Structure of QnrB1, a plasmid-mediated fluoroquinolone resistance factor.
ABSTRACT: QnrB1 is a plasmid-encoded pentapeptide repeat protein (PRP) that confers a moderate degree of resistance to fluoroquinolones. Its gene was cloned into an expression vector with an N-terminal polyhistidine tag, and the protein was purified by nickel affinity chromatography. The structure of QnrB1 was determined by a combination of trypsinolysis, surface mutagenesis, and single anomalous dispersion phasing. QnrB1 folds as a right-handed quadrilateral ?-helix with a highly asymmetric dimeric structure typical of PRP-topoisomerase poison resistance factors. The threading of pentapeptides into the ?-helical fold is interrupted by two noncanonical PRP sequences that produce outward projecting loops that interrupt the regularity of the PRP surface. Deletion of the larger upper loop eliminated the protective effect of QnrB1 on DNA gyrase toward inhibition by quinolones, whereas deletion of the smaller lower loop drastically reduced the protective effect. These loops are conserved among all plasmid-based Qnr variants (QnrA, QnrC, QnrD, and QnrS) and some chromosomally encoded Qnr varieties. A mechanism in which PRP-topoisomerase poison resistance factors bind to and disrupt the quinolone-DNA-gyrase interaction is proposed.
Project description:Since the discovery of qnrA in 1998, two additional qnr genes, qnrB and qnrS, have been described. These three plasmid-mediated genes contribute to quinolone resistance in gram-negative pathogens worldwide. A clinical strain of Proteus mirabilis was isolated from an outpatient with a urinary tract infection and was susceptible to most antimicrobials but resistant to ampicillin, sulfamethoxazole, and trimethoprim. Plasmid pHS10, harbored by this strain, was transferred to azide-resistant Escherichia coli J53 by conjugation. A transconjugant with pHS10 had low-level quinolone resistance but was negative by PCR for the known qnr genes, aac(6')-Ib-cr and qepA. The ciprofloxacin MIC for the clinical strain and a J53/pHS10 transconjugant was 0.25 microg/ml, representing an increase of 32-fold relative to that for the recipient, J53. The plasmid was digested with HindIII, and a 4.4-kb DNA fragment containing the new gene was cloned into pUC18 and transformed into E. coli TOP10. Sequencing showed that the responsible 666-bp gene, designated qnrC, encoded a 221-amino-acid protein, QnrC, which shared 64%, 42%, 59%, and 43% amino acid identity with QnrA1, QnrB1, QnrS1, and QnrD, respectively. Upstream of qnrC there existed a new IS3 family insertion sequence, ISPmi1, which encoded a frameshifted transposase. qnrC could not be detected by PCR, however, in 2,020 strains of Enterobacteriaceae. A new quinolone resistance gene, qnrC, was thus characterized from plasmid pHS10 carried by a clinical isolate of P. mirabilis.
Project description:Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. The qnr genes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6')-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat.
Project description:DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of 'poisons', namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We show that a PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis by gyrase. QnrB1 binds to the GyrB protein and stimulates ATPase activity of the isolated N-terminal ATPase domain of GyrB (GyrB43). We probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped the crosslinks to the GyrB43 protein. We propose a model in which QnrB1 binding allosterically promotes dissociation of the fluoroquinolone molecule from the cleavage complex.
Project description:Quinolones inhibit bacterial type II DNA topoisomerases (e.g. DNA gyrase) and are among the most important antibiotics in current use. However, their efficacy is now being threatened by various plasmid-mediated resistance determinants. Of these, the pentapeptide repeat-containing (PRP) Qnr proteins are believed to act as DNA mimics and are particularly prevalent in gram-negative bacteria. Predicted Qnr-like proteins are also present in numerous environmental bacteria. Here, we demonstrate that one such, Aeromonas hydrophila AhQnr, is soluble, stable, and relieves quinolone inhibition of Escherichia coli DNA gyrase, thus providing an appropriate model system for gram-negative Qnr proteins. The AhQnr crystal structure, the first for any gram-negative Qnr, reveals two prominent loops (1 and 2) that project from the PRP structure. Deletion mutagenesis demonstrates that both contribute to protection of E. coli DNA gyrase from quinolones. Sequence comparisons indicate that these are likely to be present across the full range of gram-negative Qnr proteins. On this basis we present a model for the AhQnr:DNA gyrase interaction where loop1 interacts with the gyrase A 'tower' and loop2 with the gyrase B TOPRIM domains. We propose this to be a general mechanism directing the interactions of Qnr proteins with DNA gyrase in gram-negative bacteria.
Project description:Plasmid-mediated qnr genes have been reported in bacteria worldwide and are widely associated with other relevant determinants of resistance in multiresistance plasmids. Here, we provide an update on a previously described multiplex PCR in order to detect all six qnr families (including qnrA, qnrS, qnrB, qnrC, qnrD, and qnrVC) described until now. The proposed method makes possible the screening of these genes, reducing cost and time, and it may demonstrate an underestimated prevalence of the latest variants described.
Project description:BACKGROUND: The first report on the transferable, plasmid-mediated quinolone-resistance determinant qnrA1 was in 1998. Since then, qnr alleles have been discovered worldwide in clinical strains of Gram-negative bacilli. Qnr proteins confer quinolone resistance, and belong to the pentapeptide repeat protein (PRP) family. Several PRP crystal structures have been solved, but little is known about the functional significance of their structural arrangement. RESULTS: We conducted random and site-directed mutagenesis on qnrA1 and on qnrC, a newly identified quinolone-resistance gene from Proteus mirabilis. Many of the Qnr mutants lost their quinolone resistance function. The highly conserved hydrophobic Leu or Phe residues at the center of the pentapeptide repeats are known as i sites, and loss-of-function mutations included replacement of the i site hydrophobic residues with charged residues, replacing the i-2 site, N-terminal to the i residues, with bulky side-chain residues, introducing Pro into the ?-helix coil, deletion of the N- and C-termini, and excision of a central coil. Molecular dynamics simulations and homology modeling demonstrated that QnrC overall adopts a stable ?-helix fold and shares more similarities with MfpA than with other PRP structures. Based on homology modeling and molecular dynamics simulation, the dysfunctional point mutations introduced structural deformations into the quadrilateral ?-helix structure of PRPs. Of the pentapeptides of QnrC, two-thirds adopted a type II ?-turn, while the rest adopted type IV turns. A gap exists between coil 2 and coil 3 in the QnrC model structure, introducing a structural flexibility that is similar to that seen in MfpA. CONCLUSION: The hydrophobic core and the ?-helix backbone conformation are important for maintaining the quinolone resistance property of Qnr proteins. QnrC may share structural similarity with MfpA.
Project description:In a previous study, four Salmonella isolates from humans in the Henan province of China showed reduced susceptibility to ciprofloxacin (MIC, 0.125 to 0.25 microg/ml) but were susceptible to nalidixic acid (MIC, 4 to 8 microg/ml). All isolates were negative for known qnr genes (A, B, and S), aac(6')Ib-cr, and mutations in gyrA and parC. Plasmid DNA was extracted from all four isolates and transformed into Escherichia coli TG1 and DH10B cells by electroporation, and transformants were selected on 0.06 microg/ml ciprofloxacin containing brain heart infusion agar plates. Resistance to ciprofloxacin could be transferred by electroporation, and a similar 4,270-bp plasmid was found in all transformants. By sequence analysis, the plasmid was found to carry an open reading frame that had similarities to other qnr genes and that encoded a 214-amino-acid pentapeptide repeat protein. This gene, designated qnrD, showed 48% similarity to qnrA1, 61% similarity to qnrB1, and 41% similarity to qnrS1. Further subcloning of the qnrD coding region into the constitutively expressed tetA gene of vector pBR322 showed that the gene conferred an increase in the MIC of ciprofloxacin by a factor of 32 (from an MIC of 0.002 to an MIC of 0.06 microg/ml). For comparison, qnrA1 and qnrS1 were also subcloned into pBR322 and transformed into DH10B cells, conferring MICs of 0.125 and 0.5 microg/ml, respectively. A phylogenetic analysis of all known qnr sequences was performed and showed that qnrD was more closely related to the qnrB variants but formed an independent cluster. To our knowledge, this is the first description of this qnrD gene.
Project description:Plasmid-encoded protein QnrB1 protects DNA gyrase from ciprofloxacin inhibition. Using a bacterial two-hybrid system, we evaluated the physical interactions between wild-type and mutant QnrB1, the GyrA and GyrB gyrase subunits, and a GyrBA fusion protein. The interaction of QnrB1 with GyrB and GyrBA was approximately 10-fold higher than that with GyrA, suggesting that domains of GyrB are important for stabilizing QnrB1 interaction with the holoenzyme. Sub-MICs of ciprofloxacin or nalidixic acid reduced the interactions between QnrB1 and GyrA or GyrBA but produced no reduction in the interaction with GyrB or a quinolone-resistant GyrA:S83L (GyrA with S83L substitution) mutant, suggesting that quinolones and QnrB1 compete for binding to gyrase. Of QnrB1 mutants that reduced quinolone resistance, deletions in the C or N terminus of QnrB1 resulted in a marked decrease in interactions with GyrA but limited or no effect on interactions with GyrB and an intermediate effect on interactions with GyrBA. While deletion of loop B and both loops moderately reduced the interaction signal with GyrA, deletion of loop A resulted in only a small reduction in the interaction with GyrB. The loop A deletion also caused a substantial reduction in interaction with GyrBA, with little effect of loop B and dual-loop deletions. Single-amino-acid loop mutations had little effect on physical interactions except for a ?105I mutant. Therefore, loops A and B may play key roles in the proper positioning of QnrB1 rather than as determinants of the physical interaction of QnrB1 with gyrase.
Project description:Quinolones are potent antibacterial agents that specifically target bacterial DNA gyrase and topoisomerase IV. Widespread use of these agents has contributed to the rise of bacterial quinolone resistance. Previous studies have shown that quinolone resistance arises by mutations in chromosomal genes. Recently, a multiresistance plasmid was discovered that encodes transferable resistance to quinolones. We have cloned the plasmid-quinolone resistance gene, termed qnr, and found it in an integron-like environment upstream from qacE Delta 1 and sulI. The gene product Qnr was a 218-aa protein belonging to the pentapeptide repeat family and shared sequence homology with the immunity protein McbG, which is thought to protect DNA gyrase from the action of microcin B17. Qnr had pentapeptide repeat domains of 11 and 28 tandem copies, separated by a single glycine with a consensus sequence of A/C D/N L/F X X. Because the primary target of quinolones is DNA gyrase in Gram-negative strains, we tested the ability of Qnr to reverse the inhibition of gyrase activity by quinolones. Purified Qnr-His(6) protected Escherichia coli DNA gyrase from inhibition by ciprofloxacin. Gyrase protection was proportional to the concentration of Qnr-His(6) and inversely proportional to the concentration of ciprofloxacin. The protective activity of Qnr-His(6) was lost by boiling the protein and involved neither quinolone inactivation nor independent gyrase activity. Protection of topoisomerase IV, a secondary target of quinolone action in E. coli, was not evident. How Qnr protects DNA gyrase and the prevalence of this resistance mechanism in clinical isolates remains to be determined.
Project description:The qnr genes are plasmid-borne fluoroquinolone-resistance determinants widespread in Enterobacteriaceae. Three families of qnr determinants (qnrA, B and S) have been described, but little is known about their expression and regulation. Two new determinants, qnrC and qnrD, have been found recently. Here, we describe the characterization of the qnrB2 promoter and the identification of a LexA-binding site in the promoter region of all qnrB alleles. LexA is the central regulator of the SOS response to DNA damage. We show that qnrB2 expression is regulated through the SOS response in a LexA/RecA-dependent manner, and that it can be induced by the quinolone ciprofloxacin, a known inducer of the SOS system. This is the first description of direct SOS-dependent regulation of an antibiotic-resistance mechanism in response to the antibiotic itself.