An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans.

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.

Isolation and electrophysiological recording of Ixodes ricinus synganglion neurons.

The central nervous system of hard ticks (Ixodidae) consists of a concentrated merged nerve mass known as the synganglion. Although knowledge of tick neurobiology has dramatically improved over the last two decades, this is the first time that isolation and electrophysiological recordings have been carried out on tick neurons from the synganglion. Method: We developed a simple protocol for synganglion neuron isolation and used a whole-cell patch clamp to measure ionic currents induced by acetylcholine, nicotine and muscarine. Relatively large neurons (∼ 25 µm and âˆ¼ 35 µm) were isolated and 1 mM acetylcholine was used to induce strong inward currents of -0.38 ± 0.1 nA and - 1.04 ± 0.1 nA, respectively, with the corresponding cell capacitances being at around 142 pF and 188 pF. In addition, successive application of 1 mM acetylcholine through ∼25 µm and âˆ¼ 35 µm cells for increasing amounts of time resulted in a rapid reduction in current amplitudes. We also found that acetylcholine-evoked currents were associated with a reversible increase in intracellular calcium levels for each neuronal type. In contrast, 1 mM muscarine and nicotine induced a strong and non-reversible increase in intracellular calcium levels. This study serves as a proof of concept for the mechanical isolation of tick synganglion neurons followed by their electrophysiological recording. This approach will aid investigations into the pharmacological properties of tick neurons and provides the tools needed for the identification of drug-targeted sites and effective tick control measures.

Structural Analysis of the Black-Legged Tick Saliva Protein Salp15.

Salp15 is one of the proteins in the saliva of the tick Ixodes scapularis. Together with other biomolecules injected into the mammalian host at the biting site, it helps the tick to sustain its blood meal for days. Salp15 interferes with the cellular immune response of the mammalian host by inhibiting the activation of CD4+ T-lymphocytes. This function is co-opted by pathogens that use the tick as a vector and invade the host when the tick bites, such as Borrelia burgdorferi, the causative agent of Lyme borreliosis. Because of the immunity-suppressing role of Salp15, it has been proposed as a candidate for therapeutic applications in disorders of the immune system. The protein is produced as a 135-residue long polypeptide and secreted without its N-terminal signal 1-21 sequence. Detailed structural studies on Salp15 are lacking because of the difficulty in producing large amounts of the folded protein. We report the production of Salp15 and its structural analysis by NMR. The protein is monomeric and contains a flexible N-terminal region followed by a folded domain with mixed α + ß secondary structures. Our results are consistent with a three-dimensional structural model derived from AlphaFold, which predicts the formation of three disulfide bridges and a free C-terminal cysteine.

Structural and biochemical characterization of the novel serpin Iripin-5 from Ixodes ricinus.

Iripin-5 is the main Ixodes ricinus salivary serpin, which acts as a modulator of host defence mechanisms by impairing neutrophil migration, suppressing nitric oxide production by macrophages and altering complement functions. Iripin-5 influences host immunity and shows high expression in the salivary glands. Here, the crystal structure of Iripin-5 in the most thermodynamically stable state of serpins is described. In the reactive-centre loop, the main substrate-recognition site of Iripin-5 is likely to be represented by Arg342, which implies the targeting of trypsin-like proteases. Furthermore, a computational structural analysis of selected Iripin-5-protease complexes together with interface analysis revealed the most probable residues of Iripin-5 involved in complex formation.

Mialostatin, a Novel Midgut Cystatin from Ixodes ricinus Ticks: Crystal Structure and Regulation of Host Blood Digestion.

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.

Identification and biochemical characterisation of a novel methionine aminopeptidase from the taiga tick Ixodes persulcatus.

Methionine aminopeptidases (MetAPs), which remove the initiator methionine from nascent peptides, are essential in all organisms and considered to be a valuable targets for the treatment of various diseases, including cancer, malaria, and bacterial infections. However, MetAPs have not been reported in hard ticks (family Ixodidae), and their bioinformatics characterisation in tick's genome sequences is limited. In this study, we cloned, identified, and characterised a novel MetAP from Ixodes persulcatus, a vector for pathogens causing Lyme borreliosis and tick-borne encephalitis. The sequence analysis showed that I. persulcatus MetAP was a type 1 enzyme carrying C-terminal motifs conserved in the M24A family of metallopeptidases. Protein-protein docking simulations using human MetAP revealed conservation of substrate and metal-binding residues in the catalytic site cleft of the novel enzyme, which was designated IpMetAP. Recombinant IpMetAP expressed in Escherichia coli revealed its significant enzymatic activity with the synthetic substrate H-Met-4-methyl-coumaryl-7-amide at pH 7.5 with Km of 0.014 mM, kcat of 0.25 s-1, and overall catalytic efficiency (kcat/Km) of 18.36 mM-1 s-1. The activity of IpMetAP was enhanced by the addition of divalent cations Mn2+ and Co2+ and significantly inhibited by EDTA and bestatin. Site-directed mutagenesis of conserved amino acids indicated that the substitution of metal-binding residues D226 and H288 completely abolished the IpMetAP enzymatic activity, whereas that of the other sites had only moderate effects on substrate hydrolysis. The catalytic properties of IpMetAP suggest that the enzyme behaves similar to other MetAPs and such characterization expands our knowledge of aminopeptidases and protein metabolism of tick.

BBE31 from the Lyme disease agent Borrelia burgdorferi, known to play an important role in successful colonization of the mammalian host, shows the ability to bind glutathione.

Lyme disease is a tick-borne infection caused by Borrelia burgdorferi sensu lato complex spirochetes. The spirochete is located in the gut of the tick; as the infected tick starts the blood meal, the spirochete must travel through the hemolymph to the salivary glands, where it can spread to and infect the new host organism. In this study, we determined the crystal structures of the key outer surface protein BBE31 from B. burgdorferi and its orthologous protein BSE31 (BSPA14S_RS05060 gene product) from B. spielmanii. BBE31 is known to be important for the transfer of B. burgdorferi from the gut to the hemolymph in the tick after a tick bite. While BBE31 exerts its function by interacting with the Ixodes scapularis tick gut protein TRE31, structural and mass spectrometry data revealed that BBE31 has a glutathione (GSH) covalently attached to Cys142 suggesting that the protein may have acquired some additional functions in contrast to its orthologous protein BSE31, which lacks any interactions with GSH. In the current study, in addition to analyzing the potential reasons for GSH binding, the three-dimensional structure of BBE31 provides new insights into the molecular details of the transmission process as the protein plays an important role in the initial phase before the spirochete is physically transferred to the new host. This knowledge will be potentially used for the development of new strategies to fight against Lyme disease.

The structure and function of Iristatin, a novel immunosuppressive tick salivary cystatin.

To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.

Molecular characterization of neuropeptide elevenin and two elevenin receptors, IsElevR1 and IsElevR2, from the blacklegged tick, Ixodes scapularis.

Understanding salivation in hematophagous arthropod vectors is crucial to developing novel methods to prevent vector-borne disease transmission. The interactions between the tick, host, and pathogens during salivation are highly complex, and are dynamically regulated by the tick central nervous system (synganglion). Recently, tick salivary modulation via neuropeptides was highlighted by mapping neuropeptidergic cells in the synganglion and salivary glands in hard ticks. In this study, we characterized the role of a novel neuropeptide, elevenin (IsElev), and its receptors (IsElevR1 and IsElevR2) in the innervation of the salivary glands from Ixodes scapularis female ticks. Homology-based BLAST searches of the I. scapularis genome and Sequence Read Archive (SRA), followed by gene cloning, identified candidate genes: IsElev, IsElevR1, and IsElevR2. The IsElev candidate contained common elevenin features: a signal peptide immediately before an elevenin precursor and two cysteines. During functional assays, synthetic IsElev efficiently activated both IsElevR1 and IsElevR2, as indicated by elevated calcium mobilization. IsElevR1 (EC50: 0.01 nM) was about 560 times more sensitive to synthetic IsElev than IsElevR2 (EC50: 5.59 nM). Immunoreactivity (IR) for IsElev and IsElevR1 was detected as a complex neuronal projection and several neurons in the synganglion. In salivary glands, IsElev-IR was detected in an axonal projection on the surface of the main salivary duct and in axon terminals within type II/III salivary gland acini, which are colocalized with SIFamide-IR. IsElevR1-IR was detected on the luminal surface of both type II/III acini. IsElev transcript levels were high in the synganglion and reached a peak at day 5 post-blood feeding. Salivary glands expressed IsElevR1, which gradually increased over the course of blood feeding until repletion. Here, we propose that IsElev and IsElevR1, localized in salivary gland acini types II/III, are likely involved in tick salivary secretion in the rapid engorgement phase of tick feeding. In addition, we also provide the evidences for IsElev action on the ovary by showing IsElevR1-IR and IsElevR2-IR on the surface of ovary.

Sialome diversity of ticks revealed by RNAseq of single tick salivary glands.

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.

A new lipid carrier protein in the cattle tick Rhipicephalus microplus.

Tick infestation in cattle reflects the main cause of economic loss to cattle producers. This is due to several reasons but mainly to their ability to feed on blood and generate a huge amount of eggs. Lipid transport in arthropods is achieved by highly specialized hemolymphatic lipoproteins, which resemble those described in vertebrate blood. Such lipoproteins continuously deliver lipids through the blood to growing eggs. The injection of radioactive [3H] palmitic acid into tick hemocoel showed that the gut, ovary, fat body and Gene's organ were the main organs of incorporation of this labeled fatty acid. The rate of [3H] palmitic acid incorporation by the organs was high up to 30 min after injection. The [3H] palmitic acid incorporated by these organs was later found in phospholipids and neutral lipids. Here, we describe the purification and characterization of a key player of lipid dynamics in tick hemolymph. The Rhipicephalus microplus lipid-apolipoprotein complex (RmLCP) is a new high-density lipoprotein (1.18 g/mL), which accounts for over 90% of [3H] palmitic acid present in the hemolymph. It has a native molecular weight of 420 kDa and is composed of one subunit of 122 kDa. Protein identification analysis of RmLPC subunit showed two better hits: vitellogenin 2 (23% protein coverage) and vitellogenin 5 (29% protein coverage), respectively and similarities with hemolymphatic apolipoproteins of arachnids such as the tick Ixodes scapularis (80%), the mite Galendromus occidentalis (44%) and the spider Parasteatoda tepidariorum (43%) and also for the insects Locusta migratoria (45%), Drosophila melanogaster (42%) and Manduca sexta (47%) to vitellogenin 2 and tick Ixodes scapularis (83%), the crab Limulus polyphemus (55%) and the oyster Crassostrea gigas (55%) to vitellogenin 5. Furthermore, it shows a distinct lipid composition from most arthropod lipoproteins, being composed of 40% free cholesterol, 27% phospholipids, 20% triacylglycerol and 15% hydrocarbons. In addition to binding most hemolymphatic fatty acids, this lipoprotein also binds and transports free cholesterol. In conclusion, the present study provides insight into the macromolecules involved in arachnid metabolism, which have significant potential for future use for the biological control of ticks.

Infection-derived lipids elicit an immune deficiency circuit in arthropods.

The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.

Molecular and structural characterization of novel cystatins from the taiga tick Ixodes persulcatus.

Cystatins are cysteine peptidase inhibitors that in ticks mediate processes such as blood feeding and digestion. The ixodid tick Ixodes persulcatus is endemic to the Eurasia, where it is the principal vector of Lyme borreliosis. To date, no I. persulcatus cystatin has been characterized. In the present work, we describe three novel cystatins from I. persulcatus, named JpIpcys2a, JpIpcys2b and JpIpcys2c. In addition, the potential of tick cystatins as cross-protective antigens was evaluated by vaccination of hamsters using BrBmcys2c, a cystatin from Rhipicephalus microplus, against I. persulcatus infestation. Sequence analysis showed that motifs that are characteristic of cystatins type 2 are fully conserved in JpIpcys2b, while mutations are present in both JpIpcys2a and JpIpcys2c. Protein-protein docking simulations further revealed that JpIpcys2a, JpIpcys2b and JpIpcys2c showed conserved binding sites to human cathepsins L, all of them covering the active site cleft. Cystatin transcripts were detected in different I. persulcatus tissues and instars, showing their ubiquitous expression during I. persulcatus development. Serological analysis showed that although hamsters immunized with BrBmcys2c developed a humoral immune response, this response was not adequate to protect against a heterologous challenge with I. persulcatus adult ticks. The lack of cross-protection provided by BrBmcys2c immunization is perhaps linked to the fact that cystatins cluster into multigene protein families that are expressed differentially and exhibit functional redundancy. How to target such small proteins that are secreted in low quantities remains a challenge in the development of suitable anti-tick vaccine antigens.

Activity of tick antimicrobial peptide from Ixodes persulcatus (persulcatusin) against cell membranes of drug-resistant Staphylococcus aureus.

Persulcatusin (IP), which is an antimicrobial peptide found in Ixodes persulcatus midgut, is active against Gram-positive bacteria such as Staphylococcus aureus. Multidrug-resistant bacteria in particular methicillin-resistant S. aureus (MRSA), vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) are a worldwide clinical concern. In the present study, to explore the potential of IP as a new agent against multidrug-resistant S. aureus infections, we evaluated the antimicrobial activity of IP against multidrug-resistant S. aureus strains by MIC90, morphological observation with scanning electron microscope (SEM), and the calcein leakage assay of membrane integrity. Among the six antimicrobial peptides used in this study, IP exhibited the lowest MIC90 values for both vancomycin-susceptible and -resistant S. aureus strains. The IP MIC90 against a VISA strain was equivalent to vancomycin, while the MIC90 against VRSA was relatively low. SEM observations indicated that bacterial cells exposed to IP were crumpled and showed prominent structural changes. Moreover, IP influenced the cell membranes of both MRSA and VRSA in a mere 5 min, leading to leakage of the preloaded calcein. Although a VISA strain was resistant to the action of IP on cell membrane, the MIC90 of IP was lower than that of Nisin, suggesting that IP had another bactericidal mechanism in addition to cell membrane attack. Our results indicate that the synthetic tick antimicrobial peptide, IP exhibits strong antibacterial activity against multidrug-resistant S. aureus strains, including VRSA, via both cell membrane attack and another unknown mechanism. IP represents a promising candidate for a new anti-VRSA therapy.

Tick iron and heme metabolism - New target for an anti-tick intervention.

Ticks are blood-feeding parasites and vectors of serious human and animal diseases. Ixodes ricinus is a common tick in Europe, transmitting tick-borne encephalitis, Lyme borreliosis, anaplasmosis, or babesiosis. Immunization of hosts with recombinant tick proteins has, in theory, the potential to interfere with tick feeding and block transmission of pathogens from the tick to the host. However, the efficacy of tick antigens has, to date, not been fully sufficient to achieve this. We have focused on 11 in silico identified genes encoding proteins potentially involved in tick iron and heme metabolism. Quantitative real-time PCR (qRT-PCR) expression profiling was carried out to preferentially target proteins that are up-regulated during the blood meal. RNA interference (RNAi) was then used to score the relative importance of these genes in tick physiology. Finally, we performed vaccination screens to test the suitability of these proteins as vaccine candidates. These newly identified tick antigens have the potential to improve the available anti-tick vaccines.

Comparison of synganglion neuropeptides, neuropeptide receptors and neurotransmitter receptors and their gene expression in response to feeding in Ixodes scapularis (Ixodidae) vs. Ornithodoros turicata (Argasidae).

Illumina GAII high-throughput sequencing was used to compare expressed genes for female synganglion neuropeptides, neuropeptide receptors and neurotransmitter receptors of the soft tick Ornithodoros turicata with the hard tick Ixodes scapularis. Gene ontology molecular level three mapping revealed no significant differences amongst the same categories represented in O. turicata and I. scapularis. Transcripts predicting 22 neuropeptides or their receptors in the O. turicata synganglion were similar to annotations for 23 neuropeptides or receptors previously identified from I scapularis, with minor exceptions. A transcript predicting ecdysis triggering hormone receptor was identified in O. turicata; transcripts encoding for proprotein convertase and glycoprotein B were identified in both species. Transcripts predicting the same neurotransmitter receptors were found in the synganglion of both species. Gene expression of the transcripts showed numerous differences in response to feeding. Major differences were observed in expression of genes believed important in regulating slow vs. rapid feeding, blood water elimination, cuticle synthesis plasticity and in signalling reproductive activity. Although the glutamate receptor was strongly upregulated in both species, the gamma aminobutyric acid receptor, which inhibits glutamate, was upregulated significantly only in I. scapularis. These differences are consistent with the slow vs. rapid action of the pharyngeal pump in the two species.

Comparative bioinformatics, temporal and spatial expression analyses of Ixodes scapularis organic anion transporting polypeptides.

Organic anion-transporting polypeptides (Oatps) are an integral part of the detoxification mechanism in vertebrates and invertebrates. These cell surface proteins are involved in mediating the sodium-independent uptake and/or distribution of a broad array of organic amphipathic compounds and xenobiotic drugs. This study describes bioinformatics and biological characterization of 9 Oatp sequences in the Ixodes scapularis genome. These sequences have been annotated on the basis of 12 transmembrane domains, consensus motif D-X-RW-(I,V)-GAWW-X-G-(F,L)-L, and 11 conserved cysteine amino acid residues in the large extracellular loop 5 that characterize the Oatp superfamily. Ixodes scapularis Oatps may regulate non-redundant cross-tick species conserved functions in that they did not cluster as a monolithic group on the phylogeny tree and that they have orthologs in other ticks. Phylogeny clustering patterns also suggest that some tick Oatp sequences transport substrates that are similar to those of body louse, mosquito, eye worm, and filarial worm Oatps. Semi-quantitative RT-PCR analysis demonstrated that all 9 I. scapularis Oatp sequences were expressed during tick feeding. Ixodes scapularis Oatp genes potentially regulate functions during early and/or late-stage tick feeding as revealed by normalized mRNA profiles. Normalized transcript abundance indicates that I. scapularis Oatp genes are strongly expressed in unfed ticks during the first 24h of feeding and/or at the end of the tick feeding process. Except for 2 I. scapularis Oatps, which were expressed in the salivary glands and ovaries, all other genes were expressed in all tested organs, suggesting the significance of I. scapularis Oatps in maintaining tick homeostasis. Different I. scapularis Oatp mRNA expression patterns were detected and discussed with reference to different physiological states of unfed and feeding ticks.

Understanding the evolutionary structural variability and target specificity of tick salivary Kunitz peptides using next generation transcriptome data.

BACKGROUND: Ticks are blood-sucking arthropods and a primary function of tick salivary proteins is to counteract the host's immune response. Tick salivary Kunitz-domain proteins perform multiple functions within the feeding lesion and have been classified as venoms; thereby, constituting them as one of the important elements in the arms race with the host. The two main mechanisms advocated to explain the functional heterogeneity of tick salivary Kunitz-domain proteins are gene sharing and gene duplication. Both do not, however, elucidate the evolution of the Kunitz family in ticks from a structural dynamic point of view. The Red Queen hypothesis offers a fruitful theoretical framework to give a dynamic explanation for host-parasite interactions. Using the recent salivary gland Ixodes ricinus transcriptome we analyze, for the first time, single Kunitz-domain encoding transcripts by means of computational, structural bioinformatics and phylogenetic approaches to improve our understanding of the structural evolution of this important multigenic protein family. RESULTS: Organizing the I. ricinus single Kunitz-domain peptides based on their cysteine motif allowed us to specify a putative target and to relate this target specificity to Illumina transcript reads during tick feeding. We observe that several of these Kunitz peptide groups vary in their translated amino acid sequence, secondary structure, antigenicity, and intrinsic disorder, and that the majority of these groups are subject to a purifying (negative) selection. We finalize by describing the evolution and emergence of these Kunitz peptides. The overall interpretation of our analyses discloses a rapidly emerging Kunitz group with a distinct disulfide bond pattern from the I. ricinus salivary gland transcriptome. CONCLUSIONS: We propose a model to explain the structural and functional evolution of tick salivary Kunitz peptides that we call target-oriented evolution. Our study reveals that combining analytical approaches (transcriptomes, computational, bioinformatics and phylogenetics) improves our understanding of the biological functions of important salivary gland mediators during tick feeding.

Tryptogalinin is a tick Kunitz serine protease inhibitor with a unique intrinsic disorder.

BACKGROUND: A salivary proteome-transcriptome project on the hard tick Ixodes scapularis revealed that Kunitz peptides are the most abundant salivary proteins. Ticks use Kunitz peptides (among other salivary proteins) to combat host defense mechanisms and to obtain a blood meal. Most of these Kunitz peptides, however, remain functionally uncharacterized, thus limiting our knowledge about their biochemical interactions. RESULTS: We discovered an unusual cysteine motif in a Kunitz peptide. This peptide inhibits several serine proteases with high affinity and was named tryptogalinin due to its high affinity for ß-tryptase. Compared with other functionally described peptides from the Acari subclass, we showed that tryptogalinin is phylogenetically related to a Kunitz peptide from Rhipicephalus appendiculatus, also reported to have a high affinity for ß-tryptase. Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin. The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases). CONCLUSIONS: By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level.

Bioinformatics and expression analyses of the Ixodes scapularis tick cystatin family.

The cystatins are inhibitors of papain- and legumain-like cysteine proteinases, classified in MEROPS subfamilies I25A-I25C. This study shows that 84 % (42/50) of tick cystatins are putatively extracellular in subfamily I25B and the rest are putatively intracellular in subfamily I25A. On the neighbor joining phylogeny guide tree, subfamily I25A members cluster together, while subfamily I25B cystatins segregate among prostriata or metastriata ticks. Two Ixodes scapularis cystatins, AAY66864 and ISCW011771 that show 50-71 % amino acid identity to metastriata tick cystatins may be linked to pathways that are common to all ticks, while ISCW000447 100 % conserved in I. ricinus is important among prostriata ticks. Likewise metastriata tick cystatins, Dermacentor variabilis-ACF35512, Rhipicephalus microplus-ACX53850, A. americanum-AEO36092, R. sanguineus-ACX53922, D. variabilis-ACF35514, R. sanguineus-ACX54033 and A. maculatum-AEO35155 that show 73-86 % amino acid identity may be essential to metastriata tick physiology. RT-PCR expression analyses revealed that I. scapularis cystatins were constitutively expressed in the salivary glands, midguts and other tissues of unfed ticks and ticks that were fed for 24-120 h, except for ISCW017861 that are restricted to the 24 h feeding time point. On the basis of mRNA expression patterns, I. scapularis cystatins, ISCW017861, ISCW011771, ISCW002215 and ISCW0024528 that are highly expressed at 24 h are likely involved in regulating early stage tick feeding events such as tick attachment onto host skin and creation of the feeding lesion. Similarly, ISCW018602, ISCW018603 and ISCW000447 that show 2-3 fold transcript increase by 120 h of feeding are likely associated with blood meal up take, while those that maintain steady state expression levels (ISCW018600, ISCW018601 and ISCW018604) during feeding may not be associated with tick feeding regulation. We discuss our findings in the context of advancing our knowledge of tick molecular biology.