Importance of Real-time Assays to Distinguish Multidrug Efflux Pump ...

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May 11, 2015 - Efflux Pump Inhibiting and Outer Membrane. 2 ... need for implementation of better antimicrobial stewardship, discovery of new antibiotics and. 53 ... development of novel antibiotics based on either an existing or novel chemical scaffold,. 64 ... AcrB is the inner membrane, trimeric drug-proton antiporter. 74.
JB Accepted Manuscript Posted Online 11 May 2015 J. Bacteriol. doi:10.1128/JB.02456-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Misra et al

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Importance of Real-time Assays to Distinguish Multidrug

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Efflux Pump Inhibiting and Outer Membrane

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Destabilizing Activities in Escherichia coli

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Rajeev Misra1*, Keith D. Morrison2, Hyun Jae Cho1, and Thanh Khuu1

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School of Life Sciences, Arizona State University, Tempe, Arizona 85287, USA

School of Earth and Space Exploration, Arizona State University, Tempe, Arizona 85287, USA

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Running title: Inhibition of multidrug efflux pumps

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Key word: multidrug resistance, RND-type efflux pumps, real-time efflux assays, efflux pump

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inhibitors

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*Corresponding author. Mailing address: School of Life Sciences, Arizona State University, 427 East Tyler Mall, Tempe, AZ 85287-4501, U.S.A. Phone: (480) 965-3320 Fax: (480) 965-6899 E-mail: [email protected]

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ABSTRACT

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The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high

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degree of homology with the normally silent AcrEF system. Exposure of a strain deleted for

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acrAB to antibiotic selection pressure frequently leads to the insertion sequence-mediated

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activation of the homologous AcrEF system. In this study, we used strains constitutively

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expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a

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controversy of whether phenylalanylarginine β-naphthylamide (PAβN) inhibits the activities of

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AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer

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membrane permeability barrier. Real-time efflux assays allowed for a clear distinction between

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the efflux pump-inhibiting activity of PAβN and outer membrane-destabilizing action of

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polymyxin B nonapeptide (PMXBN). When added at equal amounts, PAβN but not PMXBN

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strongly inhibited efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer

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membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a

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much greater damaging effect than PAβN. A strong action of PAβN in inhibiting efflux activity

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compared to its weak action on destabilizing the outer membrane permeability barrier suggests

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that PAβN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations,

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PAβN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high

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concentrations, PAβN in the efflux-proficient background not only inhibits the efflux pump

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activity but also destabilizes the membrane. The effects of PAβN on membrane integrity are

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compounded in cells unable to extrude PAβN.

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Importance

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The increase in multidrug resistant bacterial pathogens at an alarming rate has accelerated the

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need for implementation of better antimicrobial stewardship, discovery of new antibiotics and

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deeper understanding of the mechanism of drug resistance. Work carried out in this study

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highlights the importance of employing real-time fluorescence-based assays in differentiating

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multidrug efflux inhibitory and outer membrane destabilizing activities of antibacterial

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compounds.

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Introduction

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Multidrug resistance among human bacterial pathogens remains a grave social concern.

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Numerous strategies have been proposed to curtail the rampant increase in multidrug resistance

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among human pathogens, ranging from the effective integration of pharmacokinetic and

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pharmacodynamic parameters and implementation of antimicrobial stewardship (1) to the

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development of novel antibiotics based on either an existing or novel chemical scaffold,

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exploitation of new cellular targets and directly tackling the cellular mechanisms that confer

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multidrug resistance (2). Efflux of antibiotics from the cell is one of the common mechanisms of

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antibiotic resistance in bacteria, with resistance developing when the rate of drug efflux across

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the membrane exceeds that of drug influx (3).

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Bacterial genomes encode several membrane-bound multidrug efflux systems (4, 5). These

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systems are usually under the control of an intricate regulatory network, which, in response to the

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presence of drug and other stress molecules, increases the overall efflux activity and decreases

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influx capacity (6). One of the most extensively studied multidrug efflux systems of the

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Resistance-Nodulation-Division (RND) family is the AcrA, AcrB and TolC complex of

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Escherichia coli proteins (7, 8, 9). AcrB is the inner membrane, trimeric drug-proton antiporter

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(10, 11, 12). TolC is an outer membrane channel protein whose periplasmic aperture is critical to

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the removal of drugs from the cell (13, 14). AcrA, an inner membrane lipoprotein (15), through

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its interactions with both TolC and AcrB in the periplasm, completes the efflux pump assembly

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and catalyzes opening of the TolC channel (16, 17, 18).

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Two main reasons why AcrAB-TolC is the most studied tripartite efflux system in E. coli are:

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first, it is the only efflux system of broad substrate specificity that is constitutively expressed at

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high levels and second, it and its homologs are frequently up-regulated in drug resistant Gram-

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negative isolates of clinical relevance (5). Mutants lacking a functional copy of any of the

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corresponding genes of the AcrAB-TolC complex display increased susceptibility to a diverse

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group of antibiotics, detergents and dyes (19). The E. coli genome also encodes four other

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antibiotic efflux pump systems belonging to the RND family: AcrD, AcrEF, MdtABC, and

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MdtEF (4). Of these, only the AcrEF system appears to be functionally analogous to AcrAB (4,

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20). However, the acrEF genes are normally not expressed or expressed at low levels (21) such

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that deletion of acrEF exerts little effect on the drug susceptibility phenotype (19). Although a 3

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repressor-encoding gene, acrS, is located adjacent to acrEF, it does not regulate acrEF

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expression (22). Instead, AcrS, when artificially overexpressed from a plasmid replicon,

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represses expression of distally located acrAB (23). Thus, acrAB is under the control of two

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repressors, in addition to various global regulators (6), while acrEF expression appears to be

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under the negative control of a single global regulator, H-NS (24). Spontaneous hns mutations

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have not been obtained among drug resistant revertants of a strain lacking acrAB, presumably

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due to acute pleiotropy (25). Instead, selections demanding antibiotic resistance in the ΔacrAB

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background frequently lead to resistant colonies bearing an insertion sequence (IS) upstream of

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the acrEF coding region (26, 27, 28). These illegitimate recombination events presumably

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activate acrEF expression by creating an artificial promoter and/or eliminating H-NS-mediated

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repression of the acrEF gene. It is unclear under what physiological conditions acrEF is

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expressed or whether mutations are the only means by which acrEF expression is enhanced.

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Due to their high clinical relevance, inhibitors have been sought to reduce or abolish the

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activity of multidrug resistant efflux pumps (for reviews, see 29, 30, 31). Phenylalanylarginine β-

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naphthylamide (PAβN) was one of the first lead compounds that showed a potent inhibitory

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activity against a number of RND pumps, including AcrB (32). The authors showed that in

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strains expressing RND efflux pumps, PAβN exerts minimum damage to membrane integrity and

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potential, whereas in strains lacking a major efflux pump, it can impose some toxic effects (20,

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32). However, the conclusion that PAβN acts principally as an efflux pump inhibitor was

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questioned by three recent publications (33, 34, 35). One of the main objections stems from the

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observation that PAβN causes elevated toxicity, by damaging the membrane, in cells lacking

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major efflux pathways. AcrAB and AcrEF share high amino acid sequence identities (66.49%

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and 77.56%, respectively) and thus are expected to respond very similarly. It was therefore

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somewhat surprising that two reports concluded that PAβN is an AcrB-specific inhibitor (36, 37).

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In this paper we sought to resolve the controversy of PAβN’s cellular action by employing

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strains constitutively expressing AcrAB or AcrEF efflux pumps. We used two separate real-time

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assays designed specifically to measure efflux or outer membrane destabilizing activity in vivo.

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PAβN activities were compared with that of polymyxin B nonapeptide (PMXBN), which is

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known to destabilize the outer membrane permeability barrier (38). The data revealed that

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PAβN’s main cellular action, which is completed within 60 s after its addition, is to inhibit efflux 4

Misra et al 119

pump activities of AcrAB and AcrEF. In contrast, its outer membrane destabilizing action was

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found to be weak and occurring well after the inhibition of efflux. PMXBN acted in a fashion

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opposite to PAβN, i.e., severely disrupted the outer membrane permeability barrier without

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inhibiting efflux.

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Material and Methods

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Bacterial strains and culture conditions. Bacterial strains used in the study are listed in Table 1

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and derived from MC4100 Δara (39) and MC4100 Δara ΔacrAB::scar (40). Luria broth (LB)

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was prepared from LB Broth EZMix™ Powder (Lennox). LB agar (LBA) medium contained LB

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plus 1.5% agar (Becton Dickenson). When necessary, novobiocin and erythromycin (5 μg/ml

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each) were added to the LBA media. All cultures were grown at 37°C for the durations specified

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in the Results section. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), Nile red, N-phenyl-

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1-naphthylamine (NPN), phenylalanylarginine β-naphthylamide hydrochloride, (PAβN),

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polymyxin B nonapeptide (PMXBN), and vancomycin hydrochloride were purchased from

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Sigma-Aldrich. Nitrocefin was obtained from BioVision. All other chemicals were of analytical

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grade. NPN and nitrocefin were dissolved in 95% ethanol and dimethyl sulfoxide, respectively.

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Nile red was dissolved in methanol.

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Isolation of E. coli mutants expressing the chromosomal acrEF genes. Drug resistant

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revertants of an E. coli strain expressing a mutant AcrB protein defective in proper interaction

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with other efflux pump components (17) were isolated on medium containing erythromycin and

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novobiocin. In some instances, the reversion mutation mapped outside the acrAB and tolC loci.

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P1 transduction of the ΔtolC::Tcr or ΔacrF::Cmr allele into these drug resistant revertants

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revealed that the drug resistance phenotype was dependent on TolC and AcrF. This observation

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and the prevailing knowledge that acrEF expression can be activated by the insertion sequence

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(IS) in the absence of acrAB (22, 26, 27, 28) prompted us to examine the acrEF promoter region

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by PCR and DNA sequencing. In several revertants, PCR-amplification of the promoter region

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produced a DNA fragment that was roughly 1.3 kb larger than that amplified from the parental or

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a wild-type strain. DNA sequence analysis confirmed the presence of an IS2 element 90 bp

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upstream of the acrE start codon in three independent revertants. Interestingly, this was also the

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location of the IS2 element reported previously (26, 27, 28) indicating a likely hotspot for the

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integration of IS2. Purified envelopes were analyzed for the presence of AcrE by Western blot

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analysis using polyclonal antibodies raised against AcrA, which shares 66.49% amino acid

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sequence identity with AcrE. AcrE was readily detected from the strain carrying the IS2 element

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upstream of acrEF (data not shown). The activate acrEF allele has been referred to as acrEF.

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DNA methods. The acrSE intergenic region and the region encompassing all of acrS and part of

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acrE were amplified from the chromosome by polymerase chain reactions (PCR) using two

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different forward primers complementary to regions approximately 300 (5’-

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CACCTCATGACTATTTATACGAGAGGC-3’) and 1200 (5’-

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CAGGCTCAGGTAATGATTCGC-3’) nucleotides upstream of the acrE ATG codon, and a

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reverse primer complementary to region between the 61st and 69th codons of acrE (5’-

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GCGAACTTCGGCTATACGATAAGC-3’). DNA sequence analysis of the PCR-amplified

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products was accomplished using the acrE reverse primer.

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Protein methods. Whole cell envelopes were isolated from overnight grown cultures by the

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French press lysis method as described previously (41). Proteins were analyzed by mini sodium

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dodecyl sulfate (SDS)-polyacrylamide (11%) gel electrophoresis (PAGE) and transferred onto

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Immobilon-P PVDF membranes (Millipore). Membranes were blocked overnight in 5% (w/v)

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non-dairy cream. After blocking, membranes were incubated with primary antibodies for 1.5 h.

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Primary antibodies used were raised in rabbits against AcrAHis, and TolC-MBP (both in 1:10,000

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dilutions). After incubation with primary antibodies, membranes were washed twice for 15 min

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and incubated for 1 h with the secondary antibodies (goat anti-rabbit horseradish peroxidase-

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conjugated IgG). TolC was visualized from the same membrane blot previously used to probe

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AcrA. Detection of HRP-conjugated secondary antibodies was performed using ImmunoStar

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HRP substrate (Pierce). Protein bands were visualized by the Bio-Rad Molecular Imager

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ChemiDoc XRS System.

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Antibiotic susceptibility assay. Minimal inhibitory concentrations (MICs) against

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chloramphenicol, deoxychlolate, erythromycin, nalidixic acid, novobiocin, PAβN, SDS, and

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vancomycin were determined by two-fold serial dilution method using 96-well microtiter plates.

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Approximately 105 cells were used in each well containing 200 μl of LB or LB supplemented

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with different amounts of the inhibitor. Plates were incubated between 16 and 18 h at 37°C on a

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gently rocking platform. Optical densities were measured at 600 nm using VersaMax ELISA

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Microplate Reader from Molecular Devices. MICs were determined from at least three

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independent cultures. The MIC values were determined as the lowest concentration of

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antibiotics/inhibitors at which the bacterial culture failed to reach OD600 of 0.1. Standard errors

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were no greater than 7%, except for the ΔacrAB strain against nalidixic acid where it was 14%. 7

Misra et al 183

Efflux assays. Efflux of NPN in live bacterial cells was carried out essentially as described by

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Lomovskaya et al. (32) with some modifications. Overnight grown cultures were centrifuged and

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pellets were washed with potassium phosphate buffer (KPO4; 20 mM, pH 7.0) containing 1 mM

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MgCl2 and centrifuged again. Washed cell pellets were resuspended in KPO4/MgCl2 buffer. Cell

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suspension, at 4 x 108 cells/ml, was treated with 100 µM CCCP for 10 min at room temperature

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after which cells were pelleted and washed twice with KPO4/MgCl2 buffer and then resuspended

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in the same buffer. NPN was added to a final concentration of 10 µM, and cells were incubated

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at room temperature for 15 min then transferred into a quartz cuvette and placed in a Varian Cary

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Eclipse fluorescence spectrophotometer. For NPN, fluorescent intensity was measured every 1 s

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using excitation and emission wavelengths of 340 nm and 410 nm, respectively. Excitation and

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emission slit widths were set at 5 nm. At 100 s time point, efflux of NPN was initiated by adding

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glucose (50 mM final concentration) and changes in fluorescent intensities were measured for

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200 s. NPN efflux was blocked by adding PAβN (20 µg/ml or ≈ 40 μM final concentration) 200 s

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after the initiation of efflux. Slopes (m = y2 - y1/x2 - x1) resulting from decrease or increase in

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NPN fluorescence were calculated and expressed as fluorescence intensity per second (FI/s). Nile

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red assays were carried out essentially as described for NPN with some modifications

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incorporated as per Bohnert et al. (42), Briefly, cells were incubated with 10 µM CCCP for 15

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min at room temperature and then for additional 15 min at 37°C with 10 µM Nile red. After

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incubation with the dye, cells were pelleted by centrifugation for 5 min and then resuspended in

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KPO4/MgCl2 buffer. Fluorescence was measured immediately using excitation and emission

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wavelengths of 552 nm and 636 nm, respectively, with slit widths set at 5 mm.

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Nitrocefin hydrolysis assay. A breach in the outer membrane permeability barrier was assessed

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by an assay involving the hydrolysis of a chromogenic substrate of β-lactamase, nitrocefin (43).

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Cells, harboring bla-encoding pBR322 plasmid (44), were grown overnight in LB supplemented

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with 50 µg/ml ampicillin. On the next day, cultures were diluted 1:50 in the same medium and

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grown for two hours to mid log phase (OD600 ≈ 0.5). Cells were pelleted and washed with

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KPO4/MgCl2 buffer and centrifuged. Washed cells pellets were resuspended in KPO4/MgCl2

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buffer. Assays, in 96-well microtiter plates, were carried out in triplicate with 108 cells (0.2 ml).

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When required, PAβN or PMXBN was added to a final concentration of 40 µM, followed by

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nitrocefin (100 µM). Absorbance at 486 nm was recorded every 15 s for 30 min immediately after

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the addition of nitrocefin. 8

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RESULTS

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Drug phenotypes of AcrAB+ and AcrEF strains in the absence and presence of PAβN. We

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first determined minimal inhibitory concentrations (MICs) of various inhibitors (Table 2) using

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strains that either lack acrAB (ΔAcrAB; RAM2377), express acrAB (AcrAB+; RAM2378) or

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express acrEF in the absence of acrAB (ΔAcrAB AcrEF; RAM2379) (Table 1). Although

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both AcrAB+ and AcrEF strains behaved similarly in the presence of all seven compounds, the

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AcrEF strain consistently showed a two-fold higher MIC for chloramphenicol and nalidixic acid

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and a four-fold higher MIC for erythromycin than the isogenic AcrAB+ strain (Table 2). The only

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exception was SDS, for which the AcrAB+ strain had a two-fold higher MIC than the

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AcrEF strain (Table 2). We then determined MICs of antibiotics and SDS in the presence of 10

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µg/ml PAβN (Table 2). Note that the MIC of PAβN alone, which is also a substrate of the

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AcrAB and AcrEF pumps (Table 2), is found to be greater than 160 µg/ml in a strain expressing

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AcrAB or AcrEF. The presence of PAβN significantly reduced MICs of chloramphenicol,

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erythromycin, nalidixic acid, novobiocin and SDS, all of which are known substrates of the

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AcrAB pump. However, unlike these five inhibitors, the presence of PAβN produced no change

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in the MIC against the non-substrate antibiotic, vancomycin, in AcrAB+ and AcrEF strains. The

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presence of PAβN did, however, reduce the MIC of vancomycin two fold in a ΔacrAB strain.

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The effect of PAβN on the MIC of deoxycholate could not be determined due to the appearance

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of a white precipitate upon mixing of the two chemicals in LB.

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Based on the MIC data, we chose three antibiotics—erythromycin, nalidixic acid, and

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chloramphenicol—to further evaluate MICs in the presence of low levels of PAβN so as to

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minimize any potential side effects of PAβN on membranes. These antibiotics have different

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sizes and biochemical properties: erythromycin is a relatively large (mol wt, 733.93) and

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hydrophobic molecule compared to nalidixic acid, which is small (mol wt, 232.34), amphipathic

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and water soluble. Chloramphenicol is also small (mol wt of 323.13) but due to its hydrophobic

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nature, only moderately soluble in water. Because of their small size, nalidixic acid and

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chloramphenicol can readily penetrate the outer membrane through the porin proteins (45, 46,

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47). In contrast, passage of erythromycin through the outer membrane is expected to be impeded

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due to its large size and hydrophobicity (45, 47). (Note that since the ΔacrAB mutant displays

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hypersusceptibility to erythromycin [Table 2], it shows that entry of erythromycin into the cell is 9

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not completely blocked in the wild-type cell). Therefore, if the potentiating effect of PAβN is

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principally due to its destabilizing effect on the outer membrane permeability barrier, it is

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expected to act synergistically and preferentially with erythromycin and not necessarily with

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nalidixic acid and chloramphenicol that can already readily cross the unperturbed membrane (45,

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46, 47). On the other hand, if PAβN acts as an inhibitor of AcrAB and AcrEF, it should act

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synergistically with all three antibiotics. PAβN concentrations used in the antibiotic MIC assays

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were 1.25, 2.5 and 5 µg/ml. The MIC data showed that PAβN acted synergistically with all three

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antibiotics (Table 3). A slight difference with chloramphenicol may reflect non-overlapping

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binding sites of PAβN and chloramphenicol (48). Alternatively, rapid outer membrane

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permeation of uncharged chloramphenicol compared to charged nalidixic acid or bulky

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erythromycin makes inhibition of efflux less significant for chloramphenicol than for the latter

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two antibiotics. These results suggest that at low concentrations, PAβN’s potentiating effect on

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the three antibiotics tested is possibly due to its inhibitory action against the RND pump proteins

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and not membrane destabilization.

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Real-time efflux assays with live cells. The MIC data only indirectly reflect efflux

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activities of the two pumps and the effects of PAβN. Moreover, prolonged incubation of cells

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with PAβN, even when present in small quantities, could potentially damage the envelope

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besides inhibiting the efflux activity. Therefore, to directly measure efflux activities of the two

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pumps and the effect of PAβN on their activities, we conducted real-time assays with live cells

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by employing a fluorescent dye, N-phenyl-1-naphthylamine (NPN). NPN has been used to

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probe efflux activity (32, 49, 50).

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NPN efflux assays were carried out according to a published report (32) with some

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modifications as detailed in the Material and Methods section. Prior to carrying out NPN efflux

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assays, we performed a control experiment to ensure that the amount of NPN used does not lead

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to NPN self-quenching upon its accumulation inside the cell. For this we incubated efflux-

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deficient ΔacrAB cells, which will result in maximum accumulation of NPN inside the cell, for

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15 min and then measured fluorescence either directly (Fig. 1A main graph) or after washing

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cells with KPO4/MgCl2 buffer (Fig. 1A inset). NPN fluorescence was measured with excitation

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and emission wavelengths set at 340 nm and 410 nm, respectively. As can be seen from Fig. 1A,

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the use of NPN from 0 µM (ethanol only) to 20 µM produced a linear fluorescence output 10

Misra et al 274

proportional to the amount used, showing no self-quenching by NPN at these concentrations. In

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subsequent experiments, 10 µM of NPN was used.

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We used isogenic strains that were either deleted for acrAB (ΔacrAB), expressing wild-type

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acrAB (acrAB+) or deleted for acrAB but expressing acrEF from the chromosome due to the non-

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polar insertion of an IS2 element 90 bp upstream of the acrE ATG codon (ΔacrAB acrEF).

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Efflux of NPN, preloaded in cells de-energized by CCCP treatment, was initiated by the addition

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of glucose, which reenergizes the membrane. NPN fluoresces weakly in an aqueous environment

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but strongly in a non-polar environment of the cell (51; see below). As expected, no significant

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reduction in the NPN fluorescent intensity was observed in the ΔacrAB strain after the addition

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of glucose (Fig. 1B). A weak decline in the fluorescent intensity (m = -0.07±0.01 FI/s) after

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glucose addition is likely due to the combined efflux activities of other weakly expressed RND

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efflux pumps. The mean time for 50% NPN efflux in ΔacrAB cells was determined to be greater

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than 300 s. In contrast, a sharp decline in the NPN fluorescent intensity with m of -4.6±0.27 FI/s

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was observed in cells expressing wild-type AcrAB (Fig. 1B). The mean time for 50% NPN

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efflux was 15 s from two independent assays. Moreover, the lowest NPN fluorescent intensity, a

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drop of 91% from the pre-efflux intensity, was reached in just over 50 s after the initiation of

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efflux. In cells expressing the AcrEF pump, the average mean time for 50% NPN efflux was 18 s

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(m = -2.79±0.01 FI/s) and it took around 100 s after the initiation of efflux s to reach the lowest

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fluorescent intensity (an 85% drop from the pre-efflux intensity) (Fig. 1B). Thus cells expressing

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AcrAB consistently showed a slightly better NPN efflux activity than those expressing AcrEF.

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Different outcomes from MIC and NPN efflux assays could reflect differences in substrate

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preference, mechanism of efflux by the two pumps and/or their expression levels.

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Effect of PAβN on NPN efflux. We then examined the effects of PAβN on AcrAB- and

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AcrEF-mediated NPN efflux activities. In their original paper, Lomovskaya et al. (32) used a

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close analog of PAβN, MC-002,595 because PAβN’s own fluorescence interfered with the assay.

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We reevaluated this potential limitation under our NPN efflux assay conditions and strain

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background. We first conducted control experiments to test whether the presence of PAβN will

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quench NPN fluorescence either in KPO4/MgCl2 buffer (Fig. 2A) or in KPO4/MgCl2 buffer

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containing CCCP-treated wild-type cells (Fig. 2B). Samples, containing NPN, PAβN or both,

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were excited at 340 nm and emission spectra were measured from 370 nm to 520 nm. All 11

Misra et al 304

samples produced an extremely weak fluorescent signal peaked at 385 nm (Fig. 2A). Apart from

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this common peak, buffer containing PAβN alone produced no measurable fluorescence (Fig.

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2A). In contrast, NPN in buffer produced a modest fluorescence peak at 470 nm (Fig. 2A). The

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solution containing both chemicals produced an emission spectrum identical to that containing

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NPN alone, showing that in solution PAβN neither produces its own fluorescence nor interferes

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with that of NPN (Fig. 2A). In the presence of cells, NPN fluorescence increased dramatically

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and the peak shifted to 410 nm (Fig. 2B). In contrast, the cell suspension containing PAβN

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produced none or very weak fluorescence after incubation for 1 min or 15 min, respectively;

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however, after 30 min, a modest fluorescence signal also peaking at 410 nm emerged (Fig. 2B).

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This signal is likely generated by β-naphthylamine (β-NA) upon internalization of PAβN (52).

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Just as found in the phosphate buffer, the presence of both PAβN and NPN in the same cell

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suspension produced fluorescence spectra similar to that obtained from cells containing NPN

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alone, showing that the incubation of 40 µM PAβN with cells for as long as 15 min neither

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contributes to its own fluorescence nor interferes with that of NPN. From these control

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experiments, we concluded that when employing real-time assays, experiments involving PAβN-

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mediated inhibition of NPN efflux and lasting for 15 min can be conducted without a concern of

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interference by PAβN or its metabolite on NPN fluorescence.

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We repeated the NPN efflux experiments shown in Fig. 1B but with a modification: PAβN

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was added 200 s after the addition of glucose when NPN fluorescence was lowest in wild-type

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cells, due to steady efflux activity. The cell suspension buffer always contained 1 mM MgCl2 to

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minimize potential damaging action of PAβN on the outer membrane. The addition of 20 µg/ml

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(≈ 40 µM) PAβN increased NPN fluorescence with m =2.80±0.42 and 4.46±0.35 FI/s in cells

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expressing AcrAB and AcrEF, respectively (Fig. 3A). Even the weak decline in NPN

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fluorescence in the ΔacrAB mutant was blocked by PAβN (m = 0.63±0.08 FI/s), consistent with a

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broad inhibitory action of PAβN. Interestingly, in ΔacrAB and ΔacrAB acrEF cells the NPN

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fluorescent intensities reached pre-efflux levels 100 s after the addition of PAβN and remained

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high (Fig. 3A). By comparison, in acrAB+ cells NPN fluorescence not only failed to reach the

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pre-efflux level, it began to drop again 100 s after the addition of PAβN (Fig. 3A). Together,

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these data showed that despite responding slightly differently, both AcrAB- and AcrEF-mediated

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efflux activities were strongly inhibited by PAβN within 60 s after its addition. It is worth noting

334

that although we interpret an increase in NPN fluorescence after the addition of PAβN to reflect 12

Misra et al 335

inhibition of NPN efflux, the increase could also result from the combined effects of reduced

336

NPN efflux and increased NPN influx. These possibilities are tested in the following sections.

337

Because NPN and a metabolite of PAβN, β-NA, have overlapping fluorescence properties

338

(Fig. 2B), their fluorescence spectra in the efflux assay described in Fig. 3A could not be

339

distinguished in the same sample and at the same time. However, we overcame this limitation by

340

employing Nile red, which, like NPN, is a substrate of AcrAB (42) and fluoresces strongly at 636

341

nm when excited at 552 nm. Efflux of Nile red in CCCP-treated wild-type cells was initiated by

342

adding glucose and 200 s past efflux initiation, PAβN (40 µM or 80 µM) was added to inhibit

343

efflux (Fig. 3B). The addition of 40 µM PAβN weakly inhibited Nile red efflux and produced no

344

measurable fluorescence of its own at the emission wavelength of 410 nm. When added to 80 µM,

345

PAβN strongly inhibited Nile red efflux, and contributed two units of fluorescence, measured at

346

410 nm, 50 s after its addition. In contrast to small increase, NPN fluorescence, also measured at

347

340 nm excitation and 410 nm emission wavelengths, rose over tenfold 50 s after the addition of

348

40 µM PAβN (Fig. 3A). These data show unambiguously that PAβN or its metabolite did not

349

directly contribute to increase in fluorescence observed during the inhibition of NPN efflux in Fig.

350

3A.

351

Effects of outer membrane destabilizing agents on NPN efflux assay. Next we examined

352

NPN efflux in the presence of EDTA and polymyxin B nonapeptide (PMXBN) which are known

353

to disrupt outer membrane integrity (38). These compounds were added after the initiation of

354

NPN efflux by glucose so that their effects on efflux could be monitored in real-time. We first

355

tested EDTA, since positive charges of PAβN could potentially displace some outer membrane-

356

bound cations. However, addition of EDTA from 10 µM to 5,000 µM failed to increase NPN

357

fluorescence significantly (data not shown), indicating that chelation of outer membrane-bound

358

cations is not a critical factor in PAβN-mediated increase in NPN fluorescence.

359

We then employed PMXBN, a compound previously used to make an argument that PAβN,

360

like PMXBN, acts by destabilizing the outer membrane permeability barrier (33, 35). As was

361

done with PAβN, PMXBN was added 200 s after the initiation of NPN efflux. We used the same

362

molar concentration of PMXBN and PAβN (40 µM) for direct comparison. In the AcrAB+

363

background, addition of PMXBN only weakly elevated NPN fluorescence (Fig. 4A; m =

364

0.07±0.02 FI/s), indicating that neither NPN influx nor efflux was significantly altered. In

365

contrast, a sharp increase in NPN fluorescence occurred upon the addition of PAβN (m = 13

Misra et al 366

2.05±0.09 FI/s) presumably reflecting a complete inhibition of NPN efflux (Fig. 4A). We also

367

conducted the same analysis with the strain expressing AcrEF (Fig. 4B). Interestingly, addition

368

of PMXBN elevated NPN fluorescence at a rate roughly three times greater than that observed in

369

the AcrAB+ background (Fig. 4B). Nevertheless, PAβN-mediated increase in NPN fluorescence

370

was significantly greater than that in the presence of PMXBN (Fig. 4B).

371

The strong response of PAβN in elevating NPN fluorescence is consistent with the idea that

372

PAβN acts by inhibiting the activities of AcrAB and AcrEF. In contrast, the weak action of

373

PMXBN reflects that it neither significantly inhibits NPN efflux nor increases its influx above

374

the rate of efflux. These data show that PAβN’s effect can be readily distinguished from PMXBN

375

by employing an assay that monitors efflux in real-time.

376

Assessment of the outer membrane permeability barrier. The NPN assays described above

377

are designed to specifically monitor in vivo efflux activities of AcrAB and AcrEF and not the

378

integrity of the outer membrane permeability barrier. Therefore, we employed an assay that

379

directly measures disruption of the outer membrane permeability barrier. The assay uses

380

nitrocefin which is a chromogenic cephalosporin substrate of the periplasmically-localized β-

381

lactamase enzyme (43) and is a typical substrate of AcrB (53). A disruption of the outer

382

membrane allows β-lactamase-mediated cleavage of the β-lactam ring of nitrocefin, resulting in

383

the disappearance of the yellow color of nitrocefin and the appearance of the red color of the

384

hydrolyzed product. Nitrocefin hydrolysis assays were carried out with freshly grown cells

385

harboring the bla-encoding pBR322 plasmid (44; Table 1). Pre-incubation of buffer-washed cells

386

with PAβN or PMXBN was minimized so that their effects on the outer membrane can be

387

monitored on a real-time basis. Immediately after the addition of PAβN or PMXBN (40 µM final

388

concentration), nitrocefin was added to a final concentration of 100 µM and absorbance was

389

recorded at 486 nm for 30 min (Fig. 5). In buffer-washed control cells, hydrolysis of nitrocefin

390

was monitored without PAβN or PMXBN and OD486 values of control samples were subtracted

391

from those obtained from cells treated with PAβN or PMXBN (Fig. 5).

392

The nitrocefin hydrolysis assays revealed that PMXBN severely disrupted the outer

393

membrane permeability barrier, while PAβN had relatively little effect (Fig. 5). Quantification of

394

slope values showed that the rates of nitrocefin hydrolysis in PMXBN-treated cells were five

395

(AcrAB+) and eight (AcrEF) times higher than that seen in the PAβN-treated cells (Fig. 5). 14

Misra et al 396

Interestingly, PAβN-treated cells from the ΔacrAB strain (RAM2521) showed a nitrocefin

397

hydrolysis rate that was 1.5 and 2-fold higher than that observed in the PAβN-treated AcrAB+

398

(RAM2523) and AcrEF+ cells (RAM2525), respectively (Fig. 5). This observation is consistent

399

with the elevated vancomycin susceptibility data (Table 2) and shows that the outer membrane

400

destabilizing effect of PAβN can be compounded when it is not removed from cells lacking the

401

AcrAB or AcrEF efflux pump. Together with the NPN efflux assay, the nitrocefin hydrolysis

402

experiment showed that the primary activity of PAβN is to inhibit multidrug efflux pumps,

403

AcrAB and AcrEF.

404

15

Misra et al

405

DISCUSSION

406

In this manuscript, we determined the effects of a general RND pump inhibitor, PAβN, on

407

AcrAB and AcrEF activities. We found that PAβN, a broad efflux pump inhibitor of MexAB,

408

MexCD, and MexEF of Pseudomonas aeruginosa and AcrAB of E. coli (32), also inhibits E.

409

coli’s AcrEF activity. Our data are consistent with those of Olliver et al. (22), who also found

410

PAβN-mediated inhibition of AcrEF activity in Salmonella enterica serovar Typhimurium, but

411

inconsistent with those of Viveiros et al. (37) and Amaral et al. (36), who concluded that PAβN

412

is an AcrB-specific inhibitor. A reason for this discrepancy could be that their conclusion was

413

based on the lack of a PAβN effect on tetracycline resistance resulting from transient,

414

tetracycline-induced expression of AcrEF and other pump proteins in the absence of AcrAB (37).

415

We successfully used PAβN in the NPN efflux assay and showed, for the first time, that it is

416

an effective and potent inhibitor of both AcrAB- and AcrEF-mediated NPN efflux. Prior to this

417

study, PAβN’s use in the NPN efflux assay was avoided due to a concern of high background

418

fluorescence (32). Instead, PAβN was used to monitor its intracellular accumulation and

419

subsequent breakdown to highly fluorescent β-NA in strains expressing wild-type or mutant

420

efflux pumps (52, 54). While conversion of PAβN to β-NA is a relatively slow process, e.g.

421

taking 30 min to triple the fluorescent intensity over the background level in the wild-type strain

422

(54; Fig. 2), the inhibition of NPN efflux by PAβN is immediate, resulting in a seven-fold

423

increase in NPN fluorescence within 75 s after the addition of PAβN (Figs. 3 and 4), even when

424

only adding one-fifth the molar amount used in the accumulation studies (8). Moreover, PAβN

425

alone produced no increase in fluorescence during the same time period (Figs. 2B and 3B),

426

indicating that neither the intrinsic fluorescence properties of PAβN nor that of its cleaved

427

products, including β-NA, interfered with the NPN efflux assay. Thus, when used to measure

428

rapid changes in fluorescence due to efflux inhibition, PAβN can be employed in NPN efflux

429

studies without background fluorescence problem.

430

The robust inhibitory effects of PAβN on AcrAB and AcrEF efflux activities are compatible

431

with the notion that under our experimental conditions, PAβN first acts by blocking the RND

432

pump activities. In contrast to PAβN, PMXBN, which is expected to be retained in the outer

433

membrane, produces virtually no inhibitory effects on NPN efflux; a slight elevation in NPN

434

fluorescence likely results from increased NPN influx rather than its reduced efflux. Yet, in an 16

Misra et al 435

assay specifically deigned to monitor a breach in the outer membrane permeability barrier,

436

PMXBN acts far more destructively than PAβN. These contrasting effects of PAβN and PMXBN

437

in real-time assays reiterate their primary activities as an efflux pump inhibitor (PAβN) and a

438

destabilizer of the outer membrane permeability barrier (PMXBN). Unlike the real-time NPN

439

efflux assay, where cells are exposed to PAβN for only a brief period, prolonged incubation of

440

PAβN with cells in the MIC assay can conceivably produce an outcome stemming from both

441

efflux pump inhibition and breach in the outer membrane permeability barrier. Despite this

442

possibility, we believe that the observed synergistic effect of PAβN, when used at extremely low

443

levels of 1.25 and 2.5 µg/ml, with erythromycin and nalidixic acid (Table 3) is likely due to the

444

inhibition of efflux pump activity rather than a breach in the outer membrane permeability

445

barrier, since at these low concentrations, PAβN causes barely recordable hydrolysis of nitrocefin

446

(data not shown).

447

Highly sensitive fluorescent probes, including 8-Anilino-1-naphthalenesulfonic acid (ANS),

448

fluorescein-di-β-D-galactopyranoside (FDG) and SYTOX Green, have been used to gauge

449

PAβN’s membrane-destabilizing effects in E. coli and P. aeruginosa cells (33, 34, 35). In these

450

studies, changes in fluorescence, which indicate internalization of these normally impervious

451

fluorescent compounds, were measured 10 to 20 min (33, 35) or over an hour (34) after

452

incubation with various amounts of PAβN. In contrast, pre-incubation of cells with PAβN was

453

avoided in this study so as to minimize any membrane damage prior to the start of real-time

454

assays. Moreover, we found that ANS is a substrate of the RND-type efflux pumps (data not

455

shown); therefore, PAβN-mediated inhibition of the RND pumps is expected to reduce ANS

456

efflux from the envelope, resulting in higher fluorescent intensities, thus complicating

457

interpretation of the data. When FDG, a substrate of AcrB, was used to study the effects of PAβN

458

and other compounds in increasing membrane permeability, the authors noted that as low as 4

459

µg/ml of PAβN was enough to initiate the release of presumably intracellularly generated

460

fluorescein, the cleaved product of FDG, from a strain lacking TolC (33, 35). However, since

461

efflux-deficient cells, such as those lacking TolC, cannot remove PAβN from the cell, the

462

secondary effects of PAβN are expected to be greatly amplified. The idea that efflux pumps help

463

minimize secondary effects of PAβN is further corroborated by the fact that drastically higher

464

concentration of PAβN (128 µg/ml) was required to only moderately increase SYTOX Green

465

fluorescence in the wild-type E. coli strain (35). In contrast, 32 µg/ml of PAβN, the lowest 17

Misra et al 466

concentration used by the authors, was sufficient to significantly elevate SYTOX Green

467

fluorescence in an E. coli strain lacking AcrB or TolC (35).

468

Consistent with the idea that AcrAB and AcrEF (when expressed) help minimize secondary

469

effects of PAβN on outer membrane permeability, we found here that in strains lacking the major

470

efflux activity, PAβN causes elevated susceptibility to vancomycin and increased rate of

471

nitrocefin hydrolysis compared to efflux-proficient strains (Table 2; Fig. 5). Note that

472

vancomycin, due to its large size (molecular weight, 1485.71), is normally excluded from

473

entering Gram-negative cells. However, E. coli mutants defective in the biogenesis of the outer

474

membrane display elevated susceptibility to vancomycin (55, 56, 57, 58). Therefore, PAβN

475

clearly has the potential to breach the outer membrane permeability barrier if not expelled from

476

the cells. However, in an efflux-proficient strain, the primary effect of PAβN, when used in low

477

amounts, is not to destabilize the outer membrane permeability barrier, but instead to block the

478

activity of the efflux pump. Cells that lack acrAB or do not express acrEF display eight times

479

lower MIC for PAβN than those expressing either of the two pumps (Table 2). This shows that if

480

not removed from the cell, PAβN causes a toxic effect by disabling some essential cellular

481

function. A breach in the outer membrane permeability barrier does not appear to be the reason

482

for the observed PAβN toxicity, since we observed that the presence of PAβN at the MIC level

483

of an efflux-deficient strain (20 µg/ml) causes a modest 50% increase in the rate of nitrocefin

484

hydrolysis in the ΔacrAB strain compared to the acrAB+ strain (Fig. 5). In contrast, PAβN (32

485

µg/ml) causes a significant increase in the fluorescent intensity of a DNA binding dye SYTOX

486

Green in a ΔacrB strain, indicating a breach in the inner membrane (35). Therefore, PAβN’s

487

toxic effect in an efflux-deficient background likely stems from destabilization and/or

488

depolarization of the inner membrane potential.

489

Previous studies have ruled out any significant effect of PAβN in depolarizing the inner

490

membrane in efflux-proficient cells (32, 42). Even when used at two and half times higher

491

quantity (100 µM) than that used here (40 µM) in the NPN efflux assay, PAβN reduced the

492

transport of a proton motive force-dependent substrate by only 40% (42). The authors

493

concluded that the efflux inhibitory activity of PAβN is not the result of energy dissipation. In

494

conclusion, we suggest that PAβN, when used in low levels, specifically inhibits RND pump

495

activities. However, high levels of PAβN in efflux-proficient backgrounds and relatively low 18

Misra et al 496

levels in efflux-deficient background can lead to membrane destabilization. The data presented

497

here highlight the importance of using real-time assays to assess primary and secondary effects

498

of efflux inhibitory and membrane destabilizing compounds in vivo.

19

Misra et al

499

Acknowledgements

500

We are indebted to Phu Vuong for critically reading the manuscript, Ryan Stikeleather and Eric

501

Linden for their help in PCR analysis, and Mellecha Blake for assistance with the Nile red efflux

502

assay. Keith David Morrison is supported from the ARCS Foundation scholarship. Support for

503

this research came in part from grants from the School of Life Sciences and the National

504

Institutes of Health.

505

20

Misra et al

506

References

507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551

1. Burgess DS, Rapp RP. 2008. Bugs versus drugs: addressing the pharmacist’s challenge. Am. J. Health-Syst. Pharm. 65:S4-S15. 2. Fischbach MA, Walsh CT. 2009. Antibiotics for emerging pathogens. Science 325:10891093. 3. Nikaido H, Pagès JM. 2012. Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. FEMS Microbiol. Rev 36:340-363. 4. Nishino K, Yamaguchi A. 2001. Analysis of a complete library of putative drug transporter genes in Escherichia coli. J. Bacteriol. 183:5803-5812. 5. Piddock LJV. 2006. Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin. Microbial. Rev. 19:382-402. 6. Nishino K, Nikaido E, Yamaguchi A. 2009. Regulation and physiological function of multidrug efflux pumps in Escherichia coli and Salmonella. Biochim. Biophys. Acta 1794:834-843. 7. Misra R, Bavro VN. 2009. Assembly and transport mechanism of tripartite drug efflux systems. Biochim. Biophys. Acta 1794:817-825. 8. Nikaido H. 2011. Structure and mechanism of RND-type multidrug efflux pumps. Adv. Enzmol. Rel. Areas Mol. Biol. 77:1-60. 9. Pos KM. 2009. Drug transport mechanism of the AcrB efflux pump. Biochim. Biophys. Acta 1794:782-793. 10. Murakami S, Nakashima R, Yamashita E, Matsumoto T, Yamaguchi A. 2006. Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443:173-179. 11. Seeger MA, Schiefner A, Eicher T, Verrey F, Diederichs K, Pos KM. 2006. Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism Science 313:1295-1298. 12. Sennhauser G, Amstutz P, Briand C, Storchenegger O, Grutter MG. 2007. Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors. PLoS Biol. 5:e7. 13. Andersen C, Koronakis E, Bokma E, Eswaran J, Humphreys D, Hughes C, Koranakis V. 2002. Transition to the open state of the TolC periplasmic tunnel entrance. Proc. Natl. Acad. Sci. USA. 99:11103-11108. 14. Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C. 2000. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405:914-919. 15. Mikolosko JK, Bobyk K, Zgurskaya HI, Ghosh P. 2006. Conformational flexibility in the multidrug efflux system protein AcrA. Structure 14:577-587. 16. Bavro VN, Pietras Z, Furnham N, Pérez-Cano L, Fernández-Recio J, Pei XY, Misra R, Luisi B. 2008. Assembly and channel opening in a bacterial drug efflux machine. Mol. Cell 30:114-121. 17. Weeks JW, Bavro VN, Misra R. 2014. Genetic assessment of the role of AcrB β-hairpins in the assembly of the TolC-AcrAB multidrug efflux pump of Escherichia coli. Mol. Microbiol. 91:965-975. 18. Weeks JW, Celaya-Kolb T, Pecora S, Misra R. 2010. AcrA suppressor alterations reverse the drug hypersensitivity phenotype of a TolC mutant by inducing TolC aperture opening. Mol. Microbiol. 75:1468-1483. 19. Sulavik MC, Houseweart C, Cramer C, Jiwani N, Murgolo N, Greene J, DiDomenico B, Shaw KJ, Miller GH, Hare R, Shimer G. 2001. Antibiotic susceptibility profiles of 21

Misra et al 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595

Escherichia coli strains lacking multidrug efflux pump genes. Antimicrob. Agents Chemother. 45:1126-1136. 20. Bohnert JA, Kern WV. 2005. Selected arylpiperazines are capable of reversing multidrug resistance in Escherichia coli overexpressing RND efflux pumps. Antimicrob. Agents Chemother. 49:849-852. 21. Yi-Lau S, Zgurskaya HI. 2006. Cell division defects in Escherichia coli deficient in the multidrug efflux transporter AcrEF-TolC. J. Bacteriol. 187:7815-7825. 22. Olliver A, Vallé M, Chaslus-Dancla E, Cloeckaert A. 2005. Overexpression of the multidrug efflux operon acrEF by insertional activation with IS1 or IS10 elements in Salmonella enterica serovar Typhimurium DT204 acrB mutants selected with fluoroquinolones. Antimicrob. Agents Chemother. 49:289-301. 23. Hirakawa H, Takumi-505 Kobayashi A, Theisen U, Hirata T, Nishino K, Yamaguchi A. 2008. AcrS/EnvR represses expression of the acrAB multidrug efflux genes in Escherichia coli. J. Bacteriol. 190:6276-6279. 24. Nishino K, Yamaguchi A. 2004. Role of histone-like protein H-NS in multidrug resistance of Escherichia coli. J. Bacteriol. 186:1423-1429. 25. Hommais F, Krin E, Laurent-Winter C, Soutourina O, Malpertuny A, Le Caer J, Danchin A, Bertin P. 2001. Large-scale monitoring of pleiotropic regulation of gene expression by the prokaryotic nucleoid-associated protein, H-NS. Mol. Microbiol. 40:20-36. 26. Jellen-Ritter AS, Kern WV. 2001. Enhanced expression of the multidrug efflux pumps AcrAB and AcrEF associated with insertion element transposition in Escherichia coli mutants selected with a fluoroquinolone. Antimicrob. Agents Chemother. 45:1467-1472. 27. Klein JR, Henrich B, Plapp R. 1990. Molecular cloning of the envC gene of Escherichia coli. Curr. Microbiol. 21:341-347. 28. Kobayashi K, Tsukagoshi N, Aono R. 2001. Suppression of hypersensitivity of Escherichia coli acrB mutant to organic solvents by integrational activation of the acrEF operon with the IS1 or IS2 element. J. Bacteriol. 183:2646-2653. 29. Lomovskaya O, Bostian KA. 2006. Practical applications and feasibility of efflux pump inhibitors in the clinic—A vision for applied use. Biochem. Pharmcol. 71:910-918. 30. Marquez B. 2005. Bacterial efflux systems and efflux pumps inhibitors. Biochim. 87:11371147. 31. Pagés J.-M, Amaral L. 2009. Mechanisms of drug efflux and strategies to combat them: Challenging the efflux pump of Gram-negative bacteria. Biochim. Biophys. Acta 1794:826833. 32. Lomovskaya O, Warren MS, Lee A, Galazzo J, Fronka R, Lee M, Blais J, Cho D, Chamberland S, Renau T, Leger R, Hecker S, Watkins W, Hoshino K, Ishida H, Lee VJ. 2001. Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrob. Agents Chemother. 45:105-116. 33. Iino R, Nishino K, Noji H, Yamaguchi A, Matsumoto Y. 2012. A microfluidic device for simple and rapid evaluation of multidrug efflux pump inhibitors. Front. Microbiol. 3:1-9. 34. Lamers RP, Cavallari JF, and Burrows LL. 2013. The efflux inhibitor phenylalanine arginine-beta-naphthylamide (PAβN) permeabilizes the outer membrane of Gram-negative bacteria. PLoS One 8:e60666.

22

Misra et al 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640

35. Matsumoto Y, Hayama K, Sakakihara S, Nishino K, Noji H, Iino R, Yamaguchi A. 2011. Evaluation of multidrug efflux pump inhibitors by a new method using microfluidic channels. PLoS One 6:e18547. 36. Amaral L, Engi H, Viveiros M, Molnar J. 2007. Comparison of multidrug resistant efflux pumps of cancer and bacterial cells with respect to the same inhibitory agents. In Vivo 21:237-244. 37. Viveiros M, Jesus A, Brito M, Leandro C, Martins M, Ordway D, Molnar AM, Molnar J, Amaral J. 2005. Inducement and reversal of tetracycline resistance in Escherichia coli K12 and expression of proton gradient-dependent multidrug efflux pump genes. Antimicrob. Agents Chemother. 49:3578-3582. 38. Vaara M. 1992. Agents that increase the permeability of the outer membrane. Microbiol. Rev. 56:395-411. 39. Werner J, Misra R. 2005. YaeT (Omp85) affects the assembly of lipid-dependent and lipid independent outer membrane proteins of Escherichia coli. Mol. Microbiol. 57:1450-1459. 40. Gerken H, Misra R. 2004. Genetic evidence for functional interactions between TolC and AcrA proteins of a major antibiotic efflux pump of Escherichia coli. Mol. Microbiol. 54:620631. 41. Bennion D, Charlson ES, Coon E, Misra R. 2010. Dissection of β-barrel outer membrane 472 protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli. Mol. Microbiol. 77:1153-1171. 42. Bohnert JA, Karamian B, Nikaido H. 2010. Optimized Nile Red efflux assay of AcrAB TolC multidrug efflux system shows competition between substrates. J. Bacteriol. 54:37703777. 43. O’Callaghan CH, Morris A, Kirby SM, Shingler AH. 1972. Novel method for detection of β-lactamase by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1:283-288. 44. Boliver F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa J.H, Falkow S. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113. 45. Hirai K, Aoyama H, Tsutomu T, Iyobe S, Mitsuhashi S. 1986. Differences in susceptibility to quinolones of outer membrane mutants of Salmonella typhimurium and Escherichia coli. Antimicrob. Agents Chemother. 29:535-538. 46. Misra R, Benson SA. 1988. Isolation and characterization of OmpC porin mutants with altered pore properties. J. Bacteriol. 170:528-533. 47. Misra R, Benson SA. 1988. Genetic identification of the pore domain of the OmpC porin of Escherichia coli K-12. J. Bacteriol. 170:3611-3617. 48. Takatsuka Y, Chen C, Nikaido H. 2010. Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB of Escherichia coli. Proc. Natl. Acad. Sci. USA. 107:6559-6565. 49. Ocaktan A, Yoneyama H, Taiji N. 1997. Use of fluorescence probes to monitor function of the subunit proteins of the MexA-MexB-OprM drug extrusion machinery in Pseudomonas aeruginosa. J. Biol. Chem. 272:21964-21969. 50. Seeger MA, Ballmoos CV, Eicher T, Brandstatter L, Verrey F, Diederichs K, Pos KM. 2008. Engineered disulfide bonds support the functional rotation mechanism of multidrug efflux pump AcrB. Nat. Struct. Mol. Microbiol 15:199-2005.

23

Misra et al 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662

51. Trӓuble H, Overath P. 1973. The structure of Escherichia coli membrane studied by fluorescence measurements of lipid phase transitions. Biochim. Biophys. Acta 307:491-512. 52. Bohnert JA, Schuster S, Fähnrich E, Trittler R, Kern WV. 2007. Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND type MDR efflux pump YhiV (MdtF). J. Am. Chem. Soc. 59:1216-1222. 53. Nagano K, Nikaido H. 2009. Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli. Proc. Natl. Acad. Sci. USA. 106:5854-5858. 54. Bohnert JA, Schuster S, Seeger MA, Fähnrich E, Pos KM, Kern WV. 2008. Site-directed mutagenesis reveals putative substrate binding residues in the Escherichia coli RND efflux pump AcrB. J. Bacteriol. 190:8225-8229. 55. Eggert US, Ruiz N, Falcone BV, Branstrom AA, Goldman RC, Silhavy TJ, Kahne D. 2001. Genetic basis for activity differences between vancomycin and glycolipid derivatives of vancomycin. Science 294:361-364. 56. Leonard-Rivera M, Misra R. 2012. Conserved residues of the putative L6 loop of Escherichia coli BamA play a critical role in the assembly of β-barrel outer membrane proteins, including that of BamA itself. J. Bacteriol. 194:4662-4668. 57. Sampson SA, Misra R, Benson SA. 1989. Identification and characterization of a new gene of Escherichia coli K-12 involved in outer membrane permeability. Genetics 122:491-501. 58. Vuong P, Bennion D, Mantei J, Frost D, Misra R. 2008. Analysis of YfgL and YaeT interactions through bioinformatics, mutagenesis, and biochemistry. J. Bacteriol. 190:15071517.

24

Misra et al

663

FIGURE LEGEND

664

Fig. 1. (A) Fluorescence output at different NPN concentrations. Overnight grown and

665

KPO4/MgCl2-washed ΔacrAB cells were treated with 0, 2.5, 5, 7.5, 10, and 20 µM of NPN for 15

666

min and then fluorescence was measured either directly (main graph) or after washing cells in

667

KPO4/MgCl2 buffer (inset). The excitation and emission wavelength were set at 340 nm and 410

668

nm, respectively. (B) NPN efflux assays. Preparation of cells used in the efflux assay is

669

described in the Material and Methods section. Efflux of NPN was initiated 100 s after the start

670

of fluorescent measurement by adding 50 mM of glucose. A rapid loss in fluorescent intensity

671

(FI) shows AcrAB- or AcrEF-mediated efflux of NPN. NPN fluorescence was measured as

672

described above. Slopes (m; FI/s) were determined by Varian Cary kinetics software. Slopes and

673

standard deviation (±) were calculated from at least two independent assays.

674

Fig. 2. Comparison of spectral properties of NPN and PAβN. (A) Measurement of fluorescence

675

in KPO4/MgCl2 buffer containing NPN (10 µM), PAβN (40 µM) or both. Emission spectra (370

676

nm to 520 nm) were measured after exciting samples at 340 nm wavelength. (B) NPN (10 μM)

677

was added to one aliquot of overnight grown wild-type cells, which were treated with CCCP,

678

washed and resuspended in KPO4/MgCl2 buffer. After incubating cells at room temperature with

679

NPN for 15 min, fluorescence was measured as in (A) and then again after adding PAβN (40

680

µM) to the same aliquot and further incubating for 1, 15 and 30 min. Fluorescence was measured

681

from the second aliquot of CCCP-treated cells after incubation with just PAβN (40 µM) for 1, 15

682

and 30 min at room temperature.

683

Fig. 3. Inhibition of NPN and Nile red efflux by PAβN. (A) NPN efflux was carried out as

684

described in Fig. 1 legend. PAβN (40 μM) was added 200 s after the initiation of NPN efflux.

685

Slopes (m; FI/s) of efflux inhibition were determined by Varian Cary kinetics software. Slope

686

values and standard deviation (±) were calculated from at least two independent assays. (B) Nile

687

red efflux assays with wild-type (acrAB+) cells were carried out as described in the Materials and

688

Methods section. PAβN (40 μM or 80 µM) was added 200 s after the initiation of NPN efflux.

689

Two simultaneous fluorescence measurements were made after exciting samples at 340 nm (for

690

PAβN) and 552 nm (for Nile red) and measuring fluorescence at the emission wavelengths of

691

410 nm (for PAβN) and 636 nm (for Nile red).

25

Misra et al 692

Fig. 4. Effects of PAβN and PMXBN on NPN efflux. NPN efflux was carried out as described in

693

Fig. 1 legend. PAβN or PMXBN (both 40 μM) was added 200 s after the initiation of efflux.

694

Fluorescent intensities from experiments involving PAβN and PMXBN treatments were graphed

695

together for comparison. Slopes (m; FI/s) were determined by Varian Cary kinetics software.

696

Slopes and standard deviation (±) were calculated from at least two independent assays. Relevant

697

genotypes of cell types used in the assay are shown.

698

Fig. 5. Effects of PAβN and PMXBN on nitrocefin hydrolysis. Nitrocefin hydrolysis assays were

699

carried out as described in the Materials and Methods section. Slope values, shown as milli-OD

700

units/s, from PMXBN- and PAβN-treated cells were determined from OD486 readings obtained

701

between 60-300 s and 300-900 s, respectively. The OD486 values from PMXBN- and PAβN-

702

treated cells were deducted from those obtained from untreated control cells. Slope values and

703

standard deviation (±) were calculated from three independent experiments.

704

26

Misra et al 705

Table 1. Bacterial strains used in this study. Strain Characteristics

Reference or source

RAM1292

MC4100 Δara714

(39)

RAM2370

RAM1292 ΔacrAB::scar

(40)

RAM2371

RAM2370 acrEF (IS2; isolate #017)

This study

RAM2372

RAM2370 acrEF (IS2; isolate #045)

This study

RAM2374 RAM2375

r

This study

r

This study

r

RAM2370 x ΔacrAB::Km (via P1 transduction) RAM2371 x ΔacrAB::Km (via P1 transduction)

RAM2376

RAM2372 x ΔacrAB::Km (via P1 transduction)

This study

RAM2377

RAM2374 x zba::Tn10 (via P1 transduction)

This study

r

ΔacrAB::Km [ΔAcrAB] RAM2378

RAM2374 x zba::Tn10 (via P1 transduction) acrAB+ This study [AcrAB+]

RAM2379

RAM2376 x zba::Tn10 (via P1 transduction)

This study

r

ΔacrAB::Km [ΔAcrAB AcrEF] RAM2381

RAM2376 x zba::Tn10 (via P1 transduction) acrAB+ This study [AcrAB+ AcrEF]

RAM2521 RAM2523 RAM2525

RAM2377/pBR322 (Apr)

This study

r

This study

r

This study

RAM2378/pBR322 (Ap ) RAM2379/pBR322 (Ap )

706 707

27

Misra et al 708 709

Table 2. MIC (µg/ml) of antibiotics and detergents. a Strains

ΔacrAB

acrAB+

acrEF

1

4 (1)

8 (1)

Deoxycholate

800

≥1600

≥1600

Erythromycin

2

64 (2)

256 (4)

Nalidixic acid

1

4 (0.5)

8 (0.5)

Novobiocin

2

128 (4)

128 (4)

PAβN

20

>160

>160

SDS

≤25

1600 (≤25)

800 (≤25)

160 (80)

160 (160)

160 (160)

Inhibitors Chloramphenicol

Vancomycin 710

a

MICs in the presence of PAβN (10 µg/ml) are shown in parenthesis.

711

28

Misra et al 712

Table 3. MIC (µg/ml) of antibiotics in the presence of different amounts of PAβN. Bacterial

Antibiotics

strains acrAB+

acrEF

PAβN concentration (µg/ml) 0

1.25

2.5

5.0

Erythromycin

64

32

16

4

Nalidixic Acid

4

1

0.5

0.5

Chloramphenicol 4

2

2

2

Erythromycin

256

128

64

16

Nalidixic Acid

8

2

1

1

Chloramphenicol 8

4

2

2

713

29

A R² = 0.9771

300 250 200

250

Intensity (a.u)

Fluorescence intensity (a.u)

350

150 100

R² = 0.9933

200 150 100 50

50

0 0

5

10

15

20

25

NPN (µM)

0 0

5

10

15

20

25

NPN (µM)

B 200

Fluorescence Intensity (a.u.)

Glucose 160

ΔacrAB : m = -0.07±0.01 120

80

acrEF: m = -2.79±0.01 40

acrAB+: m = -4.60±0.27

0 0

50

100

150

Time (s)

200

250

300

Fluorescence Intensity (a.u.)

A

120

NPN NPN+PAβN PAβN

100 80 60 40 20 0 370

400

430

460

490

520

490

520

Emission wavelength (nm)

B Fluorescence Intensity (a.u.)

120

NPN NPN+PAβN (1 min) NPN+PAβN (15 min) NPN+PAβN (30 min)

100 80 60 40

PAβN (1 min) PAβN (15 min) PAβN (30 min)

20 0 370

400

430

460

Emission wavelength (nm)

A 200

Fluorescence Intensity (a.u.)

Glucose 40 µM PAβN 160 Ex: 340 nm Em: 410 nm

120

ΔacrAB: m = 0.63±0.08

80

acrEF: m = 4.46±0.35

40 acrAB+: m = 2.80±0.42 0 0

100

200

300

400

500

Time (s)

B Fluorescence Intensity (a.u.)

30

Glucose

25 20

Ex: 552 nm Em: 636 nm

40 or 80 µM PAβN

15

(80 µM)

10 (40 µM) Ex: 340 nm Em: 410 nm

5

(80 µM) (40 µM)

0 0

100

200

300

Time (s)

400

500

A 180

Glucose

acrAB+

Fluorescence Intensity (a.u.)

160 140

PAβN or PMXBN

120 100 80

PAβN: m = 2.05±0.09

60 40 PMXBN: m = 0.07±0.02

20 0 0

100

200

300

400

500

Time (s)

B 180

ΔacrAB acrEF

Fluorescence Intensity (a.u.)

160

Glucose

140

PAβN or PMXBN

120 100

PAβN: m = 2.24±0.09

80 60 40

PMXBN: m = 0.18±0.02

20 0 0

100

200

300

Time (s)

400

500

0.7

PMXBN/ΔAcrAB: m = 49.4±3.5 PMXBN/AcrAB+: m = 49.4±4.9 PMXBN/AcrEF: m = 59.2±2.9

0.6

OD486

0.5

0.4

0.3

0.2

0.1

PAβN/ΔAcrAB: m = 15.2±0.4 PAβN/AcrAB+: m = 9.9±0.5 PAβN/AcrEF: m = 7.2±0.2

0 0

300

600

900

Time (s)

1200

1500

1800