The transcriptomic response to cannabidiol of Treponema denticola, a phytocannabinoid-resistant periodontal pathogen.

AIM: The use of cannabis, which contains multiple antimicrobials, may be a risk factor for periodontitis. We hypothesized that multiple oral spirochetes would be phytocannabinoid-resistant and that cannabidiol (CBD) would act as an environmental stressor to which Treponema denticola would respond transcriptionally, thereby providing first insights into spirochetal survival strategies. MATERIALS AND METHODS: Oral spirochete growth was monitored spectrophotometrically in the presence and absence of physiologically relevant phytocannabinoid doses, the transcriptional response to phytocannabinoid exposure determined by RNAseq, specific gene activity fluxes verified using qRT-PCR and orthologues among fully sequenced oral spirochetes identified. RESULTS: Multiple strains of oral treponemes were resistant to CBD (0.1-10 µg/mL), while T. denticola ATCC 35405 was resistant to all phytocannabinoids tested (CBD, cannabinol [CBN], tetrahydrocannabinol [THC]). A total of 392 T. denticola ATCC 35405 genes were found to be CBD-responsive by RNAseq. A selected subset of these genes was independently verified by qRT-PCR. Genes found to be differentially activated by both methods included several involved in transcriptional regulation and toxin control. Suppressed genes included several involved in chemotaxis and proteolysis. CONCLUSIONS: Oral spirochetes, unlike some other periodontal bacteria, are resistant to physiological doses of phytocannabinoids. Investigation of CBD-induced transcriptomic changes provided insight into the resistance mechanisms of this important periodontal pathogen. These findings should be considered in the context of the reported enhanced susceptibility to periodontitis in cannabis users.

Oral Spirochete Treponema denticola Intraoral Infection Reveals Unique miR-133a, miR-486, miR-126-3p, miR-126-5p miRNA Expression Kinetics during Periodontitis.

miRNAs are major regulators of eukaryotic gene expression and host immunity, and play an important role in the inflammation-mediated pathways in periodontal disease (PD) pathogenesis. Expanding our previous observation with the global miRNA profiling using partial human mouth microbes, and lack of in vivo studies involving oral spirochete Treponema denticola-induced miRNAs, this study was designed to delineate the global miRNA expression kinetics during progression of periodontitis in mice infected with T. denticola by using NanoString nCounter® miRNA panels. All of the T. denticola-infected male and female mice at 8 and 16 weeks demonstrated bacterial colonization (100%) on the gingival surface, and an increase in alveolar bone resorption (p < 0.0001). A total of 70 miRNAs with at least 1.0-fold differential expression/regulation (DE) (26 upregulated and 44 downregulated) were identified. nCounter miRNA expression profiling identified 13 upregulated miRNAs (e.g., miR-133a, miR-378) and 25 downregulated miRNAs (e.g., miR-375, miR-34b-5p) in T. denticola-infected mouse mandibles during 8 weeks of infection, whereas 13 upregulated miRNAs (e.g., miR-486, miR-126-5p) and 19 downregulated miRNAs (miR-2135, miR-142-3p) were observed during 16 weeks of infection. One miRNA (miR-126-5p) showed significant difference between 8 and 16 weeks of infection. Interestingly, miR-126-5p has been presented as a potential biomarker in patients with periodontitis and coronary artery disease. Among the upregulated miRNAs, miR-486, miR-126-3p, miR-126-5p, miR-378a-3p, miR-22-3p, miR-151a-3p, miR-423-5p, and miR-221 were reported in human gingival plaques and saliva samples from periodontitis and with diabetes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed various functional pathways of DE miRNAs, such as bacterial invasion of epithelial cells, Ras signaling, Fc gamma R-mediated phagocytosis, osteoclast differentiation, adherens signaling, and ubiquitin mediated proteolysis. This is the first study of DE miRNAs in mouse mandibles at different time-points of T. denticola infection; the combination of three specific miRNAs, miR-486, miR-126-3p, and miR-126-5p, may serve as an invasive biomarker of T. denticola in PD. These miRNAs may have a significant role in PD pathogenesis, and this research establishes a link between miRNA, periodontitis, and systemic diseases.

The association between gingivitis and oral spirochetes in young cats and dogs.

Although gingivitis frequently occurs in young cats, spirochetes are often found in the early stages of periodontal disease. This study was conducted to determine the association between gingivitis and oral spirochetes in young cats and dogs. The degree of gingivitis was evaluated in a total of 68 cats and 31 dogs under one year of age, and plaques were collected from each carnassial. To detect spirochetes or Porphyromonas gulae in plaque samples, 16S rRNA gene was amplified by polymerase chain reaction (PCR) using specific primers. All data were analyzed using Fisher's exact probability test and odds ratio (OR) with a 95% confidence interval (95% CI). The prevalence of gingivitis was significantly higher in young cats (92.6%) than in young dogs (45.2%). The positive rate of spirochetes by PCR in gingivitis cases was 85.4% in young cats and 15.4% in young dogs, and the positive rate of P. gulae was 66.7% in young cats and 15.4% in young dogs. Both results were significantly higher in young cats than in young dogs. In young cats, spirochetes were significantly associated with gingivitis (OR = 7.95; 95% CI = 1.17, 53.83; P < 0.05), but P. gulae was not (OR = 2.44; 95% CI = 0.38, 15.66; P = 0.23). These results suggest that spirochetes may be associated with the early stages of periodontal disease in cats.

Transcriptional and functional characterizations of multiple flagellin genes in spirochetes.

The flagellar filament is a helical propeller for bacterial locomotion. In external flagellates, the filaments are mostly homopolymers of a single flagellin protein. By contrast, the flagellar filaments of spirochetes are mostly heteropolymers of multiple flagellin proteins. This report seeks to investigate the role of multiple flagellin proteins using the oral spirochete Treponema denticola as a model. First, biochemical and genetic studies uncover that the flagellar filaments of T. denticola mainly comprise four proteins, FlaA, FlaB1, FlaB2, and FlaB3, in a defined stoichiometry. Second, transcriptional analyses reveal that the genes encoding these four proteins are regulated by two different transcriptional factors, sigma28 and sigma70 . Third, loss-of-function studies demonstrate that each individual flagellin protein contributes to spirochete motility, but none of them is absolutely required. Last, we provide genetic and structural evidence that FlaA forms a "seam"-like structure around the core and that deletion of individual flagellin protein alters the flagellar homeostasis. Collectively, these results demonstrate that T. denticola has evolved a unique mechanism to finely regulate its flagellar filament gene expression and assembly which renders the organelle with the right number, shape, strength, and structure for its distinct motility.

Comparative Analysis of Brucepastera parasyntrophica gen. nov., sp. nov. and Teretinema zuelzerae gen. nov., comb. nov. (Treponemataceae) Reveals the Importance of Interspecies Hydrogen Transfer in the Energy Metabolism of Spirochetes.

Most members of the family Treponemataceae (Spirochaetales) are associated with vertebrate hosts. However, a diverse clade of uncultured, putatively free-living treponemes comprising several genus-level lineages is present in other anoxic environments. The only cultivated representative to date is Treponema zuelzerae, isolated from freshwater mud. Here, we describe the isolation of strain RmG11 from the intestinal tract of cockroaches. The strain represents a novel genus-level lineage of Treponemataceae and is metabolically distinct from T. zuelzerae. While T. zuelzerae grows well on various sugars, forming acetate and H2 as major fermentation products, strain RmG11 grew poorly on glucose, maltose, and starch, forming mainly ethanol and only small amounts of acetate and H2. In contrast to the growth of T. zuelzerae, that of strain RmG11 was strongly inhibited at high H2 partial pressures but improved considerably when H2 was removed from the headspace. Cocultures of strain RmG11 with the H2-consuming Methanospirillum hungatei produced acetate and methane but no ethanol. Comparative genomic analysis revealed that strain RmG11 possesses only a single, electron-confurcating hydrogenase that forms H2 from NADH and reduced ferredoxin, whereas T. zuelzerae also possesses a second, ferredoxin-dependent hydrogenase that allows the thermodynamically more favorable formation of H2 from ferredoxin via the Rnf complex. In addition, we found that T. zuelzerae utilizes xylan and possesses the genomic potential to degrade other plant polysaccharides. Based on phenotypic and phylogenomic evidence, we describe strain RmG11 as Brucepastera parasyntrophica gen. nov., sp. nov. and Treponema zuelzerae as Teretinema zuelzerae gen. nov., comb. nov. IMPORTANCE Spirochetes are widely distributed in various anoxic environments and commonly form molecular hydrogen as a major fermentation product. Here, we show that two closely related members of the family Treponemataceae differ strongly in their sensitivity to high hydrogen partial pressure, and we explain the metabolic mechanisms that cause these differences by comparative genome analysis. We demonstrate a strong boost in the growth of the hydrogen-sensitive strain and a shift in its fermentation products to acetate during cocultivation with a H2-utilizing methanogen. Our results add a hitherto unrecognized facet to the fermentative metabolism of spirochetes and also underscore the importance of interspecies hydrogen transfer in not-obligately-syntrophic interactions among fermentative and hydrogenotrophic guilds in anoxic environments.

PLSDB: advancing a comprehensive database of bacterial plasmids.

Plasmids are known to contain genes encoding for virulence factors and antibiotic resistance mechanisms. Their relevance in metagenomic data processing is steadily growing. However, with the increasing popularity and scale of metagenomics experiments, the number of reported plasmids is rapidly growing as well, amassing a considerable number of false positives due to undetected misassembles. Here, our previously published database PLSDB provides a reliable resource for researchers to quickly compare their sequences against selected and annotated previous findings. Within two years, the size of this resource has more than doubled from the initial 13,789 to now 34,513 entries over the course of eight regular data updates. For this update, we aggregated community feedback for major changes to the database featuring new analysis functionality as well as performance, quality, and accessibility improvements. New filtering steps, annotations, and preprocessing of existing records improve the quality of the provided data. Additionally, new features implemented in the web-server ease user interaction and allow for a deeper understanding of custom uploaded sequences, by visualizing similarity information. Lastly, an application programming interface was implemented along with a python library, to allow remote database queries in automated workflows. The latest release of PLSDB is freely accessible under https://www.ccb.uni-saarland.de/plsdb.

Role of the major determinant of polar flagellation FlhG in the endoflagella-containing spirochete Leptospira.

Spirochetes can be distinguished from other bacteria by their spiral-shaped morphology and subpolar periplasmic flagella. This study focused on FlhF and FlhG, which control the spatial and numerical regulation of flagella in many exoflagellated bacteria, in the spirochete Leptospira. In contrast to flhF which seems to be essential in Leptospira, we demonstrated that flhG- mutants in both the saprophyte L. biflexa and the pathogen L. interrogans were less motile than the wild-type strains in gel-like environments but not hyperflagellated as reported previously in other bacteria. Cryo-electron tomography revealed that the distance between the flagellar basal body and the tip of the cell decreased significantly in the flhG- mutant in comparison to wild-type and complemented strains. Additionally, comparative transcriptome analyses of L. biflexa flhG- and wild-type strains showed that FlhG acts as a negative regulator of transcription of some flagellar genes. We found that the L. interrogans flhG- mutant was attenuated for virulence in the hamster model. Cross-species complementation also showed that flhG is not interchangeable between species. Our results indicate that FlhF and FlhG in Leptospira contribute to governing cell motility but our data support the hypothesis that FlhF and FlhG function differently in each bacterial species, including among spirochetes.

Genetic engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete.

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

Origin of symbiotic gut spirochetes as key players in the nutrition of termites.

Termites harbour symbiotic spirochetes in their hindguts, which have long been considered treponemes, although they represent separate lines of descent from known species of Treponema. 'Termite gut treponemes' have a mutualistic relationship with the host termites with their physiological properties including CO2 -reductive acetogenesis, from which the resulting acetate fulfils most of the respiratory requirement of the host. Song and co-workers showed that a spirochetal isolate (strain RmG30) from a Madeira cockroach represents the earliest branching lineage of extremely diverse termite (Treponema) cluster I and was a simple homolactic fermenter, suggesting that CO2 -reductive acetogenesis exhibited by some members of termite cluster I originated via horizontal gene transfer. Phylogenomic and 16S rRNA sequence-based phylogenetic analyses indicated a deeply-branched sister clade containing termite cluster I was distinguishable as a family-level lineage. In this context, a new family, 'Termitinemataceae' has been proposed for this clade. Strain RmG30 has been designated as the type strain of Breznakiella homolactica gen. nov. sp. nov. named after John A. Breznak, an American microbiologist distinguished in termite gut microbiology. The study has posed important questions for the future, including the actual roles of the termite spirochetes in each termite lineage and the evolutionary process of their physiological properties.

First metagenomic report of Borrelia americana and Borrelia carolinensis in Poland - a preliminary study.

INTRODUCTION AND OBJECTIVE: Ixodes ricinus (I. ricinus) and Dermacentor reticulatus (D. reticulatus) are the most common ticks in Poland. These ticks contain many bacteria, which compose a microbiome with potential impact on humans. The aim of the study was to discover the microbiome of ticks in Poland. MATERIAL AND METHODS: Ticks were collected in The Protected Landscape Area of the Bug and Nurzec Valley, Poland, in 2016-2018 by flagging. They were cleaned in 70% ethanol and damaged in mortar with PBS (without Ca2+ and Mg2+ ions). DNA was extracted from the homogenates with spin columns kits, and used as a matrix in end-point PCR for bacterial 16S rRNA fragments amplifications, and further for next generation sequencing (NGS) by ILLUMINA. RESULTS: In 22 ticks (3 I. ricinus and 19 D. reticulatus) 38 microorganisms were detected. The most common were Francisella hispaniensis and Francisella novicida. In 17 ticks, Sphingomonas oligophenolica, and in 12 Rickettsia aeshlimanii were found. In 2, I. ricinus specific DNA of Borrelia americana and Borrelia carolinensis were found. In one female, D. reticulatus Anaplasma phagocytophilum and Anaplasma centrale were found. Pseudomonas lutea and Ps. moraviensis were detected in 9 and 8 ticks, respectively. CONCLUSIONS: Polish ticks microbiome contains not only well-known tick-borne pathogens, but also other pathogenic microorganisms. For the first time in Poland, Borrelia americana and Borrelia carolinensis in I. ricinus collected from the environment were detected. The dominant pathogenic microorganisms for humans were Francisella spp. and Rickettsia spp., and non-pathogenic - Sphingomonas oligophenolica. Knowledge of a tick microbiome might be useful in tick-borne biocontrol and tick-borne diseases prevention.

Gut microbiota resilience in horse athletes following holidays out to pasture.

Elite horse athletes that live in individual boxes and train and compete for hours experience long-term physical and mental stress that compromises animal welfare and alters the gut microbiota. We therefore assessed if a temporary period out to pasture with conspecifics could improve animal welfare and in turn, favorably affect intestinal microbiota composition. A total of 27 athletes were monitored before and after a period of 1.5 months out to pasture, and their fecal microbiota and behavior profiles were compared to those of 18 horses kept in individual boxes. The overall diversity and microbiota composition of pasture and control individuals were temporally similar, suggesting resilience to environmental challenges. However, pasture exposure induced an increase in Ruminococcus and Coprococcus that lasted 1-month after the return to individual boxes, which may have promoted beneficial effects on health and welfare. Associations between the gut microbiota composition and behavior indicating poor welfare were established. Furthermore, withdrawn behavior was associated with the relative abundances of Lachnospiraceae AC2044 group and Clostridiales family XIII. Both accommodate a large part of butyrate-producing bacterial genera. While we cannot infer causality within this study, arguably, these findings suggest that management practices maintained over a longer period of time may moderate the behavior link to the gut ecosystem beyond its resilience potential.

The starlet sea anemone, Nematostella vectensis, possesses body region-specific bacterial associations with spirochetes dominating the capitulum.

Sampling of different body regions can reveal highly specialized bacterial associations within the holobiont and facilitate identification of core microbial symbionts that would otherwise be overlooked by bulk sampling methods. Here, we characterized compartment-specific associations present within the model cnidarian Nematostella vectensis by dividing its morphology into three distinct microhabitats. This sampling design allowed us to uncover a capitulum-specific dominance of spirochetes within N. vectensis. Bacteria from the family Spirochaetaceae made up 66% of the community in the capitulum, while only representing 1.2% and 0.1% of the communities in the mesenteries and physa, respectively. A phylogenetic analysis of the predominant spirochete sequence recovered from N. vectensis showed a close relation to spirochetes previously recovered from wild N. vectensis. These sequences clustered closer to the recently described genus Oceanispirochaeta, rather than Spirochaeta perfilievii, supporting them as members of this clade. This suggests a prevalent and yet uncharacterized association between N. vectensis and spirochetes from the order Spirochaetales.

Genetic Manipulations of Oral Spirochete Treponema denticola.

There have been more than 60 different oral Treponema species identified in the oral cavity; however, only few species can be cultivated in vitro reliably. Among those cultivable species, due to its medical importance and genetic tractability, Treponema denticola, one of the keystone pathogens associated with human periodontitis, has emerged as a paradigm model organism to understanding the genetics, etiology, and pathophysiology of oral Treponema species. During the last two decades, several genetic tools have been developed, which have played an instrumental role in the study of T. denticola. This chapter describes the experimental design and procedure of genetic manipulations of T. denticola.

High conservation combined with high plasticity: genomics and evolution of Borrelia bavariensis.

BACKGROUND: Borrelia bavariensis is one of the agents of Lyme Borreliosis (or Lyme disease) in Eurasia. The genome of the Borrelia burgdorferi sensu lato species complex, that includes B. bavariensis, is known to be very complex and fragmented making the assembly of whole genomes with next-generation sequencing data a challenge. RESULTS: We present a genome reconstruction for 33 B. bavariensis isolates from Eurasia based on long-read (Pacific Bioscience, for three isolates) and short-read (Illumina) data. We show that the combination of both sequencing techniques allows proper genome reconstruction of all plasmids in most cases but use of a very close reference is necessary when only short-read sequencing data is available. B. bavariensis genomes combine a high degree of genetic conservation with high plasticity: all isolates share the main chromosome and five plasmids, but the repertoire of other plasmids is highly variable. In addition to plasmid losses and gains through horizontal transfer, we also observe several fusions between plasmids. Although European isolates of B. bavariensis have little diversity in genome content, there is some geographic structure to this variation. In contrast, each Asian isolate has a unique plasmid repertoire and we observe no geographically based differences between Japanese and Russian isolates. Comparing the genomes of Asian and European populations of B. bavariensis suggests that some genes which are markedly different between the two populations may be good candidates for adaptation to the tick vector, (Ixodes ricinus in Europe and I. persulcatus in Asia). CONCLUSIONS: We present the characterization of genomes of a large sample of B. bavariensis isolates and show that their plasmid content is highly variable. This study opens the way for genomic studies seeking to understand host and vector adaptation as well as human pathogenicity in Eurasian Lyme Borreliosis agents.

Spirochetes isolated from arthropods constitute a novel genus Entomospira genus novum within the order Spirochaetales.

Spirochetal bacteria were successfully isolated from mosquitoes (Culex pipiens, Aedes cinereus) in the Czech Republic between 1999 and 2002. Preliminary 16S rRNA phylogenetic sequence analysis showed that these strains differed significantly from other spirochetal genera within the family Spirochaetaceae and suggested a novel bacterial genus in this family. To obtain more comprehensive genomic information of these isolates, we used Illumina MiSeq and Oxford Nanopore technologies to sequence four genomes of these spirochetes (BR151, BR149, BR193, BR208). The overall size of the genomes varied between 1.68 and 1.78 Mb; the GC content ranged from 38.5 to 45.8%. Draft genomes were compared to 36 publicly available genomes encompassing eight genera from the class Spirochaetes. A phylogeny generated from orthologous genes across all taxa and the percentage of conserved proteins (POCP) confirmed the genus status of these novel spirochetes. The genus Entomospira gen. nov. is proposed with BR151 selected as type species of the genus. For this isolate and the closest related isolate, BR149, we propose the species name Entomospira culicis sp. nov. The two other isolates BR208 and BR193 are named Entomospira nematocera sp. nov. (BR208) and Entomospira entomophilus sp. nov. (BR193). Finally, we discuss their interesting phylogenetic positioning.

The BB0345 Hypothetical Protein of Borrelia burgdorferi Is Essential for Mammalian Infection.

During the natural enzootic life cycle of Borrelia burgdorferi (also known as Borreliella burgdorferi), the bacteria must sense conditions within the vertebrate and arthropod and appropriately regulate expression of genes necessary to persist within these distinct environments. bb0345 of B. burgdorferi encodes a hypothetical protein of unknown function that is predicted to contain an N-terminal helix-turn-helix (HTH) domain. Because HTH domains can mediate protein-DNA interactions, we hypothesized that BB0345 might represent a previously unidentified borrelial transcriptional regulator with the ability to regulate events critical for the B. burgdorferi enzootic cycle. To study the role of BB0345 within mammals, we generated a bb0345 mutant and assessed its virulence potential in immunocompetent mice. The bb0345 mutant was able to initiate localized infection and disseminate to distal tissues but was cleared from all sites by 14 days postinfection. In vitro growth curve analyses revealed that the bb0345 mutant grew similar to wild-type bacteria in standard Barbour-Stoenner-Kelley II (BSK-II) medium; however, the mutant was not able to grow in dilute BSK-II medium or dialysis membrane chambers (DMCs) implanted in rats. Proteinase K accessibility assays and whole-cell partitioning indicated that BB0345 was intracellular and partially membrane associated. Comparison of protein production profiles between the wild-type parent and the bb0345 mutant revealed no major differences, suggesting BB0345 may not be a global transcriptional regulator. Taken together, these data show that BB0345 is essential for B. burgdorferi survival in the mammalian host, potentially by aiding the spirochete with a physiological function that is required by the bacterium during infection.

Oral spirochetes: Pathogenic mechanisms in periodontal disease.

Periodontitis is an infectious inflammatory disease resulting from infection of biofilm forming bacteria. Several bacterial factors regulate inflammatory response and cause to tissue damage and loss of connection between gingival and tooth. Since bacterial virulence factors and also host immune responses have role, understanding of periodontal disease is complex, in overall we can say that in this disease epithelium is deleted by bacteria. Oral spirochetes are related to periodontitis, among them, Treponema denticola, have been associated with periodontal diseases such as early-onset periodontitis, necrotizing ulcerative gingivitis, and acute pericoronitis. This review will analyse mechanisms of pathogenesis of spirochetes in periodontitis. Microorganisms cause destruction of gingival tissue by two mechanisms. In one, damage results from the direct action of bacterial enzymes and cytotoxic products of bacterial metabolism. In the other, only bacterial components have role, and tissue destruction is the inevitable side effect of a subverted and exaggerated host inflammatory response to plaque antigens.

Structural analysis of the outer surface proteins from Borrelia burgdorferi paralogous gene family 54 that are thought to be the key players in the pathogenesis of Lyme disease.

Lyme disease is a tick-borne infection caused by Borrelia burgdorferi sensu lato complex spirochetes. Through a complex enzootic cycle, the bacteria transfer between two different hosts: Ixodes ticks and mammalian organisms. At the start of the tick blood meal, the spirochetes located in the tick gut upregulate the expression of several genes, mainly coding for outer surface proteins. Outer surface proteins belonging to the paralogous gene family 54 (PFam54) have been shown to be the most upregulated among the other borrelial proteins and the results clearly point to the potential importance of these proteins in the pathogenesis of Lyme disease. The significance of PFam54 proteins is confirmed by the fact that of all ten PFam54 proteins, BBA64 and BBA66 are necessary for the transfer of B. burgdorferi from infected Ixodes ticks to mammalian hosts. To enhance the understanding of the pathogenesis of Lyme disease and to promote the development of novel therapies against Lyme disease, we solved the crystal structure of the PFam54 member BBA65. Additionally, we report the structure of the B. burgdorferi BBA64 orthologous protein from B. spielmanii. Together with the previously determined crystal structures of five PFam54 members and several related proteins, we performed a comprehensive structural analysis for this important group of proteins. In addition to revealing the molecular aspects of the proteins, the structural data analysis suggests that the gene families PFam54 and PFam60, which have long been referred to as separate paralogous families, should be merged into one and designated as PFam54_60.

Isolation and Molecular Characterization of Tick-Borne Relapsing Fever Borrelia Infecting Ornithodoros (Pavlovskyella) verrucosus Ticks Collected in Ukraine.

BACKGROUND: Tick-borne relapsing fever (TBRF) is a neglected zoonotic bacterial disease known to occur on 5 continents. We report a laboratory-acquired case of TBRF caused by Borrelia caucasica, which is endemic in Ukraine and transmitted by Ornithodoros verrucosus ticks. METHODS: We isolated spirochetes and characterized them by partially sequencing the 16s ribosomal ribonucleic acid (rrs), flagellin (flaB), and deoxyribonucleic acid gyrase (gyrB) genes and conducting a phylogenetic analysis. RESULTS: These analyses revealed a close relationship of Ukrainian spirochetes with the Asian TBRF species, Borrelia persica. The taxonomic and nomenclature problems related to insufficient knowledge on the spirochetes and their vectors in the region are discussed. CONCLUSIONS: Although these findings enhance our understanding of species identities for TBRF Borrelia in Eurasia, further work is required to address the neglected status of TBRF in this part of the world. Public health practitioners should consider TBRF and include the disease into differential diagnosis of febrile illnesses with unknown etiology.