Tick saliva and its importance for tick feeding and pathogen transmission

National Institute Of Allergy And Infectious Diseases

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Abstract

The accomplishments of the Tick-Pathogen Transmission Unit in the fiscal year of 2025 are: 1. We identified and characterized ixochymostatin, an Ixodes scapularis protein belonging to the trypsin inhibitor-like (TIL) family. Ixochymostatin is expressed in multiple developmental stages and in tick salivary glands, and it acts as a slow, tight-binding inhibitor of chymase, cathepsin G, and chymotrypsin. Predictions of the tertiary structure of the ixochymostatin–chymase complex suggest a direct interaction between the inhibitor’s reactive site loop and protease active sites. In vitro, ixochymostatin protects the endothelial cell barrier against chymase-mediated degradation, thereby reducing cell permeability. In vivo, it decreases vascular permeability induced by chymase and mast cell degranulation agonists in a mouse model. Additionally, ixochymostatin inhibits the chymase-dependent generation of vasoconstrictor peptides. Antibodies against ixochymostatin neutralize its inhibitory properties, with epitope mapping identifying potential neutralization regions. Altogether, ixochymostatin emerges as a novel tick protein that modulates host responses to facilitate blood acquisition. 2. We conducted a longitudinal transcriptome analysis of the midgut in adult female Amblyomma americanum ticks across different feeding stages: unfed, slow-feeding, and rapid-feeding. Our analysis identified 15,599 putative coding sequences (CDS) across 26 functional groups. We observed dynamic transcriptional changes in the midgut, particularly during initial feeding and the transition from slow to rapid feeding. Comparative analyses with midgut transcriptomes from other hard ticks, such as I. scapularis and R. microplus, revealed transcripts commonly expressed across feeding phases. Our findings provide a detailed temporal view of metabolic pathways in A. americanum and uncover conserved transcripts with similar expression patterns across three tick species. This resource advances our understanding of tick physiology and may support the development of control strategies targeting multiple tick species. 3. We identified a secreted salivary serine protease inhibitor, IxsS7, as a novel antigenic biomarker of I. scapularis exposure in humans. The IxsS7 coding sequence is highly conserved (>90% identity) among other important Ixodes species (e.g., I. ricinus, I. persulcatus, and I. pacificus) but poorly conserved (<50% identity) with homologs from other tick genera, such as Amblyomma, Dermacentor, Rhipicephalus, and Haemaphysalis. Antibodies in sera from rabbits immunized with recombinant IxsS7 (rIxsS7) strongly recognized native IxsS7 in salivary gland homogenates (SGH) from blood-fed I. scapularis females, while showing minimal cross-reactivity with SGH from other hard tick genera. Western blot and ELISA analyses revealed that human subjects reporting recent tick exposure possessed IgG antibodies that recognized rIxsS7, highlighting its potential as a biomarker specifically for I. scapularis exposure. Further development of serological tools that measure antibody responses to Ixodes-specific salivary antigens will improve the quantification of individual- and population-level risk of tick-borne diseases such as Lyme disease. 4. We identified orthologs of canonical histone-modifying enzymes, along with components of the machinery responsible for m5C- and 6mA-DNA, and m6A-RNA methylations in a tick species. We further characterized their transcriptional profiles and enzymatic activities during embryonic development. To explore the functional consequences of epigenetic regulation in ticks, we tested the effects of various epigenetic inhibitors on the BME26 embryonic tick cell line. Molecular docking simulations were performed to predict the binding modes of these inhibitors to tick enzymes, followed by in vitro assessment of their effects on cell viability and morphology. Tick cells exposed to these inhibitors exhibited distinct phenotypic and molecular alterations. Notably, we observed higher levels of DNA methylation in the mitochondrial genome compared to nuclear DNA. Inhibition of DNA methylation using 5′-azacytidine (5′-AZA) was associated with increased activity of the mitochondrial electron transport chain and ATP synthesis, but reduced cellular proliferation. These findings highlight the importance of epigenetic regulation during tick embryogenesis and suggest that targeting these pathways may provide novel strategies for tick control. 5. We characterized Amblyostatin-1, the first salivary cystatin with immunomodulatory and anti-inflammatory properties from the Neotropical tick Amblyomma sculptum, a vector of Brazilian spotted fever. Based on its sequence and predicted three-dimensional structure, Amblyostatin-1 is classified as an I25B cystatin. Its recombinant form selectively inhibits cathepsins L, C, and S at different rates, with a low nanomolar Ki value of 0.697 ± 0.22 nM against cathepsin L. Functionally, recombinant Amblyostatin-1 partially modulates LPS-induced dendritic cell maturation by downregulating the costimulatory molecules CD80 and CD86 at higher micromolar concentrations (3 µM), while promoting IL-10 production at nanomolar concentrations (100 nM). The apparent absence of Amblyostatin-1-specific antibody responses in immunized mice suggests impairment of antigen processing and presentation in vivo. Furthermore, in a carrageenan-induced inflammation model, Amblyostatin-1 reduced edema formation and neutrophil infiltration in the skin without affecting other myeloid cells. These findings establish Amblyostatin-1 as a novel salivary cystatin with immunomodulatory and anti-inflammatory properties, underscoring its potential as an immunobiological agent.