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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: 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.

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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Related in: MedlinePlus

Characterization of the besC mutant strains.(A) Schematic representation of besC, becA and besB genes in the Borrelia burgdorferi B31 chromosome, insertion of the aadA gene cassette by homologous recombination and complementation plasmid. Arrows indicate the relative positions of the oligonucleotides used. The diagram is not drawn to scale. (B) PCR analysis of the wild-type strain, the resulting besC mutant and the complemented strain using primer pairs specific either only for the besC and aadA genes; or one primer specific for besA and the other for aadA gene. The PCR was also used to confirm the presence of the shuttle vector in the complemented strain using one primer specific for besC and another specific for the shuttle vector. (C) Immunoblotting using antiserum raised against BesA, BesB and BesC to determine the presence or absence of these proteins in wild-type, besC mutant and the complemented strain.
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ppat-1000009-g002: Characterization of the besC mutant strains.(A) Schematic representation of besC, becA and besB genes in the Borrelia burgdorferi B31 chromosome, insertion of the aadA gene cassette by homologous recombination and complementation plasmid. Arrows indicate the relative positions of the oligonucleotides used. The diagram is not drawn to scale. (B) PCR analysis of the wild-type strain, the resulting besC mutant and the complemented strain using primer pairs specific either only for the besC and aadA genes; or one primer specific for besA and the other for aadA gene. The PCR was also used to confirm the presence of the shuttle vector in the complemented strain using one primer specific for besC and another specific for the shuttle vector. (C) Immunoblotting using antiserum raised against BesA, BesB and BesC to determine the presence or absence of these proteins in wild-type, besC mutant and the complemented strain.

Mentions: In order to investigate the potential involvement of BesC in antibiotic resistance and virulence of B. burgdorferi, we constructed a besC mutant. A streptomycin resistance gene was inserted into the besC gene (Figure 2A) of low-passage infectious B. burgdorferi strain 5A4NP1 by electroporation with plasmid pOK-besC::str as described in Materials and Methods. Plasmid pCOMP (Figure 2A) was used to complement besC mutant. Strains were analyzed by PCR to confirm inactivation and complementation of besC (Figure 2B).


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)

Characterization of the besC mutant strains.(A) Schematic representation of besC, becA and besB genes in the Borrelia burgdorferi B31 chromosome, insertion of the aadA gene cassette by homologous recombination and complementation plasmid. Arrows indicate the relative positions of the oligonucleotides used. The diagram is not drawn to scale. (B) PCR analysis of the wild-type strain, the resulting besC mutant and the complemented strain using primer pairs specific either only for the besC and aadA genes; or one primer specific for besA and the other for aadA gene. The PCR was also used to confirm the presence of the shuttle vector in the complemented strain using one primer specific for besC and another specific for the shuttle vector. (C) Immunoblotting using antiserum raised against BesA, BesB and BesC to determine the presence or absence of these proteins in wild-type, besC mutant and the complemented strain.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000009-g002: Characterization of the besC mutant strains.(A) Schematic representation of besC, becA and besB genes in the Borrelia burgdorferi B31 chromosome, insertion of the aadA gene cassette by homologous recombination and complementation plasmid. Arrows indicate the relative positions of the oligonucleotides used. The diagram is not drawn to scale. (B) PCR analysis of the wild-type strain, the resulting besC mutant and the complemented strain using primer pairs specific either only for the besC and aadA genes; or one primer specific for besA and the other for aadA gene. The PCR was also used to confirm the presence of the shuttle vector in the complemented strain using one primer specific for besC and another specific for the shuttle vector. (C) Immunoblotting using antiserum raised against BesA, BesB and BesC to determine the presence or absence of these proteins in wild-type, besC mutant and the complemented strain.
Mentions: In order to investigate the potential involvement of BesC in antibiotic resistance and virulence of B. burgdorferi, we constructed a besC mutant. A streptomycin resistance gene was inserted into the besC gene (Figure 2A) of low-passage infectious B. burgdorferi strain 5A4NP1 by electroporation with plasmid pOK-besC::str as described in Materials and Methods. Plasmid pCOMP (Figure 2A) was used to complement besC mutant. Strains were analyzed by PCR to confirm inactivation and complementation of besC (Figure 2B).

Bottom Line: 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.

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