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.

Medicinsk entomologi

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Transgene Pflanzen nehmen einen wachsenden Stellenwert bei der Produktion therapeutischer Proteine, besonders von Impfstoffen, ein. Einige dieser Proteine benötigen für ihre Funktionalität verschiedenste post-translationale Modifikation und es ist nicht bekannt, ob Pflanzen zu diesen Stoffwechselleistungen befähigt sind. In der vorliegenden Arbeit sollte anhand eines bakteriellen Antigens geprüft werden, ob in Chloroplasten von Nicotiana tabacum an einem rekombinanten Protein eine besondere Form der post-translationalen Modifikation, die Lipidierung, durchgeführt wird und ob somit ein alternatives Produktionssystem für einen Lipoprotein-Impfstoff entwickelt werden kann. Basis für diese Untersuchungen war das bakterielle Lipoprotein OspA (outer surface protein A) aus Borrelia burgdorferi. Dieses Protein wird zur Prävention von Lyme-Borreliose eingesetzt und frühere Untersuchungen zeigten, dass OspA sowohl in B. burgdorferi als auch nach heterologer Expression in E. coli einer post-translationalen Lipidierung am N-Terminus unterliegt. Dabei wird das Protein unter Mitwirkung dreier Enzyme (Lgt, Lsp und Lnt) am N-Terminus mit einer Pam3Cys-Struktur versehen und die N-terminale Signalsequenz abgespalten. In dieser Arbeit konnten nach der Etablierung eines Expressionssystems mehrere unabhängige transplastome OspA-Tabakpflanzen generiert werden. Der Anteil an rekombinantem, plastidärem OspA (rpOspA) am löslichen Gesamtprotein wurde mit ca. 1% bestimmt. Dabei lag rpOspA sowohl in einer lipidierten, membrangebundenen als auch in einer nicht-modifizierten, löslichen Form vor. Strukturelle Untersuchungen an rpOspA ergaben, dass in Chloroplasten eine Bakterien-ähnliche Lipidierung an dem Protein durchgeführt wurde. Dabei wurde am N-Terminus ein Diacylglycerin über eine Thioetherbindung an das einzige in der Sequenz vorkommende Cystein gebunden. Anders als in Bakterien wurde die Signalsequenz in Chloroplasten jedoch nicht abgespalten und infolgedessen keine dritte Fettsäure an das Cystein gebunden. Die plastidäre Modifikation an rekombinantem OspA resultierte daher in einer Pam2Cys-Struktur mit Signalsequenz. Untersuchungen zur Immunogenität des rpOspA in Mäusen zeigten eindeutig, dass das rekombinante Protein aus Tabak die Bildung spezifischer Antikörper induziert und damit in seiner Wirkung vergleichbar dem bakteriellen Protein ist. Um die Lipidierungsrate des akkumulierten OspA in Chloroplasten zu erhöhen, wurden weitere transplastome Tabakpflanzen generiert. Diese wiesen neben dem ospA-Gen auch die Gene (lgt, lsp und lnt) der drei modifizierenden Enzyme aus E. coli auf. Durch die simultane Bildung von OspA und den drei modifizierenden Enzyme konnte eine quantitative Lipidierung von rpOspA mit einer Pam2Cys-Struktur erlangt werden. Als Grundlage für vergleichende Untersuchungen zwischen plastidärer und cytoplasmatischer Lipidmodifikation in Tabak wurden ebenfalls Transformationen des Zellkerns mit ospA durchgeführt, wobei jedoch keine heterologe ospA-Expression erreicht werden konnte. Erst durch die Anwendung eines transienten, viralen Expressionssystems war es möglich, ospA im Zellkern zu exprimieren. In dieser Arbeit wurde gezeigt, dass in Chloroplasten Höherer Pflanzen eine Bakterien-ähnliche Modifikation an rekombinantem OspA durchgeführt wird und das resultierende Protein eine spezifische Immunantwort in Mäusen induzieren kann. Das Potential von transgenen Pflanzen als alternatives Produktionssystem für Lipoprotein-Impfstoffe wird diskutiert. / Transgenic plants play an increasing role in the production of therapeutical proteins, especially vaccines. Some of those proteins need a posttranslational modification for their activity of immunogenicity and it is still ambiguous if plants are capable of performing these modifications. In the present study it was investigated by the use of a bacterial antigen if a particular form of posttranslational modification, the lipidation of proteins, is performed in chloroplasts of Nicotiana tabacum and if the resulting plants could be further developed into an alternative production system for lipoprotein-vaccines. For this investigation, the bacterial lipoprotein OspA (outer surface protein A) of Borrelia burgdorferi was chosen, a protein used as a vaccine against Lyme disease. Previous studies have been shown that OspA is lipidated on its N-terminus both in B. burgdorferi and when expressed in Escherichia coli. This modification is performed by three different enzymes (Lgt, Lsp and Lnt) which build up a Pam3Cys-structure at the N-terminus of the protein while the N-terminal signal sequence is cleaved. In the present study several independent transgenic plant lines could be generated by the use of a suitable expression system. Analysis showed that the fraction of recombinant plastidal OspA (rpOspA) in transplastomic plants reached approx. 1% of the total soluble protein. Moreover, a lipid modification could be detected in a fraction of the rpOspA which resulted in the membrane association of a portion of the recombinant protein. The structural analysis of rpOspA revealed that in chloroplasts, rpOspA is lipidated in a bacteria-like manner, including the attachment of a diacylglycerol moiety to the sole cysteine via a thioether linkage. Other than in bacteria, the signal peptide is not cleaved in chloroplasts; thus, no additional fatty acid is attached to the cysteine. The plastidal modification on recombinant OspA therefore resulted in a Pam2Cys-structure including the signal sequence. Furthermore, the rpOspA from tobacco showed similar immunogenic properties as the recombinant protein from bacteria when tested in mice, showing the potential of plants as a production system for lipoprotein-vaccines. To further increase and modify the lipidation rate and pattern of tobacco-derived OspA, a new set of transplastomic tobacco plants were generated. These plants not only carried the ospA gene, but also the genes encoding for the three modifying enzymes (lgt, lsp and lnt). This simultaneous expression shifted the ratio between lipidated and non-lipidated rpOspA almost quantitatively to the lipidated form. For further comparisons of the OspA-lipidation either in plastids or in the cytosol transgenic tobacco plants were established. However, after stable integration of ospA into the nuclear genome, no detectable heterologeous expression could be achieved. Only by using a transient viral expression system the nuclear ospA expression was feasible. In this study it has been shown, that in chloroplasts of higher plants a bacteria-like modification on recombinant OspA was performed and that the resulting protein induced a specific immune response in mice. The potential of transgenetic plants as an alternative production system for lipoprotein-vaccines is discussed.

Reproduction de : Thèse d'exercice : Médecine vétérinaire : Toulouse 3 : 2008. / Titre provenant de l'écran titre. Bibliogr. p 131-141.

Berlin, Freie Universität, Thesis (doctoral), 2000.

A set of studies was designed in order to better understand the exposure of horses in Canada to Ixodes-borne diseases, namely equine granulocytic anaplasmosis (EGA, caused by Anaplasma phagocytophilum) and Lyme borreliosis (LB, caused by Borrelia burgdorferi). In the first study, equine serum samples submitted to veterinary diagnostic laboratories in SK, MB and ON were tested for antibodies against A. phagocytophilum and B. burgdorferi, using the point-of-care SNAP® 4Dx® ELISA. Horses seropositive to EGA were found in SK and MB and horses seropositive to LB were found in SK, MB and ON. Overall seroprevalence according to the SNAP® 4Dx® ELISA was 0.53% for EGA and 1.6% for LB. Samples that tested positive for antibodies against A. phagocytophilum (n=2) and B. burgdorferi (n=6) by SNAP® 4Dx® ELISA and 2 randomly selected subsets of samples that tested negative (n=92 each) were then re-tested using currently recommended serologic methods, and test results were compared. A lack of agreement was found between the SNAP® 4Dx® ELISA and indirect immunofluorescent assay (IFA) for EGA (McNemar test p = 0.000001). Agreement of the SNAP® 4Dx® ELISA and ELISA confirmed with Western Blot (WB) for LB was only fair (Kappa 0.23). Due to the lack of agreement between serologic tests for EGA and LB in the first study, another study to further evaluate the agreement among available serologic tests was conducted. A set of 50 convenience serum samples submitted to the veterinary diagnostic laboratory in SK was tested by SNAP® 4Dx® Plus ELISA for antibodies against A. phagocytophilum and B. burgdorferi. Samples were also tested by IFA for antibodies against A. phagocytophilum in two referral laboratories, and by IFA, ELISA confirmed with WB and Equine Lyme multiplex assay for antibodies against B. burgdorferi in three referral laboratories. Again, test results varied between the different tests. For EGA, all 3 pair-wise test comparisons lacked agreement. For LB, agreement between tests ranged from poor to fair. Differences in test methodology and antigens used, cut-off settings between the laboratories and false positive or false negative results are likely the cause for the different assessment of the same sample as seropositive or seronegative. In the third study, the goal was to describe potential risk factors for exposure of horses in Canada to EGA and LB. Management factors in horses that tested seropositive or seronegative for EGA or LB, respectively, in the previous studies were evaluated. Horse owners were surveyed with regard to their horses’ signalment, timing of pasture housing, and province of residence, travel history, tick infestation history, history of Lyme vaccination and history of previously diagnosed tick-borne disease. Response rate (11.5%) and the number of seropositive horses available for evaluation were low, which precluded statistical analysis. The majority of seropositive horses resided in SK, was pastured in the fall, did not have a recent travel history and had not had visible tick infestation. These observations supported exposure of horses to tick-borne diseases within Canada. Potential risk factors require further investigation. As information about tick infestation in horses is scarce in general, a passive surveillance study of horse ticks in SK was conducted in 2012 and 2013. A total of 833 ticks from over 86 horses were received. All ticks were Dermacentor species, i.e. D. albipictus, D. andersoni and D. variabilis. D. albipictus ticks were mostly received in February and March, D. andersoni mainly in April and June and D. variabilis mostly in May and June. Geographic distribution of the species in SK was similar to that previously reported based on active and passive surveillance. No Ixodes species were received.

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, a multisystem disorder that can cause a variety of disorders in susceptible mammalian hosts. The immune response of infected mammals, including humans, is ineffective in clearing B. burgdorferi as demonstrated by the ability to reisolate the spirochete from naturally and experimentally infected hosts after extended periods of time. Recent evidence suggests that this pathogen evades the immune response in part through changes in antigenic reactivity.The purpose of this study was to determine if outer surface protein A (OspA) of B. burqdorferi varies in the course of infection in Syrian hamsters and thus potentially plays a role in evading the host immune response. To assess the degree of change, differences in the binding of a murine monoclonal antibody (H5332) were measured using IFA and ELISA techniques over a 9-week period of time.Results of this study suggest that OspA is persistently expressed in infected Syrian hamsters for at least 9-weeks. Moreover, this protein, or at least the epitope that H5332 binds with, is stably expressed. These results indicate OspA, or at least the epitope of OspA that I probed, does not appear to contribute to the evasive mechanisms of 8. burgdorferi in Syrian hamsters. / Department of Biology

Univ., Diss., 2005--Frankfurt (Main)

Zugl.: Giessen, Univ., Diss., 2008