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The bacterial pathogen responsible for Lyme disease ¬— Borrelia burgdorferi— is an atypical Gram-negative spirochete that is transmitted to humans via the bite of an infected Ixodes tick. Like all Gram-negative bacteria the structural portion of the cell envelope known as peptidoglycan (PG) is sandwiched between the inner and outer membranes. Unlike virtually all bacteria, this PG layer is unique in B. burgdorferi in that the amino acid structure differs from most Gram-negative and Gram-positive bacteria by the addition of an Ornithine residue to the third amino acid location in the crosslinking structure. This unique motif is hypothesized to be responsible for the unusual clinical manifestations seen in Lyme disease, specifically Lyme arthritis, the most common late stage symptom of the disease in the United States. Peptidoglycan is only one component of the cell envelope in B. burgdorferi though; other portions of the cell envelope remain understudied specifically when viewed through the lens of the immune response they may elicit in addition to that of PG. The combined immunological effect of the unique bacterial antigen found in B. burgdorferi PG, as well as other potentially associated proteins contained within the cell wall, are explored here. These studies further our understanding of the B. burgdorferi cell envelope and provide critical information that underlies the elusive pathogenesis of Lyme disease. / Master of Science in Life Sciences / Lyme disease is a growing health concern, namely for the countries in the Northern Hemisphere. The bacterium responsible for this illness is Borrelia burgdorferi. B. burgdorferi can survive in the human body and is a threat in that as it replicates in the human host, it sheds pro-inflammatory fragments of its unique cell wall into the environment. This thesis will explore the consequences of this cell wall shedding and how the human immune response differs from the response seen in other more common bacteria. Additionally, I have found that the cell envelope fragments shed from B. burgdorferi may contain more than meets the eye. There is evidence here to support the discovery of a moonlighting protein that is bound to a portion of the cell wall in B. burgdorferi. This protein acts to bolster the structural integrity of the cell while also acting to modulate the host immune response.

Laimo boreliozė (LB) yra labiausiai paplitusi erkių platinama liga. Ligos sukėlėjus perneša Ixodes rūšies erkės. Didelį borelijų paplitimą lemia platus rezervuarinių šeimininkų ratas. Šį susirgimą gali sukelti kelios B. burgdorferi s. l. komplekso bakterijų rūšys: B. afzelii, B. garinii, B. burgdorferi sensu stricto. Šiame darbe buvo tirtos 134 Ixodes ricinus erkės nuo 8 elninių gyvūnų (4 stirnų ir 4 elnių) ir 73 erkės nuo žolės (elninių teritorijoje). Naudojant dauginės polimerazės grandininės reakcijos (PGR) medodą, 9 - iose (4,3%) iš jų buvo nustatytas užsikrėtimas Borrelia burgdorferi s. l. patogenais, septyniuose mėginiuose buvo identifikuota B. afzelii, viename - B. garinii rūšis. Viename mėginyje buvo nustatyta Borrelia spp., kuri nebuvo identifikuota. Atlikta B. burgdorferi s. l. sekų analizė pagal ospA geną, naudojantis genų banko duomenimis, atskleidė polimorfizmo lygį tarp trijų, žmogui patogeniškų Borrelia rūšių ir parodė padermių pasiskirstymą skirtingose šalyse bei šeimininkuose. Variabiliausios nustatytos B. garinii ospA geno sekos, rasta 361 variabili vieta (nukleotidų įvairovė π=0,072), aptikta 18 sekų variantų. Mažesnis variabilumas nustatytas B. burgdorferi s. s. sekose – aptiktos 266 variabilios vietos (nukleotidų įvairovė π=0,16) ir 12 sekų variantų bei B. afzelii sekose – aptiktos 257 variabilios vietos (nukleotidų įvairovė π=0,17) ir 4 sekų variantai. Didesni skirtumai buvo identifikuoti tarp B. afzelii ospA geno sekų (vidutinis genetinis atstumas... [toliau žr. visą tekstą] / Lyme borreliosis (LB) is the most common tick - born disease. The pathogens are transmitted by infected ticks belonging to a species of the genus Ixodes. The high prevalence of Borrelia is determined by a wide circle of reservoir hosts. The disease can be caused by a few bacterial species of B. burgdorferi s.l. complex, such as: B. afzelii, B. garinii and B. burgdorferi sensu stricto. 134 Ixodes ricinus ticks have been explored in the thesis, collected from 8 certine animals (4 roe deer and 4 deer) and 73 ticks collected from grass (in the territory of certine animals). Using multiplex polymerase chain reaction (PCR) method, the infection with Borrelia burgdorferi s. l. pathogens was identified in 9 (4.3%), B. afzelii was identified in seven samples, while B. garinii species was found in one of them. Borrelia spp. was found in one sample, which has not been identified. A sequence analysis B. burgdorferi s. l. implemented according to the ospA gene by using a gene bank data revealed level of polymorphism among three Borrelia species pathogenic to human and showed the distribution of strains in the different countries and hosts. The most variable gene sequences are B. garinii ospA. It is found 361 variable place (nucleotide diversity π=0,072), ant detected 18 sequence variants. Less variability found in B. burgdorferi s. s. sequences: 266 variable places are found and 12 sequence variants (nucleotide diversity π=0,16) and 257 variable places are discovered in B. afzelii... [to full text]

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

The proposed thesis contributes to the basic knowledge in tick (Ixodes ricinus) and tick-borne pathogens (Borrelia burgdorferi sensu lato, tick-borne encephalitis virus) ecology in particular studying the spatial distribution, host associations and its causes and consequences in Central European habitats.

La maladie de Lyme, ou borréliose de Lyme, est une infection bactérienne causée par le spirochète Borrelia burgdorferi sensu lato et transmise à l’hôte (homme, animal) par piqûre de tique du genre Ixodes. Cette maladie, caractérisée par un polymorphisme clinique important, est la maladie à transmission vectorielle la plus répandue dans l’hémisphère nord. Un traitement par antibiotiques permet une guérison rapide, mais si la maladie est diagnostiquée tardivement, certains symptômes persistent. Actuellement, aucun vaccin n’est commercialisé pour l’homme. Dans ce contexte, nous avons développé des approches protéomiques afin d’apporter de nouveaux éléments de compréhension du mécanisme d’interactions de la triade tique / bactérie / hôte. Ces travaux, visant particulièrement le développement de nouvelles stratégies vaccinales et diagnostiques, sont articulés autour de trois parties : - L’identification, suite à de nombreuses optimisations, d’une méthode d’analyse HPLC-UV et nanoLC-MS/MS, de protéines présentes dans des extraits de glandes salivaires de tiques et possédant une activité sur la réponse immunitaire innée cutanée. Ces développements ont mis en évidence une liste restreinte de protéines potentiellement bioactives. - La mise au point, sur un modèle murin, d’une méthode de détection d’une protéine de Borrelia burgdorferi, OspC, dans des biopsies cutanées par spectrométrie de masse ciblée LC-SRM. Cette étude a ouvert des perspectives quant au développement de nouveaux outils diagnostiques. - L’évaluation de la faisabilité de la détection de Borrelia burgdorferi directement à la surface de la peau par imagerie par spectrométrie de masse MALDI-MSI. / Lyme disease, or Lyme borreliosis, is a bacterial infection caused by Borrelia burgdorferi sensu lato and transmitted to the human or animal host by an Ixodes tick bite. This disease, characterized by a huge clinical polymorphism, is the most common vector-born disease in the Northern Hemisphere. An antibiotic treatment allows a fast recovery, but if it is diagnosed too late, some symptoms persist. Currently, no vaccine is marketed for humans. In this context, we have developed proteomic approaches to bring new understanding to the interaction mechanism of the triad tick / bacteria / host. This work, aimed particularly at the development of new vaccinal and diagnostic strategies, has three parts: - Identification, after numerous optimizations, of the analytical method HPLC-UV and nanoLC-MS/MS, of proteins that are present in tick salivary gland extracts and having activity on cutaneous innate immunity response. This work has highlighted a list of proteins with a potential biological activity. - Development, with a murine model, of a method for detecting Borrelia burgdorferi protein, OspC, in cutaneous biopsies by targeted mass spectrometry LC-SRM. This study has opened up perspectives concerning new diagnostic tools. - Evaluation of the feasibility of the Borrelia burgdorferi detection directly on the skin surface by MALDI imaging mass spectrometry.

Les lymphomes T périphériques (PTCL) sont des néoplasmes rares et agressifs représentant environ 12% des lymphomes chez l’Homme. Nos travaux récents dans des souris p53-/- ont révélé une nouvelle entité de PTCL, émergeant de cellules Natural-Killer T-cell (NKT), un type particulier de lymphocyte T reconnaissant des antigènes lipidiques. Nous avons montré que ces lymphomes NKT (PTCL-NKT) présentent des caractéristiques de NKT stimulés chroniquement, et que la lymphomagenèse est initiée via l’activation chronique du TCR. Chez l’Homme, de nombreux PTCL sont suspectés pour être associés à des stimulations antigéniques chroniques, mais les mécanismes de transformation impliqués sont encore mal connus. Borrelia burgdorferi (Bb), l’agent responsable de la maladie de Lyme, provoque des infections chroniques dont l’implication dans certains lymphomes T cutanés (CTCL) a été suggérée. Cependant, cette observation manque de preuves cliniques et expérimentales. De manière intéressante, Bb est connue pour exprimer des glycolipides activateurs des NKT. Nous avons donc infecté des souris p53-/- avec des Bb vivantes, et montré que l’infection augmente significativement la fréquence des PTCL-NKT. Par traitement antibiotique précoce de souris infectées et par injections de Bb inactivées, nous avons également démontré que la chronicité de l’infection est nécessaire au développement de ces lymphomes. L’analyse phénotypique de ces PTCL-NKT a confirmé nos observations précédentes, montrant des caractéristiques de cellules NKT activées chroniquement, telles que l’expression de marqueurs d’activation et d’exhaustion (perte de NK1.1, surexpression de PD-1). Ces résultats suggèrent une implication de Borrelia dans la lymphomagenèse T. En se basant sur l’analyse de différents marqueurs phénotypiques et de leur production cytokinique, nous avons également montré que ces lymphomes présentent un profil dérégulé se rapprochant du sous-type NKT2. Une étude génomique par séquençage whole-exome sur 6 PTCL-NKT a révélé de larges pertes récurrentes du chromosome 13. Au sein de la zone minimale de délétion, nous avons identifié Jarid2, codant un facteur épigénétique impliqué dans le développement NKT par une activité histone-methytransférase. Ce gène est retrouvé altéré dans 20% des CTCL. De manière intéressante, les souris Jarid2-/- présentent une expansion périphérique de NKT au profil immature/NKT2, partageant donc des caractéristiques avec les PTCL-NKT. La perte de Jarid2 a été détectée dans presque tous les PTCL-NKT. Nous avons confirmé la perte de Jarid2 au niveau ARN et protéique. Nos résultats préliminaires montrent une hypométhylation de la lysine 9 de l’histone H3 (H3K9), la cible de Jarid2, soutenant un effet fonctionnel dans la physiopathologie des PTCL-NKT. Par conséquent, nous pensons que la perte de Jarid2 pourrait être un événement important de la lymphomagenèse NKT, puisque de plus en plus d’altérations de facteurs épigénétiques sont retrouvées dans les PTCL humains. Pour réponse à cette question, nous sommes notamment en train de générer des souris p53-/- x Jarid2-/-. En conclusion, nos données viennent renforcer le concept selon lequel certaines infections peuvent initier la transformation des cellules T par l’activation chronique du TCR. Nous avons également identifié un nouveau facteur épigénétique potentiellement impliqué dans la lymphomagenèse NKT / Peripheral T-cell lymphomas (PTCL) are aggressive and heterogeneous neoplasms that represent around 12% of Human lymphomas. Our recent work in p53-/- mice revealed a new PTCL entity, arising from Natural-Killer T-cell (NKT), a particular type of T cell recognizing lipidic antigens. We found that NKT lymphomas (NKTL) present features of chronically stimulated NKT-cells and that lymphomagenesis is driven through chronic TCR activation by microbial glycolipids. In human, many PTCL are suspected to be associated with chronic antigenic stimulation, but this transformation mechanism is still poorly understood.Borrelia burgdorferi (Bb), the causative agent of Lyme disease, induces chronic infection and has recently been suggested to be involved in cutaneous T-cell lymphomas (CTCL). However, this observation lacks clinical and experimental proofs. Interestingly, Bb is known to express NKT-activating glycolipids. We therefore infected p53-/- mice by live intradermal Bb injection and showed that Bb infection significantly increased NKTL rate. Phenotypic characterization of these NKTL confirmed our previously described features of chronically stimulated NKT-cells, with expression of activation and exhaustion markers (loss of NK1.1, upregulation of PD-1). Based on surface markers, transcription factors and cytokine production analysis, we also found that our lymphomas mostly present a NKT2 subtype profile, sometimes surprisingly mixed with NKT17 or NKT1. Genomic study by whole-exome sequencing on few of these lymphomas revealed recurrent large losses in the chromosome 13. Within the minimal deletion region, we identified Jarid2, a gene involved in NKT development by epigenetic regulation and which is found altered in 20% of CTCL. Jarid2 loss was detected in almost all NKTL. Interestingly, Jarid2-/- mice show increased NKT number in the periphery with an immature/NKT2 phenotype, sharing features with our NKTL.Thus, we believe that Jarid2 loss may be an important event in NKT lymphomagenesis, as more and more epigenetic factors are found mutated in several human PTCL. To answer this question we are currently breeding p53-/- x Jarid2-/- mice. In conclusion, our data reinforced the concept that chronic bacterial activation of T-cells through their TCR can effectively drive T-cell transformation. We also identified a new potential epigenetic factor that may be involved in lymphomagenesis

Indiana University-Purdue University Indianapolis (IUPUI) / Lyme disease, the most prevalent vector-borne illness in the United States, is a multisystem inflammatory disorder caused by infection with the spirochete Borrelia burgdorferi (Bb). This spirochete is maintained in nature through an enzootic cycle involving ticks and small mammals. The Bb genome encodes a large number of surface lipoproteins, many of which are expressed during mammalian infection. One of these lipoproteins is the major outer surface protein C (OspC) whose production is induced during transmission as spirochetes transition from ticks to mammals. OspC is required for Bb to establish infection in mice and has been proposed to facilitate evasion of innate immunity. However, the exact biological function of OspC remains elusive. Our studies show the ospC-deficient spirochete could not establish infection in NOD-scid IL2rγnull mice that lack B cells, T cells, NK cells, and lytic complement, whereas the wild-type spirochete was fully infectious in these mice. The ospC mutant also could not establish infection in SCID and C3H mice that were transiently neutropenic during the first 48 h post-challenge. However, depletion of F4/80+ phagocytes at the skin-site of inoculation in SCID mice allowed the ospC mutant to establish infection in vivo. In phagocyte-depleted SCID mice, the ospC mutant was capable to colonize the joints and triggered neutrophilia during dissemination in a similar pattern as wild-type bacteria. We then constructed GFP-expressing Bb strains to evaluate the interaction of the ospC mutant with phagocytes. Using flow cytometry and fluorometric assay for phagocytosis, we found that phagocytosis of GFP-expressing ospC mutant spirochetes by murine peritoneal macrophages and human THP-1 cells was significantly higher than parental wild-type Bb strains, suggesting that OspC has an anti-phagocytic property. This enhancement in phagocytosis was not mediated by MARCO and CD36 scavenger receptors and was not associated with changes in mRNA levels of TNFα, IL-1β, and IL-10. Phagocytosis assays with HL60 neutrophil-like cells showed that uptake of Bb strains was independent to OspC. Together, our findings reveal that F4/80+ phagocytes are important for clearance of the ospC mutant, and suggest that OspC promotes spirochetes' evasion of macrophages in the skin of mice during early Lyme borreliosis.

Many zoonotic pathogens of concern to human and veterinary health are maintained in the environment within small mammal reservoirs and vectored to new hosts by ectoparasitic arthropods. While the ecological relationships among small mammals, ectoparasites, and disease-causing symbiotic microorganisms are important to these dynamics, little is known about them across much of North America. The sylvatic cycle of Borrelia burgdorferi, the etiologic agent of Lyme disease, is of particular interest because Lyme disease is the most common vector-borne disease of humans in the United States. However, cases of Lyme disease are primarily confined to the northeastern and Midwestern United States, with only sporadic cases extending into the southeast. As a result, much of what is known of the ecology of Lyme disease comes from studies conducted in those regions. The goal of this study was to examine the ecological dynamics of the B. burgdorferi/vector/reservoir system in south-central Kentucky and gain insight into the relative paucity of Lyme disease in Kentucky. Small mammals were captured using live traps in three 200x50 m trapping grids within Western Kentucky University’s Green River Preserve from November 2014-October 2015. Captured small mammals were identified to species and standard measurements were recorded. Ectoparasites were removed and retained for identification. Collected blood and tissue were examined for B. burgdorferi DNA by polymerase chain reaction with primers specific to the OspA gene. The Bray-Curtis dissimilarity index, Schnabel population estimates, and the Shannon-Wiener diversity index were used to assess the structure of the small mammal communities. Parasite infestation was low but was affected by age and sex of the host, site, and season in different parasite taxa. Infestation by Ixodes scapularis, the primary vector for B. burgdorferi, was uncommon and prevalence of B. burgdorferi in blood was similar to the lowest prevalence previously observed in the Lyme disease endemic regions. We found that life history characteristics of hosts and ectoparasites drive their associations. We also suggest that the lack of an efficient vector for B. burgdorferi is the likely explanation for the few reported cases of Lyme disease in Kentucky.