Final Report Summary - RICYSTVACANT2010 (Exploring the salivary transcriptome of Ixodes ricinus, the Lyme disease vector in Europe, and the potential role of its cystatins in pathogen transmission) Ixodes ricinus abundance and its infection prevalence with Borrelia burgdorferi s.l. (the Lyme disease agent) have led to an increased risk of human exposure to tick bites and Borrelia infection in Europe. Tick salivary secretion assists pathogen transmission and tick salivary constituents have been proposed as a target for anti-tick vaccine development. The first objective of the project was to identify with a high-throughput approach transcripts expressed in the salivary glands of I. ricinus ticks. Prior to this project, there was a limited knowledge of I. ricinus genes expressed in the pathogen transmission interface of tick salivary glands due to the increased cost of classical sequencing methods for cDNA sequencing projects (Expressed Sequence Tags-ESTs). Also in all the cases the interplay of the ticks with the different hosts they parasitize during their complex lifecycle was not taken into consideration upon experimental design. Our work drastically improved our knowledge of I. ricinus salivary transcripts by considering the entire complex tick lifecycle and by performing massive Next Generation Sequencing (454 and Illumina pyrosequencing), a relatively novel, highly efficient and cost effective technology for the de novo sequencing and assembly of transcriptomes originating from organisms with an unknown genome (such as I. ricinus). As a result, more than 27,500 transcript sequences were deposited in the GenBank as originating from the tick I.ricinus making this tick one of the best covered as far as it concerns its gene expression description at the transcriptional level. Based on our innovative experimental methodology we now know the transcription patterns of all 27,500 genes as far as it concerns their tissue and developmental stage specificity as well as a function of tick feeding time. Moreover, the constructed sequence database (transcriptome database) provided the necessary support to identify approximately 1500 tick proteins found in the pathogen transmission interface by employing a cutting edge proteomic methodology and platform (in collaboration with the University of Mainz, Germany). Of note, all the produced data are publicly available, thus providing the community with the necessary gene expression information to support additional research projects targeting the specific tick and its pathogen-transmission lifecycle. The second objective of the project was the description, cloning and functional characterization of cystatin genes from I. ricinus salivary glands. Accordingly, and in parallel with the transcriptomic/proteomic project, library screens were carried out to identify I. ricinus secreted cystatins because cystatins were proposed -based on our previous work- as 'silent' antigens for the development of anti-tick vaccines. Fifteen I. ricinus cystatin genes were cloned and their tissue specificity was estimated with Real Time Quantitative PCR as a function of tick feeding time. While animal vaccination experiments did not reveal any contribution of the two tested cystatins in tick feeding success, four recombinant cystatins were successfully overexpressed in a prokaryotic system and subsequently 1) biochemically characterized for their inhibitory effect on different cysteine proteases and 2) tested for their role in vertebrate host immunomodulation. The preliminary pharmacological characterization of the recombinant I. ricinus cystatins suggests their immunomodulatory function as well as the potential of one I. ricinus cystatin to ameliorate the symptoms of experimental asthma in mice. Accordingly, we currently explore the possibility of a patent application to protect the pharmacological properties of the characterized cystatin(s).