Ondřej Hajdušek

Interaction of the tick immune system with transmitted pathogens.

Ticks are hematophagous arachnids transmitting a wide variety of pathogens including viruses, bacteria, and protozoans to their vertebrate hosts. The tick vector competence has to be intimately linked to the ability of transmitted pathogens to evade tick defense mechanisms encountered on their route through the tick body comprising midgut, hemolymph, salivary glands or ovaries. Tick innate immunity is, like in other invertebrates, based on an orchestrated action of humoral and cellular immune responses. The direct antimicrobial defense in ticks is accomplished by a variety of small molecules such as defensins, lysozymes or by tick-specific antimicrobial compounds such as microplusin/hebraein or 5.3-kDa family proteins. Phagocytosis of the invading microbes by tick hemocytes is likely mediated by the primordial complement-like system composed of thioester-containing proteins, fibrinogen-related lectins and convertase-like factors. Moreover, an important role in survival of the ingested microbes seems to be played by host proteins and redox balance maintenance in the tick midgut. Here, we summarize recent knowledge about the major components of tick immune system and focus on their interaction with the relevant tick-transmitted pathogens, represented by spirochetes (Borrelia), rickettsiae (Anaplasma), and protozoans (Babesia). Availability of the tick genomic database and feasibility of functional genomics based on RNA interference greatly contribute to the understanding of molecular and cellular interplay at the tick-pathogen interface and may provide new targets for blocking the transmission of tick pathogens.

Tick innate immunity.

Ticks are blood feeding parasites transmitting a wide variety of pathogens to their vertebrate hosts. The vector competence of ticks is tightly linked with their immune system. Despite its importance, our knowledge of tick innate immunity is still inadequate and the limited number of sufficiently characterized immune molecules and cellular reactions are dispersed across numerous tick species. The phagocytosis of microbes by tick hemocytes seems to be coupled with a primitive complement-like system, which possibly involves self/nonself recognition by fibrinogen-related lectins and the action of thioester-containing proteins. Ticks do not seem to possess a pro-phenoloxidase system leading to melanization and also coagulation of tick hemolymph has not been experimentally proven. They are capable of defending themselves against microbial infection with a variety of antimicrobial peptides comprising lysozymes, defensins and molecules not found in other invertebrates. Virtually nothing is known about the signaling cascades involved in the regulation of tick antimicrobial immune responses. Midgut immunity is apparently the decisive factor of tick vector competence. The gut content is a hostile environment for ingested microbes, which is mainly due to the antimicrobial activity of hemoglobin fragments generated by the digestion of the host blood as well as other antimicrobial peptides. Reactive oxygen species possibly also play an important role in the tick-pathogen interaction. The recent release of the Ixodes scapularis genome and the feasibility of RNA interference in ticks promise imminent and substantial progress in tick innate immunity research.

Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases

BackgroundTicks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets.ResultsUsing the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain), and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood.ConclusionOverall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.

IrFC - An Ixodes ricinus injury-responsive molecule related to Limulus Factor C.

Limulus Clotting Factor C is a multi-domain serine protease that triggers horseshoe crab hemolymph clotting in the presence of trace amounts of bacterial lipopolysaccharides. Here we describe and functionally characterize an homologous molecule, designated as IrFC, from the hard tick Ixodes ricinus. Tick Factor C consists of an N-terminal cysteine-rich domain, four complement control protein (sushi) modules, an LCCL domain, a truncated C-lectin domain and a C-terminal trypsin-type domain. Developmental expression profiling by quantitative real-time PCR revealed that the irfc mRNA is expressed in all stages including eggs. In tissues dissected from adult I. ricinus females, the irfc mRNA is present mainly in tick hemocytes and accordingly, indirect immunofluorescence microscopy localized IrFC intracellularly, in tick hemocytes. Irfc mRNA levels were markedly increased upon injection of sterile saline, or different microbes, demonstrating that the irfc gene transcription occurs in response to injury. This indicates a possible role of IrFC in hemolymph clotting and/or wound healing, although these defense mechanisms have not been yet definitely demonstrated in ticks. RNAi silencing of irfc expression resulted in a significant reduction in phagocytic activity of tick hemocytes against the Gram-negative bacteria Chryseobacterium indologenes and Escherichia coli, but not against the yeast, Candida albicans. This result suggests that IrFC plays a role in the tick primordial complement system and as such possibly mediates transmission of tick-borne pathogens.

Thioester-containing proteins of the tick Ixodes ricinus: Gene expression, response to microbial challenge and their role in phagocytosis of the yeast Candida albicans

The ability of ticks to act as vectors for a wide range of serious human and animal infectious diseases is apparently linked to the insufficiency of the tick immune system to effectively eliminate pathogens they transmit. At the tick-pathogen interface, an important role is presumably played by components of an ancient complement system that includes a repertoire of thioester-containing proteins (TEPs), which in Ixodes sp. comprises three α2-macroglobulins (A2M), three C3 complement component-related molecules (C3), two macroglobulin complement-related (Mcr) and one insect-type TEPs (Tep). In order to assess the function of TEPs in tick immunity, a quantitative real-time PCR expression analysis of tick TEPs was performed at various developmental stages of Ixodes ricinus, and in tissues dissected from adult females. Expression of TEP genes was mostly tissue specific; IrA2M1, IrC3-1, IrC3-3 were found to be expressed in cells of tick fat body adjacent to the tracheal trunks, IrA2M2 in hemocytes, IrTep in ovaries, IrMcr1 in salivary glands and only IrA2M3, IrC3-2 and IrMcr2 mRNAs were present in multiple organs. Expression of tick TEPs was further examined in response to injection of model microbes representing Gram-negative, Gram-positive bacteria and yeast. The greatest expression induction was observed for IrA2M1 and IrC3-1 after challenge with the yeast Candida albicans. Phagocytosis of the yeast was strongly dependent on an active thioester bond and the subsequent silencing of individual tick TEPs by RNA interference demonstrated the involvement of IrC3-1 and IrMcr2. This result suggests the existence of a distinct complement-like pathway, different from that leading to phagocytosis of Gram-negative bacteria. Understanding of the tick immune response against model microbes should provide new concepts for investigating interactions between ticks and relevant tick-borne pathogens.

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.

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

ABSTRACT 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. Graphical abstract Figure. No Caption available. HighlightsIrC2/Bf ‐ a molecule related to the complement components C2 and/or Bf was characterized in the tick Ixodes ricinus.IrC2/Bf expression is responsive to the injection of the Borrelia spirochetes and the yeast Candida albicans.RNAi‐mediated silencing of IrC2/Bf reduced phagocytosis of Borrelia afzelii and C. albicans by tick hemocytes.IrC2/Bf functions likely as a convertase of I. ricinus C3‐related molecules in the same complement‐like pathway.