Comparative proteomics of the vector Dermacentor reticulatus revealed differentially regulated proteins associated with pathogen transmission in response to laboratory infection with Rickettsia slovaca
Parasites & Vectors volume 12, Article number: 318 (2019)
Tick-borne rickettsial diseases are caused by pathogens acquired from hard ticks. In particular, Rickettsia slovaca, a zoonotic infectious bacterium causing tick-borne lymphadenopathy (TIBOLA), is transmitted by the vectors Dermacentor spp. that can be found all over Europe. Although recent studies point out the extreme complexity of bacteria-induced effects in these blood-feeding vectors, the knowledge of individual molecules involved in the preservation and transmission of the pathogen is still limited. System biology tools, including proteomics, may contribute greatly to the understanding of pathogen-tick-host interactions.
Herein, we performed a comparative proteomics study of the tick vector Dermacentor reticulatus that was experimentally infected with the endosymbiotic bacterium R. slovaca. Rickettsia-free ticks, collected in the southern region of Slovakia, were infected with the bacterium by a capillary tube-feeding system, and the dynamics of infection was assessed by quantitative PCR method after 5, 10, 15 and 27 days.
At the stage of controlled proliferation (at 27 dpi), 33 (from 481 profiled) differentially abundant protein spots were detected on a two-dimensional gel. From the aforementioned protein spots, 21 were successfully identified by tandem mass spectrometry.
Although a few discovered proteins were described as having structural or housekeeping functions, the vast majority of the affected proteins were suggested to be essential for tick attachment and feeding on the host, host immune system evasion and defensive response modulation to ensure successful pathogen transmission.
Ticks are obligate haematophagous ectoparasites of a wide range of vertebrate hosts, including humans and other mammals, which are invading new territories due to global climate change. They are considered one of the most important vectors of zoonotic diseases and epidemics caused by viruses, bacteria and protozoans [1, 2]. These pathogens represent a serious public health risk as well as an undesirable source of economic loss due to reduced meat and milk production or impaired reproductive function in livestock, including late-term abortions and stillbirths . Thus, it is essential to increase protection against these vectors, which represent a significantly growing burden on human and animal health worldwide.
Dermacentor reticulatus (Fabricius, 1794) is a widely and abundantly spread tick species in Europe [3,4,5]. It has a high reproduction rate, and is very resistant against unfavourable environmental conditions [4, 5]. This species is responsible for transmitting various tick-borne pathogens: (i) viruses, e.g. Omsk haemorrhagic fever virus or tick-borne encephalitis virus; and (ii) bacteria, e.g. Rickettsia slovaca (tick-borne lymphadenopathy agent, TIBOLA), Coxiella burnetii (responsible for Q fever) and Francisella tularensis (the causative agent of tularemia). These ticks also represent a major vector for blood parasites such as Babesia canis (responsible for canine babesiosis), Babesia cabalii and Theileria equi (agent of equine piroplasmosis) [4,5,6].
Rickettsia slovaca is a spotted fever group rickettsia that was isolated in 1968 for the first time from the tick Dermacentor marginatus collected in central Slovakia . Subsequently, it has been detected or isolated from D. marginatus and D. reticulatus throughout Europe [8,9,10,11,12,13,14]. The bacterium has a typical rod shape with a diameter of 0.37–0.45 μm and a length of 0.8–1.2 μm . It is a causative agent of the mild human disease TIBOLA, which has been confirmed in many European countries. The infection is accompanied by tick bite-related skin lesions and cervical lymphadenopathies [13, 14, 16,17,18,19,20].
Next-generation sequencing showed considerable diversity of the tick inner microbiome . Due to evolved control mechanisms, it is plausible to regulate the proliferation of the commensal microbes without causing a notable impact on the fitness of the vector . Tick innate immunity is based on the coordinated action of humoral and cellular immune responses. The invading microbes are phagocytosed by tick haemocytes, which are navigated by the primordial complement-like system composed of thioester-containing proteins, fibrinogen-related lectins and convertase-like factors. The midgut is a major organ where the microbes that are ingested via the blood meal encounter the vector’s internal tissues. Direct antimicrobial action is carried out by a variety of specialised molecules including defensins, lysozymes, microplusin, hebraein, 5.3 kDa family polypeptides, reactive oxygen species, etc. However, this dynamic process is more complex. Apart from the vector and host immune response, the bacterial symbionts and pathogens also have their own molecular tools to manipulate the defence response in order to induce infection in both tick and mammalian host.
Recently, advancements in the understanding of tick-pathogen interaction have been achieved by several transcriptomics or proteomics studies. To verify the hypothesis that the tick immune system may control the preservation of pathogens, Jaworski et al.  traced the expression of Dermacentor variabilis tick genes induced by a bacterial infection. Adjustments in the expression of genes, which are likely encoding tick immune-related proteins, clearly demonstrated the complexity of the defence system of arachnids. Further proteomics studies allowed identifying novel secreted proteins in Dermacentor andersoni  as well as revealing molecules associated with the adaptive stress response to rickettsial infection in unfed D. reticulatus larvae . Furthermore, Rachinsky et al.  established a proteome database containing proteins involved in successful pathogen transmission. Among others, proteins implicated in signalling processes were discovered using comparative proteomics [25, 26]. Interestingly, it has also been reported that proteins from apoptotic signalling pathways of Ixodes scapularis were more heavily regulated in the midguts than in salivary glands in response to Anaplasma phagocytophilum infection .
Indeed, the application of proteomics seems to be essential for understanding the intimate details in tick-host-pathogen interaction. New discoveries related to vector competence and biological processes associated with the transmission of tick-borne diseases are needed. Furthermore, proteomics studies might even result in the identification of candidate protective antigens that are capable of blocking the transmission of pathogens. Identifying these proteins may lead to the development of protective vaccines [28, 29] aiming to reduce the vector capacity for tick-borne pathogens. In this study, we investigated the differences in the abundance of D. reticulatus proteins in response to R. slovaca infection.
Collecting the questing ticks
Questing D. reticulatus adult ticks were collected in Gabčíkovo during 2016 and 2017. The locality (47°53′N, 17°32′E) is situated in southwestern Slovakia, at 110 m above sea level. Ticks were collected in an alluvial habitat near the River Danube by dragging a woollen flag over the lower vegetation.
Collected ticks were rinsed with 70% ethanol and sterile water and then dried. Subsequently, a small part of the leg was excised from each individual. Total genomic DNA was isolated by boiling the legs in 0.7 M ammonium hydroxide  and analysed by PCR. Female ticks that tested negative for Rickettsia spp. were split into two (infected and control) groups and kept in the laboratory during the experiment.
Laboratory infection of ticks
Rickettsia slovaca strain 13-B, originating from the collection of the Department of Rickettsiology (Institute of Virology, Biomedical Research Center, Bratislava, Slovakia) was revived and propagated in Vero cell line (Cercopithecus aethiops monkey epithelial cells ATCC CCL-81) in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 3% fetal bovine serum (FBS) at 34 °C.
Rickettsia spp.-negative ticks divided into three biological replicate groups of five ticks (per time point) were infected with the bacterium by capillary tube feeding (infected groups) (Fig. 1). Briefly, the drawn-out glass microcapillary pipettes were loaded with approximately 2 μl of the medium containing 104/μl R. slovaca (assessed by quantitative real-time PCR described below). The pipettes were then placed over hypostomes of ticks immobilised in Petri dishes as described previously for infection with Borrelia and Rickettsia helvetica [31, 32]. The ticks were then allowed to feed for 1 h at room temperature (RT). As a control group, Rickettsia spp.-negative ticks were fed bacteria-free medium (control group).
Protein and complete RNA extractions and cDNA synthesis
After 5, 10, 15 and the 27 days post-infection (dpi), the ticks from both the infected and the control group were frozen in liquid nitrogen and ground down to a fine powder using a mortar and a pestle. Subsequently, genomic DNA, complete RNA and total protein were extracted using a DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany), following the instructions of the manufacturer. Samples were stored at − 80 °C until further use.
The fraction containing RNA was treated with RQ1 RNA-free DNAase (Promega, Madison, USA) for 4 h in order to eliminate possible contamination with genomic DNA. The samples were then cleaned by a RNAeasy miniElute cleanup kit (Qiagen) and spectrophotometrically quantified by a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, USA). Aliquots of 1 μg RNA isolated from the different conditions were amplified using a First Strand cDNA synthesis kit (Thermo Fisher Scientific, Carlsbad, USA), following the manufacturer’s instructions (product stored at − 20 °C until used).
Molecular identification of pathogens in ticks
Tick samples were screened prior the experiment by PCR-based methods for the presence of Rickettsia spp. Amplification of the gltA gene  was used for detection. Real-time PCR assays based on the ompB gene were applied for specific identification of R. slovaca  and other species (Babesia spp. (conventional PCR), Coxiella burnetti and R. raoultii [34,35,36]) presented in the samples to exclude concurrent infection. All primers and probes (Table 1) were synthesised by Microsynth AG (Balbach, Switzerland).
The real-time PCR mixtures contained 10 μl of the 2× SuperHot Master mix (Bioron, Ludwigshafen, Germany), 100 nM of each primer, 100 nM probe, 0.5 μl of 100 mM MgCl2 and 3 μl of the template DNA/cDNA, the total volume being 20 μl. The PCR programme consisted of an initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 20 s and annealing at 60 °C for 40 s. The real-time PCR assays were carried out using a CFX96™ Real-Time System (Bio-Rad, Hercules, USA). The DNA from rickettsiae-negative ticks and nuclease-free water were used as negative controls in each reaction. Genetic material from R. slovaca, R. raoultii  and Babesia canis purified from ticks as well as the in-house reference strains (R. slovaca, C. burnetii) were used as positive controls. The analyses were performed in two technical replicates.
For absolute quantification of R. slovaca in capillary-fed ticks, the standard curve was generated as described by Jiang et al. ; the limit of detection for qPCR assay was determined at 3 × 102 copies of the target gene (Fig. 2). A one-way ANOVA calculator was used to compare copy numbers in each time point throughout the study (http://www.socscistatistics.com). The significance threshold was set at P = 0.05.
Protein separation and quantification by gel electrophoresis
Total tick protein was obtained after the RNA extraction step of the DNA/RNA/Protein Mini Kit (see above). The precipitated proteins were reconstituted in a buffer which contained 7 M urea, 2 M thiourea, 40 mM Tris, 1% amidosulfobetaine-14 and 1% Triton X-100; the protein concentration was determined by a 2-D Quant Kit (GE Healthcare, Diegem, Luxembourg). For initial screening, polyacrylamide gel electrophoresis was performed in the presence of sodium dodecyl sulphate (SDS-PAGE). Briefly, aliquots containing 15 μg of solubilised proteins were boiled for 5 min, and the volume was adjusted with 4× Laemmli buffer (8% SDS, 240 mM Tris, pH 6.8, 40% glycerol, 2% 2-mercaptoethanol and 0.04% bromophenol blue). Separation was carried out in 15% polyacrylamide gels with Tris-glycine running buffer, pH 8.3, using Protean Tetra mini Cell apparatus (Bio-Rad) at 60 V for 15 min, followed by 200 V until the tracking dye reached the bottoms of the gels. After separation, the proteins were visualised by staining with colloidal Coomassie Blue G-250 . Several apparently different bands were excised manually and digested by trypsin as described below.
At 27 dpi, control group and infected group protein samples, prepared in triplicates, were used for quantitative comparison using 2-DE. Protein aliquots of 150 μg were adjusted to 340 μl with isoelectric focusing buffer (7 M urea, 2 M thiourea, 40 mM Tris 1% amidosulfobetaine-14, 1% Triton X-100, 1% ampholytes pH 3–10 and 1% DeStreak). After centrifugation at 10,000× g for 10 min, the supernatant was transferred to 18 cm immobilised non-linear pH gradient strips 3–10 (GE Healthcare). Following overnight passive rehydration, first-dimensional separation was carried out using an Ettan IPGphor 3 unit (GE Healthcare). The strips were reduced for 15 min in 4 ml of equilibration buffer (0.1 M Tris pH 6.8, 30% glycerol, 6 M urea and 3% SDS) containing 2% dithiothreitol and then alkylated in the dark for 15 min in the same buffer with 2.5% iodoacetamide. After subsequent washing in running buffer (25 mM Tris, 192 mM glycine and 0.1% SDS), the strips were placed on top of 13% polyacrylamide gels and sealed with 0.5% agarose containing 0.002% bromophenol blue in the running buffer. The second-dimensional separation was carried out in Protean II xi Cell (Bio-Rad) at 10 mA per gel for 1 h and 30 mA per gel until the tracking dye disappeared. The gels were fixed two times for 30 min in 50% methanol containing 7% acetic acid and then stained in the dark overnight with SYPRO Ruby (Invitrogen, Carlsbad, USA). This was followed by two washing steps for 30 min, the first in 10% methanol with 7% acetic acid and the second in milliQ H2O. The gels were then scanned on a PharosFX Molecular Imager (Bio-Rad) and stained in colloidal Coomassie blue for manual excision of target spots .
Quantitative software-assisted gel analysis was performed using SameSpots v.5.1 software (TotalLab, Manchester, UK). The images were aligned, and dust particles, stain particles, streaks or damaged gel areas were filtered out. The protein spots were then manually validated, and the determined relative volumes were normalised to the median distribution of reference gel in order to compensate minor differences in sample loading. Differentially abundant protein spots found in the infected and control samples were chosen based on an ANOVA P-value ≤ 0.05 and an abundance ratio ≥ 1.5.
In-gel protein digestion
The excised protein spots were washed with agitation in 50 mM ammonium bicarbonate (ABC; Sigma-Aldrich, Darmstadt, Germany) containing 50% acetonitrile (ACN; Merck, Darmstadt, Germany) at RT. After complete destaining, the gel pieces were dehydrated for 10 min at RT in 100% ACN, reduced for 30 min at 50 °C with 10 mM dithiothreitol (DTT), and alkylated for 30 min in the dark with 50 mM iodoacetamide in 100 mM ABC. The gel plugs were then rewashed three times with 50 mM ABC for 10 min at RT, dehydrated with 100% ACN, and incubated for 14 h at 37 °C in digestion solution (40 ng of lyophilised sequencing grade modified trypsin (Promega) per 5 µl of 10 mM ABC and 10% ACN). The resulting peptide mixtures were extracted twice by 50 µl of 1% trifluoroacetic acid in 70% ACN. The total volume of samples was reduced to 20 µl by vacuum evaporation using a Concentrator plus (Eppendorf, Hamburg, Germany). The solution was then stored at − 20 °C.
Mass spectrometry and bioinformatics for protein identification
The peptides were analysed by LC-MS/MS using nanoAcquity UHPLC (Waters, Manchester, UK) and Q-TOF Premier (Waters). Briefly, the samples were loaded onto the trap column and desalted. Subsequently, they were separated on a BEH130 C18 analytical column (200 mm length, 75 μm diameter, 1.7 μm particle size), using a 20 min gradient of 5–40% ACN containing 0.1% formic acid at a 300 nl/min flow rate. The column outlet was connected to the PicoTip emitter (360 μm outer diameter, 20 μm inner diameter, 10 μm tip diameter) and the eluted peptides were nanosprayed (3.4 kV capillary voltage) to the mass spectrometer. Spectra were recorded in the MSE mode (parallel high and low energy traces without precursor ion selection). Ions with 50–1950 m/z were detected in both channels. The external mass calibrant Glu1-Fibrinopeptide B (500 fmol/ml) was infused through the reference line at a 500 nl/min flow rate and used for mass correction.
Data were processed by ProteinLynx Global Server v.3.0 (Waters). For peak picking, the following threshold parameters were applied: low energy 140 counts and high energy 30 counts. Precursors and fragment ions were coupled, using correlations of chromatographic elution profiles in low/high energy traces. Spectra were searched against Ixodidae protein sequences supplemented with R. slovaca and R. raoultii proteomes downloaded from UniProt in June 2017 (137,542 entries, http://www.uniprot.org) and BLAST-ed against the SwissProt database (http://www.uniprot.org/, containing 545,536 entries downloaded in June 2017). Workflow parameters for the protein identification searches were: (i) maximum one possible trypsin miscleavage; (ii) a fixed modification (carbamidomethyl cysteine) and a variable modification (oxidised methionine and deamidated asparagine or glutamine); (iii) the mass tolerance of precursors and fragments was automatically determined by the software; and (iv) peptide matching was limited to less than 4% false discovery rate against the randomised database. Identifications were accepted whenever at least two distinct reliable peptides matched the protein sequence. The protein localisations were predicted with PSORT algorithm (https://psort.org).
Confirmation of successful infection of ticks
The collection of ticks was performed by dragging a woollen flag over the lower vegetation in Gabčíkovo (southern Slovakia). Subsequently, D. reticulatus ticks were analysed for rickettsial infection using PCR. The Rickettsia-negative ticks were infected with R. slovaca by the capillary tube feeding method, and the presence of the bacterium was assessed after 5, 10, 15 and 27 days post-infection (dpi) by quantitative PCR (qPCR). This analysis confirmed the successful infection of ticks with the bacterium, showing a range of 7.6 × 104 to 4.9 × 106 average copy numbers (P < 0.05) (Fig. 3). All samples were PCR-negative for R. raoultii, Babesia spp. and Coxiella burnetii.
Monitoring the response to infection by SDS-PAGE
The ticks were frozen in liquid nitrogen, ground in a mortar, and subjected to lysis followed by protein extraction and separation of the mixtures by SDS-PAGE (Fig. 4) after 5, 10, 15 and 27 dpi. In comparison with the control group, the three bands that appeared differentially abundant were selected and excised. Proteins present in the gel pieces were digested by trypsin and identified by tandem mass spectrometry. The abundance of band #1, containing an unknown tick host protein, increased gradually due to the presence of R. slovaca. The main protein in band #3 was identified as paramyosin, an immunomodulatory protein playing an important role in immune reactions against parasites in vertebrate hosts . Finally, the major protein in band #2 was recognised as putative defensin. This protein was present in practically all samples, including the control ones (in lower abundance). However, it almost completely disappeared from the sample collected at 27 dpi.
Tick-pathogen interaction during “steady-state” infection at 27 dpi
To reveal the intimate details of the tick-host-pathogen interaction and to figure out how the tick immune system maintains the presence of pathogens during “steady-state” infection, we tracked changes in the protein abundance of D. reticulatus tick proteins compared to control, during the “steady-state” rickettsial infection at the latest infection time point (27 dpi). After two-dimensional gel electrophoresis (2-DE) (Fig. 5 and Additional file 1: Figure S1) was performed; 481 reproducibly quantified gel spots were detected. All data, including descriptive statistics, are shown in Additional file 2: Table S1. Comparison with the control group of samples discovered 33 (6.9%) differentially abundant spots (Fig. 5), 21 of which were successfully identified by LC-MS/MS (Table 2). Following the acquisition of relevant functional information, we described 6 secreted proteins, 5 proteins functioning in the cytoplasm, 4 nuclear proteins, 2 proteins localised in mitochondria and 1 unknown function protein, which is predicted to have a membrane localisation (Table 2). Two gel spots (spots 1482 and 1489) that were more abundant in response to R. slovaca challenge at 27 dpi were identified as salivary serine protease inhibitors, serpins; one spot (spot 1976) as a zinc-binding oxidoreductase and one spot as a putative secreted protein (ISCW gene, spot 3035). We discovered three gel spots that corresponded with secreted glycine-rich (GRP; spots 1897 and 1945) and glycine-proline rich (spot 5154) proteins that were accumulated in response to the infection. Troponin I-like protein (spot 2707), glutathione S-transferase (GST; spot 2818), heat-hock protein 20 (HSP20; spot 2715) and a putative ML-domain protein (spot 2791) were more abundant in response to rickettsial infection.
The putative myosin regulatory light chain (spot 2833) and the putative myosin class I heavy chain (spot 305) were regulated differently in infected vector D. reticulatus. Finally, we identified several differentially abundant proteins with housekeeping functions. The mitochondrial subunit of ATP synthase (spot 2917), ubiquinone oxidoreductase (spot 1602), phosphoglycerate kinase (spot 1601) and histone H4 (spot 4628) were accumulated in response to R. slovaca infection. On the other hand, an ubiquitin-conjugating enzyme E2 (spot 3871) and a putative eukaryotic translation initiation factor 4 γ (spot 1808) were less abundant in infected ticks. Apart from these housekeeping proteins, we also identified a structural constituent of the cuticle (spot 5046) and an uncharacterised protein that is an integral component of the membrane (spot 3025), which were either less or more abundant in infected ticks, respectively.
In Slovakia, tick vectors D. reticulatus and D. marginatus are generally infected with R. slovaca and R. raoultii [8, 9, 15, 39]. Gabčíkovo is a locality with a prevalence of Rickettsia-positive questing D. reticulatus adult ticks of 49.12% (95% CI: 36.14–62.10%). While R. raoultii was detected as a dominant species (45.61%, 95% CI: 32.68–58.54%), R. slovaca, Coxiella burnetii and Babesia spp. were found in only a few ticks (3.51%, 95% CI: 0–8.29%; 1.75%, 95% CI: 0–5.16%; 1.83%, 95% CI: 0.76–2.91%, respectively) . In this study, Rickettsia-negative D. reticulatus ticks were successfully infected with R. slovaca by a capillary tube-feeding system, and the infection was maintained in the range of 7.6 × 104 to 4.9 × 106 copies throughout the duration of the experiment. These findings correspond with our previous result  in which we proved that the number of R. slovaca in D. marginatus increased from 1 × 104 to 1 × 106–4 × 106 copies between 6 and 21 dpi. This observation may indicate tight control of R. slovaca proliferation with the tick vector.
Although, we realise that the ticks collected in the field might be subjected to an unknown random set of conditions including time elapsed since moulting, nutrient availability and the presence of other pathogens and symbionts, the identification of putative defensin (Fig. 4, band #2) in the preliminary SDS-PAGE analysis may indicate a response to an ongoing infection. Many of these molecules and their isoforms have been identified so far in both hard and soft ticks where they act either against Gram-positive or some Gram-negative-bacteria [40,41,42,43,44,45,46,47,48]. For instance, defensin-like peptide (DLP) from Haemaphysalis longicornis has been shown to have antimicrobial activity against drug-resistant microorganisms, including the fungus Candida albicans . Interestingly, the putative defensin was detected in almost all samples in this study, including the control ones, although it was present in lower abundance. However, it almost completely vanished from the sample collected at 27 dpi. If we consider the antimicrobial activity of this molecule [49, 50], the observed findings may suggest a presence of “steady-state” infection with controlled bacterial proliferation.
Among the 21 identified proteins that were found to be differentially presented in response to R. slovaca challenge at 27 dpi, we recognised salivary serine protease inhibitors: serpins and their closely related  zinc-binding oxidoreductase. A similar result was reported by Ayllón et al.  and Liu et al.  who showed that the expression of protease inhibitors in salivary glands and midguts of adult female ticks was altered due to infection. Cystatins and serpins were also upregulated in I. scapularis that were infected with A. phagocytophilum and in Ixodes ricinus challenged with Bartonella hensela. On the other hand, several serpins, identified as components of the D. variabilis immune system, were found to be unregulated on a transcription level in response to bacterial infection . This discrepancy, as shown by the results of these studies, can be explained by the fact that ticks produce numerous serpins. For instance, 45 serpins were detected in the genome of I. scapularis, 120 in Amblyomma americanum and 22 in Rhipicephalus microplus. There is also evidence of serpin expression, as 36 transcripts were detected in the transcriptome of I. ricinus and 10 in Hyalomma excavatum . Despite numerous occurrences of these inhibitors in ticks, little is known about their biological function. It has been suggested that their key function is related to the modulation of the host immune system. Serpins, as well as a putative secreted protein (ISCW gene)  which was also accumulated in response to rickettsial infection, probably play a role in the mechanism that suppresses the defence reaction of vertebrate hosts. Apparently, it seems that R. slovaca possesses molecular tools for manipulating the defence response of the tick in order to induce an infection.
In this study, we also discovered glycine-proline rich proteins that accumulated in response to the infection. This corresponds with the result of Macaluso et al. , who showed that the genes encoding GRPs in H. longicornis were regulated upon tick infestation as a consequence of feeding. Indeed, these proteins were found to be essential for tick attachment and feeding on the host. Specifically, they were involved in the host’s immune system evasion . Because the high expression of GRPs has been demonstrated in adult female ticks, these proteins have been proposed as potential candidate molecules in the formulation of an anti-tick vaccine cocktail for the biological control of ticks . Likewise, a specific protective anti-tick immune response was shown in mice immunised by a recombinant troponin I-like protein from H. longicornis . However, the main function of this protein is to regulate the contraction of striated muscle , and it has also been demonstrated to be expressed in salivary glands , where it can act as a potent inhibitor of angiogenesis . Troponin I-like protein was found to be more abundant in our study in response to rickettsial infection.
Detoxification of free haem liberated upon host blood digestion is a special challenge for ticks. Thus, it was suggested that GST in the tick midgut might function as an intracellular buffer of labile haem pool to ameliorate its cytotoxic effects upon intracellular hydrolysis of haemoglobin . The authors showed by RNA-seq analysis of I. ricinus midguts that expression of this protein was upregulated in the presence of red blood cells in the diet. Interestingly, GST was also more abundant in our study. Additionally, HSP20, accumulated due to rickettsial infection, might be associated with the tick cell response to blood-feeding stress, pathogen infection and questing behaviour . Similarly to myosin-like proteins, HSP20 has been shown to have immunogenic properties in saliva . In our study, putative myosin regulatory light chain and putative myosin class I heavy chain were discovered to be regulated differently in infected D. reticulatus. Myosin is a motor protein, best known for its role in ATP-dependent muscle contraction. Non-muscle myosin was reported as highly abundant in the saliva of I. scapularis ticks and has been associated with different aspects of wound healing . Increased abundance of two myosin subunits has even been detected in ovaries of R. microplus infected with Babesia, and for this reason, the molecules have been proposed to be important in successful pathogen transmission . Furthermore, a putative ML-domain protein that was more abundant in response to rickettsial infection in our study might be involved in pathogen recognition and immune response. The gene encoding this protein in I. ricinus was found among the genes that are strongly induced by blood meals .
Finally, we identified the mitochondrial subunit of ATP synthase, ubiquinone oxidoreductase, and phosphoglycerate kinase, which was accumulated in response to R. slovaca infection. These enzymes are essential for glycolysis and electron transport. The ATP synthase was found to be upregulated in the midgut of Babesia bovis-infected R. microplus ticks . In this study, we also discovered the histone H4 to be more abundant. On the other hand, an ubiquitin-conjugating enzyme E2 and a putative eukaryotic translation initiation factor 4 γ were found to be less abundant in infected ticks. These proteins play a vital role in protein degradation, protein biosynthesis, DNA replication or chromosomal stability. It has been shown that histone H2B has a key role in mediating Rickettsia felis internalisation into a tick cell line. It directly interacts with outer-membrane protein B (OmpB), a major rickettsial adhesin molecule. When this protein was depleted, the rickettsial infection in the ISE6 cell line was reduced .
This initial comparative proteomic analysis based on 2-DE of the Dermacentor reticulatus vector infected with a pathogenic bacterium Rickettsia slovaca has revealed 33 differentially abundant proteins in the samples isolated at 27 dpi. The majority have been previously described in other studies in the context of feeding, bacterial infection or defence reaction of the tick (Fig. 6). Some proteins have even been proposed as potential protective antigens or putative immune modulators. Among the proteins discovered as more abundant in infected ticks, we found an uncharacterised protein (UniProt accession B7Q9B4) that may represent a new target for further functional validation. This protein has been bioinformatically predicted to have membrane localisation and porin domain, plausibly playing an important role in the tick-host-pathogen interaction. Considering the functional role of identified differentially abundant proteins, we hypothesise that bacterial infection induces changes that may increase the efficacy of pathogen transmission to the vertebrate host using the tick vector. Thus, this newly acquired knowledge contributes greatly to the clarification of the nature of the vector-pathogen interaction and opens the door to further experiments in the future.
Availability of data and materials
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via PRIDE partner repository with dataset identifier PXD011442.
two-dimensional gel electrophoresis
liquid chromatography coupled to tandem mass spectrometry
secreted glycine-rich protein
heat-shock protein 20
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
de la Fuente J, Antunes S, Bonnet S, Cabezas-Cruz A, Domingos AG, Estrada-Peña A, et al. Tick-pathogen interactions and vector competence: identification of molecular drivers for tick-borne diseases. Front Cell Infect Microbiol. 2017;7:114.
Jongejan F, Uilenberg G. The global importance of ticks. Parasitology. 2004;129(Suppl.):S3–14.
Rubel F, Brugger K, Pfeffer M, Chitima-Dobler L, Didyk YM, Leverenz S, et al. Geographical distribution of Dermacentor marginatus and Dermacentor reticulatus in Europe. Ticks Tick Borne Dis. 2016;7:224–33.
Biernat B, Karbowiak G, Werszko J, Stanczak J. Prevalence of tick-borne encephalitis virus (TBEV) RNA in Dermacentor reticulatus ticks from natural and urban environment, Poland. Exp Appl Acarol. 2014;64:543–51.
Földvári G, Široký P, Szekeres S, Majoros G, Sprong H. Dermacentor reticulatus: a vector on the rise. Parasit Vectors. 2016;9:314.
Villar M, Popara M, Ayllón N, De Fernández Mera IG, Mateos-Hernández L, Galindo RC, et al. A systems biology approach to the characterization of stress response in Dermacentor reticulatus tick unfed larvae. PLoS ONE. 2014;9:e89564.
Brezina R, Řeháček J, Áč P, Majerská M. Two strains of rickettsiae of Rocky Mountain SFG recovered from D. marginatus ticks in Czechoslovakia. Results of preliminary serological identification. Acta Virol. 1969;13:142–5.
Špitalská E, Štefanidesová K, Kocianová E, Boldiš V. Rickettsia slovaca and Rickettsia raoultii in Dermacentor marginatus and Dermacentor reticulatus ticks from Slovak Republic. Exp Appl Acarol. 2012;57:189–97.
Špitalská E, Sparagano O, Stanko M, Schwarzová K, Špitálsky Z, Škultéty Ľ, et al. Diversity of Coxiella-like and Francisella-like endosymbionts, and Rickettsia spp, Coxiella burnetii as pathogens in the tick populations of Slovakia, Central Europe. Ticks Tick Borne Dis. 2018;9:1207–11.
Marquez FJ, Rojas A, Ibarra V, Cantero A, Rojas J, Oteo JA, Muniain MA. Prevalence data of Rickettsia slovaca and other SFG Rickettsiae species in Dermacentor marginatus in the Southeastern Iberian Peninsula. Ann N Y Acad Sci. 2006;1078:328–30.
Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin Microbiol Rev. 2005;18:719–56.
Pluta S, Tewald F, Hartelt K, Oehme R, Kimmig P, Mackenstedt U. Rickettsia slovaca in Dermacentor marginatus ticks, Germany. Emerg Infect Dis. 2009;15:2077–8.
Raoult D, Lakos A, Fenollar F, Beytout J, Brouqui P, Fournier PE. Spotless rickettsiosis caused by Rickettsia slovaca and associated with Dermacentor ticks. Clin Infect Dis. 2002;34:1331–6.
Selmi M, Bertolotti L, Tomassone L, Mannelli A. Rickettsia slovaca in Dermacentor marginatus and tick-borne lymphadenopathy, Tuscany, Italy. Emerg Infect Dis. 2008;14:817–20.
Řeháček J. Rickettsia slovaca, the organism and its ecology. Acta Sci Nat Acad Sci Bohem Brno. 1984;18:1–50.
Raoult D, Berbis P, Roux V, Xu W, Maurin M. A new tick-transmitted disease due to Rickettsia slovaca. Lancet. 1997;350:112–3.
Lakos A. Tick-borne lymphadenopathy (TIBOLA). Wien Klin Wochenschr. 2002;114:648–54.
Ibarra V, Oteo JA, Portillo A, Santibáñez S, Blanco JR, Metola L, et al. Rickettsia slovaca infection: DEBONEL/TIBOLA. Ann N Y Acad Sci. 2006;1078:206–14.
Parola P, Rovery C, Rolain JM, Brouqui P, Davoust B, Raoult D. Rickettsia slovaca and R. raoultii in tick-borne rickettsioses. Emerg Infect Dis. 2009;15:1105–8.
Oteo JA, Portillo A. Tick-borne rickettsioses in Europe. Ticks Tick Borne Dis. 2012;3:271–8.
Andreotti R, De León AAP, Dowd SE, Guerrero FD, Bendele KG, Scoles GA. Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing. BMC Microbiol. 2011;11:6.
Hajdušek O, Šíma R, Ayllón N, Jalovecká M, Perner J, de la Fuente J, Kopácek P. Interaction of the tick immune system with transmitted pathogens. Front Cell Infect Microbiol. 2013;4:26.
Jaworski DC, Zou Z, Bowen CJ, Wasala NB, Madden R, Wang Y, et al. Pyrosequencing and characterization of immune response genes from the American dog tick, Dermacentor variabilis (L.). Insect Mol Biol. 2010;19:617–30.
Mudenda L, Pierlé SA, Turse JE, Scoles GA, Purvine SO, Nicora CD, et al. Proteomics informed by transcriptomics identifies novel secreted proteins in Dermacentor andersoni saliva. Int J Parasitol. 2014;44:1029–37.
Rachinsky A, Guerrero FD, Scoles GA. Proteomic profiling of Rhipicephalus (Boophilus) microplus midgut responses to infection with Babesia bovis. Vet Parasitol. 2008;152:294–313.
Villar M, Torina A, Nuñez Y, Zivkovic Z, Marina A, Alongi A, et al. Application of highly sensitive saturation labeling to the analysis of differential protein expression in infected ticks from limited samples. Proteome Sci. 2010;8:43.
Ayllón N, Villar M, Galindo RC, Kocan KM, Šíma R, López JA, et al. Systems biology of tissue-specific response to Anaplasma phagocytophilum reveals differentiated apoptosis in the tick vector Ixodes scapularis. PLoS Genetics. 2015;11:e1005120.
Kocan KM, de la Fuente J, Blouin EF. Advances toward understanding the molecular biology of the Anaplasma-tick interface. Front Biosci. 2008;13:7032–45.
Contreras M, Alberdi P, Fernández De Mera IG, Krull C, Nijhof A, Villar M, de la Fuente J. Vaccinomics approach to the identification of candidate protective antigens for the control of tick vector infestations and Anaplasma phagocytophilum infection. Front Cell Infect Microbiol. 2017;7:360.
Rijpkema S, Golubić D, Molkenboer M, Verbeek-de Kruif N, Schellekens J. Identification of four genomic groups of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in a Lyme borreliosis endemic region of northern Croatia. Exp Appl Acarol. 1996;20:23–30.
Korshus JB, Munderloh UG, Bey RG, Kurtti TJ. Experimental infection of dogs with Borrelia burgdorferi sensu stricto using Ixodes scapularis ticks artificially infected by capillary feeding. Med Microbiol Immunol. 2004;193:27–34.
Baldridge GD, Kurtti TJ, Burkhardt N, Baldridge AS, Nelson CM, Oliva AS, Munderloh UG. Infection of Ixodes scapularis ticks with Rickettsia monacensis expressing green fluorescent protein: a model system. J Invert Pathol. 2007;94:163–74.
Boretti FS, Perreten A, Meli MM, Cattori V, Willi B, Wengi N, et al. Molecular investigation of Rickettsia helvetica infection in dogs, foxes, humans, and Ixodes ticks. Appl Environ Microbiol. 2009;75:3230–7.
Jiang J, You BJ, Liu E, Apte A, Yarina TR, Myers TE, et al. Development of three quantitative real-time PCR assays for the detection of Rickettsia raoultii, Rickettsia slovaca, and Rickettsia aeschlimannii and their validation with ticks from the country of Georgia and the Republic of Azerbaijan. Ticks Tick Borne Dis. 2012;3:327–31.
Casati S, Sager H, Gern L, Piffaretti JC. Presence of potentially pathogenic Babesia sp. for human in Ixodes ricinus in Switzerland. Ann Agric Environ Med. 2006;13:65–70.
Melničáková J, Derdáková M, Barák I. A system to simultaneously detect tick-borne pathogens based on the variability of the 16S ribosomal genes. Parasit Vectors. 2013;6:269.
Dyballa N, Metzger S. Fast and sensitive colloidal Coomassie G-250 staining for proteins in polyacrylamide gels. J Vis Exp. 2009;30:1431.
Zhang TT, Zhang JC, Cui XJ, Zheng JJ, Li R, Wang F, et al. Evaluation of immune protection induced by DNA vaccines from Haemaphysalis longicornis paramyosin in rabbits. Parasit Vectors. 2017;10:325.
Boldiš V, Špitalská E. Dermacentor marginatus and Ixodes ricinus ticks versus L929 and Vero cell lines in Rickettsia slovaca life cycle evaluated by quantitative real time PCR. Exp Appl Acarol. 2010;50:353–9.
Nakajima Y, Ishibashi J, Yukuhiro F, Asaoka A, Taylor D, Yamakawa M. Antibacterial activity and mechanism of action of tick defensin against Gram-positive bacteria. Biochim Biophys Acta. 2003;1624:125–30.
Lai R, Lomas LO, Jonczy J, Turner PC, Rees HH. Two novel non-cationic defensin-like antimicrobial peptides from haemolymph of the female tick, Amblyomma hebraeum. Biochem J. 2004;379:681–5.
Rudenko N, Golovchenko M, Grubhoffer L. Gene organization of a novel defensin of Ixodes ricinus: first annotation of an intron/exon structure in a hard tick defensin gene and first evidence of the occurrence of two isoforms of one member of the arthropod defensin family. Insect Mol Biol. 2007;16:501–7.
Rudenko N, Golovchenko M, Edwards MJ, Grubhoffer L. Differential expression of Ixodes ricinus tick genes induced by blood feeding or Borrelia burgdorferi infection. J Med Entomol. 2005;42:36–41.
Zhou J, Liao M, Ueda M, Gong H, Xuan X, Fujisaki K. Sequence characterization and expression patterns of two defensin-like antimicrobial peptides from the tick Haemaphysalis longicornis. Peptides. 2007;28:1304–10.
Wang Y, Zhu S. The defensin gene family expansion in the tick Ixodes scapularis. Dev Comp Immunol. 2011;35:1128–34.
Wang J, Bian G, Pan W, Feng T, Dai J. Molecular characterization of a defensin gene from a hard tick, Dermacentor silvarum. Parasit Vectors. 2015;8:25.
Chrudimska T, Slaninova J, Rudenko N, Ruzek D, Grubhoffer L. Functional characterization of two defensin isoforms of the hard tick Ixodes ricinus. Parasit Vectors. 2011;4:63.
Chrudimska T, Cerovsky V, Slaninova J, Rego RO, Grubhoffer L. Defensin from the ornate sheep tick Dermacentor marginatus and its effect on Lyme borreliosis spirochetes. Dev Comp Immunol. 2014;46:165–70.
Lu X, Che Q, Lv Y, Wang M, Lu Z, Feng F, et al. A novel defensin-like peptide from salivary glands of the hard tick, Haemaphysalis longicornis. Protein Sci. 2010;19:392–7.
Zasloff M. Antimicrobial peptides of multicellular organisms. Nature. 2002;415:389–95.
Anderson JM, Sonenshine DE, Valenzuela JG. Exploring the mialome of ticks: an annotated catalogue of midgut transcripts from the hard tick, Dermacentor variabilis (Acari: Ixodidae). BMC Genomics. 2008;9:552.
Liu XY, de la Fuente J, Cote M, Galindo RC, Moutailler S, Vayssier-Taussat M, et al. IrSPI, a tick serine protease inhibitor involved in tick feeding and Bartonella henselae infection. PLoS Negl Trop Dis. 2014;8:e2993.
Chmelař J, Kotál J, Langhansová H, Kotsyfakis M. Protease inhibitors in tick saliva: The role of serpins and cystatins in tick-host-pathogen interaction. Front Cell Infect Microbiol. 2017;7:216.
Grabowski JM, Perera R, Roumani AM, Hedrick VE, Inerowicz HD, Hill CA, Kuhn RJ. Changes in the proteome of langat-infected Ixodes scapularis ISE6 cells: metabolic pathways associated with flavivirus infection. PLoS Negl Trop Dis. 2016;10:e0004180.
Macaluso KR, Mulenga A, Simser JA, Azad AF. Differential expression of genes in uninfected and Rickettsia-infected Dermacentor variabilis ticks as assessed by differential-display PCR. Infect Immun. 2003;71:6165–70.
Leal BF, Alzugaray MF, Seixas A, Da Silva Vaz I, Ferreira CAS. Characterization of a glycine-rich protein from Rhipicephalus microplus: tissue expression, gene silencing and immune recognition. Parasitology. 2017;145:927–38.
You MJ. Immunization of mice with recombinant P27/30 protein confers protection against hard tick Haemaphysalis longicornis (Acari: Ixodidae) infestation. J Vet Sci. 2005;6:47–51.
Leavis PC, Gergely J. Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction. CRC Crit Rev Biochem. 1984;16:235–305.
You M, Xuan X, Tsuji N, Kamio T, Igarashi I, Nagasawa H, et al. Molecular characterization of a troponin I-like protein from the hard tick Haemaphysalis longicornis. Insect Biochem Mol Biol. 2001;32:67–73.
Fukumoto S, Sakaguchi T, You M, Xuan X, Fujisaki K. Tick troponin I-like molecule is a potent inhibitor for angiogenesis. Microvasc Res. 2006;71:218–21.
Perner J, Kotál J, Hatalová T, Urbanová V, Bartošová-Sojková P, Brophy PM, Kopáček P. Inducible glutathione S-transferase (IrGST1) from the tick Ixodes ricinus is a haem-binding protein. Insect Biochem Mol Biol. 2018;95:44–54.
Busby AT, Ayllón N, Kocan KM, Blouin EF, de la Fuente G, Galindo RC, et al. Expression of heat shock proteins and subolesin affects stress responses, Anaplasma phagocytophilum infection and questing behavior in the tick, Ixodes scapularis. Med Vet Entomol. 2012;26:92–102.
Lewis LA, Radulović TM, Kim TK, Porter LM, Mulenga A. Identification of 24h Ixodes scapularis immunogenic tick saliva proteins. Ticks Tick Borne Dis. 2015;6:424–34.
Kim TK, Tirloni L, Pinto AFM, Moresco J, Yates JR III, da Silva Vaz I, Jr Mulenga A. Ixodes scapularis tick saliva proteins sequentially secreted every 24 h during blood feeding. PLoS Negl Trop Dis. 2016;10:e0004323.
Rachinsky A, Guerrero FD, Scoles GA. Differential protein expression in ovaries of uninfected and Babesia-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus. Insect Biochem Mol Biol. 2007;37:1291–308.
Horáčková J, Rudenko N, Golovchenko M, Havlíková S, Grubhoffer L. IrML– a gene encoding a new member of the ML protein family from the hard tick, Ixodes ricinus. J Vector Ecol. 2010;35:410–8.
Thepparit C, Bourchookarn A, Petchampai N, Barker SA, Macaluso KR. Interaction of Rickettsia felis with histone H2B facilitates the infection of a tick cell line. Microbiology. 2010;156:2855–63.
The authors are grateful for the financial support of the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and Slovak Academy of Sciences (Grant VEGA 2/0068/17 and 2/0052/19) and the Research & Development Operational Programme funded by the European Regional Development Fund (grant no. 26240220096).
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All analytical quality gels included in the comparative proteomic analysis. Abbreviations: C, control set; I, infected set.
Quantitative data for all gel spots. Statistical test and ratio are shown (positive value spot is more abundant in gels from infected samples, negative value is less abundant). Abbreviations: C, control; I, infected; SD, standard deviation.
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Flores-Ramirez, G., Sallay, B., Danchenko, M. et al. Comparative proteomics of the vector Dermacentor reticulatus revealed differentially regulated proteins associated with pathogen transmission in response to laboratory infection with Rickettsia slovaca. Parasites Vectors 12, 318 (2019). https://doi.org/10.1186/s13071-019-3564-y
- Tick vector
- Comparative proteomics
- Protective antigens
- Immune modulation
- Bacterial transmission