The Kunitz-Like Modulatory Protein Haemangin Is Vital for Hard Tick Blood-Feeding Success

Stanford University, United States of America
PLoS Pathogens (Impact Factor: 7.56). 08/2009; 5(7):e1000497. DOI: 10.1371/journal.ppat.1000497
Source: PubMed


Ticks are serious haematophagus arthropod pests and are only second to mosquitoes as vectors of diseases of humans and animals. The salivary glands of the slower feeding hard ticks such as Haemaphysalis longicornis are a rich source of bioactive molecules and are critical to their biologic success, yet distinct molecules that help prolong parasitism on robust mammalian hosts and achieve blood-meals remain unidentified. Here, we report on the molecular and biochemical features and precise functions of a novel Kunitz inhibitor from H. longicornis salivary glands, termed Haemangin, in the modulation of angiogenesis and in persistent blood-feeding. Haemangin was shown to disrupt angiogenesis and wound healing via inhibition of vascular endothelial cell proliferation and induction of apoptosis. Further, this compound potently inactivated trypsin, chymotrypsin, and plasmin, indicating its antiproteolytic potential on angiogenic cascades. Analysis of Haemangin-specific gene expression kinetics at different blood-feeding stages of adult ticks revealed a dramatic up-regulation prior to complete feeding, which appears to be functionally linked to the acquisition of blood-meals. Notably, disruption of Haemangin-specific mRNA by a reverse genetic tool significantly diminished engorgement of adult H. longicornis, while the knock-down ticks failed to impair angiogenesis in vivo. To our knowledge, we have provided the first insights into transcriptional responses of human microvascular endothelial cells to Haemangin. DNA microarray data revealed that Haemangin altered the expression of 3,267 genes, including those of angiogenic significance, further substantiating the antiangiogenic function of Haemangin. We establish the vital roles of Haemangin in the hard tick blood-feeding process. Moreover, our results provide novel insights into the blood-feeding strategies that enable hard ticks to persistently feed and ensure full blood-meals through the modulation of angiogenesis and wound healing processes.

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    • "To date, about 15 single Kunitz-domain peptides from ticks have been functionally characterized. Classical serine protease inhibitors were analyzed from the hard ticks Rhipicephalus microplus (BMCL) [25], Rhipicephalus appendiculatus (TdPI) [26], Rhipicephalus haemaphysaloides (Rhipilin-1) [27], Haemaphysalis longicornis (HlChl, HIMKI and Haemangin) [28-30], Amblyomma cajennense (Amblyomin-X) [31], and Ixodes scapularis (Tryptogalinin) [32]. Protease inhibitors were also characterized from the soft ticks Ornithodoros moubata (TAP) [33] and Ornithodoros savignyi (FXaI) that mainly function as anti-clotting agents. "
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    ABSTRACT: 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. 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. 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.
    Full-text · Article · Jan 2014 · BMC Evolutionary Biology
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    • "Ticks modulate neutrophil attraction by modifying chemokine and cytokine responses; reducing activation and down regulating reactive oxygen intermediates and nitric oxide; and, reducing surface integrin expression as well as endothelium expression of adhesion molecules (Brossard and Wikel, 2008; Kazimirova and Stibraniova, 2013). Wound healing is also modulated by tick saliva binding and inhibition of growth factors, reducing angiogenesis, impairing fibroblast migration, and by the presence of metalloproteases that remodel extracellular matrix (Fukumoto et al., 2006; Ribeiro et al., 2006; Kramer et al., 2008; Francischetti et al., 2009; Islam et al., 2009; Kazimirova and Stibraniova, 2013). Saliva binding and inhibition of transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) reduces wound healing. "
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    ABSTRACT: Ticks are unique among hematophagous arthropods by continuous attachment to host skin and blood feeding for days; complexity and diversity of biologically active molecules differentially expressed in saliva of tick species; their ability to modulate the host defenses of pain and itch, hemostasis, inflammation, innate and adaptive immunity, and wound healing; and, the diverse array of infectious agents they transmit. All of these interactions occur at the cutaneous interface in a complex sequence of carefully choreographed host defense responses and tick countermeasures resulting in an environment that facilitates successful blood feeding and establishment of tick-borne infectious agents within the host. Here, we examine diverse patterns of tick attachment to host skin, blood feeding mechanisms, salivary gland transcriptomes, bioactive molecules in tick saliva, timing of pathogen transmission, and host responses to tick bite. Ticks engage and modulate cutaneous and systemic immune defenses involving keratinocytes, natural killer cells, dendritic cells, T cell subpopulations (Th1, Th2, Th17, Treg), B cells, neutrophils, mast cells, basophils, endothelial cells, cytokines, chemokines, complement, and extracellular matrix. A framework is proposed that integrates tick induced changes of skin immune effectors with their ability to respond to tick-borne pathogens. Implications of these changes are addressed. What are the consequences of tick modulation of host cutaneous defenses? Does diversity of salivary gland transcriptomes determine differential modulation of host inflammation and immune defenses and therefore, in part, the clades of pathogens effectively transmitted by different tick species? Do ticks create an immunologically modified cutaneous environment that enhances specific pathogen establishment? Can tick saliva molecules be used to develop vaccines that block pathogen transmission?
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