A doença de Lyme é uma doença causada pelas bactérias do complexo Borrelia burgdorferi sensu lato que são transmitidas por carrapatos do complexo Ixodes ricinus. A distribuição geográfica dessas bactérias é confirmada no Hemisfério Norte (America do Norte, Europa e Ásia). Suspeita-se que uma enfermidade, compatível com doença de Lyme no Brasil, esteja provavelmente relacionada a uma espécie de Borrelia diferente das espécies causadoras de doença de Lyme no hemisfério Norte e que o vetor sejam carrapatos do gênero Amblyomma. Por estas razões, a doença brasileira passou a ser chamada de Doença de Lyme-Símile (DLS). A borreliose aviária é causada pela Borrelia anserina, que apresenta distribuição cosmopolita. É transmitida por carrapatos argasideos do gênero Argas. O presente trabalho foi dividido em dois capítulos: o primeiro investigou a presença de Borrelia spp em áreas onde foram relatados casos humanos de DLS no Estado de São Paulo. No segundo capítulo, é relatado o primeiro isolamento em meio BSK e a caracterização molecular de uma cepa de espiroqueta aviária presumidamente identificada como B. anserina, no Brasil. Para o primeiro capítulo, foram processados um total de 349 carrapatos Amblyomm cajennense adultos coletados em áreas com suspeita de DLS, nove amostras de sangue ou tecidos de pacientes humanos com diagnóstico clínico e sorológico de DLS, duas amostras de caldas de meio BSK previamente inoculado com tecidos ou sangue de pacientes com suspeita de DLS, e três amostras de caldas de meio BSK previamente inoculado com carrapatos coletados de áreas com suspeita de DLS. Essas amostras tiveram seu DNA extraído e testado pela nested-PCR com primers específicos para porções do gene flagelina B (flaB), aptos a amplificar porções deste gene de qualquer espécie de Borrelia. Todas as amostras foram negativas pela nested-PCR, não evidenciando a presença de Borrelia sp nas amostras avaliadas. Para o segundo capítulo, foi utilizada uma amostra de espiroqueta aviária originária de carrapatos Argas miniatus, colhidos em um galinheiro no Município de Pedro Leopoldo, MG. O isolamento in vitro e caracterização molecular da espiroqueta aviária foi feito a partir da inoculação de soro infectado, contendo espiroquetas viáveis, em meio BSK. O soro, baço, fígado e o próprio cultivo foram utilizado para amplificação na PCR para os genes rrs e flaB, seguido de seqüenciamento dos mesmos. O isolamento da espiroqueta foi obtido com sucesso, com várias passagens realizadas. A análise genética das seqüências do isolado mostrou 99.8% (483 de 484-bp) e 98.7% (754 de 764-bp) de similaridade às seqüências correspondentes dos genes rrs e flaB de B. anserina, respectivamente, disponíveis no GenBank. Pela análise filogenética inferida pela seqüência parcial do gene flaB, a cepa Brasileira agrupou-se com a seqüência de B. anserina dos EUA. Os resultados indicam a cepa brasileira estudada, designada de cepa PL, pertence à espécie B.anserina. / Lyme disease is caused by bacteria belonging to the complex Borrelia burgdorferi sensu lato, which are transmitted by ticks of the Ixodes ricinus complex. The distribution of these bacteria are restricted to the northern hemisphere (North America, Europe, and Asia). Lyme disease-like cases have been reported in Brazil, but it is possible that another Borrelia species is involved in these cases, and ticks of the genus Amblyomma have been implicated as vectors. Due to these reasons, the disease in Brazil has been referred as Lyme Disease-Simile (LDS). Borrelia anserina, the agent of avian spirochetosis, has a wordwide distribution, where it is transmitted primarily by ticks of the genus Argas. The present study was divided in two chapters: the first one evaluated the presence of Borrelia spp in areas of the state of São Paulo where LDS have been reported. The second chapter reports the first in vitro isolation in BSK medium and molecular characterization of a spirochete strain from Brazil, presumably identified as B. anserina. For the first chapter, a total of 349 adult ticks (Amblyomma cajennense) collected in areas where LDS cases have been reported were processed. In addition, nine human blood or tissue samples from patients with clinical and serological diagnostic of LDS, two samples from BSK medium previously inoculated with samples of skin or blood of LDS patients, and three samples of BSK medium previously inoculated with tick samples were also processed. All these samples were processed for DNA extraction and then tested by nested-PCR employing primers targeting a portion of the flagelin B gene (flaB),which amplify a flaB fragment in all known Borrelia species. All samples were negative by this nested-PCR, showing no evidence of Borrelia sp in the tested samples. The second chapter evaluated an avian spirochete strain originated from Argas miniatus ticks from Pedro Leopoldo municipality, state of Minas Gerais. DNA fragments of the rrs (16S rRNA) and flab genes were amplified by PCR and sequenced to determine phylogenetic similarities. The resulting sequences were 99.8% (483 of 484) and 98.7% (754 of 764) similar to GenBank corresponding sequences of B. anserina rrs and flaB genes, respectively. By neighbor-joining phylogenetic analysis, the flaB sequence of the Brazilian strain clustered in a monophyletic group with the sequence of B. anserina under 100% bootstrap support. The isolate was successfully isolated in BSK medium, with seven passages performed. The spirochete crude antigen, fixed in glass slides, showed strong immunfluorescence reactivity with sera from chickens previously inoculated with the isolate. The spirochete strain isolated in the present study was genetically identified as B. anserina, labeled as strain PL.

University, Diss., 2006--Würzburg. / Erscheinungsjahr an der Haupttitelstelle: 2005.

A doença de Lyme é uma doença causada pelas bactérias do complexo Borrelia burgdorferi sensu lato que são transmitidas por carrapatos do complexo Ixodes ricinus. A distribuição geográfica dessas bactérias é confirmada no Hemisfério Norte (America do Norte, Europa e Ásia). Suspeita-se que uma enfermidade, compatível com doença de Lyme no Brasil, esteja provavelmente relacionada a uma espécie de Borrelia diferente das espécies causadoras de doença de Lyme no hemisfério Norte e que o vetor sejam carrapatos do gênero Amblyomma. Por estas razões, a doença brasileira passou a ser chamada de Doença de Lyme-Símile (DLS). A borreliose aviária é causada pela Borrelia anserina, que apresenta distribuição cosmopolita. É transmitida por carrapatos argasideos do gênero Argas. O presente trabalho foi dividido em dois capítulos: o primeiro investigou a presença de Borrelia spp em áreas onde foram relatados casos humanos de DLS no Estado de São Paulo. No segundo capítulo, é relatado o primeiro isolamento em meio BSK e a caracterização molecular de uma cepa de espiroqueta aviária presumidamente identificada como B. anserina, no Brasil. Para o primeiro capítulo, foram processados um total de 349 carrapatos Amblyomm cajennense adultos coletados em áreas com suspeita de DLS, nove amostras de sangue ou tecidos de pacientes humanos com diagnóstico clínico e sorológico de DLS, duas amostras de caldas de meio BSK previamente inoculado com tecidos ou sangue de pacientes com suspeita de DLS, e três amostras de caldas de meio BSK previamente inoculado com carrapatos coletados de áreas com suspeita de DLS. Essas amostras tiveram seu DNA extraído e testado pela nested-PCR com primers específicos para porções do gene flagelina B (flaB), aptos a amplificar porções deste gene de qualquer espécie de Borrelia. Todas as amostras foram negativas pela nested-PCR, não evidenciando a presença de Borrelia sp nas amostras avaliadas. Para o segundo capítulo, foi utilizada uma amostra de espiroqueta aviária originária de carrapatos Argas miniatus, colhidos em um galinheiro no Município de Pedro Leopoldo, MG. O isolamento in vitro e caracterização molecular da espiroqueta aviária foi feito a partir da inoculação de soro infectado, contendo espiroquetas viáveis, em meio BSK. O soro, baço, fígado e o próprio cultivo foram utilizado para amplificação na PCR para os genes rrs e flaB, seguido de seqüenciamento dos mesmos. O isolamento da espiroqueta foi obtido com sucesso, com várias passagens realizadas. A análise genética das seqüências do isolado mostrou 99.8% (483 de 484-bp) e 98.7% (754 de 764-bp) de similaridade às seqüências correspondentes dos genes rrs e flaB de B. anserina, respectivamente, disponíveis no GenBank. Pela análise filogenética inferida pela seqüência parcial do gene flaB, a cepa Brasileira agrupou-se com a seqüência de B. anserina dos EUA. Os resultados indicam a cepa brasileira estudada, designada de cepa PL, pertence à espécie B.anserina. / Lyme disease is caused by bacteria belonging to the complex Borrelia burgdorferi sensu lato, which are transmitted by ticks of the Ixodes ricinus complex. The distribution of these bacteria are restricted to the northern hemisphere (North America, Europe, and Asia). Lyme disease-like cases have been reported in Brazil, but it is possible that another Borrelia species is involved in these cases, and ticks of the genus Amblyomma have been implicated as vectors. Due to these reasons, the disease in Brazil has been referred as Lyme Disease-Simile (LDS). Borrelia anserina, the agent of avian spirochetosis, has a wordwide distribution, where it is transmitted primarily by ticks of the genus Argas. The present study was divided in two chapters: the first one evaluated the presence of Borrelia spp in areas of the state of São Paulo where LDS have been reported. The second chapter reports the first in vitro isolation in BSK medium and molecular characterization of a spirochete strain from Brazil, presumably identified as B. anserina. For the first chapter, a total of 349 adult ticks (Amblyomma cajennense) collected in areas where LDS cases have been reported were processed. In addition, nine human blood or tissue samples from patients with clinical and serological diagnostic of LDS, two samples from BSK medium previously inoculated with samples of skin or blood of LDS patients, and three samples of BSK medium previously inoculated with tick samples were also processed. All these samples were processed for DNA extraction and then tested by nested-PCR employing primers targeting a portion of the flagelin B gene (flaB),which amplify a flaB fragment in all known Borrelia species. All samples were negative by this nested-PCR, showing no evidence of Borrelia sp in the tested samples. The second chapter evaluated an avian spirochete strain originated from Argas miniatus ticks from Pedro Leopoldo municipality, state of Minas Gerais. DNA fragments of the rrs (16S rRNA) and flab genes were amplified by PCR and sequenced to determine phylogenetic similarities. The resulting sequences were 99.8% (483 of 484) and 98.7% (754 of 764) similar to GenBank corresponding sequences of B. anserina rrs and flaB genes, respectively. By neighbor-joining phylogenetic analysis, the flaB sequence of the Brazilian strain clustered in a monophyletic group with the sequence of B. anserina under 100% bootstrap support. The isolate was successfully isolated in BSK medium, with seven passages performed. The spirochete crude antigen, fixed in glass slides, showed strong immunfluorescence reactivity with sera from chickens previously inoculated with the isolate. The spirochete strain isolated in the present study was genetically identified as B. anserina, labeled as strain PL.

Peptidoglycan (PG) is an essential cell-wall biopolymer in virtually all bacteria. It is composed of glycan strands of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) crosslinked by peptide chains of alternating D- and L- amino acids and diamines. PG plays an important role in 1) cell elongation and division, 2) cell strength and morphology, 3) antibiotic susceptibility, and 4) host immune detection and modulation. While differences in peptide chains are common, deviations in glycan strand composition were not previously known to occur. Here, we provide characterization of the first known deviation to bacterial glycan strand composition — GlcNAc-GlcNAc-anhMurNAc (G-G- anhM) in Borrelia burgdorferi, the causative agent of Lyme disease. B. burgdorferi with less G-G-anhM were found to be significantly less motile, flexible, and stress-tolerant while possessing gross morphological defects and less overall PG. Our studies also characterized the muropeptide profile of Borrelia afzelii, Borrelia garinii, and Borrelia hermsii — species of Borrelia associated with causing different disease manifestations of Lyme disease, and relapsing fever, respectively. These species were found to incorporate appreciable amounts of G-G-anhM into their PG, suggesting an evolutionary adaptation to life inside a tick that predates the differentiation of Lyme disease and relapsing fever Borrelia. Finally, we provide partial characterization of a putative penicillin-binding protein in B. burgdorferi — a class of highly conserved PG synthesis enzymes present in the vast majority of bacteria. Collectively, the work in this thesis furthers our understanding of the structure, function, and synthesis of PG in Borrelia. / Master of Science in Life Sciences / Peptidoglycan (PG) is the main cell-wall component in the vast majority of bacteria. PG is composed of strong, rigid sugars linked together by short, flexible amino acid chains, and resembles a mesh-like bag that surrounds the cell. In nearly all bacteria that have PG, it plays an important role in how 1) the cell grows and divides, 2) the cell dictates its shape, 3) antibiotics treat bacterial infections, and 4) the human body detects and responds to a bacterial infection. While the amino acids that make up PG are known to differ between bacterial species, deviations in sugar organization are not known to occur. Here, we characterize the first known deviation to sugar organization in bacterial PG in Borrelia burgdorferi — the bacteria that causes Lyme disease. B. burgdorferi with less of this deviation possess defects absent in their normal counterparts. In addition, we show that other Borrelia species that cause a variety of different diseases around the world mimic this sugar deviation, suggesting the majority, if not all, of Borrelia may do so. Finally, we provide partial characterization of the function of an enzyme thought to synthesize PG in B. burgdorferi. Collectively, the work in this thesis furthers our understanding of the structure, function, and synthesis of PG in Borrelia.

Borrelia burgdorferi — the causative agent of Lyme borreliosis — accounts for ~500,000 infections in the United States per year. Relative to other bacteria, B. burgdorferi is highly unusual in many regards. For instance, the synthesis and composition of B. burgdorferi cell wall is extremely unique and plays a critical role in Lyme pathogenesis. The cell wall is made up of peptidoglycan (PG) - a mesh-like structure, composed of long rigid glycan strands of repeating sugars GlcNAc and MurNAc, and flexible peptide stems, interlinked by amino acid cross-bridges. PG is an essential component for survival of the bacterial cell, protecting it from the osmotic stress and environmental threats, as well as defining the shape of the bacterium and aiding in the motility. One unique feature of the B. burgdorferi PG is the chemical composition of stem peptide, which involves the atypical cross-link between Ornithine and Glycine. We identified gene bb0586 as a femX homologue in borrelial genome and hypothesize that it encodes a glycyl transferase enzyme responsible for synthesis of glycine cross-bridges, that hold together glycan strands in the peptidoglycan cell wall. Here, we predicted the structure of FemXBb, identified and characterized the substrate-binding site, and proposed a novel mechanism for substrate recognition and recruitment, involving previously uncharacterized elements of the structure. We have also determined the ability of recombinant FemXBb to add Glycine bridges to mDAP in E. coli and investigated the effect that femX knock-out can have on the B. burgdorferi. In addition, we have investigated the steps of PG biosynthesis in B. burgdorferi. The results of our research suggest the existence of a highly unusual mechanism of PG synthesis in Lyme disease spirochete, which has a potential to be used for development of targeted antibacterial therapies. / Master of Science in Life Sciences / Borrelia burgdorferi — the causative agent of Lyme borreliosis — accounts for ~500,000 infections in the United States per year. Relative to other bacteria, B. burgdorferi is highly unusual in many regards. For instance, the synthesis and composition of B. burgdorferi cell wall is extremely unique and plays a critical role in Lyme pathogenesis. The cell wall is a mesh-like structure, a sacculus, enclosing the vulnerable inside contents of a bacterial cell. It is composed of long rigid glycan strands of repeating sugars, and flexible peptide stems, interlinked by cross-bridges, holding the whole structure together. PG is an essential component for survival of the bacterial cell, protecting it from the outside stress and environmental threats, as well as defining the shape of the bacterium and aiding in the motility. B. burgdorferi PG is known to be highly atypical compared to other bacteria. One of its features is the unusual cross-link between peptides, made up of single Glycine amino acid. We identified a gene encoding a protein responsible for the addition of this amino acid during the cell wall biosynthesis. Here, we predicted the structure of this protein, its substrate-binding site, and proposed a novel mechanism for substrate recognition and recruitment. We have also expressed the borrelial protein in E. coli and confirmed its activity and the impact it has on the bacterium and investigated the effect that gene knock-out can have on the B. burgdorferi. In addition, we have investigated the steps of PG biosynthesis in B. burgdorferi. The results of our research suggest the existence of a highly unusual mechanism of PG synthesis in Lyme disease spirochete, which has a potential to be used for development of targeted antibacterial therapies.

No description available.

Laimo liga arba Laimo boreliozė yra labiausiai Europoje, Šiaurės Amerikoje, Rusijoje ir Tolimuosiuose Rytuose plintanti, erkių pernešama zoonozė (Masuzawa, 2004, Wang ir kt., 1999, Sinski ir kt., 2006, Barbour, 1998). Tai yra dažniausia Ixodes ricinus erkių pernešama infekcija (Krupka ir kt., 2007, Žakovska ir kt., 2008). Daugiausia jos atvejų užfiksuojama Centrinėje-Rytų Europoje (Derdakova, Lenčakova, 2005). Laimo boreliozė yra vis didėjanti visuomenės sveikatos problema (Piesman, Gern, 2004, Hillyard, 1996). Kasmet tiek Europoje, tiek Šiaurės Amerikoje užfiksuojama tūktančiai ligos atvejų (Wang ir kt., 1999, Ostfeld, Keesing, 2000, Barbour, 1998). Pastaraisiais metais vykstant klimato pokyčiams, žmogui vis labiau keičiant jį supančią aplinką, Ixodes ricinus erkių gausumas Europoje sparčiai auga. To pasėkoje daugėja ir erkių pernešamų susirgimų (Tsao, 2009, Derdakova, Lenčakova, 2005). Gamtoje enzootinį Laimo ligos ciklą sudaro: sukėlėjas – B. burgdorferi sensu lato (s.l.), serkė - vektorius, ir rezervuarinis šeimininkas (Humair, Gern, 2000). B. burgdorferi s.l. tyrimai sukėlėją platinančiose erkėse ir rezervuariniuose šeimininkuose, kaip ir tikslus B. burgdorferi s.l. genotipų nustatymas padėtų tiek suprasti šios ligos ekologiją ir epidemiologiją, tiek nustatyti efektyvias prevencijos priemones (Derdakova, Lenčakova, 2005). Šio darbo tikslas – įvertinti dviejų pagrindinių rezervuarinių šeimininkų geltonkaklės pelės (Apodemus flavicollis) ir rudojo pelėno (Myodes glareolus)... [toliau žr. visą tekstą] / SUMMARY The aim of present study was to assess the importance of two main reservoir hosts A. flavicollis and M. gareolus, and their ectoparasites I. ricinus immature ticks in Lyme disease agent B. burgdorferi s.l. circulation in natural foci. The following research tasks were formulated: 1. To establish the infectivity level of questing immature I. ricinus ticks with B. burgdorferi s.l. and to determine it’s genotypes composition. 2. To investigate the infectivity level of two main Lithuania rodent species A. flavicollis and M. glareolus with B. burgdorferi s.l. and to determine it’s genotypes composition. 3. To estimate prevalence and abundant of infestation with immature I. ricinus ticks in A. flavicollis and M. glareolus species rodents. To calculate any differences between rodents in species, age and sex level. 4. To evaluate transmition efficient of B. burgdorferi s.l. from rodent to feeding I. ricinus larvae. The infectivity level in questing I. ricinus nymphs in fragmented mixed forests of Lithuania was relatively high, and varied in different districts from 14% to 24%. The most prevalent genotype was B. afzelii, and only in one district B. garinii was found. In case of I. ricinus larvae the overall infectivity level with Lyme disease spirochete was 3,5%. In order to establish the infectivity level with B. burgdorferi s.l. in total 302 rodents comprising 3 species - A. flavicollis, M. glareolus and A. agrarius were investigated. The results have showed that in all... [to full text]

Borrelia hermsii, an etiologic agent of tick-borne relapsing fever, binds negative complement regulator factor H (FH) via its FhbA protein. Direct demonstration of the role of FhbA in the disease process has been hindered by the lack of genetic manipulation systems for the relapsing fever Borrelia. Here, we demonstrate successful generation of a B. hermsii strain YOR fhbA deletion mutant (Bh YORΔfhbA) that constitutively produces green fluorescent protein (GFP). Genetic manipulation did not affect growth rate or plasmid composition. Bh YORΔfhbA lost factor H-binding and C3b-inactivation capabilities, but retained resistance to killing in human serum and infectivity in mice. Stable production of GFP was demonstrated in vitro and in vivo. Collectively, these results suggest that B. hermsii employs an unidentified mechanism of complement evasion that is FH-independent and sufficient for persistence within the host. Additionally, this study represents a significant methodological advancement in the molecular characterization of relapsing fever spirochetes.

Lyme disease is the most common tick-borne illness in North America. It can be hard to detect using blood tests, especially in the early stage. Due to the number of significant complications from untreated or undertreated Lyme disease, better methods need to be found to detect the disease. Some surface lipoproteins may be used to detect early disease due to their early expression. Others are maintained for the duration of the infection and can be used to detect chronic Lyme disease. Antibody responses to OspA, OspC and OspE were measured in sera from experimentally infected dogs. The response to OspA was only detectable in a few samples and did not appear to be sensitive for mammalian infections. A protein construct based on multiple epitopes of OspC was used to detect an antibody response starting three weeks after initial infection, and remained detectable for three months. As the response to OspC decreased, OspE could be used to detect an antibody response from three to six months after the initial infection. Because of the strength and the differences in timing of the antibody responses, OspC and OspE could be used to design an accurate blood test for Lyme disease that also indicates early and late stages of infection based on the results.

Lyme disease is an emerging infection that is caused by the Borrelia burgdorferi sensu lato complex. These bacteria exist in nature through an enzootic life cycle involving Ixodes ticks and various reservoir hosts. One way that this bacterium adapts to the different hosts in the enzootic cycle is through the expression of outer surface protein C (OspC). OspC is a surface exposed lipoprotein encoded on circular plasmid 26 that forms homodimers on the bacterial surface and has distinct conserved and variable portions of sequence. When ospC is deleted, the spirochetes are unable to cause mammalian infection although the mechanism of this is unknown. Additionally, OspC is thought to be involved in reservoir host specificity/association and in tissue dissemination. In order to better understand the functional domains of OspC, the different conserved and variable portions of this protein were investigated. Three conserved portions of OspC were investigated: (1) the conserved cysteine residue at position 130 (C130), (2) the last ten C-terminal amino acids (C10), and (3) ligand binding domain 1 (LBD1). The C130 residue was mutated and this substitution disrupted OspC oligomerization in vitro and in vivo. A B. burgdorferi strain lacking the C10 retained full infectivity and plasminogen binding. The mutation of a single residue within LBD1 rendered B. burgdorferi noninfectious, indicating the importance of this domain in infection establishment. The variable portion of OspC was investigated by: (1) altering the surface charge of ligand binding domain 2 (LBD2), (2) inserting different OspC types into a constant genetic background, and (3) creating OspC hybrids. Alteration of the surface charge of LBD2 by site directed mutagenesis resulted in a lack of persistence in mice. By inserting an OspC type known to be noninfectious in mice into an infectious strain, infectivity was abolished. Strains expressing OspC hybrids indicated that multiple domains of OspC are involved in species specificity. Together these analyses demonstrated that OspC is as important protein that plays multiple roles in pathogenesis. The work presented here helps to increase the understanding of this crucial protein and the strains described can be used to decipher the full function of OspC.