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An RND-type efflux system in Borrelia burgdorferi is involved in virulence and resistance to antimicrobial compounds.

Bunikis I, Denker K, Ostberg Y, Andersen C, Benz R, Bergström S - PLoS Pathog. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, Umeå, Sweden.

ABSTRACT
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.

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Channel-forming activities of FPLC purified BesC.(A) Single-channel recording of a diphytanoyl phosphatidylcholine/n-decane membrane observed in the presence of 100 ng/ml of fraction 35 of the MonoQ-FPLC of the B-fraction of B. burgdorferi B31-A p66::str. The aqueous phase contained 1 M KCl; Vm  =  20 mV; T  =  20°C. (B) Histogram of the probability P(G) for the occurrence of a given conductivity unit observed with membranes formed of 1% diphytanoyl phosphatidylcholine/n-decane in the presence of fraction 35 of the MonoQ-FPLC of the B-fraction of B. burgdorferi B31-A p66::str. P(G) is the probability that a given conductance increment G is observed in the single-channel experiments. It was calculated by dividing the number of fluctuations with a given conductance increment by the total number of conductance fluctuations. The average single-channel conductance for 122 single-channel events was 300 pS.
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ppat-1000009-g003: Channel-forming activities of FPLC purified BesC.(A) Single-channel recording of a diphytanoyl phosphatidylcholine/n-decane membrane observed in the presence of 100 ng/ml of fraction 35 of the MonoQ-FPLC of the B-fraction of B. burgdorferi B31-A p66::str. The aqueous phase contained 1 M KCl; Vm  =  20 mV; T  =  20°C. (B) Histogram of the probability P(G) for the occurrence of a given conductivity unit observed with membranes formed of 1% diphytanoyl phosphatidylcholine/n-decane in the presence of fraction 35 of the MonoQ-FPLC of the B-fraction of B. burgdorferi B31-A p66::str. P(G) is the probability that a given conductance increment G is observed in the single-channel experiments. It was calculated by dividing the number of fluctuations with a given conductance increment by the total number of conductance fluctuations. The average single-channel conductance for 122 single-channel events was 300 pS.

Mentions: To analyze the channels formed by BesC, single-channel experiments were performed with purified protein. FPLC fractions containing BesC resulted in a step-wise increase of the membrane conductance in a lipid bilayer assay. Figure 3A shows a single-channel recording of a lipid bilayer membrane in the presence of a very low concentration of BesC at a membrane voltage of 20 mV. Figure 3B shows a histogram of 122 conductance steps observed in single-channel experiments with BesC-containing FPLC fractions. This protein forms obvious channels with an average single-channel conductance of 300 pS in 1 M KCl. Interestingly the 300 pS pore did not display any voltage dependence even at voltages as high as ± 150 mV (data not shown).


An RND-type efflux system in Borrelia burgdorferi is involved in virulence and resistance to antimicrobial compounds.

Bunikis I, Denker K, Ostberg Y, Andersen C, Benz R, Bergström S - PLoS Pathog. (2008)

Channel-forming activities of FPLC purified BesC.(A) Single-channel recording of a diphytanoyl phosphatidylcholine/n-decane membrane observed in the presence of 100 ng/ml of fraction 35 of the MonoQ-FPLC of the B-fraction of B. burgdorferi B31-A p66::str. The aqueous phase contained 1 M KCl; Vm  =  20 mV; T  =  20°C. (B) Histogram of the probability P(G) for the occurrence of a given conductivity unit observed with membranes formed of 1% diphytanoyl phosphatidylcholine/n-decane in the presence of fraction 35 of the MonoQ-FPLC of the B-fraction of B. burgdorferi B31-A p66::str. P(G) is the probability that a given conductance increment G is observed in the single-channel experiments. It was calculated by dividing the number of fluctuations with a given conductance increment by the total number of conductance fluctuations. The average single-channel conductance for 122 single-channel events was 300 pS.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279261&req=5

ppat-1000009-g003: Channel-forming activities of FPLC purified BesC.(A) Single-channel recording of a diphytanoyl phosphatidylcholine/n-decane membrane observed in the presence of 100 ng/ml of fraction 35 of the MonoQ-FPLC of the B-fraction of B. burgdorferi B31-A p66::str. The aqueous phase contained 1 M KCl; Vm  =  20 mV; T  =  20°C. (B) Histogram of the probability P(G) for the occurrence of a given conductivity unit observed with membranes formed of 1% diphytanoyl phosphatidylcholine/n-decane in the presence of fraction 35 of the MonoQ-FPLC of the B-fraction of B. burgdorferi B31-A p66::str. P(G) is the probability that a given conductance increment G is observed in the single-channel experiments. It was calculated by dividing the number of fluctuations with a given conductance increment by the total number of conductance fluctuations. The average single-channel conductance for 122 single-channel events was 300 pS.
Mentions: To analyze the channels formed by BesC, single-channel experiments were performed with purified protein. FPLC fractions containing BesC resulted in a step-wise increase of the membrane conductance in a lipid bilayer assay. Figure 3A shows a single-channel recording of a lipid bilayer membrane in the presence of a very low concentration of BesC at a membrane voltage of 20 mV. Figure 3B shows a histogram of 122 conductance steps observed in single-channel experiments with BesC-containing FPLC fractions. This protein forms obvious channels with an average single-channel conductance of 300 pS in 1 M KCl. Interestingly the 300 pS pore did not display any voltage dependence even at voltages as high as ± 150 mV (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, Umeå, Sweden.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus