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.

La borréliose de Lyme est une maladie à transmission vectorielle en forte progression ces dernières années. Après une infection par la bactérie appartenant au complexe Borrelia burgdorferi sensu lato via une piqûre de tique, de multiples troubles (cardiaques, rhumatologiques…) peuvent apparaître. Il n’existe à l’heure actuelle aucun vaccin contre la maladie chez l’homme. De plus, les méthodes actuelles de diagnostic souffrent d’un manque de sensibilité, de spécificité ou de rapidité. Nous avons développé différentes approches protéomiques pour l’étude de cette maladie. Dans un premier temps, nous avons mis en évidence de nouveaux candidats vaccinaux par une approche Ge-LC-MS/MS de type label free. Dans un second temps, nous avons mis au point une méthode de détection de la bactérie dans des biopsies cutanées par spectrométrie de masse ciblée SRM. Nous avons également caractérisé les modifications post-traductionnelles d’une protéine identifiée dans les glandes salivaires de tique, et capable de lyser les fibroblastes. Un dernier volet a concerné l’évaluation de deux instruments et de l’apport de modes d’acquisition originaux pour l’analyse protéomique / Lyme borreliosis has been rising strongly for the last twenty years. After an infection by the bacterium belonging to the Borrelia burgdorferi sensu lato complex through a tick bite, multiple disorders (cardiac, rhumatological…) may appear. There is no current vaccine available for human being. Moreover, actual diagnosis methods lack of sensitivity, specificity and quickness. We developed various proteomic approaches to study the Lyme disease. Firstly, we discovered new vaccine candidates by using a Ge-LC-MS/MS label free approach. Secondly, we set up the detection of the bacteria in human cutaneous biopsies by targeted SRM mass spectrometry. We also characterized the post-translational modifications of a lytic protein present in tick salivary glands. Finally, we evaluated the performances of two instruments and the contribution of original acquisition modes for proteomic analyses.

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

En Afrique, les fièvres récurrentes causées par différentes espèces bactériennes du genre Borrelia sont des infections négligées transmises par les arthropodes et sont responsables de manifestations cliniques variant d’une septicémie mortelle à des formes plus bénignes et d'autres manifestations cliniques, en particulier d'avortement chez les femmes enceintes. Quatre espèces différentes de Borrelia, initialement séparées les unes des autres sur la base de leur répartition géographique et de leur vecteur, sont actuellement cultivées de prélèvements cliniques et de vecteurs : Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis et Borrelia hispanica. Ces différentes espèces circulent sur le continent africain en parallèle avec au moins six espèces non encore cultivées et détectées dans des vecteurs. Notre travail est une contribution à l’investigation des fièvres récurrentes à Borrelia en Afrique. Dans cette perspective, nous avons mis au point la détection rapide en spectrométrie de masse MALDI-TOF des Borrelia dans les tiques en créant au préalable une base de données Borrelia MALDI-TOF-MS. La base de données de Borrelia et un logiciel de soustraction IHU ont été utilisés pour détecter B. crocidurae dans 20 tiques Ornithodoros sonrai, y compris huit tiques qui ont été testées positives pour B. crocidurae par PCR-séquençage, ce qui ouvre la voie à l'utilisation du MALDI-TOF-MS pour la double identification des vecteurs et des agents pathogènes vectorisés, dont il s’agissait du premier exemple maintenant étendu à d’autres modèles dans notre laboratoire. / In Africa, relapsing fever borreliae are neglected arthropod-borne pathogens causing mild to deadly septicemia and other clinical manifestations, particularly abortion in pregnant women. Four different species of Borrelia, initially distinguished one from another on the basis of geography and vector, are currently cultured causative agents in Africa: Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis et Borrelia hispanica. These different species are circulating in parallel to at least six not-yet cultured species in vectors. Our work consisted in the investigation of recurrent fevers borreliosis in Africa. We have developed rapid detection in MALDI-TOF mass spectrometry of Borrelia in ticks by creating a prior a Borrelia MALDI-TOF-MS database. The Borrelia database and a custom software program that subtracts the uninfected O. sonrai profile were used to detect B. crocidurae in 20 O. sonrai ticks, including eight ticks that tested positive for B. crocidurae by PCR-sequencing; which paves the way for the use of MALDI-TOF-MS for the dual identification of vectors and vectorized pathogens. We have also illustrates a non-specialized circulation of B. crocidurae borreliae within a collection of 35 O. sonrai ticks in West Africa. These ticks were genotyped by 16S rRNA mitochondrial gene sequencing while B. crocidurae was genotyped by Multispacer Sequence Typing (MST). The 35 ticks were grouped into 12 genotypes strong geographic structuring and 35 B. crocidurae into 29 genotypes without strict geographic structure. One O. sonrai genotype carried several B. crocidurae genotypes and one B. crocidurae genotype was found in different O. sonrai genotypes.

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.

Lyme disease is a tick-transmitted illness caused by Borrelia burgdorferi sensu lato (s.l.), a group of spirochetes with at least three human pathogenic species, B. burgdorferi sensu stricto, B. afzelii and B. garinii. These spirochetes cycle between vertebrate reservoirs, mainly rodents, and ixodid ticks. Both terrestrial birds and seabirds can be infected with B. burgdorferi s.l. but the function of birds as reservoirs is largely unknown, even though they are potentially important epidemiologically due to their ability to carry ectoparasites and microorganisms over long distances. This thesis describes the role of birds in Lyme disease Borrelia biology in general and Borrelia ecology and epidemiology in particular. B. burgdorferi s.l. has previously been detected in the seabird tick Ixodes uriae and an enzootic Borrelia cycle distinct from terrestrial Borrelia cycles has been described. In this study B. garinii was isolated from the proposed seabird reservoirs and the tick I. uriae infesting them. The strains isolated did not show evident differences from human pathogenic B. garinii strains, indeed 7/8 strains had an ospC allele associated with Borrelia causing disseminated Lyme disease. Antibodies against B. burgdorferi s.l. were detected in people frequently bitten by I. uriae. Thus the marine enzootic Borrelia cycle may be a risk for humans, either by direct transfer of the spirochete from /. uriae or via introduction of Borrelia into a terrestrial enzootic Borrelia cycle. In order to investigate the role of passerine (Passeriformes) birds as amplification hosts in the terrestrial Borrelia cycle, experimental infections of canary finches (Serinus canaria) and redwing thrushes (Turdus iliacus) were carried out. The result showed that B. burgdorferi s.l. can persist for several months in passerine birds and the infection in redwing thrushes can be reactivated in response to migration. Thus, birds may be more infectious to ticks during their migration and therefore important long-range disseminators of B. burgdorferi s.l. Migration in birds is associated with elevated stress hormones that in turn can cause reactivation of latent infections. Lyme disease in humans could perhaps be activated when the immune response is modulated by stress. Herein I describe a patient with a stress activated latent Borrelia infection, which supports this hypothesis. The seabird tick I. uriae has a circumpolar distribution in both the northern and southern hemispheres and in this study identical B. garinii flagellin gene (flaB) sequences were detected in I. uriae from these hemispheres, indicating a transequatorial transport of B. garinii. Parsimony analysis of I. uriae ITS2 and 16S rDNA sequences suggested that northern and southern I. uriae might be reproductively separated. Therefore passive transport of infected ticks between the polar regions is unlikely and instead seabirds probably carry an active Borrelia infection during their migration. In conclusion, this work shows that migrating seabirds and passerine birds probably are important for the long-range dispersal of B. burgdorferi s.l., and that this mechanism of dispersal could be important for the distribution of human Lyme disease. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 2001, härtill 6 uppsatser</p> / digitalisering@umu

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.

<p>In Europe, Lyme disease<em> </em>is caused by the species <em>Borrelia (B.) burgdorferi</em> sensu stricto,<em> B. garinii </em>and <em>B. afzelii.</em> The disease is the most common vector-borne infection in Europe and the United States,<em> </em>and the resulting manifestation can involve the skin, nervous system, heart and joints. The symptoms that arise are associated with the <em>Borrelia </em>species causing the infection. The species most associated with neuroborreliosis is <em>B. garinii</em> whilst <em>B. burgdorferi </em>sensu stricto is associated with arthritis and <em>B. afzelii </em>is associated with dermatological symptoms. Lyme disease normally has three phases in untreated patients. The first phase is characterised by erythema migrans, a reddening of the skin around the area of the tick bite. If the disease develops to the second phase the patient will suffer from neuroborreliosis which is characterised by neurological symptoms such as headache and peripheral facial paralysis. Cerebrospinal fluid (CSF) analysis is used to diagnose neuroborreliosis. The diagnosis is complicated by variations between the different <em>Borrelia</em> species and that many healthy individuals have antibodies directed against <em>Borrelia</em>. Antibodies in CSF can be found in different diseases. The antibodies can be produced in the central nervous system or come across the blood-brain barrier and thus derive originally from the blood. By measuring the concentration of total albumin in serum and in CSF it can be determined if the antibodies present in the CSF have been produced in the central nervous system or if they originate from the blood. The typical manifestation in the last phase of Lyme disease is severe arthritis. The aim of this examination project was to compare two ELISAs for detection of antibodies directed to <em>Borrelia</em>. Indirect ELISAs from DAKO and Euroimmun were compared for the diagnosis of neuroborreliosis in 100 individuals. <em>Borrelia </em>specific antibodies of class IgM or IgG were found in 16 of 100 patients by DAKO’s ELISA and in 20 of the same 100 patients by Euroimmun’s ELISA. The reason that Euroimmun’s method detected more cases of neuroborreliosis is probably that this method detects antibodies directed to all three pathological species of <em>Borrelia </em>while DAKO’s method only detects antibodies directed to <em>B. burgdorferi</em>. In conclusion, this study indicates that Euroimmun’s method to detect antibodies of class IgM and IgG directed to <em>Borrelia </em>is superior to DAKO’s method. The obtained results were confirmed by Western blot analysis which gave results in accordance with those of Euroimmun’s ELISA.</p>

The Lyme disease spirochete, B. burgdorferi is atypical in that a large portion of its genome is in the form of plasmids. Many of the plasmid-carried genes form extensive paralogous gene families and encode outer-surface proteins. In this report we have assessed the humoral immune response to proteins belonging to the paralogous protein family, family 163. We have cloned and expressed BBP39, BBO40, BBQ47 and BBN39 and used these recombinant proteins to monitor the temporal nature of the antibody response to these antigens during experimental infection of mice. The immunodominant regions of each protein have also been assessed through immunoblot analyses of a series of truncations of each protein. These analyses have led to the delineation of the targets of the antibody response during infection and of the specificity of the antibody response to family 163 proteins. In addition, we quantified the expression of each gene using real time RT-PCR.

Lyme disease is the most common tick-borne disease in North America, with approximately 35,000 cases reported to the Centers for Disease Control in 2008. The genome of its causative agent, Borrelia burgdorferi, encodes for a set of genes involved in the metabolism and regulatory activities of the second messenger nucleotide, cyclic-di-GMP (c-di-GMP). Rrp1 is a response regulatory-diguanylate cyclase, and its regulatory capability is likely mediated via production of c-di-GMP, as it lacks a DNA-binding domain. One known class of c-di-GMP effector/binding proteins are those that harbor a PIlZ domain. The genome of B. burgdorferi strain 5A4 encodes for one chromosomally-carried PilZ domain, which we have designated PlzA. Additionally, certain B. burgdorferi strains encode for a second PilZ domain-containing protein (PlzB) which is plasmid-carried. Both PlzA and PlzB were found to bind specifically to c-di-GMP, and c-di-GMP binding by PlzA was found to be dependant upon arginine residues in the c-di-GMP binding region. Additionally, expression of PlzA was found to be upregulated by tick feeding and was constitutive in the mammalian host. We next constructed two deletion/allelic exchange mutants – one with the targeted deletion of PlzA, and on ethat replaced PlzA with PlzB in a strain lacking the plzB gene. Our studies demonstrated that ΔplzA was deficient in motility and was also non-infectious in the mouse model of B. burgdorferi infection. Additionally, this strain remained viable in larval Ixodes ticks. Also, B31-plzB KI was deficient in motility, as well as infectivity, demonstrating that PlzB is unable to complement for functions fo PlzA in vitro and in vivo and that it may play other roles in the biology of B. burgdorferi strains carrying the plzB gene. These studies represent the first identification of a c-di-GMP binding protein in any spirochete, but also represent the first demonstration of the importance of PilZ domain proteins in a spirochetal system. We additionally examined the effects of c-di-GMP synthesis and breakdown in the related bacterium, B. hermsii, a causative agent of tick-borne relapsing fever (TBRF). Deletion mutants in Rrp1 (B. hermsii’s sole diguanylate cyclase) and PdeA (B. hermsii’s only EAL domain-containing phosphodiesterase) were created. These strains were analyzed in order to determine: 1) the effect(s) of the losse of Rrp1/PdeA on intracellular spirochete c-di-GMP levels, and 2) the effects of Rrp1/PdeA on the establishment of murine infection and on gross motility/chemotaxis. It was demonstrated that c-di-GMP accumulates intracellularly in the cells lacking PdeA. Additionally, spirochetes were shown to chemotax towards N-acetyl-glucosamine (NAG) and they did not form soft agar swarms. In contrast, cells lacking Rrp1 did not accumulate detectable levels of c-di-GMP, demonstrated a reduced ability to chemotax towards NAG, and swarmed on soft agar in a fashion indistinguishable from wild type. Despite these differences in phenotype, both mutant strains display an attenuated murine infectivity. These results indicate that c-di-GMP is indeed important in the TBRF spirochete, B. hermsii and this vital second messenger plays key roles in virulence, motility, and chemotaxis. These studies also pave the way for future investigation of B. hermsii through use of targeted genetic manipulation.