203 sequences). These were classified into 10 functional classes: biogenesis (49 sequences), defense (9 sequences), development (36 sequences), signal transduction (28 sequences), transport (15 sequences), protein modification (33 sequences), homeostasis (6 sequences), metabolism (45 sequences) and miscellaneous/uncharacterized (11 sequences). A total of 60 sequences were shared between H. dromedarii SMaT, the sialotransciptome and six other hard tick species. The peptide fragments of these sequences that aligned to proteins from the UniProtKB/Swiss-Prot database were predicted to be promising epitopes and mapped to 10 functional classes at different ratios.

Conclusions: Our immuno-informatics analysis identified 60 sequences common among hard tick species and encoded by H. dromedarii salivary glands. These annotated SMaT sequences of H. dromedarii will pave the way for the identification and discovery of novel potential protective antigens that are either secreted, membrane-associated or transmembrane.