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Borrelia miyamotoi infection is an emerging tick-borne disease that causes hard tick-borne relapsing fever. B. miyamotoi is transmitted through the bite of ticks, including Ixodes persulcatus. Although accumulating evidence suggests that tick salivary proteins enhance the infectivity of other tick-borne pathogens, the association of B. miyamotoi with tick-derived proteins remains unknown. In this study, the effect of I. persulcatus sialostatin L2 (Ip-sL2), a tick-derived cystatin, on specific immunity to B. miyamotoi was preliminarily investigated in vitro. Mice were immunized with heat-killed B. miyamotoi and in vitro analyses of the splenocytes of the immunized mice indicated that the expression levels of the activation markers of CD11c + and CD3 + cells were significantly upregulated by B. miyamotoi stimulation. Spleen cells from B. miyamotoi-immunized mice were used to determine whether Ip-sL2 regulates murine immune responses against B. miyamotoi. Treatment with Ip-sL2 in vitro inhibited the activation of CD11c + and CD3 + cells as well as inflammatory cytokine production by cultured splenocytes. These findings show that Ip-sL2 has modulatory effects on murine immune responses to B. miyamotoi. Therefore, it is necessary to clarify in the future whether Ip-sL2 is involved in the enhanced infectivity of B. miyamotoi., (Copyright © 2022. Published by Elsevier GmbH.)

Ferritin 2 (FER2) is an iron storage protein, which has been shown to be critical for iron homeostasis during blood feeding and reproduction in ticks and is therefore suitable as a component for anti-tick vaccines. In this study, we identified the FER2 of Ixodes persulcatus, a major vector for zoonotic diseases such as Lyme borreliosis and tick-borne relapsing fever in Japan, and investigated its functions. Ixodes persulcatus-derived ferritin 2 (Ip-FER2) showed concentration-dependent iron-binding ability and high amino acid conservation, consistent with FER2s of other tick species. Vaccines containing the recombinant Ip-FER2 elicited a significant reduction of the engorgement weight of adult I. persulcatus. Interestingly, the reduction of engorgement weight was also observed in Ixodes ovatus, a sympatric species of I. persulcatus. In silico analyses of FER2 sequences of I. persulcatus and other ticks showed a greater similarity with I. scapularis and I. ricinus and lesser similarity with Hyalomma anatolicum, Haemaphysalis longicornis, Rhipicephalus microplus, and R. appendiculatus. Moreover, it was observed that the tick FER2 sequences possess conserved regions within the primary structures, and in silico epitope mapping analysis revealed that antigenic regions were also conserved, particularly among Ixodes spp ticks. In conclusion, the data support further protective tick vaccination applications using the Ip-FER2 antigens identified herein., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Tick saliva contains immunosuppressants which are important to obtain a blood meal and enhance the infectivity of tick-borne pathogens. In Japan, Ixodes persulcatus is a major vector for Lyme borreliosis pathogens, such as Borrelia garinii, as well as for those causing relapsing fever, such as B. miyamotoi. To date, little information is available on bioactive salivary molecules, produced by this tick. Thus, in this study, we identified two proteins, I. persulcatus derived sialostatin L1 (Ip-sL1) and sL2 (Ip-sL2), as orthologs of I. scapularis derived sL1 and sL2. cDNA clones of Ip-sL1 and Ip-sL2 shared a high identity with sequences of sL1 and sL2 isolated from the salivary glands of I. scapularis. Semi-quantitative PCR revealed that Ip-sL1 and Ip-sL2 were expressed in the salivary glands throughout the life of the tick. In addition, Ip-sL1 and Ip-sL2 were expressed even before the ticks started feeding, and their expression continued during blood feeding. Recombinant Ip-sL1 and Ip-sL2 were developed to characterize the proteins via biological and immunological analyses. These analyses revealed that both Ip-sL1 and Ip-sL2 had inhibitory effects on cathepsins L and S. Ip-sL1 and Ip-sL2 inhibited the production of IP-10, TNFα, and IL-6 by LPS-stimulated bone-marrow-derived dendritic cells (BMDCs). Additionally, Ip-sL1 significantly impaired BMDC maturation. Taken together, these results suggest that Ip-sL1 and Ip-sL2 confer immunosuppressive functions and appear to be involved in the transmission of pathogens by suppressing host immune responses, such as cytokine production and dendritic cell maturation. Therefore, further studies are warranted to investigate the immunosuppressive functions of Ip-sL1 and Ip-sL2 in detail to clarify their involvement in pathogen transmission via I. persulcatus., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Cystatins are cysteine peptidase inhibitors that in ticks mediate processes such as blood feeding and digestion. The ixodid tick Ixodes persulcatus is endemic to the Eurasia, where it is the principal vector of Lyme borreliosis. To date, no I. persulcatus cystatin has been characterized. In the present work, we describe three novel cystatins from I. persulcatus, named JpIpcys2a, JpIpcys2b and JpIpcys2c. In addition, the potential of tick cystatins as cross-protective antigens was evaluated by vaccination of hamsters using BrBmcys2c, a cystatin from Rhipicephalus microplus, against I. persulcatus infestation. Sequence analysis showed that motifs that are characteristic of cystatins type 2 are fully conserved in JpIpcys2b, while mutations are present in both JpIpcys2a and JpIpcys2c. Protein-protein docking simulations further revealed that JpIpcys2a, JpIpcys2b and JpIpcys2c showed conserved binding sites to human cathepsins L, all of them covering the active site cleft. Cystatin transcripts were detected in different I. persulcatus tissues and instars, showing their ubiquitous expression during I. persulcatus development. Serological analysis showed that although hamsters immunized with BrBmcys2c developed a humoral immune response, this response was not adequate to protect against a heterologous challenge with I. persulcatus adult ticks. The lack of cross-protection provided by BrBmcys2c immunization is perhaps linked to the fact that cystatins cluster into multigene protein families that are expressed differentially and exhibit functional redundancy. How to target such small proteins that are secreted in low quantities remains a challenge in the development of suitable anti-tick vaccine antigens., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Ixodes ricinus immunosuppressor (Iris) is a tick salivary gland protein derived from I. ricinus. In this study, Iris homolog was identified in the salivary glands of Ixodes persulcatus, which is the specific vector of the Lyme disease agent in Japan. The homolog was named Ipis-1. To investigate the function of Ipis-1, we prepared a recombinant Ipis-1 expressed in COS-7 cells as a rabbit IgG Fc-fused protein (Ipis-1-Ig). Cell proliferation assay and IFN-γ ELISA showed that Ipis-1-Ig inhibits the proliferation and IFN-γ production of bovine peripheral blood mononuclear cells (PBMCs). Notably, Ipis-1-Ig inhibited the cell proliferation and production of IFN-γ in bovine PBMCs even when CD14(+) cells were depleted, suggesting that Ipis could directly interact with T cells and inhibit their functions. In conclusion, Ipis could contribute to the establishment of environments suitable for tick blood feeding and pathogen transmission by suppressing the function of immune cells., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Among arthropods, ticks lead as vectors of animal diseases and rank second to mosquitoes in transmitting human pathogens. Cyclin-dependent kinases (CDK) participate in cell cycle control in eukaryotes. CDKs are serine/threonine protein kinases and these catalytic subunits are activated or inactivated at specific stages of the cell cycle. To determine the potential of using CDKs as anti-tick vaccine antigens, hamsters were immunized with recombinant Ixodes persulcatus CDK10, followed by a homologous tick challenge. Though it was not exactly unexpected, IpCDK10 vaccination significantly impaired tick blood feeding and fecundity, which manifested as low engorgement weights, poor oviposition, and a reduction in 80% of hatching rates. These findings may underpin the development of more efficacious anti-tick vaccines based on the targeting of cell cycle control proteins., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Salp15, a 15-kDa tick salivary gland protein, has several suppressive modes of activity against host immunity and plays a critical role in the transmission of Lyme disease spirochetes in Ixodes scapularis and Ixodes ricinus, major vectors of Lyme disease in North America and Western Europe. Salp15 adheres to Borrelia burgdorferi and specifically interacts with its outer surface protein C (OspC), protecting the spirochete from antibody-mediated cytotoxicity and facilitating infection in the mice. Recently, we identified two Salp15 homologues, IperSalp15-1 and IperSalp15-2, in Ixodes persulcatus, a vector for Lyme disease in Japan. Here we describe the function of IperSalp15 in the transmission of Lyme borreliosis. To investigate the function of IperSalp15, recombinant IperSalp15-1 and IperSalp15-2 were prepared in bacterial and insect cells. Both were identified in the sera of tick-immunized hamsters, indicating that these are secretory proteins in exposed host animals. Solid-phase overlay and indirect fluorescence assays showed that IperSalp15 binds to OspC from B. burgdorferi, Borrelia garinii, and Borrelia afzelii. Importantly, this binding likely protected the spirochete from antibody-mediated cytotoxicity in vitro. In addition, IperSalp15 tended to facilitate infection in mice. Thus, further characterization of tick molecules, including IperSalp15, could lead to the development of new strategies to prevent the transmission of tick-borne diseases., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Salp16, a 16-kDa tick salivary gland protein, is known to be the molecule involved in the transmission of Anaplasma phagocytophilum, an obligate intracellular pathogen causing zoonotic anaplasmosis, from its mammalian hosts to Ixodes scapularis. Recently, the presence of A. phagocytophilum was documented in Japan and Ixodes persulcatus was identified as one of its vectors. The purpose of this study was to identify Salp16 genes in I. persulcatus and characterize their function. Two cDNA clones encoding the Salp16-like sequences were obtained from the salivary glands of fed female I. persulcatus ticks and designated Salp16 Iper1 and Iper2. Gene expression analyses showed that the Salp16 Iper genes were expressed specifically in the salivary glands and were up-regulated by blood feeding. These proteins attenuated the oxidative burst of activated bovine neutrophils and inhibited their migration induced by the chemoattractant interleukin-8 (IL-8). These results demonstrate that Salp16 Iper proteins contribute to the establishment of blood feeding as an immunosuppressant of neutrophil, an essential factor in innate host immunity. Further examination of the role of Salp16 Iper in the transmission of pathogens, including A. phagocytophilum, will increase our understanding of the tick-host-pathogen interface., (© 2014 The Royal Entomological Society.)