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

Abstract

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.

TrackingBorrelia afzeliifrom infectedIxodes ricinusnymphs to mice suggests a direct ‘gut-to-mouth’ route of Lyme disease transmission

“…Instead, we assume that regurgitation, or rather active reverse migration of motile spirochetes from midgut to the mouthpart (for which we coin the term ‘gut-to-mouth’ route) is the most probable way of transmission of B. afzelii from I. ricinus nymph to the host. This alternative way of B. afzelii transmission avoiding I. ricinus hemocoel and salivary glands is indirectly supported also by our recent research showing that silencing of tick immune molecules or elimination of phagocytosis in tick hemocoel by injection of latex beads had no obvious impact on B. afzelii transmission (33–35).…”

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

“…By using the tick hemocytes of semi-engorged females we tested the effects of individual ixoderin KDs on the phagocytosis of B. afzelii CB43. For this purpose we used a phagocytic assay (modified from Urbanova et al, 2017 ) that can reliably distinguish between spirochetes located outside of the hemocytes, sticking to their surface, and those, which were certainly phagocytosed (Figure 6 ). The phagocytosis of Borrelia can be reduced by pre-incubation of the hemolymph with the thioester-blocking reagent methylamine (Figure 7A ).…”

“…Similarly, the tick salivary protein TSLPI inhibits the host complement system and thus facilitates Borrelia transmission (Schuijt et al, 2011a , b ). By using the Borrelia -phagocytic assay and Borrelia -transmission system (Urbanova et al, 2017 ) we tested whether tick ixoderins, expressed both in the hemolymph and salivary glands, are able to activate the tick complement-like system and kill the spirochetes or bind to the surface of pathogens to protect them in the tick vector or the vertebrate host. In these assays we used in vitro cultivated Borrelia (BSK-H complete medium, 33°C), which express similar surface proteins as the activated spirochetes in the tick hemolymph during tick feeding and are infectious to the vertebrate host (Obonyo et al, 1999 ; Ohnishi et al, 2001 ; Dunham-Ems et al, 2009 ).…”

“…The in vitro phagocytic assays with Chryseobacterium indologenes, Escherichia coli, Staphylococcus aureus , and Candida albicans and the methylamine (MA) pre-treatment assay were carried out as described previously (Buresova et al, 2011 ; Urbanova et al, 2014 ). For the phagocytic assay with Borrelia (modified from Urbanova et al, 2017 ), spirochetes of B. afzelii CB43 were cultivated in BSK-H complete medium (Sigma) at 33°C for 5–7 days to a concentration of 10 8 spirochetes per ml. The hemocytes (~4 × 10 4 ) from ixoderin or gfp dsRNA-injected semi-engorged females, collected in 240 μl of L15-BOFES medium, were incubated with 10 μl of B. afzelii CB43 (10 6 spirochetes) for 120 min at 28°C.…”

Borrelia bavariensis: Vector Switch, Niche Invasion, and Geographical Spread of a Tick-Borne Bacterial Parasite

“…possess a number of immune effectors and modulators such as recognition molecules, among them thioester-containing proteins (T-TEPS, that serve as lectins labeling cells for immune attack), phagocytotic hemocytes, defensins, lysozymes, antimicrobial peptides, a dityrosine network (DTN), and (atypical) signaling pathways. The signaling pathways regulating the immune system include Toll, IMD (Immunodeficiency) and JAK-STAT (Janus Kinase/Signal Transducers and Activators of Transcription) as well as an indirect, cross-species signaling pathway that recognizes the cytokine interferon gamma in the blood of the vertebrate host (Johns et al, 2001;Hajdusek et al, 2013;Yang et al, 2014;Sonenshine and Macaluso, 2017;Urbanova et al, 2017). RNA interference studies on genes involved in the tick's immune response have shown that depletion of expression may lead to suppression of Borrelia colonization in ticks (Narasimhan et al, 2017) suggesting that Borrelia can exploit tick molecules to its own advantage.…”