IrC2/Bf - A yeast and Borrelia responsive component of the complement system from the hard tick Ixodes ricinus.

Ticks possess components of a primordial complement system that presumably play a role in the interaction of the tick immune system with tick-borne pathogens and affect their transmission. Here we characterized a novel complement component, tagged as IrC2/Bf, from the hard tick Ixodes ricinus, the principal vector of Lyme disease in Europe. IrC2/Bf is a multi-domain molecule composed of 5-7 CCP modules, varied by alternative splicing, followed by a von Willebrand factor A domain and a C-terminal trypsin-like domain. The primary structure and molecular architecture of IrC2/Bf displays the closest homology to the C3-complement component convertases described in horseshoe crabs. The irc2/bf gene is mainly expressed in the tick fat body associated with the trachea and, as determined by western blotting, the protein is present in low amounts in tick hemolymph. Expression of irc2/bf mRNA was significantly up-regulated in response to the intra-hemocoelic injection of the yeast Candida albicans and all tested Borrelia sp. strains (B. burgdorferi NE5264, B. burgdorferi CB26, B. garinii MSLB, B. afzelii CB43), but was not affected by injection of model Gram-negative and Gram-positive bacteria or the aseptic injection control. In-line with these results, RNAi-mediated silencing of irc2/bf inhibited phagocytosis of B. afzelii and C. albicans but not the other bacteria. Tissue expression profiles, specific responses to microbial challenges, and patterns of phagocytic phenotypes upon RNAi silencing observed for IrC2/Bf match well with the previously reported characteristics of I. ricinus C3-related molecule 1 (IrC3-1). Therefore we presume that IrC2/Bf functions as a convertase in the same complement activation pathway protecting ticks against yeast and Borrelia infection.

Characterization of Ixodes ricinus Fibrinogen-Related Proteins (Ixoderins) Discloses Their Function in the Tick Innate Immunity.

Ticks are important vectors of serious human and animal disease-causing organisms, but their innate immunity can fight invading pathogens and may have the ability to reduce or block transmission to mammalian hosts. Lectins, sugar-binding proteins, can distinguish between self and non-self-oligosaccharide motifs on pathogen surfaces. Although tick hemolymph possesses strong lectin activity, and several lectins have already been isolated and characterized, little is known about the implementation of these molecules in tick immunity. Here, we have described and functionally characterized fibrinogen-related protein (FReP) lectins in Ixodes ticks. We have shown that the FReP family contains at least 27 genes (ixoderins, ixo) that could, based on phylogenetic and expression analyses, be divided into three groups with differing degrees of expansion. By using RNA interference-mediated gene silencing (RNAi) we demonstrated that IXO-A was the main lectin in tick hemolymph. Further, we found that ixoderins were important for phagocytosis of Gram-negative bacteria and yeasts by tick hemocytes and that their expression was upregulated upon injection of microbes, wounding, or after blood feeding. However, although the tick hemocytes could swiftly phagocytose Borrelia afzelii spirochetes, their transmission and burst of infection in mice was not altered. Our results demonstrate that tick ixoderins are crucial immune proteins that work as opsonins in the tick hemolymph, targeting microbes for phagocytosis or lysis.

Tick Thioester-Containing Proteins and Phagocytosis Do Not Affect Transmission of Borrelia afzelii from the Competent Vector Ixodes ricinus.

The present concept of the transmission of Lyme disease from Borrelia-infected Ixodes sp. ticks to the naïve host assumes that a low number of spirochetes that manage to penetrate the midgut epithelium migrate through the hemocoel to the salivary glands and subsequently infect the host with the aid of immunomodulatory compounds present in tick saliva. Therefore, humoral and/or cellular immune reactions within the tick hemocoel may play an important role in tick competence to act as a vector for borreliosis. To test this hypothesis we have examined complement-like reactions in the hemolymph of the hard tick Ixodes ricinus against Borrelia afzelii (the most common vector and causative agent of Lyme disease in Europe). We demonstrate that I. ricinus hemolymph does not exhibit borreliacidal effects comparable to complement-mediated lysis of bovine sera. However, after injection of B. afzelii into the tick hemocoel, the spirochetes were efficiently phagocytosed by tick hemocytes and this cellular defense was completely eliminated by pre-injection of latex beads. As tick thioester-containing proteins (T-TEPs) are components of the tick complement system, we performed RNAi-mediated silencing of all nine genes encoding individual T-TEPs followed by in vitro phagocytosis assays. Silencing of two molecules related to the C3 complement component (IrC3-2 and IrC3-3) significantly suppressed phagocytosis of B. afzelii, while knockdown of IrTep (insect type TEP) led to its stimulation. However, RNAi-mediated silencing of T-TEPs or elimination of phagocytosis by injection of latex beads in B. afzelii-infected I. ricinus nymphs had no obvious impact on the transmission of spirochetes to naïve mice, as determined by B. afzelii infection of murine tissues following tick infestation. This result supports the concept that Borrelia spirochetes are capable of avoiding complement-related reactions within the hemocoel of ticks competent to transmit Lyme disease.