BACKGROUND: In recent years, there have been several sialome projects revealing transcripts expressed in the salivary glands of ticks, which are important vectors of several human diseases. Here, we focused on the sialome of the European vector of Lyme disease, Ixodes ricinus. RESULTS: In the attempt to describe expressed genes and their dynamics throughout the feeding period, we constructed cDNA libraries from four different feeding stages of Ixodes ricinus females: unfed, 24 hours after attachment, four (partially fed) and seven days (fully engorged) after attachment. Approximately 600 randomly selected clones from each cDNA library were sequenced and analyzed. From a total 2304 sequenced clones, 1881 sequences forming 1274 clusters underwent subsequent functional analysis using customized bioinformatics software. Clusters were sorted according to their predicted function and quantitative comparison among the four libraries was made. We found several groups of over-expressed genes associated with feeding that posses a secretion signal and may be involved in tick attachment, feeding or evading the host immune system. Many transcripts clustered into families of related genes with stage-specific expression. Comparison to Ixodes scapularis and I. pacificus transcripts was made. CONCLUSION: In addition to a large number of homologues of the known transcripts, we obtained several novel predicted protein sequences. Our work contributes to the growing list of proteins associated with tick feeding and sheds more light on the dynamics of the gene expression during tick feeding. Additionally, our results corroborate previous evidence of gene duplication in the evolution of ticks.

• MeSH
• Arachnid Vectors genetics   metabolism   MeSH
• DNA Primers genetics   MeSH
• Expressed Sequence Tags MeSH
• Phylogeny MeSH
• Gene Library MeSH
• Ixodes genetics   metabolism   MeSH
• DNA, Complementary genetics   MeSH
• Molecular Sequence Data MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Salivary Proteins and Peptides chemistry   genetics   MeSH
• Saliva metabolism   MeSH
• Gene Expression Profiling MeSH
• Computational Biology MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• DNA Primers MeSH
• DNA, Complementary MeSH
• Salivary Proteins and Peptides MeSH

Ticks salivate while feeding on their hosts. Saliva helps blood feeding through host anti-hemostatic and immunomodulatory components. Previous transcriptomic and proteomic studies revealed the complexity of tick saliva, comprising hundreds of polypeptides grouped in several multi-genic families such as lipocalins, Kunitz-domain containing peptides, metalloproteases, basic tail secreted proteins, and several other families uniquely found in ticks. These studies also revealed that the composition of saliva changes with time; expression of transcripts from the same family wax and wane as a function of feeding time. Here, we examined whether host immune factors could influence sialome switching by comparing sialomes of ticks fed naturally on a rabbit, to ticks artificially fed on defibrinated blood depleted of immune components. Previous studies were based on transcriptomes derived from pools of several individuals. To get an insight into the uniqueness of tick sialomes, we performed transcriptomic analyses of single salivary glands dissected from individual adult female I. ricinus ticks. Multivariate analysis identified 1,279 contigs differentially expressed as a function of time and/or feeding mode. Cluster analysis of these contigs revealed nine clusters of differentially expressed genes, four of which appeared consistently across several replicates, but five clusters were idiosyncratic, pointing to the uniqueness of sialomes in individual ticks. The disclosure of tick quantum sialomes reveals the unique salivary composition produced by individual ticks as they switch their sialomes throughout the blood meal, a possible mechanism of immune evasion.

• MeSH
• Ixodes genetics   metabolism   MeSH
• Rabbits MeSH
• Humans MeSH
• Sequence Analysis, RNA MeSH
• Salivary Glands metabolism   MeSH
• Saliva metabolism   MeSH
• Gene Expression Profiling MeSH
• Transcriptome * MeSH
• Computational Biology MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In the next generation sequencing era we are encountering hundreds of thousands of sequences from specific organisms. Such massive data must be accurately classified both functionally and structurally. Determining appropriate sequences with a specific function from next generation sequencing, however, is a daunting experimental task. A recent salivary gland transcriptome from the hard tick Ixodes ricinus, a European disease vector, has been made publicly available. Among the protein families sequenced by the salivary gland transcriptome of I. ricinus, the Kunitz-domain is one of the highly represented protein families. Thus far, recent tick transciptomes solely classify (computationally) Kunitz sequences as putative serine protease inhibitors. We present here a novel method using a machine-learning algorithm to "fish" for candidate ion-channel effectors and loss of serine protease inhibitor function within the Kunitz-domain protein family of the I. ricinus salivary gland transcriptome. The models, data and scripts used in this work are available online from http://life.bsc.es/pid/web/imoal/kunitz-classification.html.

• Keywords
• Function prediction, Kunitz, Machine learning, Tick, Transcriptome,
• MeSH
• Algorithms MeSH
• Protease Inhibitors MeSH
• Ion Channels genetics   MeSH
• Ixodes genetics   MeSH
• Protein Domains MeSH
• Arthropod Proteins genetics   MeSH
• Amino Acid Sequence MeSH
• Sequence Analysis, DNA MeSH
• Cluster Analysis MeSH
• Salivary Proteins and Peptides genetics   MeSH
• Transcriptome * MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protease Inhibitors MeSH
• Ion Channels MeSH
• Arthropod Proteins MeSH
• Salivary Proteins and Peptides MeSH

'Omics' technologies have facilitated the identification of hundreds to thousands of tick molecules that mediate tick feeding and play a role in the transmission of tick-borne diseases. Deep sequencing methodologies have played a key role in this knowledge accumulation, profoundly facilitating the study of the biology of disease vectors lacking reference genomes. For example, the nucleotide sequences of the entire set of tick salivary effectors, the so-called tick 'sialome', now contain at least one order of magnitude more transcript sequences compared to similar projects based on Sanger sequencing. Tick feeding is a complex and dynamic process, and while the dynamic 'sialome' is thought to mediate tick feeding success, exactly how transcriptome dynamics relate to tick-host-pathogen interactions is still largely unknown. The identification and, importantly, the functional analysis of the tick 'sialome' is expected to shed light on this 'black box'. This information will be crucial for developing strategies to block pathogen transmission, not only for anti-tick vaccine development but also the discovery and development of new, pharmacologically active compounds for human diseases.

• Keywords
• proteome, salivary gland, sialome, tick, transcriptome,
• MeSH
• Genome physiology   MeSH
• Host-Pathogen Interactions MeSH
• Ticks genetics   physiology   MeSH
• Humans MeSH
• Proteomics * MeSH
• Salivary Glands physiology   MeSH
• Transcriptome physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Tick-borne diseases pose a global medical problem. As transmission of tick-borne pathogens to their hosts occurs during tick feeding, development of vaccines thwarting this process could potentially prevent transmission of multiple tick-borne pathogens. The idea of tick vaccines is based on the phenomenon of acquired tick immunity, rejection of ticks feeding on hosts which were repeatedly infested by ticks. Recently, we demonstrated that saliva of the blacklegged tick Ixodes scapularis, which is the main vector of tick-borne pathogens in northeast USA, is sufficient for induction of tick immunity in the guinea pig model and that immunity directed against tick glycoproteins is important in this phenomenon. Nevertheless, immunity elicited against individual tick salivary antigens, which have been identified and tested so far, provided only modest tick rejection. We therefore now tested fractions of tick saliva produced by liquid chromatography for their ability to induce tick immunity in the guinea pig model. Immunization with all individual fractions elicited antibodies that reacted with tick saliva, however only some fractions displayed the ability to induce robust protective tick immunity. Mass spectrometry analysis led to identification of 24 proteins present only in saliva fractions which were able to induce tick immunity, suggesting suitable candidates for development of a tick vaccine.

• Keywords
• Fractionation, Ixodes scapularis, Saliva, Sialome, Tick, Vaccine,
• MeSH
• Chromatography, Liquid MeSH
• Glycoproteins MeSH
• Ixodes * MeSH
• Guinea Pigs MeSH
• Saliva MeSH
• Animals MeSH
• Check Tag
• Guinea Pigs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Glycoproteins MeSH

Tick saliva facilitates tick feeding and infection of the host. Gene expression analysis of tick salivary glands and other tissues involved in host-pathogen interactions has revealed a wide range of bioactive tick proteins. Transcriptomic analysis has been a milestone in the field and has recently been enhanced by next-generation sequencing (NGS). Furthermore, the application of quantitative proteomics to ticks with unknown genomes has provided deeper insights into the molecular mechanisms underlying tick hematophagy, pathogen transmission, and tick-host-pathogen interactions. We review current knowledge on the transcriptomics and proteomics of tick tissues from a systems-biology perspective and discuss future challenges in the field.

• Keywords
• next-generation sequencing, sialomes, systems biology, tick-borne pathogens,
• MeSH
• Host-Parasite Interactions genetics   physiology   MeSH
• Ticks genetics   metabolism   MeSH
• Humans MeSH
• Proteome * MeSH
• Systems Biology * MeSH
• Transcriptome * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Proteome * MeSH

The publication of the first tick sialome (salivary gland transcriptome) heralded a new era of research of tick protease inhibitors, which represent important constituents of the proteins secreted via tick saliva into the host. Three major groups of protease inhibitors are secreted into saliva: Kunitz inhibitors, serpins, and cystatins. Kunitz inhibitors are anti-hemostatic agents and tens of proteins with one or more Kunitz domains are known to block host coagulation and/or platelet aggregation. Serpins and cystatins are also anti-hemostatic effectors, but intriguingly, from the translational perspective, also act as pluripotent modulators of the host immune system. Here we focus especially on this latter aspect of protease inhibition by ticks and describe the current knowledge and data on secreted salivary serpins and cystatins and their role in tick-host-pathogen interaction triad. We also discuss the potential therapeutic use of tick protease inhibitors.

• Keywords
• cystatins, immunomodulation, protease inhibitors, serpins, tick-host interaction,
• MeSH
• Cystatins physiology   therapeutic use   MeSH
• Immunomodulation MeSH
• Protease Inhibitors classification   metabolism   therapeutic use   MeSH
• Serine Proteinase Inhibitors physiology   therapeutic use   MeSH
• Host-Parasite Interactions MeSH
• Ticks metabolism   MeSH
• Humans MeSH
• Serpins physiology   therapeutic use   MeSH
• Saliva enzymology   metabolism   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Cystatins MeSH
• Protease Inhibitors MeSH
• Serine Proteinase Inhibitors MeSH
• Serpins MeSH

BACKGROUND: Triatoma infestans is the main vector of Chagas disease in South America. As in all hematophagous arthropods, its saliva contains a complex cocktail that assists blood feeding by preventing platelet aggregation and blood clotting and promoting vasodilation. These salivary components can be immunologically recognized by their vector's hosts and targeted with antibodies that might disrupt blood feeding. These antibodies can be used to detect vector exposure using immunoassays. Antibodies may also contribute to the fast evolution of the salivary cocktail. METHODOLOGY: Salivary gland cDNA libraries from nymphal and adult T. infestans of breeding colonies originating from different locations (Argentina, Chile, Peru and Bolivia), and cDNA libraries originating from F1 populations of Bolivia, were sequenced using Illumina technology. Coding sequences (CDS) were extracted from the assembled reads, the numbers of reads mapped to these CDS, sequences were functionally annotated and polymorphisms determined. MAIN FINDINGS/SIGNIFICANCE: Over five thousand CDS, mostly full length or near full length, were publicly deposited on GenBank. Transcripts that were over 10-fold overexpressed from different geographical regions, or from different developmental stages were identified. Polymorphisms were mapped to derived coding sequences, and found to vary between developmental instars and geographic origin of the biological material. This expanded sialome database from T. infestans should be of assistance in future proteomic work attempting to identify salivary proteins that might be used as epidemiological markers of vector exposure, or proteins of pharmacological interest.

• MeSH
• Gene Library * MeSH
• Salivary Proteins and Peptides genetics   metabolism   MeSH
• Saliva chemistry   MeSH
• Transcriptome genetics   MeSH
• Triatoma genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Geographicals
• South America MeSH
• Names of Substances
• Salivary Proteins and Peptides MeSH

BACKGROUND: Malaria is a devastating infectious disease caused by Plasmodium parasites transmitted through the bites of infected Anopheles mosquitoes. Salivary glands are the only mosquito tissue invaded by Plasmodium sporozoites, being a key stage for the effective parasite transmission, making the study of Anopheles sialome highly relevant. METHODS: RNA-sequencing was used to compare differential gene expression in salivary glands of uninfected and Plasmodium berghei-infected Anopheles coluzzii mosquitoes. RNA-seq results were validated by quantitative RT-PCR. The transmembrane glucose transporter gene AGAP007752 was selected for functional analysis by RNA interference. The effect of gene silencing on infection level was evaluated. The putative function and tertiary structure of the protein was assessed. RESULTS: RNA-seq data showed that 2588 genes were differentially expressed in mosquitoes salivary glands in response to P. berghei infection, being 1578 upregulated and 1010 downregulated. Metabolism, Immunity, Replication/Transcription/Translation, Proteolysis and Transport were the mosquito gene functional classes more affected by parasite infection. Endopeptidase coding genes were the most abundant within the differentially expressed genes in infected salivary glands (P < 0.001). Based on its putative function and expression level, the transmembrane glucose transporter gene, AGAP007752, was selected for functional analysis by RNA interference. The results demonstrated that the number of sporozoites was 44.3% lower in mosquitoes fed on infected mice after AGAPP007752 gene knockdown when compared to control (P < 0.01). CONCLUSIONS: Our hypothesis is that the protein encoded by the gene AGAPP007752 may play a role on An. coluzzii salivary glands infection by Plasmodium parasite, working as a sporozoite receptor and/or promoting a favorable environment for the capacity of sporozoites.

• MeSH
• Anopheles physiology   MeSH
• Host-Pathogen Interactions MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Mice MeSH
• Plasmodium berghei growth & development   MeSH
• Glucose Transport Proteins, Facilitative antagonists & inhibitors   MeSH
• Receptors, Cell Surface antagonists & inhibitors   MeSH
• Sequence Analysis, RNA MeSH
• Salivary Glands parasitology   MeSH
• Gene Expression Profiling * MeSH
• Gene Silencing MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glucose Transport Proteins, Facilitative MeSH
• Receptors, Cell Surface MeSH

During feeding on vertebrate hosts, ticks secrete saliva composed of a rich cocktail of bioactive molecules modulating host immune responses. Although most of the proteinaceous fraction of tick saliva is of little immunogenicity, repeated feeding of ticks on mammalian hosts may lead to impairment of tick feeding, preventing full engorgement. Here, we challenged rabbits with repeated feeding of both Ixodes ricinus nymphs and adults and observed the formation of specific antibodies against several tick salivary proteins. Repeated feeding of both I. ricinus stages led to a gradual decrease in engorged weights. To identify the salivary antigens, isolated immunoglobulins from repeatedly infested rabbits were utilized for a protein pull-down from the saliva of pilocarpine-treated ticks. Eluted antigens were first identified by peptide mass fingerprinting with the aid of available I. ricinus salivary gland transcriptomes originating from early phases of tick feeding. To increase the authenticity of immunogens identified, we also performed, for the first time, de novo assembly of the sialome from I. ricinus females fed for six days, a timepoint used for pilocarpine-salivation. The most dominant I. ricinus salivary immunogens identified in our study were zinc-dependent metalloproteases of three different families. To corroborate the role of metalloproteases at the tick/host interface, we fed ticks micro-injected with a zinc metalloprotease inhibitor, phosphoramidon, on a rabbit. These ticks clearly failed to initiate feeding and to engorge. However, neither feeding to ticks immune blood of repeatedly infested rabbits, nor phosphoramidon injection into ticks, prevented their engorgement when fed in vitro on an artificial membrane system. These data show that Zn metalloproteases play a decisive role in the success of tick feeding, mediated by complex molecular interactions between the host immune, inflammatory, and hemostatic processes, which are absent in in vitro feeding. This basic concept warrants further investigation and reconsideration of the current strategies towards the development of an effective "anti-tick" vaccine.

• Keywords
• Ixodes ricinus, acquired resistance, antigen, immunoprecipitation, metalloprotease, ticks, vaccine,
• MeSH
• Tick Infestations * MeSH
• Ixodes * MeSH
• Rabbits MeSH
• Metalloproteases MeSH
• Arthropod Proteins MeSH
• Salivary Proteins and Peptides MeSH
• Salivary Glands MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Metalloproteases MeSH
• Arthropod Proteins MeSH
• Salivary Proteins and Peptides MeSH