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

Immunomodulatory effects of tick saliva on dermal cells exposed to \u3cem\u3eBorrelia burgdorferi\u3c/em\u3e, the agent of Lyme disease

Background: The prolonged feeding process of ixodid ticks, in combination with bacterial transmission, should lead to a robust inflammatory response at the blood-feeding site. Yet, factors present in tick saliva may down-regulate such responses, which may be beneficial to spirochete transmission. The primary goal of this study was to test the hypothesis that tick saliva, in the context of Borrelia burgdorferi, can have widespread effects on the production of immune mediators in skin. Methods: A cross-section of tick feeding on skin was examined histologically. Human THP-1 cells stimulated with B. burgdorferi and grown in the presence or absence of tick saliva were examined by human DNA microarray, cytokine bead array, sandwich ELISA, and qRT-PCR. Similar experiments were also conducted using dermal fibroblasts. Results: Tick feeding on skin showed dermal infiltration of histiocytes and granulocytes at the bite location. Changes in monocytic transcript levels during co-culture with B. burgdorferi and saliva indicated that tick saliva had a suppressive effect on the expression of certain pro-inflammatory mediators, such as IL-8 (CXCL8) and TLR2, but had a stimulatory effect on specific molecules such as the Interleukin 10 receptor, alpha subunit (IL-10RA), a known mediator of the immunosuppressive signal of IL-10. Stimulated cell culture supernatants were analyzed via antigen-capture ELISA and cytokine bead array for inflammatory mediator production. Treatment of monocytes with saliva significantly reduced the expression of several key mediators including IL-6, IL-8 and TNF-alpha. Tick saliva had an opposite effect on dermal fibroblasts. Rather than inhibiting, saliva enhanced production of pro-inflammatory mediators, including IL-8 and IL-6 from these sentinel skin cells. Conclusions: The effects of ixodid tick saliva on resident skin cells is cell type-dependent. The response to both tick and pathogen at the site of feeding favors pathogen transmission, but may not be wholly suppressed by tick saliva

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

Towards a new phenotype for tick resistance in beef and dairy cattle:a review

About 80% of the world's cattle are affected by ticks and tick-borne diseases, both of which cause significant production losses. Cattle host resistance to ticks is the most important factor affecting the economics of tick control, but it is largely neglected in tick-control programs due to technical difficulties and costs associated with identifying individual-animal variation in resistance. The present paper reviews the scientific literature to identify factors affecting resistance of cattle to ticks and the biological mechanisms of host tick resistance, to develop alternative phenotype(s) for tick resistance. If new cost-effective phenotype(s) can be developed and validated, then tick resistance of cattle could be genetically improved using genomic selection, and incorporated into breeding objectives to simultaneously improve cattle productive attributes and tick resistance. The phenotype(s) could also be used to improve tick control by using cattle management. On the basis of the present review, it is recommended that three possible phenotypes (haemolytic analysis measures of skin hypersensitivity reactions simplified artificial tick infestations) be further developed to determine their practical feasibility for consistently, cost-effectively and reliably measuring cattle tick resistance in thousands of individual animals in commercial and smallholder farmer herds in tropical and subtropical areas globally. During evaluation of these potential new phenotypes, additional measurements should be included to determine the possibility of developing a volatile-based resistance phenotype, to simultaneously improve cattle resistance to both ticks and biting flies. Because the current measurements of volatile chemistry do not satisfy the requirements of a simple, cost-effective phenotype for use in commercial cattle herds, consideration should also be given to inclusion of potentially simpler measures to enable indirect genetic selection for volatile-based resistance to ticks

A Deep Insight into the Sialotranscriptome of the Gulf Coast Tick, Amblyomma maculatum

Background: Saliva of blood sucking arthropods contains compounds that antagonize their hosts ’ hemostasis, which include platelet aggregation, vasoconstriction and blood clotting; saliva of these organisms also has anti-inflammatory and immunomodullatory properties. Perhaps because hosts mount an active immune response against these compounds, the diversity of these compounds is large even among related blood sucking species. Because of these properties, saliva helps blood feeding as well as help the establishment of pathogens that can be transmitted during blood feeding. Methodology/Principal Findings: We have obtained 1,626,969 reads by pyrosequencing a salivary gland cDNA library from adult females Amblyomma maculatum ticks at different times of feeding. Assembly of this data produced 72,441 sequences larger than 149 nucleotides from which 15,914 coding sequences were extracted. Of these, 5,353 had.75 % coverage to their best match in the non-redundant database from the National Center for Biotechnology information, allowing for the deposition of 4,850 sequences to GenBank. The annotated data sets are available as hyperlinked spreadsheets. Putative secreted proteins were classified in 133 families, most of which have no known function. Conclusions/Significance: This data set of proteins constitutes a mining platform for novel pharmacologically activ

A high affinity serotonin- and histamine-binding lipocalin from tick saliva

To overcome the inflammatory response in its host, the cattle-feeding, brown ear tick secretes histamine-binding proteins into the feeding site. These proteins are β-barrels with two internal binding sites: a high-affinity (H) site for histamine and a site (L) for which the natural ligand is unknown. Here we report a related protein (SHBP), secreted by a rodent- and cattle-feeding tick, that traps both histamine and serotonin. The histamine-binding H site is well conserved in SHBP, whereas residue changes in the L-like site are consistent with binding of the bulkier serotonin molecule. As histamine is a key inflammatory mediator in cattle, while serotonin takes on this role in rodents, the diversification of these tick proteins may reflect host adaptatio

Exosites mediate the anti-inflammatory effects of a multifunctional serpin from the saliva of the tick Ixodes ricinus.

Serine protease inhibitors (serpins) are a structurally related but functionally diverse family of ubiquitous proteins. We previously described Ixodes ricinus immunosuppressor (Iris) as a serpin from the saliva of the tick I. ricinus displaying high affinity for human leukocyte elastase. Iris also displays pleotropic effects because it interferes with both the immune response and hemostasis of the host. It thus inhibits lymphocyte proliferation and the secretion of interferon-gamma or tumor necrosis factor-alpha by peripheral blood mononuclear cells, and also platelet adhesion, coagulation and fibrinolysis. Its ability to interfere with coagulation and fibrinolysis, but not platelet adhesion, depends on the integrity of its antiproteolytic reactive center loop domain. Here, we dissect the mechanisms underlying the interaction of recombinant Iris with peripheral blood mononuclear cells. We show that Iris binds to monocytes/macrophages and inhibits their ability to secrete tumor necrosis factor-alpha. Recombinant Iris also has a protective role in endotoxemic shock. The anti-inflammatory ability of Iris does not depend on its antiprotease activity. Moreover, we pinpoint the exosites involved in this activity.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

Structure, Function, and Evolution of Biogenic Amine-binding Proteins in Soft Ticks*S⃞

Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to the lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins