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.

Host cell attachment by pathogenic bacteria can play very different roles in the course of infection. The pathogenic spirochetes Borrelia hermsii and Borrelia burgdorferi sensu lato which cause relapsing fever and Lyme disease, respectively, are transmitted by the bite of infected ticks. After transmission, these spirochetes can cause systemic infection. Relapsing fever spirochetes remain largely in the bloodstream causing febrile episodes, while Lyme disease will often colonize a variety of tissues, such as the heart, joint and nervous system, resulting in a chronic multisystemic disorder. Borrelia species have the ability to bind to various cell types, a process which plays a crucial role in pathogenesis and may influence spirochetal clearance from the bloodstream. Colonization of multiple tissues and cell types is likely promoted by the ability to bind to components found in target tissues, and many B. burgdorferi adhesins have been shown to promote attachment to a wide variety of cells and extracellular matrix components. Different Lyme disease strains have been shown to preferentially colonize certain tissues, although the basis of this tissue tropism is not well understood. In this study we found that among different Lyme disease strains, allelic variation of the adhesin DbpA contributes to variation in its in vitro binding activities raising the possibility that this variation contributes to tissue tropism in vivo. In studying B. hermsii infection, we found evidence by both histological and fluorescence in situ hybridization (FISH) analysis of tissues that indicated that red blood cells were removed by tissue resident macrophages in infected mice. Spirochetes in the spleen and liver were often visualized associated with RBCs, lending support to the hypothesis that direct interaction of B. hermsii spirochetes with RBCs leads to clearance of bacteria from the bloodstream. Our findings indicate that host cell attachment play a key role in the establishment of Lyme disease infection, and in contrast contributes to the clearance of relapsing fever infection.

Les maladies à transmission vectorielle, qui représentent dix-sept pour cent de l’ensemble des maladies infectieuses, sont un réel problème de santé publique. Parmi elles, la borréliose de Lyme est la maladie vectorielle la plus répandue de l’hémisphère nord. Elle est causée par la transmission d’une bactérie, Borrelia burgdorferi sensu lato, par une tique dure du genre Ixodes. Le premier contact entre l’hôte vertébré et la tique, et donc a fortiori entre l’hôte vertébré et la bactérie, se fait au niveau cutané. La peau est donc une interface essentielle dans le développement précoce de l’immunité envers Borrelia. La tique effectue un repas de plusieurs jours qui induit une lésion cutanée par ses pièces piqueuses et sa salive, créant une cavité lui permettant de se nourrir. Ce processus facilite le repas sanguin, mais aussi la transmission de Borrelia. Nous avons caractérisé une protéine présente dans la salive de tique responsable de la formation de la cavité : l’histone H4. Celle-ci lyse les fibroblastes et possède une activité bactéricide envers les bactéries commensales de la peau. Ces deux activités pourraient être exploitées par Borrelia afin de favoriser sa transmission et son développement dans la peau. Une fois injectées, Borrelia et la salive interagissent avec les cellules de la peau telles que les kératinocytes de l’épiderme, les fibroblastes et les cellules immunitaires du derme. Nous avons montré que l’inflammation induite par la lésion augmente la réponse inflammatoire des kératinocytes contre Borrelia. Cependant, la salive de tique inhibe efficacement cette coopération inflammatoire dont la mise en place dépend des voies inflammatoires TLR3/TRIFF et TLR2/MyD88. Une fois la tique détachée de l’hôte vertébré et la salive de tique disparue, la coopération inflammatoire ne serait plus inhibée et pourrait expliquer en partie l’apparition de l’érythème migrans. L’inflammation cutanée précoce implique d’autres cellules que les kératinocytes et les fibroblastes telles que les macrophages, les cellules dendritiques ou encore les neutrophiles. Nous avons étudié l’implication d’un autre type cellulaire peu exploré dans le contexte de la borréliose de Lyme : les mastocytes. Ces cellules sont capables de répondre efficacement contre Borrelia par la sécrétion d’IL-6 et la dégranulation. Les antigènes bactériens nécessaires à l’activation des mastocytes semblent liés à la bactérie intacte et vivante. La salive de tique réduit la sécrétion d’IL-6 induite par Borrelia, mais ne l’inhibe pas complètement. A ce stade de nos études, les mastocytes ne semblent pas jouer un rôle majeur. Lors de la transmission, Borrelia exprime différents gènes qui lui permettront notamment de disséminer vers différents organes cibles de l’hôte vertébré : le système nerveux, l’articulation et la peau à distance. Nous avons identifié chez un clone de B. burgdorferi sensu stricto aux propriétés disséminatrices importantes, l'expression de deux gènes, bb0347 et bb0213, qui semblent associés à cette virulence. bb0347 code une protéine d’adhésion à la matrice extracellulaire, ce qui pourrait favoriser la migration de la bactérie à travers les tissus cutanés de l’hôte vertébré et donc sa dissémination. Le rôle de bb0213 n’est pas connu à ce jour. / Vector-borne diseases account for seventeen percent of world-wide infectious diseases. They are amajor threat to public health. Lyme borreliosis is the first vector-borne disease of the northernhemisphere. It is caused by a bacteria, Borrelia burgdorferi sensu lato, inoculated by a hard tickbelonging to the Ixodes genus. The first contact between the vertebrate host and the tick, and sobetween the vertebrate host and the bacteria, occurs at the skin interface. The skin is then of majorimportance for the early development of the immune response against Borrelia.The tick bite induces a skin injury owing to its biting pieces, the hypostome and two chelicerae. The ticksaliva also creates a feeding pool allowing the tick to feed efficiently. This process also facilitates Borreliatransmission. We have characterized a tick saliva protein which might participate to the formation ofthe feeding pool: histone H4. This protein lyses fibroblasts and harbors bactericidal properties againstcommensal bacteria. These two activities might help Borrelia to infect the vertebrate host by sustainingits development in the skin. Once bacteria have been injected into the skin, they interact with residentskin cells, keratinocytes and fibroblasts, and immune cells. We have shown that the inflammationinduced by the tick bite increases the keratinocyte inflammatory response against Borrelia. However,the saliva inhibits this cross-talk which depends on TLR3/TRIFF and TLR2/MyD88 pathways. Once thetick has detached and the saliva has disappeared, the cross-talk might explained the inflammationobserved during the erythema migrans.Other skin cells than keratinocytes and fibroblasts are involved in the early inflammatory responseagainst Borrelia such as dendritic cells, macrophages and neutrophils. We have explored theinvolvement of another poorly-studied cell-type: mast cells. We have shown that these cells can secreteIL-6 and degranulate in response to Borrelia. Bacteria antigens responsible of the activation mightdepends on the living state of Borrelia. The tick saliva is able to negatively control the secretion of IL-6,but not to completely inhibit it. At this point, we cannot conclude in a WSH mouse model deficient inmast cells, to a major role of these cells in the inflammatory response against Borrelia.While in the skin, Borrelia expresses many genes which will facilitate the dissemination across thevertebrate host, to reach different target organs (brain, joint, distant skin). We have characterized twogenes potentially involved in the dissemination of a virulent clone of B. burgdorferi sensu stricto: bb0347and bb0213. bb0347 encodes for an adhesion which can specifically interact with the extracellularmatrix of the skin while the role of bb0213 is unknown. bb0347 might help the bacteria to migratethrough skin tissues and then increases the infection rate.

Borrelios är den vanligaste fästingburna infektionen på norra halvklotet, och orsakas av spiroketer tillhörande Borrelia burgdorferi sensu lato-komplexet. Dessa bakterier kan spridas till flera organ och ge upphov till olika symptom i bland annat hud, nervsystem, leder och hjärta. Omkring 15 % utvecklar neurologiska symptom, så kallad neuroborrelios. Den bästa indikatorn på aktiv neuroborrelios är framförallt karakteristiska neurologiska symptom samt tecken på en inflammatorisk förändring i cerebrospinalvätskan (CSV) i kombination med lokalt producerade antikroppar mot Borrelia burgdorferi s.l. i CSV. Nuvarande metod för diagnostik av neuroborrelios är en immunokemisk metod, en ELISA (enzyme-linked immunosorbent assay) som bygger på en jämförelse av Borrelia-antikroppsnivåer i CSV och i serum genom beräkning av antikroppsindex (AI). Beräkning av AI kompenserar för en eventuell ospecifik överföring av antikroppar från serum, till följd av en skada på blod-hjärnbarriären. Det finns dock tecken på att den nuvarande analysmetoden har för låg sensitivitet med falskt negativa resultat, framförallt tidigt i infektionsförloppet. För diagnostik av andra former av borrelios än neuroborrelios används en typ av ELISA baserad på C6-peptid. C6-peptid ELISA visar god känslighet för detektion av B. burgdorferi s.l.-specifika antikroppar i serum. C6-antigenet utgör en starkt immunogen och konserverad region av bakteriens VlsE-ytprotein. Syftet med den här studien var att undersöka om detektion av antikroppar mot C6-peptid i CSV kan komplettera den nuvarande använda metoden och därmed förbättra den totala sensitiviteten för diagnostik av neuroborrelios. I studien analyserades 169 patientprover från unga personer, samt 18 oklara patientfall som tidigare bedömts negativa med den nuvarande metoden. Antikroppar mot C6-peptid detekterades hos åtta unga patienter samt två oklara patientfall. Av dessa hade åtminstone tre unga patienter sannolikt neuroborrelios. Resultat från den här studien tyder på att C6-peptid-ELISA på CSV-prover kan fungera som ett komplement till befintlig metod för diagnostik av neuroborrelios. En kombination av båda metoderna kan sannolikt ge en betydligt högre sensitivitet. Vid tolkning av resultat från C6-peptid-baserade analysmetoder på CSV ska hänsyn tas till eventuell ospecifik överföring av B. burgdorferi s.l.-specifika antikroppar genom blod-hjärnbarriären. / Lyme Borreliosis, caused by spirochetes of the Borrelia burgdorferi sensu lato-complex, is the most common tick-borne infection in the temperate regions of the northern hemisphere. The bacteria can infect many different organs, this can give rise to a variety of symptoms in skin, the nervous system, joints and heart. Approximately 15 % of the infected individuals show neurological symptoms referred to as neuroborreliosis. An active neuroborreliosis is indicated by inflammatory changes in the cerebrospinal fluid (CSF) and local synthesis of anti-Borrelia antibodies in CSF. The current method to diagnose neuroborreliosis is an enzyme-linked immunosorbent assay (ELISA) which compares levels of anti-Borrelia antibodies in CSF and serum by calculating an antibody index (AI). Calculations of AI compensate for unspecific leakage of antibodies from serum to CSF following an injury of the blood-brain barrier. The drawback of the current method is a low sensitivity with a high rate of false negative results in samples collected early during an infection. Another type of ELISA, based on the use of a C6 peptide, has earlier shown good sensitivity for detection of B. burgdorferi s.l.-specific antibodies in serum. The C6 antigen corresponds to a highly immunogenic and conserved region of the bacterial surface protein VlsE. The aim of this study was to investigate whether a detection of antibodies against the C6 peptide in CSF could improve the total sensitivity for the diagnostics of neuroborreliosis. In the current study, 169 samples with negative AI from young patients and 18 samples from special cases were analyzed. Antibodies against the C6 peptide were found in 8 young patients and in 2 samples from special cases. Out of these, 3 young patients were stated positive for neuroborreliosis. Results of this study show that the C6 peptide ELISA on CSF samples could act as a complement to the current serological method for diagnosing neuroborreliosis. A combination of both methods could possibly increase the overall sensitivity. However, the blod-brain barrier injury issue is a problem in the analysis and interpretation of the results of the C6 peptide-based method on CSF should take into consideration a possible dysfunction of the blood-brain barrier. In conclusion, a combination of both the current method and the C6 peptide ELISA could give a markedly improved sensitivity in diagnostics of neuroborreliosis.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 5 uppsatser.

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.

The tick Ixodes ricinus is one of the most important vectors of human pathogens like the spirochete Borrelia burgdorferi and TBE virus. The effect of repeated infestations with pathogen-free I. ricinus nymphs and the effect of vaccination with ticks saliva of the tick I. ricinus on the transmision Borrelia burgdorferi and on the production of the cytokines were studied. The draining lymph nodes of C3H/HeN mice were examined for IL-2, IFN-? and IL-10 production by antigen capture enzyme-linked immunosorbent assay. Spirochete load in skin, heart and bladder was examined by real-time polymerase chain reaction.

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.