Ixodes ricinus Tick Lipocalins: Identification, Cloning, Phylogenetic Analysis and Biochemical Characterization

BACKGROUND: During their blood meal, ticks secrete a wide variety of proteins that interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: Screening a cDNA library in association with RT-PCR and RACE methodologies allowed us to identify 14 new lipocalin genes in the salivary glands of the Ixodes ricinus hard tick. A computational in-depth structural analysis confirmed that LIRs belong to the lipocalin family. These proteins were called LIR for "Lipocalin from I. ricinus" and numbered from 1 to 14 (LIR1 to LIR14). According to their percentage identity/similarity, LIR proteins may be assigned to 6 distinct phylogenetic groups. The mature proteins have calculated pM and pI varying from 21.8 kDa to 37.2 kDa and from 4.45 to 9.57 respectively. In a western blot analysis, all recombinant LIRs appeared as a series of thin bands at 50-70 kDa, suggesting extensive glycosylation, which was experimentally confirmed by treatment with N-glycosidase F. In addition, the in vivo expression analysis of LIRs in I. ricinus, examined by RT-PCR, showed homogeneous expression profiles for certain phylogenetic groups and relatively heterogeneous profiles for other groups. Finally, we demonstrated that LIR6 codes for a protein that specifically binds leukotriene B4. CONCLUSIONS/SIGNIFICANCE: This work confirms that, regarding their biochemical properties, expression profile, and sequence signature, lipocalins in Ixodes hard tick genus, and more specifically in the Ixodes ricinus species, are segregated into distinct phylogenetic groups suggesting potential distinct function. This was particularly demonstrated by the ability of LIR6 to scavenge leukotriene B4. The other LIRs did not bind any of the ligands tested, such as 5-hydroxytryptamine, ADP, norepinephrine, platelet activating factor, prostaglandins D2 and E2, and finally leukotrienes B4 and C4.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Ir-CPI, a coagulation contact phase inhibitor from the tick Ixodes ricinus, inhibits thrombus formation without impairing hemostasis

Blood coagulation starts immediately after damage to the vascular endothelium. This system is essential for minimizing blood loss from an injured blood vessel but also contributes to vascular thrombosis. Although it has long been thought that the intrinsic coagulation pathway is not important for clotting in vivo, recent data obtained with genetically altered mice indicate that contact phase proteins seem to be essential for thrombus formation. We show that recombinant Ixodes ricinus contact phase inhibitor (Ir-CPI), a Kunitz-type protein expressed by the salivary glands of the tick Ixodes ricinus, specifically interacts with activated human contact phase factors (FXIIa, FXIa, and kallikrein) and prolongs the activated partial thromboplastin time (aPTT) in vitro. The effects of Ir-CPI were also examined in vivo using both venous and arterial thrombosis models. Intravenous administration of Ir-CPI in rats and mice caused a dose-dependent reduction in venous thrombus formation and revealed a defect in the formation of arterial occlusive thrombi. Moreover, mice injected with Ir-CPI are protected against collagen- and epinephrine-induced thromboembolism. Remarkably, the effective antithrombotic dose of Ir-CPI did not promote bleeding or impair blood coagulation parameters. To conclude, our results show that a contact phase inhibitor is an effective and safe antithrombotic agent in vivo

Ir-LBP, an Ixodes ricinus Tick Salivary LTB4-Binding Lipocalin, Interferes with Host Neutrophil Function

BACKGROUND: During their blood meal, ticks secrete a wide variety of proteins that can interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: We previously identified 14 new lipocalin genes in the tick Ixodes ricinus. One of them codes for a protein that specifically binds leukotriene B4 with a very high affinity (Kd: +/-1 nM), similar to that of the neutrophil transmembrane receptor BLT1. By in silico approaches, we modeled the 3D structure of the protein and the binding of LTB4 into the ligand pocket. This protein, called Ir-LBP, inhibits neutrophil chemotaxis in vitro and delays LTB4-induced apoptosis. Ir-LBP also inhibits the host inflammatory response in vivo by decreasing the number and activation of neutrophils located at the tick bite site. Thus, Ir-LBP participates in the tick's ability to interfere with proper neutrophil function in inflammation. CONCLUSIONS/SIGNIFICANCE: These elements suggest that Ir-LBP is a "scavenger" of LTB4, which, in combination with other factors, such as histamine-binding proteins or proteins inhibiting the classical or alternative complement pathways, permits the tick to properly manage its blood meal. Moreover, with regard to its properties, Ir-LBP could possibly be used as a therapeutic tool for illnesses associated with an increased LTB4 production.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Variability and Action Mechanism of a Family of Anticomplement Proteins in Ixodes ricinus

Background: Ticks are blood feeding arachnids that characteristically take a long blood meal. They must therefore counteract host defence mechanisms such as hemostasis, inflammation and the immune response. This is achieved by expressing batteries of salivary proteins coded by multigene families. Methodology/Principal Findings: We report the in-depth analysis of a tick multigene family and describe five new anticomplement proteins in ixodes ricinus. Compared to previously described Ixodes anticomplement proteins, these segregated into a new phylogenetic group or subfamily. These proteins have a novel action mechanism as they specifically bind to properdin, leading to the inhibition of C3 convertase and the alternative complement pathway. An excess of non-synonymous over synonymous changes indicated that coding sequences had undergone diversifying selection. Diversification was not associated with structural, biochemical o, functional diversity, adaptation to host species or stage specificity but rather to differences in antigenicity. Conclusion/Significance: Anticomplement proteins from I. ricinus are the first inhibitors that specifically target a positive regulator of complement, properdin. They may provide new tools for the investigation of role of properdin in physiological and pathophysiological mechanisms. They may also be useful in disorders affecting the alternative complement pathway, Looking for and detecting the different selection pressures involved will help in understanding the evolution of multigene families and hematophagy in arthropods. © 2008 Couveur et al.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Etude de l'inhibition des mécanismes d'hémostase par des protéines de glandes salivaires de la tique Ixodes ricinus

Les tiques du genre Ixodes englobent un grand nombre d’espèces dont Ixodes ricinus, objet de cette étude. Ixodes ricinus peuple les sous-bois de nos régions et la plupart des grandes forêts de l’Europe centrale et de l’est. Au cours de leur cycle parasitaire, les tiques réalisent un repas sanguin au détriment de petits mammifères (mulots, lièvres,…), mais aussi de grands mammifères tels que le cerf, la biche ou le sanglier. Elles deviennent particulièrement voraces au début du printemps et en automne, c’est pourquoi à ces époques de l’année, les promeneurs et les animaux domestiques (principalement, le chien) peuvent en être infestés. Pour mener à bien leur repas sanguin, les tiques mettent en place une série de mécanismes les rendant non seulement indécelables par l’animal parasité mais aussi leur donnant la capacité de contrecarrer les défenses de l’hôte. En effet, lors de sa fixation et durant tout le repas sanguin, la tique injecte un cocktail de facteurs bioactifs qui inhibent la sensation de douleur au moment de l’ancrage dans le derme, mais également les défenses immunitaires de l’hôte parasité. De plus, certains de ces facteurs possèdent des propriétés anti-hémostatiques garantissant la fluidité du sang pendant tout le repas.Le Service de Biologie Moléculaire des Ectoparasites (anciennement Service de Génétique Appliquée) a identifié un grand nombre de séquences d’ARNm induites spécifiquement dans les glandes salivaires de la tique au cours de son repas sanguin. Deux d’entres-elles ont servi de point de départ à ce travail :il s’agit de seq16 (renommée Metis1) et de seq7 (renommée Ir-CPI).Le travail sur la séquence seq16 a conduit à l’identification d’une nouvelle famille de 5 métalloprotéases (nommée Metis pour Metalloprotease from Ixodes ricinus). L’analyse des séquences a permis de les associer à certaines métalloprotéases hémorragiques de venin de serpent. Bien que les 5 métalloprotéases décrites possèdent toutes les mêmes caractéristiques au niveau de leur séquence et de leur profil d’expression dans les glandes salivaires, les résultats d’analyse phylogénétique, d’étude de variation antigénique, de leur mode d’activation et de leur spécificité d’action permettent de les diviser en 3 sous-familles. L’utilisation de la technique d’ARN interférence en in vivo et l’analyse vaccinale soulignent le rôle essentiel de la famille Metis dans les premières heures du repas de la tique et de manière générale dans la réussite du repas sanguin. Enfin, des études d’activité ont montré que certains membres de la famille ont une activité fibrino(gèno)lytique ;plus particulièrement, la protéine Metis4 possède une activité protéolytique envers la gélatine, la caséine, la fibronectine et le fibrinogène. Ces études ont montré également un mode d’activation original ;plutôt que d’être activée par une pro-séquence en amino-terminal, Metis4 s’active spontanément en perdant un peptide à son extrémité carboxy-terminale. Le travail sur la séquence seq7 a conduit à la caractérisation d’un nouvel inhibiteur de sérines protéases. Cette protéine, nommée Ir-CPI pour « Ixodes ricinus - Contact Phase Inhibitor », possède un motif kunitz et est capable d’inhiber de manière importante la voie intrinsèque de la coagulation et dans une moindre mesure la fibrinolyse. Grâce à la technique d’ARN interférence, l’activité de la protéine recombinante a été corrélée à l’activité de la protéine native exprimée dans les glandes salivaires. Ir-CPI inhibe dans un plasma humain l’activation réciproque du facteur XII, de la prékallikréine et du facteur XI, et se fixe spécifiquement à la forme activée de ces trois facteurs. De plus, Ir-CPI est capable de bloquer la fibrinolyse en inhibant spécifiquement la plasmine. Enfin, les résultats obtenus sur deux modèles animaux indépendants établissent l’effet d’Ir-CPI comme agent anti-thrombotique en empêchant la formation de caillot et de manière remarquable sans altérer l'équilibre hémostatique.En conclusion, ce travail a identifié des protéines anti-hémostatiques qui agissent soit comme inhibiteur de protéases empêchant la coagulation soit comme protéases facilitant la lyse du caillot. Cette redondance de protéines anti-hémostatiques illustre remarquablement la capacité de la tique à agir en synergie sur des facteurs essentiels aux mécanismes de défense de l’hôte.Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Electrochemical DNA hybridization detection using peptide nucleic acids and [Ru(NH3)6]3+ on gold electrodes.

An electrochemical DNA hybridization detection method based on the electrostatic interactions of [Ru(NH3)6]3+ cations with the anionic phosphate backbone of DNA is proposed. PNA molecules are immobilized as capture probes on the gold substrate. The cationic ruthenium complexes do not interact electrostatically with the PNA probes due to the absence of the anionic phosphate groups on the PNA probes. But after hybridization, [Ru(NH3)6]3+ is adsorbed on the DNA backbone, giving a clear hybridization detection signal in ac voltammetry. The analytical parameters (sensitivity, selectivity and reproducibility) are evaluated. Very good discrimination against the single-base mismatch A2143G, internal to the 23S rRNA gene of Helicobacter pylori, is observed. Moreover the system is successfully applied to the detection of complementary PCR amplicons.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe