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
2
Efflux Pump Inhibiting and Outer Membrane
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Destabilizing Activities in Escherichia coli
4 5 6
Rajeev Misra1*, Keith D. Morrison2, Hyun Jae Cho1, and Thanh Khuu1
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2
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
17
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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Misra et al
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ABSTRACT
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The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high
31
degree of homology with the normally silent AcrEF system. Exposure of a strain deleted for
32
acrAB to antibiotic selection pressure frequently leads to the insertion sequence-mediated
33
activation of the homologous AcrEF system. In this study, we used strains constitutively
34
expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a
35
controversy of whether phenylalanylarginine β-naphthylamide (PAβN) inhibits the activities of
36
AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer
37
membrane permeability barrier. Real-time efflux assays allowed for a clear distinction between
38
the efflux pump-inhibiting activity of PAβN and outer membrane-destabilizing action of
39
polymyxin B nonapeptide (PMXBN). When added at equal amounts, PAβN but not PMXBN
40
strongly inhibited efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer
41
membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a
42
much greater damaging effect than PAβN. A strong action of PAβN in inhibiting efflux activity
43
compared to its weak action on destabilizing the outer membrane permeability barrier suggests
44
that PAβN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations,
45
PAβN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high
46
concentrations, PAβN in the efflux-proficient background not only inhibits the efflux pump
47
activity but also destabilizes the membrane. The effects of PAβN on membrane integrity are
48
compounded in cells unable to extrude PAβN.
49 50
Importance
51 52
The increase in multidrug resistant bacterial pathogens at an alarming rate has accelerated the
53
need for implementation of better antimicrobial stewardship, discovery of new antibiotics and
54
deeper understanding of the mechanism of drug resistance. Work carried out in this study
55
highlights the importance of employing real-time fluorescence-based assays in differentiating
56
multidrug efflux inhibitory and outer membrane destabilizing activities of antibacterial
57
compounds.
58
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Misra et al
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Introduction
60
Multidrug resistance among human bacterial pathogens remains a grave social concern.
61
Numerous strategies have been proposed to curtail the rampant increase in multidrug resistance
62
among human pathogens, ranging from the effective integration of pharmacokinetic and
63
pharmacodynamic parameters and implementation of antimicrobial stewardship (1) to the
64
development of novel antibiotics based on either an existing or novel chemical scaffold,
65
exploitation of new cellular targets and directly tackling the cellular mechanisms that confer
66
multidrug resistance (2). Efflux of antibiotics from the cell is one of the common mechanisms of
67
antibiotic resistance in bacteria, with resistance developing when the rate of drug efflux across
68
the membrane exceeds that of drug influx (3).
69
Bacterial genomes encode several membrane-bound multidrug efflux systems (4, 5). These
70
systems are usually under the control of an intricate regulatory network, which, in response to the
71
presence of drug and other stress molecules, increases the overall efflux activity and decreases
72
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
74
Escherichia coli proteins (7, 8, 9). AcrB is the inner membrane, trimeric drug-proton antiporter
75
(10, 11, 12). TolC is an outer membrane channel protein whose periplasmic aperture is critical to
76
the removal of drugs from the cell (13, 14). AcrA, an inner membrane lipoprotein (15), through
77
its interactions with both TolC and AcrB in the periplasm, completes the efflux pump assembly
78
and catalyzes opening of the TolC channel (16, 17, 18).
79
Two main reasons why AcrAB-TolC is the most studied tripartite efflux system in E. coli are:
80
first, it is the only efflux system of broad substrate specificity that is constitutively expressed at
81
high levels and second, it and its homologs are frequently up-regulated in drug resistant Gram-
82
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
84
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
Misra et al 89
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,
91
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
93
under the negative control of a single global regulator, H-NS (24). Spontaneous hns mutations
94
have not been obtained among drug resistant revertants of a strain lacking acrAB, presumably
95
due to acute pleiotropy (25). Instead, selections demanding antibiotic resistance in the ΔacrAB
96
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
98
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.
101
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
104
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
115
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
121
opposite to PAβN, i.e., severely disrupted the outer membrane permeability barrier without
122
inhibiting efflux.
123
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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
126
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
129
each) were added to the LBA media. All cultures were grown at 37°C for the durations specified
130
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
134
grade. NPN and nitrocefin were dissolved in 95% ethanol and dimethyl sulfoxide, respectively.
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Nile red was dissolved in methanol.
136
Isolation of E. coli mutants expressing the chromosomal acrEF genes. Drug resistant
137
revertants of an E. coli strain expressing a mutant AcrB protein defective in proper interaction
138
with other efflux pump components (17) were isolated on medium containing erythromycin and
139
novobiocin. In some instances, the reversion mutation mapped outside the acrAB and tolC loci.
140
P1 transduction of the ΔtolC::Tcr or ΔacrF::Cmr allele into these drug resistant revertants
141
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
143
(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
145
produced a DNA fragment that was roughly 1.3 kb larger than that amplified from the parental or
146
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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Misra et al 153
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
155
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’-
159
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-
169
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
180
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
182
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
184
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
186
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
188
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
190
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
200
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
203
wavelengths of 552 nm and 636 nm, respectively, with slit widths set at 5 mm.
204
Nitrocefin hydrolysis assay. A breach in the outer membrane permeability barrier was assessed
205
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
210
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
213
the addition of nitrocefin. 8
Misra et al
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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
222
exception was SDS, for which the AcrAB+ strain had a two-fold higher MIC than the
223
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
226
AcrAB or AcrEF. The presence of PAβN significantly reduced MICs of chloramphenicol,
227
erythromycin, nalidixic acid, novobiocin and SDS, all of which are known substrates of the
228
AcrAB pump. However, unlike these five inhibitors, the presence of PAβN produced no change
229
in the MIC against the non-substrate antibiotic, vancomycin, in AcrAB+ and AcrEF strains. The
230
presence of PAβN did, however, reduce the MIC of vancomycin two fold in a ΔacrAB strain.
231
The effect of PAβN on the MIC of deoxycholate could not be determined due to the appearance
232
of a white precipitate upon mixing of the two chemicals in LB.
233
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
235
minimize any potential side effects of PAβN on membranes. These antibiotics have different
236
sizes and biochemical properties: erythromycin is a relatively large (mol wt, 733.93) and
237
hydrophobic molecule compared to nalidixic acid, which is small (mol wt, 232.34), amphipathic
238
and water soluble. Chloramphenicol is also small (mol wt of 323.13) but due to its hydrophobic
239
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,
241
47). In contrast, passage of erythromycin through the outer membrane is expected to be impeded
242
due to its large size and hydrophobicity (45, 47). (Note that since the ΔacrAB mutant displays
243
hypersusceptibility to erythromycin [Table 2], it shows that entry of erythromycin into the cell is 9
Misra et al 244
not completely blocked in the wild-type cell). Therefore, if the potentiating effect of PAβN is
245
principally due to its destabilizing effect on the outer membrane permeability barrier, it is
246
expected to act synergistically and preferentially with erythromycin and not necessarily with
247
nalidixic acid and chloramphenicol that can already readily cross the unperturbed membrane (45,
248
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
250
were 1.25, 2.5 and 5 µg/ml. The MIC data showed that PAβN acted synergistically with all three
251
antibiotics (Table 3). A slight difference with chloramphenicol may reflect non-overlapping
252
binding sites of PAβN and chloramphenicol (48). Alternatively, rapid outer membrane
253
permeation of uncharged chloramphenicol compared to charged nalidixic acid or bulky
254
erythromycin makes inhibition of efflux less significant for chloramphenicol than for the latter
255
two antibiotics. These results suggest that at low concentrations, PAβN’s potentiating effect on
256
the three antibiotics tested is possibly due to its inhibitory action against the RND pump proteins
257
and not membrane destabilization.
258
Real-time efflux assays with live cells. The MIC data only indirectly reflect efflux
259
activities of the two pumps and the effects of PAβN. Moreover, prolonged incubation of cells
260
with PAβN, even when present in small quantities, could potentially damage the envelope
261
besides inhibiting the efflux activity. Therefore, to directly measure efflux activities of the two
262
pumps and the effect of PAβN on their activities, we conducted real-time assays with live cells
263
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).
265
NPN efflux assays were carried out according to a published report (32) with some
266
modifications as detailed in the Material and Methods section. Prior to carrying out NPN efflux
267
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-
269
deficient ΔacrAB cells, which will result in maximum accumulation of NPN inside the cell, for
270
15 min and then measured fluorescence either directly (Fig. 1A main graph) or after washing
271
cells with KPO4/MgCl2 buffer (Fig. 1A inset). NPN fluorescence was measured with excitation
272
and emission wavelengths set at 340 nm and 410 nm, respectively. As can be seen from Fig. 1A,
273
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
275
subsequent experiments, 10 µM of NPN was used.
276
We used isogenic strains that were either deleted for acrAB (ΔacrAB), expressing wild-type
277
acrAB (acrAB+) or deleted for acrAB but expressing acrEF from the chromosome due to the non-
278
polar insertion of an IS2 element 90 bp upstream of the acrE ATG codon (ΔacrAB acrEF).
279
Efflux of NPN, preloaded in cells de-energized by CCCP treatment, was initiated by the addition
280
of glucose, which reenergizes the membrane. NPN fluoresces weakly in an aqueous environment
281
but strongly in a non-polar environment of the cell (51; see below). As expected, no significant
282
reduction in the NPN fluorescent intensity was observed in the ΔacrAB strain after the addition
283
of glucose (Fig. 1B). A weak decline in the fluorescent intensity (m = -0.07±0.01 FI/s) after
284
glucose addition is likely due to the combined efflux activities of other weakly expressed RND
285
efflux pumps. The mean time for 50% NPN efflux in ΔacrAB cells was determined to be greater
286
than 300 s. In contrast, a sharp decline in the NPN fluorescent intensity with m of -4.6±0.27 FI/s
287
was observed in cells expressing wild-type AcrAB (Fig. 1B). The mean time for 50% NPN
288
efflux was 15 s from two independent assays. Moreover, the lowest NPN fluorescent intensity, a
289
drop of 91% from the pre-efflux intensity, was reached in just over 50 s after the initiation of
290
efflux. In cells expressing the AcrEF pump, the average mean time for 50% NPN efflux was 18 s
291
(m = -2.79±0.01 FI/s) and it took around 100 s after the initiation of efflux s to reach the lowest
292
fluorescent intensity (an 85% drop from the pre-efflux intensity) (Fig. 1B). Thus cells expressing
293
AcrAB consistently showed a slightly better NPN efflux activity than those expressing AcrEF.
294
Different outcomes from MIC and NPN efflux assays could reflect differences in substrate
295
preference, mechanism of efflux by the two pumps and/or their expression levels.
296
Effect of PAβN on NPN efflux. We then examined the effects of PAβN on AcrAB- and
297
AcrEF-mediated NPN efflux activities. In their original paper, Lomovskaya et al. (32) used a
298
close analog of PAβN, MC-002,595 because PAβN’s own fluorescence interfered with the assay.
299
We reevaluated this potential limitation under our NPN efflux assay conditions and strain
300
background. We first conducted control experiments to test whether the presence of PAβN will
301
quench NPN fluorescence either in KPO4/MgCl2 buffer (Fig. 2A) or in KPO4/MgCl2 buffer
302
containing CCCP-treated wild-type cells (Fig. 2B). Samples, containing NPN, PAβN or both,
303
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
305
this common peak, buffer containing PAβN alone produced no measurable fluorescence (Fig.
306
2A). In contrast, NPN in buffer produced a modest fluorescence peak at 470 nm (Fig. 2A). The
307
solution containing both chemicals produced an emission spectrum identical to that containing
308
NPN alone, showing that in solution PAβN neither produces its own fluorescence nor interferes
309
with that of NPN (Fig. 2A). In the presence of cells, NPN fluorescence increased dramatically
310
and the peak shifted to 410 nm (Fig. 2B). In contrast, the cell suspension containing PAβN
311
produced none or very weak fluorescence after incubation for 1 min or 15 min, respectively;
312
however, after 30 min, a modest fluorescence signal also peaking at 410 nm emerged (Fig. 2B).
313
This signal is likely generated by β-naphthylamine (β-NA) upon internalization of PAβN (52).
314
Just as found in the phosphate buffer, the presence of both PAβN and NPN in the same cell
315
suspension produced fluorescence spectra similar to that obtained from cells containing NPN
316
alone, showing that the incubation of 40 µM PAβN with cells for as long as 15 min neither
317
contributes to its own fluorescence nor interferes with that of NPN. From these control
318
experiments, we concluded that when employing real-time assays, experiments involving PAβN-
319
mediated inhibition of NPN efflux and lasting for 15 min can be conducted without a concern of
320
interference by PAβN or its metabolite on NPN fluorescence.
321
We repeated the NPN efflux experiments shown in Fig. 1B but with a modification: PAβN
322
was added 200 s after the addition of glucose when NPN fluorescence was lowest in wild-type
323
cells, due to steady efflux activity. The cell suspension buffer always contained 1 mM MgCl2 to
324
minimize potential damaging action of PAβN on the outer membrane. The addition of 20 µg/ml
325
(≈ 40 µM) PAβN increased NPN fluorescence with m =2.80±0.42 and 4.46±0.35 FI/s in cells
326
expressing AcrAB and AcrEF, respectively (Fig. 3A). Even the weak decline in NPN
327
fluorescence in the ΔacrAB mutant was blocked by PAβN (m = 0.63±0.08 FI/s), consistent with a
328
broad inhibitory action of PAβN. Interestingly, in ΔacrAB and ΔacrAB acrEF cells the NPN
329
fluorescent intensities reached pre-efflux levels 100 s after the addition of PAβN and remained
330
high (Fig. 3A). By comparison, in acrAB+ cells NPN fluorescence not only failed to reach the
331
pre-efflux level, it began to drop again 100 s after the addition of PAβN (Fig. 3A). Together,
332
these data showed that despite responding slightly differently, both AcrAB- and AcrEF-mediated
333
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
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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