Tick saliva and its importance for tick feeding and pathogen transmission

National Institute Of Allergy And Infectious Diseases

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Abstract

The ongoing research at Tick-Pathogen Transmission Unit focuses on the following projects: 1- Transcriptomic and proteomic analysis of tick salivary glands As blood feeding arthropods, ticks salivate while they puncture host skin in their search of blood. Tick saliva contains hundreds of compounds that have anti-coagulant, vasodilatory, anti-inflammatory, and immunomodulatory functions. While helping the vector to feed, tick saliva also modifies the site where pathogens are injected and, in many cases, facilitates the infection process. In recent years, we have contributed to the analysis of several salivary proteomes and the functional characterization of tick saliva proteins. A deeper understanding of tick feeding biology is needed to discover weak links that can be targeted for effective anti-tick vaccine development. To further obtain insights into the transcriptome and proteome of ticks, we used RNA-seq followed by proteomics to explore the transcriptome and proteome of salivary glands of Rhipicephalus sanguineus adult female ticks, a vector of Rickettsia rickettsii in the U.S. The correlations between transcriptional and translational expression in the different groups were evaluated, confirming that not all transcripts are expressed at the same time, expression profile switches at intervals, characterizing the phenomenon of sialome switching, which could serve as a mechanism of escape from the host immunity. Another remarkable observation from this study was the identification of host-derived proteins as a component of tick salivary gland content. These results and disclosed sequences that were deposited at NCBI contribute to our understanding of tick feeding biology, to the development of novel anti-tick methods, and to the discovery of novel pharmacologically active products from tick saliva. 2- How ticks evade and exploit host keratinocytes: relevance to tick feeding and Borrelia burgdorferi transmission. The skin epidermis is the first host tissue that the tick mouthparts, tick saliva, and a tick-borne pathogen contact during the tick feeding process. This interface is crucial for influencing the initial host response to tick feeding and pathogen transmission. Keratinocytes are of particular interest since they constitute the majority of cells in the skin, mediating skin homeostasis and responding rapidly to mechanical damage via secretion of soluble factors (e.g. cytokines and chemokines) by permanent cross-talk with immune cells. The role of non-professional immune cells such as epidermal keratinocytes is somehow a neglected topic in the context of tick feeding and tick-borne pathogen biology, and our research is attempting to fill this gap. Several serine proteases, including several members of the kallikrein-related proteases, are expressed by keratinocytes and are involved in the maintenance of structural integrity and barrier function of the epidermis, mediating keratinocyte proliferation and differentiation, inflammation, pain, and wound healing mechanisms in the skin. We have expressed active forms of mouse kallikrein-related proteases and a system to report mouse protease-activated receptor (mPAR-1, mPAR-2, mPAR-3, and mPAR-4) activation is also established in CHO-K1 cells. We have isolated and identified inhibitors of kallikrein-related proteases from salivary glands of Ixodes scapularis nymphs. Biochemical and biophysical analysis have been carried out to understand the mechanism of protease inhibition by these tick saliva inhibitors. These proteins are being tested for anti-inflammatory activity in animal models and production of pro- and anti-inflammatory cytokines in cultured mouse keratinocytes. A system to evaluated the wound healing process using mouse keratinocytes have been stablished and some kallikrein-related proteases that accelerated this process and its respective inhibitors from tick saliva have been identified. Given the important pro-inflammatory and pro-nociceptive actions of skin-derived proteases and PARs, utilization of PAR- and protease-knockout mice and RNA silencing of tick inhibitors are being used in tick feeding experiments to test the hypothesis that keratinocyte modulation contributes to the ticks ability to successfully acquire a blood meal and transmit Borrelia burgdorferi. 3- A tick salivary protein that inhibits the contact phase of blood coagulation: The contact system, also named as plasma kallikrein-kinin system, consists of three serine proteinases, coagulation factors XII (FXII) and XI (FXI), and plasma prekallikrein (PK), and the non-enzymatic cofactor high molecular weight kininogen (HK). PK and FXI through HK assemble on a co-localized, multiprotein receptor complex on endothelial cells. We have isolated from tick saliva an inhibitor of this system. The protein is inhibits kallikrein and binds glycosaminoglycans in vitro. Using endothelial cells to activate the contact phase, we have shown that this inhibitor binds to endothelial cell surface. We are proposing to use animal models of blood coagulation to test its antithrombotic properties in vivo.