Diss. (sammanfattning) Umeå : Umeå universitet, 2007. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on June 28, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / "December 2006" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

Made available in DSpace on 2014-08-20T13:32:50Z (GMT). No. of bitstreams: 1 dissertacao_nubia_seyffert.pdf: 4325642 bytes, checksum: 2836f7bcdef6a962ef1e00c38936c413 (MD5) Previous issue date: 2007-02-05 / Leptospiral immunoglobulin-like (Lig)proteins are exposed on membrane surface of pathogenic Leptospira and may play a role in host cell attachment and invasion during infection.A pair of Ligs,named LigA and LigB,with identical and non-identical polypeptide regions has recently been identified.The two proteins elicit a strong humoral immune response during acute host infection and have been suggested as targets for development of vaccines and diagnostic tests for leptospirosis.This study reports the production and characterization of monoclonal antibodies (Mabs)against recombinant non-identical fragments of LigA (rLigANI)and LigB (rLigBNI).The recombinant fragments were used for mice immunization and screening hybridomas by indirect ELISA.Four Mabs obtained against rLigANI and six Mabs obtained against rLigBNI were isotyped and evaluated regarding their potential for use in studies of immunoprotection and diagnostic test development.The Mabs were of the IgM (1),Ig 2b (3)and Ig 1 (6)isotypes and reacted with the native proteins from a pathogenic strain of Leptospira i terroga s serovar Copenhageni L1 130 in an indirect ELISA and immunofluorescence.Mabs affinity constants felt between 4x10 7 M -1 and 2x10 8 M -1 ,and epitope mapping by additive ELISA has shown that each Mab either react with the same epitope in the recombinant molecule or cause steric hindrance. Tissue sections from kidneys of hamsters experimentally infected with leptospires and probed with the Mabs reacted positively by immunohistochemistry.These findings suggest that the Mabs obtained can be useful for the studies of immunoprotection and development of diagnostic tests. / As proteínas leptospiral immu oglobuli -like (Lig)estão expostas na superfície da membrana das leptospiras patogênicas e podem estar envolvidas no ataque e penetração às células do hospedeiro durante a infecção.Recentemente,foi identificado um par de Ligs,nomeadas LigA e LigB,com regiões polipeptídicas idênticas e não-idênticas.As duas proteínas estimulam uma resposta humoral forte durante a infecção aguda no hospedeiro e têm sido sugeridas como alvos para o desenvolvimento de vacinas e testes diagnósticos para leptospirose.Este estudo relata a produção e caracterização de anticorpos monoclonais (Mabs)contra fragmentos recombinantes não idênticos de LigA (rLigANI)e LigB (rLigBNI).Os fragmentos recombinantes foram utilizados para a imunização dos camundongos e triagem dos hibridomas por ELISA indireto.Quatro Mabs obtidos contra rLigANI e 6 Mabs contra rLigBNI foram isotipados e avaliados quanto ao seu potencial para uso em estudos de imunoproteção e desenvolvimento de testes diagnósticos.Os Mabs foram dos isotipos IgM (1),Ig 2b (3)and Ig 1 (6)e reagiram com as proteínas nativas de Leptospira i terroga s sorovar Copenhageni cepa L1 130 em ELISA indireto e imunofluorescência.A constante de afinidade dos Mabs manteve-se entre 4x10 7 M -1 and 2x10 8 M -1 ,e o mapeamento de epitopos por ELISA de aditividade demonstrou que cada Mab reage com o mesmo epitopo na molécula recombinante ou causa um impedimento espacial (steric hi dra ce ).Finalmente,os Mabs reagiram positivamente em testes imuno-histoquímicos de seções do tecido renal de hamsters experimentalmente infectados com leptospiras.Esses dados sugerem que os Mabs obtidos podem ser usados para estudos de imunoproteção e desenvolvimento de testes de diagnóstico de leptospirose.

Enterohemorrhagic E. coli (EHEC) has emerged as an important agent of diarrheal disease in the developed countries. Attachment to host cells, an essential step during intestinal colonization by EHEC, is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed attaching and effacing (A/E) lesion, directly beneath bound bacteria. The outer membrane protein, intimin, is required for the formation of this structure, as is Tir, a bacterial protein that is translocated into the host cell and thought to function as a receptor for intimin. In this thesis, we characterized A/E lesion formation by in vivo and in vitro-grown EHEC, aimed at testing whether bacterial adaptation to the mammalian host included up regulation of A/E lesion formation. Our results showed that actin signaling by EHEC was induced upon bacterial growth in vivo, and this induction was likely due to the up regulation of multiple activities by in vivo-grown EHEC. We also focused on the interaction between intimin and the host cell, an interaction that triggers actin condensation of A/E lesion formation. We evaluated the role of β1 integrins, one of the proposed receptors of intimin, in A/E lesion formation, and demonstrated that β1 integrins are not essential for intimin-mediated cell binding and actin condensation. To better understand intimin function, we mapped the functional domains of intimin, showed that the minimal cell binding domain of intimin correlates with the minimal Tir-binding domain. This minimal Tir-binding domain, when purified and coated on latex beads, was sufficient to trigger actin condensation on preinfected mammalian cells, suggesting that Tir-binding by intimin is critical in the final step of A/E lesion formation. To further demonstrate the significance of the interaction between intimin and Tir in A/E lesion formation, we developed a yeast two-hybrid system to identify intimin mutants diminished in Tir-binding, and then characterized those mutants for the ability to trigger actin condensation, the final step of A/E lesion formation. Finally, as a first step to study the downstream actin signaling pathway after Tir-binding, we mapped the domain of Tir involved in intimin-binding, and showed that the N-terminus and C-terminus of Tir are likely to be localized in the host cell cytoplasm, available to interact with downstream effectors in actin signaling.

No description available.

The pathogenic members of the genus Yersinia, Y. pseudotuberculosis, Y. pestis and Y. enterocolitica are transmitted from animals to man and may give rise to disease with a variety of symptoms. These bacteria possess related plasmids necessary for virulence. In this study, gene products encoded by the virulence plasmid have been identified and characterized. A temperature-inducible outer membrane protein YOP1, is encoded by the virulence plasmid. YOP1 is expressed by Y. pseudotuberculosis and Y. enterocolitica at 37°C. The genetic locale of trie structural gene for YOPl on the virulence plasmid was determined. A mutant that was unable to express this protein, remained fully virulent, showing that YOP1 is not a virulence determinant. Several other proteins encoded by the virulence plasmid are induced at 37°C in a medium lacking Ca2+. These proteins are not expressed at 26°C and expression is repressed by Ca2+-concentrations in excess of 2.5 mM. In Ca2+-deficient medium, the induced proteins can be found extracellu- larly as well as in the outer membrane. However, in the presence of Ca at 37°C they are only found in the outer membrane. The released proteins consist of eight polypeptides as revealed by two-dimensional electro­phoresis. These proteins, Y0P2a and 2b, YOP3, Y0P4a and 4b, the V-antigen and a small uncharacterized polypeptide, are expressed by all three pathogenic Yersinia species, both in vivo and in vitro. The Ca2+-controlled expression of the YOP proteins is regulated by genes in the Ca2+ -region, which are conserved in the three species. Mutations in this region repress the expression of the Ca2+-regulated YOPs. The genetic loci identified for five of these proteins revealed that only the structural gene of the Y0P4b protein is part of the Ca2+ -region. The other genes were found at separate locations outside this region. The structural genes for YOP4b, YOP3 and the V-antigen, together with the genes for two additional polypeptides, were localized to a common region conserved on the plasmids of the Yersinia species. The structural genes for Y0P2b (yopH) and Y0P5 (yopE) are located in different positions on the plasmid from Y. enterocolitica, compared to the other two species. This plasmid has Been rearranged so that these genes are located close to one another. The DNA sequence of the yopH gene shows that it is a singly transcrip­tional unit. Transcription of this gene is regulated by Ca2+-concentra­tion and by temperature. A mutant strain of Y. pseudo tuberculosis, de­leted for the yopH gene on the virulence plasmid, is avirulent In mice. Virulence is restored by trans-complementation with the cloned yopH gene. The mutant strain is also’ unable to inhibit phagocytosis of macrophages as compared to the wild-type strain. The trans-compleroented strain shows inhibition comparable to that of the wild-type. Therefore, the YOP2b protein is considered to be an essential virulence determinant. / digitalisering@umu.se

Beta-barrel outer membrane proteins (OMPs) present on the outer membrane of Gram-negative bacteria are vital to cell survival. Their biogenesis is a challenging process which is tightly regulated by protein-chaperone interactions at various stages. Upon secretion from the inner membrane, OMPs are solubilized by periplasmic chaperones seventeen kilodalton protein (Skp) and survival factor A (SurA) and maintained in a folding competent state until they reach the outer membrane. As periplasm has an energy deficient environment, thermodynamics plays an important role in fine tuning these chaperone-OMP interactions. Thus, a complete understanding of such associations necessitates an investigation into both structural and thermodynamic aspects of the underlying intercommunication. Yet, they have been difficult to discern because of the conformational heterogeneity of the bound substrates, fast chain dynamics and the aggregation prone nature of OMPs. This demands for use of single molecule spectroscopy techniques, specifically, single molecule Förster resonance energy transfer (smFRET). In this thesis, upon leveraging the conformational and temporal resolution offered by smFRET, an exciting insight is obtained into the mechanistic and functional features of unfolded and Skp/SurA - bound states of two differently sized OMPs: OmpX (8 β-strands) and outer membrane phospholipase A (OmpLA – 12 β-strands). First, it was elucidated that the unfolded states of both the proteins exhibit slow interconversion within their sub-populations. Remarkably, upon complexing with chaperones, irrespective of the chosen OMP, the bound substrates expanded with localised chain reconfiguration on a sub-millisecond timescale. Yet, due to the different interaction mechanisms employed by Skp (encapsulation) and SurA (multivalent binding), their clients were found to be characterised by distinct conformational ensembles. Importantly, the extracted thermodynamic parameters of change in enthalpy and entropy exemplified the mechanistically dissimilar functionalities of the two chaperones. Furthermore, both Skp and SurA were found to be capable of disintegrating aggregated OMPs rather cooperatively, highlighting their multifaceted chaperone activity. This work is of significant fundamental value towards understanding the ubiquitous chaperone-protein interactions and opens up the possibility to design drugs targeting the chaperone-OMP complex itself, one step ahead of the OMP assembly on the outer membrane.

Host cell binding is an essential step in colonization by many bacterial pathogens, and the Lyme disease agent, Borrelia burgdorferi, which colonizes multiple tissues, is capable of attachment to diverse cell types. Glycosaminoglycans (GAGs) are ubiquitously expressed on mammalian cells and are recognized by multiple B. burgdorferi surface proteins. We previously showed that B. burgdorferi strains differ in the particular spectrum of GAGs that they recognize, leading to differences in the cultured mammalian cell types that they efficiently bind. The molecular basis of these binding specificities remains undefined, due to the difficulty of analyzing multiple, potentially redundant cell attachment pathways and to the paucity of genetic tools for this pathogen. Complementation of a high-passage non-adherent B. burgdorferi strain reveals that the expression of DbpA, DbpB, or BBK32, is sufficient to confer efficient spirochete attachment to 293 epithelial cells. Epithelial cell attachment by DbpA and B was mediated by dermatan sulfate, while BBK32 recognized dermatan and heparan sulfate. The GAG binding properties of bacteria expressing DbpB or DbpA were distinguishable in that DbpB, but not DbpA, promoted spirochetal attachment to C6 glial cells. Furthermore, DbpA alleles from diverse Lyme disease spirochetes exhibit allelic variation with respect to binding decorin, dermatan sulfate, and epithelial cells. Targeted disruption of bbk32 resulted in decreased spirochete binding to fibronectin, GAGs, and mammalian cells. Thus, DbpA, DbpB, and BBK32 may play central but distinct roles in cell type-specific binding by Lyme disease spirochetes. This study illustrates that transformation of high-passage B. burgdorferi strains and targeted gene disruption provide a comprehensive genetic approach to analyze virulence-associated phenotypes conferred by multiple bacterial factors.