An RND-Type Efflux System in Borrelia burgdorferi Is Involved in Virulence and Resistance to Antimicrobial Compounds

Department of Molecular Biology, Umeå University, Umeå, Sweden.
PLoS Pathogens (Impact Factor: 7.56). 03/2008; 4(2):e1000009. DOI: 10.1371/journal.ppat.1000009
Source: PubMed


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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    • "This conforms to the view they play a key role in pump assembly and function, particularly in binding to the large periplasmic extension of the IM transporters. As would be expected in this case, primary sequence analysis indicates the cognate interacting transporter domains of BesB and other transporters are likewise structurally conserved (Supplemental Table 2) [16]. In both the data-based assembly model of AcrAB-TolC [7] and the in vitro co-crystal structure of an adaptor-transporter (CusBA) subcomplex [35] tripartite pump assembly is facilitated by the flexibility of the adaptor's serially-linked domains [11] [13], which was confirmed by MD analysis of the closely related MexA [36]. "
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    ABSTRACT: Periplasmic adaptor proteins are essential components of bacterial tripartite multidrug efflux pumps. Here we report the 2.35Å resolution crystal structure of the BesA adaptor from the spirochete Borrelia burgdorferi solved using selenomethionine derivatized protein. BesA shows the archetypal linear, flexible, multi-domain architecture evident among proteobacteria and retains the lipoyl, β-barrel and membrane-proximal domains that interact with the periplasmic domains of the inner membrane transporter. However, it lacks the α-hairpin domain shown to establish extensive coiled-coil interactions with the periplasmic entrance helices of the outer membrane-anchored TolC exit duct. This has implications for the modelling of assembled tripartite efflux pumps.
    FEBS letters 07/2013; 587(18). DOI:10.1016/j.febslet.2013.06.056 · 3.17 Impact Factor
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    • "Conceivably, the second hairpin only engages with MtrE after it has bound to MtrD, to stabilize the MtrE channel in the open conformation. The role of the hairpin in unlocking of the aspartate gate can also be implied by comparative studies of the Borellia efflux pump BesABC (Bunikis et al., 2008), where the MFP BesA uncharacteristically lacks a hairpin. It is notable however that the corresponding second selectivity gate in the OMP BesC is also not present. "
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    ABSTRACT: The MtrCDE multidrug pump, from Neisseria gonorrhoeae, is assembled from the inner and outer membrane proteins MtrD and MtrE, which are connected by the periplasmic membrane fusion protein MtrC. Although it is clear that MtrD delivers drugs to the channel of MtrE, it remains unclear how drug delivery and channel opening are connected. We used a vancomycin sensitivity assay to test for opening of the MtrE channel. Cells expressing MtrE or MtrE-E434K were insensitive to vancomycin; but became moderately and highly sensitive to vancomycin respectively, when coexpressed with MtrC, suggesting that the MtrE channel opening requires MtrC binding and is energy-independent. Cells expressing wild-type MtrD, in an MtrCE background, were vancomycin-insensitive, but moderately sensitive in an MtrCE-E434K background. The mutation of residues involved in proton translocation inactivated MtrD and abolished drug efflux, rendered both MtrE and MtrE-E434K vancomycin-insensitive; imply that the pump-component interactions are preserved, and that the complex is stable in the absence of proton flux, thus sealing the open end of MtrE. Following the energy-dependent dissociation of the tripartite complex, the MtrE channel is able to reseal, while MtrE-E434K is unable to do so, resulting in the vancomycin-sensitive phenotype. Thus, our findings suggest that opening of the OMP via interaction with the MFP is energy-independent, while both drug export and complex dissociation require active proton flux.
    Molecular Microbiology 04/2013; 88(3). DOI:10.1111/mmi.12211 · 4.42 Impact Factor
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    • "Another protein, formerly identified as porin Oms28 [45] was shown not to be a porin as it is localized in the periplasmic space [46]. Besides the porins, the channel-tunnel BesC, a TolC-homologue, which is a component of the Borrelia multi-drug-efflux systems, was identified in the outer membrane, forming channels of 300 pS in 1 M KCl [47]. "
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