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Decorin binding proteins (Dbps) mediate attachment of spirochetes in host organisms during the early stages of Lyme disease infection. Previously, different binding mechanisms of Dbps to glycosaminoglycans have been elucidated for the pathogenic species Borrelia burgdorferi sensu stricto and B. afzelii. We are investigating various European Borrelia spirochetes and their interactions at the atomic level using NMR. We report preparative scale recombinant expression of uniformly stable isotope enriched B. afzelii DbpA in Escherichia coli, its chromatographic purification, and solution NMR assignments of its backbone and sidechain 1H, 13C, and 15N atoms. This data was used to predict secondary structure propensity, which we compared to the North American B. burgdorferi sensu stricto and European B. garinii DbpA for which solution NMR structures had been determined previously. Backbone dynamics of DbpA from B. afzelii were elucidated from spin relaxation and heteronuclear NOE experiments. NMR-based secondary structure analysis together with the backbone dynamics characterization provided a first look into structural differences of B. afzelii DbpA compared to the North American species and will serve as the basis for further investigation of how these changes affect interactions with host components.

Lyme borreliosis is a vector-borne infection caused by bacteria under theBorrelia burgdorferisensu lato complex, both in Europe and North America. Differential gene expression at different times throughout its infectious cycle allows the spirochete to survive very diverse environments within different mammalian hosts as well as the tick vector. To date, the vast majority of data about spirochetal proteins and their functions are from genetic studies carried out on North American strains of a single species, i.e.B. burgdorferi sensu stricto. The whole-genome sequences recently obtained for several European species/strains make it feasible to adapt and use genetic techniques to study inherent differences between them. This review highlights the crucial need to undertake independent studies of genospecies within Europe, given their varying genetic content and pathogenic potential, and differences in clinical manifestation.

Hematophagous arthropods are responsible for the transmission of a variety of pathogens that cause disease in humans and animals. Ticks of the Ixodes ricinus complex are vectors for some of the most frequently occurring human tick-borne diseases, particularly Lyme borreliosis and tick-borne encephalitis virus (TBEV). The search for vaccines against these diseases is ongoing. Efforts during the last few decades have primarily focused on understanding the biology of the transmitted viruses, bacteria and protozoans, with the goal of identifying targets for intervention. Successful vaccines have been developed against TBEV and Lyme borreliosis, although the latter is no longer available for humans. More recently, the focus of intervention has shifted back to where it was initially being studied which is the vector. State of the art technologies are being used for the identification of potential vaccine candidates for anti-tick vaccines that could be used either in humans or animals. The study of the interrelationship between ticks and the pathogens they transmit, including mechanisms of acquisition, persistence and transmission have come to the fore, as this knowledge may lead to the identification of critical elements of the pathogens' life-cycle that could be targeted by vaccines. Here, we review the status of our current knowledge on the triangular relationships between ticks, the pathogens they carry and the mammalian hosts, as well as methods that are being used to identify anti-tick vaccine candidates that can prevent the transmission of tick-borne pathogens.

The different components of the mouthparts of hard ticks (Ixodidae) enable these parasites to penetrate host skin, secrete saliva, embed, and suck blood. Moreover, the tick’s mouthparts represent a key route for saliva-assisted pathogen transmission as well as pathogen acquisition from blood meal during the tick feeding process. Much has been learned about the basic anatomy of the tick’s mouthparts and in the broad outlines of how they function in previous studies. However, the precise mechanics of these functions are little understood. Here, we propose for the first time an animated model of the orchestration of the tick mouthparts and associated structures during blood meal acquisition and salivation. These two actions are known to alternate during tick engorgement. Specifically, our attention has been paid to the mechanism underlining the blood meal uptake into the pharynx through the mouth and how ticks prevent mixing the uptaken blood with secreted saliva. We animated function of muscles attached to the salivarium and their possible opening /closing of the salivarium, with a plausible explanation of the movement of saliva within the salivarium and massive outpouring of saliva.

Abstract Vector-borne diseases constitute 17% of all infectious diseases in the world; among the blood-feeding arthropods, ticks transmit the highest number of pathogens. Understanding the interactions between the tick vector, the mammalian host and the pathogens circulating between them is the basis for the successful development of vaccines against ticks or the tick-transmitted pathogens as well as for the development of specific treatments against tick-borne infections. A lot of effort has been put into transcriptomic and proteomic analyses; however, the protein-carbohydrate interactions and the overall glycobiology of ticks and tick-borne pathogens has not been given the importance or priority deserved. Novel (bio)analytical techniques and their availability have immensely increased the possibilities in glycobiology research and thus novel information in the glycobiology of ticks and tick-borne pathogens is being generated at a faster pace each year. This review brings a comprehensive summary of the knowledge on both the glycosylated proteins and the glycan-binding proteins of the ticks as well as the tick-transmitted pathogens, with emphasis on the interactions allowing the infection of both the ticks and the hosts by various bacteria and tick-borne encephalitis virus.

Lyme borreliosis is a bacterial infection that can be spread to humans by infected ticks and may severely affect many organs and tissues. Nearly four decades have elapsed since the discovery of the disease agent called Borrelia burgdorferi. Although there is a plethora of knowledge on the infectious agent and thousands of scientific publications, an effective way on how to combat and prevent Lyme borreliosis has not been found yet. There is no vaccine for humans available, and only one active vaccine program in clinical development is currently running. A spirited search for possible disease interventions is of high public interest as surveillance data indicates that the number of cases of Lyme borreliosis is steadily increasing in Europe and North America. This review provides a condensed digest of the history of vaccine development up to new promising vaccine candidates and strategies that are targeted against Lyme borreliosis, including elements of the tick vector, the reservoir hosts, and the Borrelia pathogen itself.

Numerous methods exist for fluorescently labeling proteins either as direct fusion proteins (GFP, RFP, YFP, etc.—attached to the protein of interest) or utilizing accessory proteins to produce fluorescence (SNAP-tag, CLIP-tag), but the significant increase in size that these accompanying proteins add may hinder or impede proper protein folding, cellular localization, or oligomerization. Fluorescently labeling proteins with biarsenical dyes, like FlAsH, circumvents this issue by using a short 6-amino acid tetracysteine motif that binds the membrane-permeable dye and allows visualization of living cells. Here, we report the successful adaptation of FlAsH dye for live-cell imaging of two genera of spirochetes, Leptospira and Borrelia, by labeling inner or outer membrane proteins tagged with tetracysteine motifs. Visualization of labeled spirochetes was possible by fluorescence microscopy and flow cytometry. A subsequent increase in fluorescent signal intensity, including prolonged detection, was achieved by concatenating two copies of the 6-amino acid motif. Overall, we demonstrate several positive attributes of the biarsenical dye system in that the technique is broadly applicable across spirochete genera, the tetracysteine motif is stably retained and does not interfere with protein function throughout the B. burgdorferi infectious cycle, and the membrane-permeable nature of the dyes permits fluorescent detection of proteins in different cellular locations without the need for fixation or permeabilization. Using this method, new avenues of investigation into spirochete morphology and motility, previously inaccessible with large fluorescent proteins, can now be explored.

Lyme borreliosis is the most common zoonotic disease transmitted by ticks in Europe and North America. Despite having multiple tick vectors, the causative agent, Borrelia burgdorferi sensu lato , is vectored mainly by Ixodes ricinus in Europe. In the present study, we aimed to review and summarize the existing data published from 2010 to 2016 concerning the prevalence of B. burgdorferi sensu lato spirochetes in questing I. ricinus ticks. The primary focus was to evaluate the infection rate of these bacteria in ticks, accounting for tick stage, adult tick gender, region, and detection method, as well as to investigate any changes in prevalence over time. The data obtained were compared to the findings of a previous metastudy. The literature search identified data from 23 countries, with 115,028 ticks, in total, inspected for infection with B. burgdorferi sensu lato . We showed that the infection rate was significantly higher in adults than in nymphs and in females than in males. We found significant differences between European regions, with the highest infection rates in Central Europe. The most common genospecies were B. afzelii and B. garinii , despite a negative correlation of their prevalence rates. No statistically significant differences were found among the prevalence rates determined by conventional PCR, nested PCR, and real-time PCR. IMPORTANCE Borrelia burgdorferi sensu lato is a pathogenic bacterium whose clinical manifestations are associated with Lyme borreliosis. This vector-borne disease is a major public health concern in Europe and North America and may lead to severe arthritic, cardiovascular, and neurological complications if left untreated. Although pathogen prevalence is considered an important predictor of infection risk, solitary isolated data have only limited value. Here we provide summarized information about the prevalence of B. burgdorferi sensu lato spirochetes among host-seeking Ixodes ricinus ticks, the principal tick vector of borreliae in Europe. We compare the new results with previously published data in order to evaluate any changing trends in tick infection.

To understand the response of the Lyme disease spirochete Borrelia burgdorferi exposed to stress conditions and assess the viability of this spirochete, we used a correlative cryo-fluorescence and cryo-scanning microscopy approach. This approach enables simple exposition of bacteria to various experimental conditions that can be stopped at certain time intervals by cryo-immobilization, examination of cell viability without necessity to maintain suitable culture conditions during viability assays, and visualization of structures in their native state at high magnification. We focused on rare and transient events e.g., the formation of round bodies and the presence of membranous blebs in spirochetes exposed to culture medium, host sera either without or with the bacteriolytic effect and water. We described all crucial steps of the workflow, particularly the influence of freeze-etching and accelerating voltage on the visualization of topography. With the help of newly designed cryo-transport device, we achieved greater reproducibility.

The presented article introduces methods and some results of a project aiming to improve the protection of landscape permeability for migration of large mammals. The main aim of the project was delimitation of migration corridors, representing an ecological network connecting areas of existing, or potential presence of focal species. Based on mapping of current and historical occurrence of selected species of large predators and ungulates, areas of potential presence of such species were defined, including further analysis of connectivity of these areas. At the same time, all categories of anthropogenic and natural barriers were described. Migration corridors were designed over detailed topographic datasets, according to the results of habitat analyses of both groups of animals, and subsequently were tested in the field. The project resulted in a coherent network of existing and proposed migration corridors, with sections categorised according to permeability, as well as methodical protection and a management plan

Correlative light and electron microscopy is an imaging technique that enables identification and targeting of fluorescently tagged structures with subsequent imaging at near-to-nanometer resolution. We established a novel correlative cryo-fluorescence microscopy and cryo-scanning electron microscopy workflow, which enables imaging of the studied object of interest very close to its natural state, devoid of artifacts caused for instance by slow chemical fixation. This system was tested by investigating the interaction of the zoonotic bacterium Borrelia burgdorferi with two mammalian cell lines of neural origin in order to broaden our knowledge about the cell-association mechanisms that precedes the entry of the bacteria into the cell. This method appears to be an unprecedentedly fast (

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Background]: Ixodes scapularis is the most common tick species in North America and a vector of important pathogens that cause diseases in humans and animals including Lyme disease, anaplasmosis and babesiosis. Tick defensins have been identified as a new source of antimicrobial agents with putative medical applications due to their wide-ranging antimicrobial activities. Two multigene families of defensins were previously reported in I. scapularis. The objective of the present study was to characterise the potential antimicrobial activity of two defensins from I. scapularis with emphasis on human pathogenic bacterial strains and important phytopathogenic fungi. [Methods]: Scapularisin-3 and Scapularisin-6 mature peptides were chemically synthesised. In vitro antimicrobial assays were performed to test the activity of these two defensins against species of different bacterial genera including Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Listeria spp. as well as Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa along with two plant-pathogenic fungi from the genus Fusarium. In addition, the tissue-specific expression patterns of Scapularisin-3 and Scapularisin-6 in I. scapularis midgut, salivary glands and embryo-derived cell lines were determined using PCR. Finally, tertiary structures of the two defensins were predicted and structural analyses were conducted. [Results]: Scapularisin-6 efficiently killed L. grayi, and both Scapularisin-3 and Scapularisin-6 caused strong inhibition (IC value: ∼1 μM) of the germination of plant-pathogenic fungi Fusarium culmorum and Fusarium graminearum. Scapularisin-6 gene expression was observed in I. scapularis salivary glands and midgut. However, Scapularisin-3 gene expression was only detected in the salivary glands. Transcripts from the two defensins were not found in the I. scapularis tick cell lines ISE6 and ISE18. [Conclusion]: Our results have two main implications. Firstly, the anti-Listeria and antifungal activities of Scapularisin-3 and Scapularisin-6 suggest that these peptides may be useful for (i) treatment of antibiotic-resistant L. grayi in humans and (ii) plant protection. Secondly, the antimicrobial properties of the two defensins described in this study may pave the way for further studies regarding pathogen invasion and innate immunity in I. scapularis., Miray Tonk is a Marie Curie Early Stage Researcher supported by the POSTICK ITN (Post-graduate training network for capacity building to control ticks and tick-borne diseases) within the FP7- PEOPLE – ITN programme (EU Grant No. 238511). This project was partially supported by the Grant Agency of the Czech Republic (GACR P302/11/1901) and with institutional support RVO: 60077344 from Biology Centre, Institute of Parasitology as well as grant ANTIGONE (EU-7FP; 278976). James J. Valdés was sponsored by project CZ.1.07/2.3.00/30.0032, co-financed by the European Social Fund and the state budget of the Czech Republic. Radek Šíma was supported by the Grant 13-12816P (GA CR). Mohammad Rahnamaeian and Andreas Vilcinskas acknowledge the Ministry for Science and Art of the State of Hesse (Germany) for funding the LOEWE Center of Insect Biotechnology and Bioresources. Zdeněk Franta was supported by Alexander von Humboldt Research Fellowship for Postdoctoral Researchers.

In this study the idea of the topological similarity index has been generalized so that its applicability reaches beyond a simple HMO model to semi- empirical or even ab initio methods. The greatest advantage of this new index is its complete positional invariance, which makes it possible to avoid time consuming optimization with respect to the mutual position of molecules. As a consequence of this invariance, the index can find a number of interesting applications, especially in the design of compounds with desired biological properties. The applicability of the index has been numerically tested on a series of isosteric and isoelectronic molecules.