Quinolones have been very useful antimicrobial agents because they are highly potent, active against a wide range of bacteria, and relatively non-toxic. Their broad use, however, has been followed by rising rates of resistance. Quinolone resistance has traditionally been understood to arise either by mutations that alter DNA gyrase and topoisomerase IV, enzymes that are the targets for quinolone action, or by mutations that increase expression of efflux pumps that actively eliminate the agents from the cell. Neither type of resistance has been transmissible since both are due to mutations on the bacterial chromosome. Hence, it came as a surprise when plasmid-mediated quinolone resistance was discovered. Three distinct mechanisms for such resistance are known: target protection by pentapeptide repeat proteins of the QnrA, QnrB, and QnrS families that may act in part as DNA mimics, quinolone inactivation by mutant aminoglycoside 6'N-acetyltransferase [Aac(6')-Ib- cr], and provision of new systems for quinolone efflux. Each mechanism confers low-level resistance but facilitates selection of higher level, clinically significant resistance. Although plasmid-mediated quinolone resistance was discovered only 11 years ago, subsequent studies have shown the genes to be broadly distributed in gram-negative bacteria from around the world and to be typically incorporated into integrons on multiresistance plasmids. This resubmission application builds on our prior studies to obtain a deeper and more detailed understanding of the resistance due to Qnr proteins.
Under Specific Aim 1, we propose to identify essential regions and amino acid residues in QnrB1 via alanine-scanning mutagenesis and deletion analysis. Cloned mutant genes will be screened for ability to confer quinolone resistance and to inhibit bacterial growth. Candidate mutant proteins will be overexpressed, purified, and tested for protection and inhibition of purified gyrase and ability to block DNA binding to gyrase.
Under Specific Aim 2, we propose to evaluate the native functions of qnrA, qnrB, and qnrS. We have found a LexA recognition sequence upstream from plasmid-mediated qnrB alleles and have shown that qnrB expression is under SOS control. In Shewanella algae, a reservoir of qnrA, we have further found cold shock to trigger qnrA expression, and we propose to test further conditions of expression in S. algae, Vibrio splendidus, a reservoir of qnrS-like genes, Stenotrophomonas maltophilia, a reservoir of qnrB-like genes, and we will determine the effect of quinolones and other DNA damaging agents, such as ultraviolet light (as well as other conditions of environmental stress) on qnr expression. We will also directly test the hypothesis that Qnr proteins protect against the natural gyrase-targeting toxin microcin B17. In addition we will screen for proteins other than gyrase that interact with Qnr by use of bacterial and yeast two-hybrid systems.
Under Specific Aim 3, we propose to explore Qnr/gyrase interaction as revealed by isothermal titration calorimetry or surface plasmon resonance and by x- ray crystallography.

Public Health Relevance

Quinolones are widely used antimicrobial agents that have been compromised by bacterial resistance, which was originally thought only to occur from chromosomal mutation. Plasmid-encoded transferable resistance has now been shown to have emerged and spread to many gram-negative human pathogens and to have a diversity of mechanisms, apparently co-opting chromosomal proteins that interact with topoisomerases, the quinolone target enzymes. Thus, understanding of these mechanisms of resistance and how the genes encoding them have been mobilized and modified to confer resistance is of importance for public health and for understanding of the range of bacterial adaptation strategies.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Special Emphasis Panel (ZRG1-IDM-R (02))
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Huntley, Clayton C
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Massachusetts General Hospital
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Tavío, María M; Jacoby, George A; Hooper, David C (2014) QnrS1 structure-activity relationships. J Antimicrob Chemother 69:2102-9
Liao, Chun-Hsing; Hsueh, Po-Ren; Jacoby, George A et al. (2013) Risk factors and clinical characteristics of patients with qnr-positive Klebsiella pneumoniae bacteraemia. J Antimicrob Chemother 68:2907-14
Jacoby, George A; Corcoran, Marian A; Mills, Debra M et al. (2013) Mutational analysis of quinolone resistance protein QnrB1. Antimicrob Agents Chemother 57:5733-6
Jacoby, George A; Hooper, David C (2013) Phylogenetic analysis of chromosomally determined qnr and related proteins. Antimicrob Agents Chemother 57:1930-4
Kwak, Yee Gyung; Jacoby, George A; Hooper, David C (2013) Induction of plasmid-carried qnrS1 in Escherichia coli by naturally occurring quinolones and quorum-sensing signal molecules. Antimicrob Agents Chemother 57:4031-4
Okumura, Ryo; Liao, Chun-Hsing; Gavin, Mariah et al. (2011) Quinolone induction of qnrVS1 in Vibrio splendidus and plasmid-carried qnrS1 in Escherichia coli, a mechanism independent of the SOS system. Antimicrob Agents Chemother 55:5942-5
Kim, Hong Bin; Park, Chi Hye; Gavin, Mariah et al. (2011) Cold shock induces qnrA expression in Shewanella algae. Antimicrob Agents Chemother 55:414-6
Vetting, Matthew W; Hegde, Subray S; Wang, Minghua et al. (2011) Structure of QnrB1, a plasmid-mediated fluoroquinolone resistance factor. J Biol Chem 286:25265-73
Jacoby, George A; Griffin, Caitlin M; Hooper, David C (2011) Citrobacter spp. as a source of qnrB Alleles. Antimicrob Agents Chemother 55:4979-84
Kim, Hong Bin; Wang, Minghua; Ahmed, Sabeena et al. (2010) Transferable quinolone resistance in Vibrio cholerae. Antimicrob Agents Chemother 54:799-803

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