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Pleiotropic effects of inactivating a carboxyl-terminal protease, CtpA, in Borrelia burgdorferi
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Uhlin)
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Bergström)
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2004 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 7, 2074-2084 p.Article in journal (Refereed) Published
Abstract [en]

The aetiological agent of Lyme disease, Borrelia burgdorferi cycles between its tick vector and mammalian hosts, implying that it can sense different environments and consequently change the expression of genes encoding several surface-associated proteins. The genome of the type strain B. burgdorferi B31 has revealed 175 different gene families. The p13 gene, situated on the chromosome, encodes a channel-forming protein that belongs to the gene family 48 consisting of eight additional paralogous genes. The heterogeneity of the P13 protein from different Lyme disease Borrelia strains was investigated. The predicted surface-exposed domains are the most heterogeneous regions and contain probable epitopes of P13. The membrane-spanning architecture of P13 was determined and a model for the location of this protein in the outer membrane is presented. The transcription of the paralogues of gene family 48 during in vitro culturing and in a mouse infection model was also analysed. The bba01 gene is the only p13 paralogue present in all three Lyme-disease-causing genospecies; it is stable during cultivation in vitro and the BBA01 protein was expressed in all Borrelia strains investigated. Conversely, paralogues bbi31, bbq06 and bbh41 were only detected in B. burgdorferi and the corresponding plasmids harbouring bbi31 and bbh41 were lost during in vitro passage. Finally, p13 and bbi31 are the only members of gene family 48 that are transcribed in mice, suggesting their importance during mammalian infection.

Place, publisher, year, edition, pages
2004. Vol. 186, no 7, 2074-2084 p.
Keyword [en]
Amino Acid Sequence, Bacterial Proteins/chemistry/genetics/metabolism, Borrelia burgdorferi/*enzymology/genetics, Carboxypeptidases/chemistry/*genetics/*metabolism, Electrophoresis; Polyacrylamide Gel, Immunoblotting, Molecular Sequence Data, Mutation, Sequence Analysis; DNA, Sequence Homology; Amino Acid, Spectrometry; Mass; Matrix-Assisted Laser Desorption-Ionization, Substrate Specificity
National Category
Cell and Molecular Biology
URN: urn:nbn:se:umu:diva-12965DOI: 10.1099/mic.0.26728-0PubMedID: 15028692OAI: diva2:152636
Available from: 2007-10-03 Created: 2007-10-03 Last updated: 2011-10-26Bibliographically approved
In thesis
1. Porins of Borrelia burgdorferi
Open this publication in new window or tab >>Porins of Borrelia burgdorferi
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Borrelia burgdorferi is a pathogenic spirochete which cycles between its arthropod vector and vertebrate host. If transmitted to humans, B. burgdorferi causes Lyme disease, an infection which can impair different organs, such as the skin, joints, nervous system and heart. Alterations in protein expression due to the different environments Borrelia encounters during its complicated life cycle require advanced adaptation mechanisms. The outer surface-exposed proteins play a critical role in survival and pathogenesis of Borrelia in different hosts and tissues, being involved in avoiding the host immune response, adhesion to different tissues and nutrient acquisition. This thesis aimed to characterize integral outer membrane proteins which play a role in solute and nutrient uptake, and provides support for their role in the environmental adaptation of Borrelia.

In this thesis, three B. burgdorferi proteins, P13, BBA01 and P66, were shown to be porins, and characterized structurally and functionally using a combination of biochemical, biophysical and genetic methods. The channel-forming function of the 13 kDa protein, P13, was elucidated by a lipid bilayer assay. Post-translational processing of P13 occurred at the C-terminus by C-terminal processing protease (CtpA)-dependent cleavage. The membrane-spanning architecture of P13 was determined by epitope mapping and computer-based structural predictions which revealed that P13 is an unusual porin, not possessing the structural properties of conventional porins: rather than forming β-barrels, it is predicted to span the membrane with hydrophobic α-helices.

p13 belongs to a paralogous gene family. The transcription of p13 and other gene family members during in vitro growth and in a mouse infection model was therefore investigated. The paralog BBA01, which has the highest sequence homology to P13, is expressed during in vitro growth in all three Lyme disease causing species, although at very low levels. Like P13, BBA01 is also processed by CtpA and exhibits very similar channel-forming activity. Furthermore, in the absence of P13, a proportion of total BBA01 protein is relocated to the bacterial surface with strong indications that BBA01 and P13 are functionally interchangeable.

P66, an integrin binding protein, was also determined to be a porin. The oligomeric state of native P66, elucidated by chemical cross-linking, indicated that P66 forms trimers, as do the majority of conventional porins. Electron crystallography and a projection map of P66 crystals at 2.2 nm resolution revealed tetragonal unit cell symmetry with the area intercalated between the assembled protein structures consistent with the approximate expected size of the channel formed by P66. Finally, the biological relevance of two porins, P13 and P66, was demonstrated in a double mutant displaying a stress response as revealed by increased sensitivity to high osmolarity and elevated expression of the B. burgdorferi heat-shock protein HtrA homolog.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi, 2006. 84 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1019
Lyme disease, P13, paralog, P66, channel-forming activity, porin
National Category
Microbiology in the medical area
urn:nbn:se:umu:diva-740 (URN)91-7264-059-6 (ISBN)
Public defence
2006-04-21, Major Groove, 6L, Umeå Universitet, Umeå, 09:00 (English)
Available from: 2006-03-31 Created: 2006-03-31 Last updated: 2009-10-28Bibliographically approved

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Östberg, YngvePinne, MarijaBergström, Sven
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