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Borrelia channel-forming proteins: structure and function
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Borrelia is a Gram-negative, corkscrew-shaped bacterium transmitted by infected ticks or lice. Borreliae are subdivided into pathogens of two diseases: Lyme disease, caused mainly by B. burgdorferi, B. afzelii and B. garinii; and relapsing fever caused primarily by B. duttonii, B. hermsii, B. recurrentis or B. crocidurae. Both diseases differ in their manifestations, duration times and dissemination patterns. Antibiotics are the major therapeutics, although unfortunately antibiotic treatment is not always beneficial. To date, drug resistance mechanisms in B. burgdorferi are unknown. Transporters of the resistance-nodulation-division (RND) family appear to be involved in drug resistance, especially in Gram-negative bacteria. They consist of three components: a cytoplasmic membrane export system, a membrane fusion protein (MFP), and an outer membrane factor (OMP). The major antibiotic efflux activity of this type in Escherichia coli is mediated by the tripartite multidrug resistance pump AcrAB-TolC. Based on the sequence homology we conclude that the besA (bb0140), besB (bb0141) and besC (bb0142) genes code for a similar efflux system in B. burgdorferi. We created a deletion mutant of besC. The minimal inhibitory concentration (MIC) values of B. burgdorferi carrying an inactive besC gene were 4- to 8-fold lower than in the wild type strain. Animal experiments showed that the besC mutant was unable to infect mice. Black lipid bilayer experiments were carried out to determine the biophysical properties of purified BesC. This study showed the importance of BesC protein for B. burgdorferi pathogenicity and resistance to antibiotics, although its importance in clinical isolates is not known.

Due to its small genome, Borrelia is metabolically and biosynthetically deficient, thereby making it highly dependent on nutrients provided by their hosts. The uptake of nutrients by Borrelia is not yet completely understood. We describe the purification and characterization of a 36-kDa protein that functions as a putative dicarboxylate-specific porin in the outer membrane of Borrelia. The protein was designated as DipA, for dicarboxylate-specific porin A. DipA was biophysically characterized using the black lipid bilayer assay. The permeation of KCl through the channel could be partly blocked by titrating the DipA-mediated membrane conductance with increasing concentrations of different organic dicarboxylic anions. The obtained results imply that DipA does not form a general diffusion pore, but a porin with a binding site specific for dicarboxylates which play important key roles in the deficient metabolic and biosynthetic pathways of Borrelia species.

The presence of porin P66 has been shown in both Lyme disease and relapsing fever spirochetes. In our study, purified P66 homologues from Lyme disease species B. burgdorferi, B. afzelii and B. garinii and relapsing fever species B. duttonii, B. recurrentis and B. hermsii were compared and their biophysical properties were further characterized in black lipid bilayer assay. Subsequently, the channel diameter of B. burgdorferi P66 was investigated in more detail. For this study, different nonelectrolytes with known hydrodynamic radii were used. This allowed us to determine the effective diameter of the P66 channel lumen. Furthermore, the blockage of the channel after addition of nonelectrolytes revealed seven subconducting states and indicated a heptameric structure of the P66 channel. These results may give more insight into the functional properties of this important porin.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2010. , 77 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1340
Keyword [en]
Borrelia, Lume disease, relapsing fever, BesC, drug efflux, DipA, P66, porins
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-32777ISBN: 978-91-7264-971-2 (print)OAI: oai:DiVA.org:umu-32777DiVA: diva2:305826
Public defence
2010-04-16, Betula Lecture Hall, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2010-03-26 Created: 2010-03-25 Last updated: 2010-03-26Bibliographically approved
List of papers
1. An RND-type efflux system in Borrelia burgdorferi is involved in virulence and resistance to antimicrobial compounds
Open this publication in new window or tab >>An RND-type efflux system in Borrelia burgdorferi is involved in virulence and resistance to antimicrobial compounds
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2008 (English)In: PLoS Pathogenicity, ISSN 1553-7374, Vol. 4, no 2, e1000009- p.Article in journal (Refereed) Published
Abstract [en]

Borrelia burgdorferi is remarkable for its ability to thrive in widely different environments due to its ability to infect various organisms. In comparison to enteric Gram-negative bacteria, these spirochetes have only a few transmembrane proteins some of which are thought to play a role in solute and nutrient uptake and excretion of toxic substances. Here, we have identified an outer membrane protein, BesC, which is part of a putative export system comprising the components BesA, BesB and BesC. We show that BesC, a TolC homolog, forms channels in planar lipid bilayers and is involved in antibiotic resistance. A besC knockout was unable to establish infection in mice, signifying the importance of this outer membrane channel in the mammalian host. The biophysical properties of BesC could be explained by a model based on the channel-tunnel structure. We have also generated a structural model of the efflux apparatus showing the putative spatial orientation of BesC with respect to the AcrAB homologs BesAB. We believe that our findings will be helpful in unraveling the pathogenic mechanisms of borreliae as well as in developing novel therapeutic agents aiming to block the function of this secretion apparatus.

Keyword
Amino Acid Sequence, Animals, Anti-Bacterial Agents/pharmacology, Bacterial Outer Membrane Proteins/genetics/*metabolism, Bacterial Proteins/genetics/*metabolism, Borrelia burgdorferi/genetics/metabolism/*pathogenicity, DNA; Bacterial, Drug Resistance; Microbial/*physiology, Gene Expression Regulation; Bacterial/genetics, Gene Silencing, Ion Channels/drug effects/metabolism, Membrane Transport Proteins/genetics/*metabolism, Mice, Mice; Inbred C3H, Microbial Sensitivity Tests, Models; Molecular, Molecular Sequence Data, Organisms; Genetically Modified, RNA; Messenger/metabolism, Virulence
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-10115 (URN)18389081 (PubMedID)
Available from: 2008-06-17 Created: 2008-06-17 Last updated: 2011-10-26Bibliographically approved
2. Oms38 is the first identified pore-forming protein in the outer membrane of relapsing fever spirochetes
Open this publication in new window or tab >>Oms38 is the first identified pore-forming protein in the outer membrane of relapsing fever spirochetes
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2008 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 190, no 21, 7035-7042 p.Article in journal (Refereed) Published
Abstract [en]

Relapsing fever is a worldwide, endemic disease caused by several spirochetal species belonging to the genus Borrelia. During the recurring fever peaks, borreliae proliferate remarkably quickly compared to the slow dissemination of Lyme disease Borrelia and therefore require efficient nutrient uptake from the blood of their hosts. This study describes the identification and characterization of the first relapsing fever porin, which is present in the outer membranes of B. duttonii, B. hermsii, B. recurrentis, and B. turicatae. The pore-forming protein was purified by hydroxyapatite chromatography and designated Oms38, for outer membrane-spanning protein of 38 kDa. Biophysical characterization of Oms38 was done by using the black lipid bilayer method, demonstrating that Oms38 forms small, water-filled channels of 80 pS in 1 M KCl that did not exhibit voltage-dependent closure. The Oms38 channel is slightly selective for anions and shows a ratio of permeability for cations over anions of 0.41 in KCl. Analysis of the deduced amino acid sequences demonstrated that Oms38 contains an N-terminal signal sequence which is processed under in vivo conditions. Oms38 is highly conserved within the four studied relapsing fever species, sharing an overall amino acid identity of 58% and with a strong indication for the presence of amphipathic beta-sheets.

National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-20788 (URN)10.1128/JB.00818-08 (DOI)18757545 (PubMedID)
Available from: 2009-03-25 Created: 2009-03-25 Last updated: 2013-11-20Bibliographically approved
3. DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates
Open this publication in new window or tab >>DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Lyme disease Borrelia are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borrelia. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2‑oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.

Keyword
Lyme disease, spirochete, outer membrane, black lipid bilayer, porin
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-32781 (URN)
Note
Authors two and three contributed equally to the paperAvailable from: 2010-03-25 Created: 2010-03-25 Last updated: 2010-03-26Bibliographically approved
4. P66 porins are present in both Lyme disease and relapsing fever spirochetes: a comparison of the biophysical properties of P66 porins from six Borrelia species
Open this publication in new window or tab >>P66 porins are present in both Lyme disease and relapsing fever spirochetes: a comparison of the biophysical properties of P66 porins from six Borrelia species
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2010 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1798, no 6, 1197-1203 p.Article in journal (Refereed) Published
Abstract [en]

The genus Borrelia is the cause of the two human diseases: Lyme disease (LD) and relapsing fever (RF). BothLD and RF Borrelia species are obligate parasites and are dependent on nutrients provided by their hosts. Thefirst step of nutrient uptake across the outer membrane of these Gram-negative bacteria is accomplished bywater-filled channels, so-called porins. The knowledge of the porin composition in the outer membranes ofthe different pathogenic Borrelia species is limited. Only one porin has been described in relapsing feverspirochetes to date, whereas four porins are known to be present in Lyme disease agents. From these, theBorrelia burgdorferi outer membrane channel P66 is known to act as an adhesin and was well studied as aporin. To investigate if P66 porins are expressed and similarly capable of pore formation in other Borreliacausing Lyme disease or relapsing fever three LD species (B. burgdorferi, B. afzelii, B. garinii) and three RFspecies (B. duttonii, B. recurrentis and B. hermsii) were investigated for outer membrane proteins homologousto P66. A search in current published RF genomes, comprising the ones of B. duttonii, B. recurrentis and B.hermsii, indicated that they all contained P66 homologues. The P66 homologues of the six Borrelia specieswere purified to homogeneity and their pore-forming abilities as well as the biophysical properties of thepores were analyzed using the black lipid bilayer assay.

Place, publisher, year, edition, pages
Elsevier, 2010
Keyword
Borrelia, P66, porin, outer membrane
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-32771 (URN)10.1016/j.bbamem.2010.02.011 (DOI)000278321200021 ()20188698 (PubMedID)
Available from: 2010-03-25 Created: 2010-03-25 Last updated: 2012-05-15Bibliographically approved
5. Use of nonelectrolytes reveals the channel size and oligomeric constitution of the Borrelia burgdorferi P66 porin
Open this publication in new window or tab >>Use of nonelectrolytes reveals the channel size and oligomeric constitution of the Borrelia burgdorferi P66 porin
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The outer membrane protein P66 of the Lyme disease spirochete Borrelia burgdorferi is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl. We studied in a non-theoretical manner the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. Furthermore, we calculated the filling of the channel with these nonelectrolytes and the results revealed that nonelectrolytes with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. Thus, the diameter of the P66 entrance was determined to be ≤ 1.9 nm with a constriction site diameter of about 0.7 nm. Furthermore, the P66-induced membrane conductance could be blocked by 80-90% after addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose in the low millimolar range. Interestingly, the analysis of the power density spectra of P66 after blockage with nonelectrolytes revealed no chemical interaction responsible for channel block. The blockage of one P66 single-channel conductance unit of 11 nS occurred by seven subconducting states, thus indicating a heptameric organization of the P66 oligomer. This organization of P66 as a heptamer was confirmed by Blue Native PAGE and immunoblot analysis, which demonstrated that P66 forms a complex with a mass of approximately 460 kDa.

Keyword
nutrient uptake, outer membrane transport, Lyme disease, porin radius determination, black lipid bilayer
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-32783 (URN)
Note

Ignas Bunikis är ej medförfattare till den publicerade versionen i PLoS ONE 2013, 8:11, Article Number: e78272, DOI: 10.1371/journal.pone.0078272

Ignas Bunikis is not co-author in the published version in PLoS ONE 2013, 8:11, Article Number: e78272, DOI: 10.1371/journal.pone.0078272

Available from: 2010-03-25 Created: 2010-03-25 Last updated: 2013-12-11Bibliographically approved

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