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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
University of Würzburg.
Max Planck Institute for Developmental Biology.
German Cancer Research Center.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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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. Vol. 1798, no 6, 1197-1203 p.
Keyword [en]
Borrelia, P66, porin, outer membrane
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
URN: urn:nbn:se:umu:diva-32771DOI: 10.1016/j.bbamem.2010.02.011ISI: 000278321200021PubMedID: 20188698OAI: diva2:305726
Available from: 2010-03-25 Created: 2010-03-25 Last updated: 2012-05-15Bibliographically approved
In thesis
1. Borrelia channel-forming proteins: structure and function
Open this publication in new window or tab >>Borrelia channel-forming proteins: structure and function
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.
Umeå University medical dissertations, ISSN 0346-6612 ; 1340
Borrelia, Lume disease, relapsing fever, BesC, drug efflux, DipA, P66, porins
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
urn:nbn:se:umu:diva-32777 (URN)978-91-7264-971-2 (ISBN)
Public defence
2010-04-16, Betula Lecture Hall, Umeå University, Umeå, 10:00 (English)
Available from: 2010-03-26 Created: 2010-03-25 Last updated: 2010-03-26Bibliographically approved

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Bunikis, IgnasBonde, MariBergström, Sven
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