Tick saliva and its importance for tick feeding and pathogen transmission

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

The accomplishments of the Tick-Pathogen Unit are: 1- We identified and performed in silico characterization of neuropeptides present in different life stages and organs of the tick Rhipicephalus microplus, generating transcriptomes from ovary, salivary glands, fat body, midgut, and embryo. Annotation of synganglion transcripts led to the identification of 32 functional categories of proteins. Neuropeptide precursors are among the sequences over-represented in R. microplus synganglion, with at least 5-fold higher transcription levels compared with other stages/organs. A total of 52 neuropeptide precursors were identified, several of these neuropeptides being reported in ticks for the first time. 2- We described the transcriptome of Rhipicephalus microplus ticks lacking its Coxiella endosymbiont. Coxiella sp. is a maternally inherited symbiont in most tick species that is required for essential functions, including feeding, reproduction, and/or molting. In a previous report we showed that Rhipicephalus microplus larvae with reduced levels of Coxiella had their development interrupted at the metanymph life stage. A comparative transcriptomic analysis of these metanymphs was used to investigate tick physiology in the absence and/or presence of the symbiont. Results revealed a global alteration of transcripts from several functional categories, with a marked impact in those coding for proteins that are critical to blood-feeding capacity during development. The data presented in this study contribute significantly to the arthropod-bacteria symbiosis research field while highlighting a novel aspect of the complex and largely unknown tick-symbiont-vertebrate host interaction. 3- We performed a characterization of two small salivary peptides from the rat flea, XC-42 and XC-43, which inhibit thrombin, the main protease of the blood coagulation cascade. XC-43, the smaller of the two peptides, is a fast, tight-binding inhibitor of thrombin with a dissociation constant of less than 10 pM; XC-42 exhibits similar selectivity as well as kinetic and binding properties. The crystal structure of XC-43 in complex with thrombin shows that despite its substrate-like binding mode, XC-43 is not detectably cleaved by thrombin and that it interacts with the thrombin surface from the enzyme catalytic site through the fibrinogen-binding exosite I. The low rate of hydrolysis was verified in solution experiments with XC-43, which show the substrate to be largely intact after 2 h of incubation with thrombin at 37 C. The low rate of XC-43 cleavage by thrombin may be attributable to specific changes in the catalytic triad observable in the crystal structure of the complex or to extensive interactions in the prime sites that may stabilize the binding of cleavage products. Based on the increased arterial occlusion time, tail bleeding time, and blood coagulation parameters in rat models of thrombosis, XC-43 could be valuable as an anticoagulant. 4- We identified a new mechanism by which a specific skin-derived protease contributes to wound healing. In a scratch-induced wound using keratinocyte monolayers, a strong acceleration of wound closure was observed when cells were supplemented with this protease. This effect was dose-dependent and accompanied by an increase in protease expression on cell extracts. The scratch per se, without previous protease treatment, was also able to induce mainly the expression of this protease on the cell surface, and the wound closure is accelerated in wounded cells when comparing to non-wounded cells. Migrating cells showing increased proteases expression are located close to the wound edges. After 24 h of scratch-induced wound, protease expression and activity were detected in culture media, indicating that the protease is secreted to the extracellular medium. Soluble factors released by wounded cells promoted cell migration, being this effect partially inhibited by a specific protease inhibitor. By proteomics, we also identified several growth factor-related proteins secreted by these wounded cells treated with the protease. Of significant interest, an antagonist of IGFR inhibits the wound closure response promoted by this protease. The wound closure phenotype promoted by this protease is related to cell migration and not proliferation. The protease-induced signaling involved ERK1/2 phosphorylation and consequently FAK, paxillin, RhoA, RAC-1 and CDC-42 effectors activation. This process ultimately is involved in actin assembly and polymerization, which facilitates cell migration. Further experiments applying 3D inserts of skin and in vivo models of wound healing using protease and knock-out animals are underway to validate our in vitro findings. We also identified and characterized a new tick salivary inhibitor for this protease. We have demonstrated that antibodies against this tick salivary inhibitor block its function and we have identified putative epitopes that may be used as antigen to compose an anti-tick vaccine. 5- We identified a new mechanism by which a specific plasmatic protease contributes to wound healing. We demonstrated for the first time that keratinocyte surface support high molecular weight-dependent pre-kallikrein activation and kinin generation. This event was modulated and completely blocked by a tick salivary inhibitor that binds to keratinocyte surface through a specific phospholipid. This unique mechanism allows kallikrein inhibition locally at the keratinocyte membrane, reducing cell migration and wound closure induced by the protease. The inhibitor also reduced intracellular nitric oxide formation by a mechanism independent of NOS, bradykinin receptor B2, and the reactive oxygen species generated by the protease. The keratinocyte migration seems to be modulated by the inhibitor through a pathway dependent of EGF receptor phosphorylation, ERK1/2 and paxillin activation, which ultimately coordinates MMP2 secretion, actin polymerization and stress fiber formation. These results highlighted interesting new strategies used by tick salivary proteins to avoid host skin barriers against blood feeding. 6- We performed the characterization of Alboserpin, the major salivary anticoagulant from the mosquito vector Aedes albopictus, a specific inhibitor of the human coagulation factor Xa (FXa). In this study, we investigated the anti-inflammatory properties of Alboserpin, in vitro and in vivo. In vitro, Alboserpin inhibited FXa-induced protease-activated receptor (PAR)-1, PAR-2, PAR-3, VCAM, ICAM, and NF-B gene expression in primary dermal microvascular endothelial cells. Alboserpin also prevented FXa-stimulated ERK1/2 gene expression and subsequent inflammatory cytokine release (MCP-1, TNF-, IL-6, IL-8, IL-1, IL-18). In vivo, Alboserpin reduced paw edema induced by FXa and subsequent release of inflammatory cytokines (CCL2, MCP-1, IL-1, IL-6, IL-1). Alboserpin also reduced FXa-induced endothelial permeability in vitro and in vivo. These findings show that Alboserpin is a potent anti-inflammatory molecule, in vivo and in vitro, and may play a significant role in blood feeding.