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An RND-type efflux system in Borrelia burgdorferi is involved in virulence and resistance to antimicrobial compounds
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). (Bergström)
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
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2008 (Engelska)Ingår i: PLoS Pathogenicity, ISSN 1553-7374, Vol. 4, nr 2, s. e1000009-Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2008. Vol. 4, nr 2, s. e1000009-
Nyckelord [en]
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
Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-10115PubMedID: 18389081Scopus ID: 2-s2.0-40349106166OAI: oai:DiVA.org:umu-10115DiVA, id: diva2:149786
Tillgänglig från: 2008-06-17 Skapad: 2008-06-17 Senast uppdaterad: 2023-03-24Bibliografiskt granskad
Ingår i avhandling
1. Borrelia channel-forming proteins: structure and function
Öppna denna publikation i ny flik eller fönster >>Borrelia channel-forming proteins: structure and function
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå University, 2010. s. 77
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1340
Nyckelord
Borrelia, Lume disease, relapsing fever, BesC, drug efflux, DipA, P66, porins
Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-32777 (URN)978-91-7264-971-2 (ISBN)
Disputation
2010-04-16, Betula Lecture Hall, Umeå University, Umeå, 10:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2010-03-26 Skapad: 2010-03-25 Senast uppdaterad: 2018-01-12Bibliografiskt granskad

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Bunikis, IgnasÖstberg, YngveBergström, Sven

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