Ixodes ricinus Salivary Serpin Iripin-8 Inhibits the Intrinsic Pathway of Coagulation and Complement.

Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P' side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity.

Mosquito vector proteins homologous to α1-3 galactosyl transferases of tick vectors in the context of protective immunity against malaria and hypersensitivity to vector bites.

BACKGROUND: An epitope, Galα1-3Galß1-4GlcNAc-R, termed α-gal, is present in glycoconjugates of New World monkeys (platyrrhines) and other mammals but not in hominoids and Old World monkeys (catarrhines). The difference is due to the inactivation of α1-3 galactosyl transferase (α1-3 GT) genes in catarrhines. Natural antibodies to α-gal are therefore developed in catarrhines but not platyrrhines and other mammals. Hypersensitivity reactions are commonly elicited by mosquito and tick vector bites. IgE antibodies against α-gal cause food allergy to red meat in persons who have been exposed to tick bites. Three enzymes synthesising the terminal α1-3-linked galactose in α-gal, that are homologous to mammalian α and ß1-4 GTs but not mammalian α1-3 GTs, were recently identified in the tick vector Ixodes scapularis. IgG and IgM antibodies to α-gal are reported to protect against malaria because mosquito-derived sporozoites of malaria parasites express α-gal on their surface. This article explores the possibility that the α-gal in sporozoites are acquired from glycoconjugates synthesised by mosquitoes rather than through de novo synthesis by sporozoites. METHODS: The presence of proteins homologous to the three identified tick α1-3 GTs and mammalian α1-3 GTs in two important mosquito vectors, Aedes aegypti and Anopheles gambiae, as well as Plasmodium malaria parasites, was investigated by BLASTp analysis to help clarify the source of the α-gal on sporozoite surfaces. RESULTS: Anopheles gambiae and Ae. aegypti possessed several different proteins homologous to the three I. scapularis proteins with α1-3 GT activity, but not mammalian α1-3 GTs. The putative mosquito α1-3 GTs possessed conserved protein domains characteristic of glycosyl transferases. However, the genus Plasmodium lacked proteins homologous to the three I. scapularis proteins with α1-3 GT activity and mammalian α1-3 GTs. CONCLUSIONS: The putative α1-3 GTs identified in the two mosquito vectors may synthesise glycoconjugates containing α-gal that can be transferred to sporozoite surfaces before they are inoculated into skin during blood feeding. The findings merit further investigation because of their implications for immunity against malaria, hypersensitivity to mosquito bites, primate evolution, and proposals for immunisation against α-gal.

An Ixodes scapularis glutathione S-transferase plays a role in cell survival and viability during Langat virus infection of a tick cell line.

Ticks are important vectors of diseases affecting both humans and animals. To be an efficient vector, ticks have to survive infection by pathogens such as the Langat virus (LGTV). One method utilized by ticks is their complex antioxidant mechanism. Included in the vast antioxidant processes are several enzymes involved in redox homeostasis. The ubiquitous glutathione S-transferases (GSTs) belong to the antioxidant family of enzymes. In this study, we evaluated the role of a GST during LGTV infection. ISE6 cells were infected with LGTV with a multiplicity of infection (MOI) of 0.01 and observed daily. The infection success was monitored via indirect immunofluorescent antibody test (IFAT) for LGTV for up to 4 days. The gene expression of IsGST1 was determined by real-time polymerase chain reaction (PCR) using IsGST1 gene-specific primers. Knockdown of the IsGST1 gene with subsequent LGTV infection was also performed. Afterward, ISE6 cell mortality and viability were checked daily until the fourth day. The virus titer from supernatants of IsGST1-knockdown cells was quantified using a focus-formation assay. IFAT data showed that LGTV infects ISE6 cells in a time-dependent manner with increasing infection from day 0 to day 4. The IsGST1 genes showed an increasing expression until day 2 of infection, while decreased expression was observed from day 3 to day 4 post-infection. Knockdown of the IsGST1 resulted in increased mortality on the third day of infection, while the cell viability was also negatively affected by the knockdown of the IsGST1 genes from day 0 to day 4 post-infection. Knockdown of the IsGST1 genes also resulted in a decreased viral titer from the supernatants of the ISE6 cells infected with LGTV. Based on the results, GSTs are possibly utilized both by cells and the virus for mutual survival and proliferation.

An Ixodes scapularis Protein Disulfide Isomerase Contributes to Borrelia burgdorferi Colonization of the Vector.

Borrelia burgdorferi causes Lyme disease, the most common tick-transmitted illness in North America. When Ixodes scapularis feed on an infected vertebrate host, spirochetes enter the tick gut along with the bloodmeal and colonize the vector. Here, we show that a secreted tick protein, I. scapularisprotein disulfide isomerase A3 (IsPDIA3), enhances B. burgdorferi colonization of the tick gut. I. scapularis ticks in which ispdiA3 has been knocked down using RNA interference have decreased spirochete colonization of the tick gut after engorging on B. burgdorferi-infected mice. Moreover, administration of IsPDIA3 antiserum to B. burgdorferi-infected mice reduced the ability of spirochetes to colonize the tick when feeding on these animals. We show that IsPDIA3 modulates inflammatory responses at the tick bite site, potentially facilitating spirochete survival at the vector-host interface as it exits the vertebrate host to enter the tick gut. These data provide functional insights into the complex interactions between B. burgdorferi and its arthropod vector and suggest additional targets to interfere with the spirochete life cycle.

Newly identified DNA methyltransferases of Ixodes ricinus ticks.

DNA methylation at the fifth position of cytosine (5mC) and at the sixth position of adenine (6 mA) plays an important role in the regulation of the gene expression and, in eukaryotes, is essential for normal development. For Ixodes ricinus, the most common European arthropod vector of human and animal pathogens, the DNA methylation profile and the role of DNA methylation in tick development are still under discussion. Our goal was to analyze the status of I. ricinus DNA methylation at different life stages and identify enzymes that produce this type of DNA modification. We found that 5mC and 6mA are present in I. ricinus genomic DNA at all life stages. In the transcriptome of I. ricinus, we identified the sequences of the putative IrDNMT1, IrDNMT3, and IrDAMT enzymes, and bioinformatic analysis and three-dimensional modeling predicted their DNA methylation activity. This confirms that I. ricinus possesses a complete DNA methylation toolkit. Our results suggest that DNA methylation is important for the physiology and transstadial development of ticks.

Ixodes scapularis Src tyrosine kinase facilitates Anaplasma phagocytophilum survival in its arthropod vector.

Anaplasma phagocytophilum, the agent of human anaplasmosis, is an obligate intracellular bacterium that uses multiple survival strategies to persist in Ixodes scapularis ticks. Our previous study showed that A. phagocytophilum efficiently induced the tyrosine phosphorylation of several Ixodes proteins that includes extended phosphorylation of actin at tyrosine residue Y178. In order to identify the tyrosine kinase responsible for the A. phagocytophilum induced tyrosine phosphorylation of proteins, we combed the I. scapularis genome and identified a non-receptor Src tyrosine kinase ortholog. I. scapularis Src kinase showed high degree of amino acid sequence conservation with Dsrc from Drosophila melanogaster. We noted that at different developmental stages of I. scapularis ticks, larvae expressed significantly higher levels of src transcripts in comparison to the other stages. We found that A. phagocytophilum significantly reduced Src levels in unfed nymphs and in nymphs while blood feeding (48 h during feeding) in comparison to the levels noted to relative uninfected controls. However, A. phagocytophilum increased Src levels in fully engorged larvae and nymphs (48 h post feeding) and in vitro tick cells in comparison to the relative uninfected controls. Inhibition of Src kinase expression and activity by treatment with src-dsRNA or Src-inhibitor, respectively, significantly reduced A. phagocytophilum loads in ticks and tick cells. Overall, our study provides evidence for the important role of I. scapularis Src kinase in facilitating A. phagocytophilum colonization and survival in the arthropod vector.

Inducible glutathione S-transferase (IrGST1) from the tick Ixodes ricinus is a haem-binding protein.

Blood-feeding parasites are inadvertently exposed to high doses of potentially cytotoxic haem liberated upon host blood digestion. Detoxification of free haem is a special challenge for ticks, which digest haemoglobin intracellularly. Ticks lack a haem catabolic mechanism, mediated by haem oxygenase, and need to dispose of vast majority of acquired haem via its accumulation in haemosomes. The knowledge of individual molecules involved in the maintenance of haem homeostasis in ticks is still rather limited. RNA-seq analyses of the Ixodes ricinus midguts from blood- and serum-fed females identified an abundant transcript of glutathione S-transferase (gst) to be substantially up-regulated in the presence of red blood cells in the diet. Here, we have determined the full sequence of this encoding gene, ir-gst1, and found that it is homologous to the delta-/epsilon-class of GSTs. Phylogenetic analyses across related chelicerates revealed that only one clear IrGST1 orthologue could be found in each available transcriptome from hard and soft ticks. These orthologues create a well-supported clade clearly separated from other ticks' or mites' delta-/epsilon-class GSTs and most likely evolved as an adaptation to tick blood-feeding life style. We have confirmed that IrGST1 expression is induced by dietary haem(oglobin), and not by iron or other components of host blood. Kinetic properties of recombinant IrGST1 were evaluated by model and natural GST substrates. The enzyme was also shown to bind haemin in vitro as evidenced by inhibition assay, VIS spectrophotometry, gel filtration, and affinity chromatography. In the native state, IrGST1 forms a dimer which further polymerises upon binding of excessive amount of haemin molecules. Due to susceptibility of ticks to haem as a signalling molecule, we speculate that the expression of IrGST1 in tick midgut functions as intracellular buffer of labile haem pool to ameliorate its cytotoxic effects upon haemoglobin intracellular hydrolysis.

Multiple legumain isoenzymes in ticks.

By searching nucleotide databases for the North American Lyme disease vector, Ixodes scapularis, we have complemented the previously characterized European Ixodes ricinus legumain IrAE1 with a full set of nine analogous genes (isae1-9). Six of these were PCR confirmed as genes present in all tick genomes tested. The absolute mRNA copy number examined by quantitative (q)PCR enabled expression profiling and an absolute comparison of mRNA levels for individual I. scapularis (Is)AEs in tick tissues. Four IsAEs (1, 2, 4, 9) were expressed solely in the gut and thus are proposed to be involved in host blood digestion. Expression qPCR profiling over developmental stages confirmed IsAE1, the direct analogue of previously characterized I. ricinus IrAE1, as the principle legumain transcript in partially engorged females, and demonstrated its strong regulation by on-host feeding in larvae, nymphs and females. In contrast, IsAE2 was the predominant gut legumain in unfed nymphs, unfed females and males. In-silico, IsAE1 and IsAE2 protein three-dimensional structural models displayed minimal differences in overall proenzyme structures, even in comparison with recently resolved crystal structures of mammalian prolegumain. Three functional studies were performed in I. ricinus with IsAE1/IsAE2 analogues: double IrAE1/IrAE2 RNA interference silencing, feeding of ticks on IrAE1+IrAE2 immunized hosts and in vitro membrane tick feeding on blood containing a legumain-specific inhibitor. The latter experiment led to reduced weights of fully engorged ticks and limited oviposition, and indicated the potential of legumain inhibitors for novel anti-tick interventions.

Anaplasma phagocytophilum Infection Subverts Carbohydrate Metabolic Pathways in the Tick Vector, Ixodes scapularis.

The obligate intracellular pathogen, Anaplasma phagocytophilum, is the causative agent of human, equine, and canine granulocytic anaplasmosis and tick-borne fever (TBF) in ruminants. A. phagocytophilum has become an emerging tick-borne pathogen in the United States, Europe, Africa, and Asia, with increasing numbers of infected people and animals every year. It has been recognized that intracellular pathogens manipulate host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. However, our current knowledge on how A. phagocytophilum affect these processes in the tick vector, Ixodes scapularis is limited. In this study, a genome-wide search for components of major carbohydrate metabolic pathways was performed in I. scapularis ticks for which the genome was recently published. The enzymes involved in the seven major carbohydrate metabolic pathways glycolysis, gluconeogenesis, pentose phosphate, tricarboxylic acid cycle (TCA), glyceroneogenesis, and mitochondrial oxidative phosphorylation and ß-oxidation were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis major carbohydrate metabolic pathway components in response to A. phagocytophilum infection of tick tissues and cultured cells. The results showed that major carbohydrate metabolic pathways are conserved in ticks. A. phagocytophilum infection inhibits gluconeogenesis and mitochondrial metabolism, but increases the expression of glycolytic genes. A model was proposed to explain how A. phagocytophilum could simultaneously control tick cell glucose metabolism and cytoskeleton organization, which may be achieved in part by up-regulating and stabilizing hypoxia inducible factor 1 alpha in a hypoxia-independent manner. The present work provides a more comprehensive view of the major carbohydrate metabolic pathways involved in the response to A. phagocytophilum infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.

Multienzyme degradation of host serum albumin in ticks.

Host blood proteins, represented mainly by hemoglobin and serum albumin, serve as the ultimate source of amino acids needed for de novo protein synthesis during tick development and reproduction. While uptake and processing of hemoglobin by tick gut cells have been studied in detail, molecular mechanisms of host serum albumin degradation remain unknown. In this work, we have used artificial membrane feeding of Ixodes ricinus females on a hemoglobin-free diet in order to characterize the proteolytic machinery involved in albuminolysis. Morphological comparisons of ticks fed on whole blood (BF) and serum (SF) at microscopic and ultrastructural levels showed that albumin and hemoglobin have different trafficking routes in tick gut cells. Analysis in vitro with selective inhibitors demonstrated that albumin is degraded at an acidic pH by a network of cysteine and aspartic peptidases with predominant involvement of cysteine cathepsins having endo- and exopeptidase activities. The cleavage map of albumin and the roles of individual peptidases in albumin degradation were determined. These results indicate that the albuminolytic pathway is controlled by the same proteolytic system that is responsible for hemoglobinolysis. This was further supported by the overall similarity of gut peptidase profiles in SF and BF ticks at the transcriptional and enzymatic activity levels. In conclusion, our work provides evidence that although hemoglobin and albumin are transported differentially during heterophagy they are digested by a common multienzyme proteolytic network. This central digestive system, critical for successful blood feeding in tick females, thus represents a valuable target for novel anti-tick interventions.

Natural hybridization between Ixodes ricinus and Ixodes persulcatus ticks evidenced by molecular genetics methods.

The recently shown phenomenon of natural hybridization between Ixodes persulcatus and Ixodes pavlovskyi ticks (Kovalev et al., 2015) stimulated similar studies in the sympatric zones of other tick species. In the present paper, 265 Ixodes ricinus and I. persulcatus ticks from Estonia were subjected to a search for interspecific hybrids based on nuclear (ITS2) and mitochondrial (cox1) markers as well as morphological features. Surprisingly, only 72.1% of ticks morphologically identified as I. ricinus actually were I. ricinus both at nuclear and mitochondrial markers, while the accuracy of morphological species identification for I. persulcatus was 99.3%. Among ticks morphologically identified as I. ricinus, 24.6% turned out to be interspecific hybrids and 3.3% were I. persulcatus. Generally, about 11% of the individuals studied were shown to be interspecific hybrids with different levels of nuclear DNA introgression. The analysis of hybrid populations proved the mating pair female I. ricinus×male I. persulcatus to form hybrids more efficiently, then female I. persulcatus×male I. ricinus. The same trend can be observed for backcrosses preferentially mating with I. ricinus. Hybridization between I. ricinus and I. persulcatus proved the existing view about their reproductive isolation to be untenable. Interspecific hybridization occurring between both closely (I. persulcatus and I. pavlovskyi) and more distantly (I. ricinus and I. persulcatus) related Ixodes species could introduce novel alleles that modify vector competence, host use or the ability to exploit diverse microhabitats.

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.

Structural and functional analyses of a glutaminyl cyclase from Ixodes scapularis reveal metal-independent catalysis and inhibitor binding.

Glutaminyl cyclases (QCs) from mammals and Drosophila are zinc-dependent enzymes that catalyze N-terminal pyroglutamate formation of numerous proteins and peptides. These enzymes have been found to be critical for the oviposition and embryogenesis of ticks, implying that they are possible physiological targets for tick control. Here, 1.10-1.15 Šresolution structures of a metal-independent QC from the black-legged tick Ixodes scapularis (Is-QC) are reported. The structures exhibit the typical scaffold of mammalian QCs but have two extra disulfide bridges that stabilize the central ß-sheet, resulting in an increased thermal stability. Is-QC contains ~0.5 stoichiometric zinc ions, which could be removed by 1 mM EDTA. Compared with the Zn-bound form, apo-Is-QC has a nearly identical active-site structure and stability, but unexpectedly possesses significantly increased QC activities towards both synthetic and physiological substrates. Enzyme-kinetic analysis revealed that apo-Is-QC has a stronger substrate-binding affinity, suggesting that bound zinc interferes with substrate binding during catalysis. The structures of Is-QC bound to the inhibitor PBD150 revealed similar binding modes to both forms of Is-QC, with the exception of the inhibitor imidazole ring, which is consistent with the comparable inhibition activities of the inhibitor towards both forms of Is-QC. These findings have implications for the design of new QC inhibitors.

A blood meal-induced Ixodes scapularis tick saliva serpin inhibits trypsin and thrombin, and interferes with platelet aggregation and blood clotting.

Ixodes scapularis is a medically important tick species that transmits causative agents of important human tick-borne diseases including borreliosis, anaplasmosis and babesiosis. An understanding of how this tick feeds is needed prior to the development of novel methods to protect the human population against tick-borne disease infections. This study characterizes a blood meal-induced I. scapularis (Ixsc) tick saliva serine protease inhibitor (serpin (S)), in-house referred to as IxscS-1E1. The hypothesis that ticks use serpins to evade the host's defense response to tick feeding is based on the assumption that tick serpins inhibit functions of protease mediators of the host's anti-tick defense response. Thus, it is significant that consistent with hallmark characteristics of inhibitory serpins, Pichia pastoris-expressed recombinant IxscS-1E1 (rIxscS-1E1) can trap thrombin and trypsin in SDS- and heat-stable complexes, and reduce the activity of the two proteases in a dose-responsive manner. Additionally, rIxscS-1E1 also inhibited, but did not apparently form detectable complexes with, cathepsin G and factor Xa. Our data also show that rIxscS-1E1 may not inhibit chymotrypsin, kallikrein, chymase, plasmin, elastase and papain even at a much higher rIxscS-1E1 concentration. Native IxscS-1E1 potentially plays a role(s) in facilitating I. scapularis tick evasion of the host's hemostatic defense as revealed by the ability of rIxscS-1E1 to inhibit adenosine diphosphate- and thrombin-activated platelet aggregation, and delay activated partial prothrombin time and thrombin time plasma clotting in a dose-responsive manner. We conclude that native IxscS-1E1 is part of the tick saliva protein complex that mediates its anti-hemostatic, and potentially inflammatory, functions by inhibiting the actions of thrombin, trypsin and other yet unknown trypsin-like proteases at the tick-host interface.

Purification of a serine protease and evidence for a protein C activator from the saliva of the tick, Ixodes scapularis.

The saliva of ticks is critical to their survival as parasites and hematophagous animals. In this study, we have purified an enzyme with trypsin-like activity from the saliva of the tick vector of Lyme Disease, Ixodes scapularis. This enzyme, named as IXOSP (I. scapularis salivary serine protease), is a 29.9 kDa molecule with N-terminus FPxMVxLRIKxR. A BLAST search identified IXOSP as a secreted serine protease (AAY66740) with a conserved catalytic triad His, Asp, and Ser. In vitro studies demonstrated that IXOSP cleaves chromogenic substrates with arginine in the P1 position, by a mechanism inhibited by PMSF or aprotinin. Gene expression studies revealed that IXOSP is expressed at different tick developmental stages, including eggs, and unfed or fed adult tick salivary glands, but not in nymphs or in the midgut. While the physiological substrate for IXOSP remains to be identified, we demonstrated that I. scapularis saliva activate protein C (PC) resulting in the production of activated PC, a potent anticoagulant that also regulates a myriad of inflammatory responses through protease activated receptors. In contrast, the salivary glands of Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Aedes aegypti, Lutzomyia longipalpis, and Phlebotomus ariasi did not activate protein C. These discoveries are discussed in the context of blood coagulation, inflammation and vector-host interactions.

Identification of new sphingomyelinases D in pathogenic fungi and other pathogenic organisms.

Sphingomyelinases D (SMases D) or dermonecrotic toxins are well characterized in Loxosceles spider venoms and have been described in some strains of pathogenic microorganisms, such as Corynebacterium sp. After spider bites, the SMase D molecules cause skin necrosis and occasional severe systemic manifestations, such as acute renal failure. In this paper, we identified new SMase D amino acid sequences from various organisms belonging to 24 distinct genera, of which, 19 are new. These SMases D share a conserved active site and a C-terminal motif. We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site. Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus. Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms.

The E3 ubiquitin ligase XIAP restricts Anaplasma phagocytophilum colonization of Ixodes scapularis ticks.

Ubiquitination is a posttranslational modification that regulates protein degradation and signaling in eukaryotes. Although it is acknowledged that pathogens exploit ubiquitination to infect mammalian cells, it remains unknown how microbes interact with the ubiquitination machinery in medically relevant arthropods. Here, we show that the ubiquitination machinery is present in the tick Ixodes scapularis and demonstrate that the E3 ubiquitin ligase named x-linked inhibitor of apoptosis protein (XIAP) restricts bacterial colonization of this arthropod vector. We provide evidence that xiap silencing significantly increases tick colonization by the bacterium Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis. We also demonstrate that (i) XIAP polyubiquitination is dependent on the really interesting new gene (RING) catalytic domain, (ii) XIAP polyubiquitination occurs via lysine (K)-63 but not K-48 residues, and (iii) XIAP-dependent K-63 polyubiquitination requires zinc for catalysis. Taken together, our data define a role for ubiquitination during bacterial colonization of disease vectors.

Molecular characterization of tick salivary gland glutaminyl cyclase.

Glutaminyl cyclase (QC) catalyzes the cyclization of N-terminal glutamine residues into pyroglutamate. This post-translational modification extends the half-life of peptides and, in some cases, is essential in binding to their cognate receptor. Due to its potential role in the post-translational modification of tick neuropeptides, we report the molecular, biochemical and physiological characterization of salivary gland QC during the prolonged blood feeding of the black-legged tick (Ixodes scapularis) and the gulf-coast tick (Amblyomma maculatum). QC sequences from I. scapularis and A. maculatum showed a high degree of amino acid identity to each other and other arthropods and residues critical for zinc binding/catalysis (D159, E202, and H330) or intermediate stabilization (E201, W207, D248, D305, F325, and W329) are conserved. Analysis of QC transcriptional gene expression kinetics depicts an upregulation during the bloodmeal of adult female ticks prior to fast-feeding phases in both I. scapularis and A. maculatum suggesting a functional link with bloodmeal uptake. QC enzymatic activity was detected in saliva and extracts of tick salivary glands and midguts. Recombinant QC was shown to be catalytically active. Furthermore, knockdown of QC transcript by RNA interference resulted in lower enzymatic activity, and small, unviable egg masses in both studied tick species as well as lower engorged tick weights for I. scapularis. These results suggest that the post-translational modification of neurotransmitters and other bioactive peptides by QC is critical to oviposition and potentially other physiological processes. Moreover, these data suggest that tick-specific QC-modified neurotransmitters/hormones or other relevant parts of this system could potentially be used as novel physiological targets for tick control.

Use of DNA sequences for Rickettsia identification in Ixodes ricinus ticks: the first detection of Rickettsia monacensis in Poland.

The cosmopolitan tick Ixodes ricinus inhabiting Europe, including Poland, is a vector for many pathogens, such as various Rickettsia species, which spread to new territories. They are present mainly in the Mediterranean countries, but have also been found in Central Europe at increasing frequency. In the present study, the gltA gene, encoding citrate synthase, and an internal transcribed spacer (ITS) were employed to detect the DNA and identify the species of tick-borne pathogens of the Rickettsia genus. The presence of bacterial DNA was detected in 9.5% of the examined I. ricinus individuals. Based on the nucleotide sequences of the analysed genomic fragments, most pathogens were identified as Rickettsia helvetica, while Rickettsia monacensis was revealed in one case. We have described for the first time, to our knowledge, the occurrence of this species in Poland. Both markers employed in the experiments were successful in species identification of R. helvetica. The newly described species R. monacensis may be identified by the protein-coding gene, but the ITS nucleotide sequences proved insufficient.

Seasonal and spatial occurrence of glycerol-3-phosphate dehydrogenase variability in Ixodes ricinus (Acari: Ixodidae) populations.

Previous results indicate that glycerol-3-phosphate dehydrogenase variability represents the adaptation of Ixodes ricinus L. (Acari: Ixodidae) to fluctuations of environmental conditions, particularly to temperature. Analysis of crucial polymorphisms in I. ricinus Gpdh gene was done by the restriction method, and three different haplotypes were obtained (GPDH441 1, GPDH441 2, and GPDH441 3), corresponding to GPDH alleles detected by allozyme electrophoresis. Differences in GPDH441 haplotype and genotype frequencies were found between samples from open and forest habitats. Significant seasonal variations of GPDH441 haplotype and genotype frequencies were detected in samples from the open habitats. No seasonal variations were observed at forest localities, probably because of the less pronounced amplitude of environmental factors. The possible role of host availability was discussed as an important factor that affects seasonal dynamics and genetic composition of tick populations.