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Porins of Borrelia burgdorferi
Umeå University, Faculty of Medicine, Molecular Biology.
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.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1019
Keyword [en]
Lyme disease, P13, paralog, P66, channel-forming activity, porin
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-740ISBN: 91-7264-059-6 (print)OAI: oai:DiVA.org:umu-740DiVA: diva2:144383
Public defence
2006-04-21, Major Groove, 6L, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2006-03-31 Created: 2006-03-31 Last updated: 2009-10-28Bibliographically approved
List of papers
1. Molecular analysis of the channel-forming protein P13 and its paralogue family 48 from different Lyme disease Borrelia species
Open this publication in new window or tab >>Molecular analysis of the channel-forming protein P13 and its paralogue family 48 from different Lyme disease Borrelia species
2004 (English)In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 150, no Pt 3, 549-559 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.

Keyword
Amino Acid Sequence, Animals, Bacterial Outer Membrane Proteins/chemistry/genetics/physiology, Bacterial Proteins/chemistry/*genetics/physiology, Base Sequence, Borrelia/*genetics/pathogenicity/physiology, DNA; Bacterial/genetics, Epitope Mapping, Genes; Bacterial, Humans, Ion Channels/chemistry/genetics/physiology, Lyme Disease/microbiology, Mice, Mice; Inbred C3H, Models; Molecular, Molecular Sequence Data, Multigene Family, Sequence Homology; Amino Acid, Species Specificity
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-12966 (URN)10.1099/mic.0.26728-0 (DOI)14993304 (PubMedID)
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2017-12-14Bibliographically approved
2. Elimination of channel-forming activity by insertional inactivation of the p13 gene in Borrelia burgdorferi
Open this publication in new window or tab >>Elimination of channel-forming activity by insertional inactivation of the p13 gene in Borrelia burgdorferi
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2002 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, no 24, 6811-6819 p.Article in journal (Refereed) Published
Abstract [en]

P13 is a chromosomally encoded 13-kDa integral outer membrane protein of the Lyme disease agent, Borrelia burgdorferi. The aim of this study was to investigate the function of the P13 protein. Here, we inactivated the p13 gene by targeted mutagenesis and investigated the porin activities of outer membrane proteins by using lipid bilayer experiments. Channel-forming activity was lost in the p13 mutant compared to wild-type B. burgdorferi, indicating that P13 may function as a porin. We purified native P13 to homogeneity by fast performance liquid chromatography and demonstrated that pure P13 has channel-forming activity with a single-channel conductance in 1 M KCl of 3.5 nS, the same as the porin activity that was lost in the p13 mutant. Further characterization of the channel formed by P13 suggested that it is cation selective and voltage independent. In addition, no major physiological effects of the inactivated p13 gene could be detected under normal growth conditions. The inactivation of p13 is the first reported inactivation of a gene encoding an integral outer membrane protein in B. burgdorferi. Here, we describe both genetic and biophysical experiments indicating that P13 in B. burgdorferi is an outer membrane protein with porin activity.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-5029 (URN)10.1128/JB.184.24.6811-6819.2002 (DOI)12446631 (PubMedID)
Available from: 2006-03-31 Created: 2006-03-31 Last updated: 2017-12-14Bibliographically approved
3. Pleiotropic effects of inactivating a carboxyl-terminal protease, CtpA, in Borrelia burgdorferi
Open this publication in new window or tab >>Pleiotropic effects of inactivating a carboxyl-terminal protease, CtpA, in Borrelia burgdorferi
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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.

Keyword
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
Identifiers
urn:nbn:se:umu:diva-12965 (URN)10.1099/mic.0.26728-0 (DOI)15028692 (PubMedID)
Available from: 2007-10-03 Created: 2007-10-03 Last updated: 2017-12-14Bibliographically approved
4. The BBA01 protein, a member of paralog family 48 from Borrelia burgdorferi, is potentially interchangeable with the channel-forming protein P13.
Open this publication in new window or tab >>The BBA01 protein, a member of paralog family 48 from Borrelia burgdorferi, is potentially interchangeable with the channel-forming protein P13.
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2006 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 188, no 12, 4207-4217 p.Article in journal (Refereed) Published
Abstract [en]

The Borrelia burgdorferi genome exhibits redundancy, with many plasmid-carried genes belonging to paralogous gene families. It has been suggested that certain paralogs may be necessary in various environments and that they are differentially expressed in response to different conditions. The chromosomally located p13 gene which codes for a channel-forming protein belongs to paralog family 48, which consists of eight additional genes. Of the paralogous genes from family 48, the BBA01 gene has the highest homology to p13. Herein, we have inactivated the BBA01 gene in B. burgdorferi strain B31-A. This mutant shows no apparent phenotypic difference compared to the wild type. However, analysis of BBA01 in a C-terminal protease A (CtpA)-deficient background revealed that like P13, BBA01 is posttranslationally processed at its C terminus. Elevated BBA01 expression was obtained in strains with the BBA01 gene introduced on the shuttle vector compared to the wild-type strain. We could further demonstrate that BBA01 is a channel-forming protein with properties surprisingly similar to those of P13. The single-channel conductance, of about 3.5 nS, formed by BBA01 is comparable to that of P13, which together with the high degree of sequence similarity suggests that the two proteins may have similar and interchangeable functions. This is further strengthened by the up-regulation of the BBA01 protein and its possible localization in the outer membrane in a p13 knockout strain, thus suggesting that P13 can be replaced by BBA01.

Keyword
Bacterial Outer Membrane Proteins/*metabolism, Borrelia burgdorferi/*metabolism/physiology, Carboxypeptidases/metabolism, Ion Channels/*metabolism, Multigene Family, Peptide Hydrolases/metabolism
Identifiers
urn:nbn:se:umu:diva-12925 (URN)doi:10.1128/JB.00302-06 (DOI)16740927 (PubMedID)
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2017-12-14Bibliographically approved
5. The P66 protein of Borrelia burgdorferi demonstrates dual functions of channel-forming and integrin-binding activity
Open this publication in new window or tab >>The P66 protein of Borrelia burgdorferi demonstrates dual functions of channel-forming and integrin-binding activity
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Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5032 (URN)
Available from: 2006-03-31 Created: 2006-03-31 Last updated: 2010-01-13Bibliographically approved
6. Structural characterization of Borrelia burgdorferi porins by electron crystallography
Open this publication in new window or tab >>Structural characterization of Borrelia burgdorferi porins by electron crystallography
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(English)Manuscript (Other academic)
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-5033 (URN)
Available from: 2006-03-31 Created: 2006-03-31 Last updated: 2010-01-13Bibliographically approved

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