Download Periplasmic adaptor protein AcrA has a distinct

Journal of Antimicrobial Chemotherapy (2009) 64, 965– 972
doi:10.1093/jac/dkp311
Advance Access publication 10 September 2009
Periplasmic adaptor protein AcrA has a distinct role in the antibiotic
resistance and virulence of Salmonella enterica serovar Typhimurium
Jessica M. A. Blair1, Roberto M. La Ragione2, Martin J. Woodward2 and Laura J. V. Piddock1*
1
School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham,
Birmingham, UK; 2Department of Food & Environmental Safety, Veterinary Laboratories Agency,
Weybridge, Surrey, UK
Received 24 June 2009; returned 21 July 2009; revised 3 August 2009; accepted 4 August 2009
Objectives: AcrA can function as the periplasmic adaptor protein (PAP) in several RND tripartite efflux
pumps, of which AcrAB-TolC is considered the most important. This system confers innate multiple
antibiotic resistance. Disruption of acrB or tolC impairs the ability of Salmonella Typhimurium to colonize and persist in the host. The aim of this study was to investigate the role of AcrA alone in multidrug
resistance and pathogenicity.
Methods: The acrA gene was inactivated in Salmonella Typhimurium SL1344 by insertion of the aph
gene and this mutant complemented with pWKS30acrA. The antimicrobial susceptibility of the mutant
to six antibiotics as well as various dyes and detergents was determined. In addition, efflux activity
was quantified. The ability of the mutant to adhere to, and invade, tissue culture cells in vitro was
measured.
Results: Following disruption of acrA, RT–PCR and western blotting confirmed that acrB/AcrB was
still expressed when acrA was disrupted. The acrA mutant was hypersusceptible to antibiotics, dyes
and detergents. In some cases, lower MICs were seen than for the acrB or tolC mutants. Efflux of the
fluorescent dye Hoechst H33342 was less than in wild-type following disruption of acrA. acrA was also
required for adherence to, and invasion of, tissue culture cells.
Conclusions: Inactivation of acrA conferred a phenotype distinct to that of acrB::aph and tolC::aph.
These data indicate a role for AcrA distinct to that of other protein partners in both efflux of substrates
and virulence.
Keywords: AcrAB-TolC, efflux, pathogenicity
Introduction
Salmonella enterica is a Gram-negative bacterium capable of
infecting a range of animals and causing a variety of diseases in
humans and animals, including bacteraemia, enteric fever and
enterocolitis, the most common manifestation. Enteric fever is
caused by Salmonella Typhi and Salmonella Paratyphi, while nontyphoidal Salmonella serovars, such as Salmonella Enteritidis and
Salmonella Typhimurium, are most commonly responsible for an
enterocolitis with symptoms including headache, severe diarrhoea,
abdominal pain and vomiting. Annually, there are 350000
reported cases in Europe1,2 and 200000 in the USA, although it
has been suggested that the true number of cases is probably
around three times higher as infection is usually self-limiting, does
not require treatment and therefore goes unreported.3,4
Resistance to antibiotics is a growing clinical concern, with
an increasing number of multiply resistant strains being isolated.5 Active efflux, where antimicrobial compounds are
pumped out of the cell through membrane-spanning efflux
pumps such as AcrAB-TolC, has been shown to be a clinically
relevant mechanism of resistance to multiple antibiotics.6 – 8
Efflux can be responsible for resistance to multiple, structurally
distinct, antibiotics and also for the innate resistance of some
bacteria to whole classes of antibiotics.9,10 Overexpression of
these pumps has been detected in human and animal clinical isolates of Salmonella Typhimurium, where it has been associated
with multiple drug resistance (MDR).6 – 8,11,12 In clinical isolates
shown to overexpress AcrB, the MICs of nalidixic acid, tetracycline and chloramphenicol were above the concentrations used
to define resistance according to the BSAC.13 In addition,
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*Corresponding author. Tel: þ44-121-414-6966; Fax: þ44-121-414-6819; E-mail: [email protected]
.....................................................................................................................................................................................................................................................................................................................................................................................................................................
965
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Blair et al.
inactivation of acrB led to increased susceptibility to a range of
antibiotics, dyes and detergents, including the quinolones, tetracycline, chloramphenicol, bile salts, SDS, ethidium bromide and
triclosan.8,12,14
AcrAB-TolC is a tripartite efflux pump found in
Enterobacteriaceae and is a member of the resistance nodulation
division (RND) family of efflux pumps. It has a broad substrate
range, including various antibiotics, dyes and detergents.12,15
The three components are AcrB, the inner membrane transporter
protein, AcrA, the periplasmic adaptor protein (PAP), and TolC,
the outer membrane channel. AcrA from Escherichia coli is an
elongated sickle-shaped protein,16 which has been shown to
interact with both AcrB and TolC17 – 20 as well as being bound to
the inner membrane by an N-terminal lipid moiety.21 Suggested
roles for AcrA include assembly and maintenance of a stable
complex in vivo,19,22 and transmitting conformational changes in
AcrB to TolC, facilitating the opening of the outer membrane
channel.16 Although the components of the AcrAB-TolC pump
function together as a tripartite pump, the individual components
can also function in conjunction with other efflux systems. For
example, TolC, in E. coli, has been shown to be required for the
function of many efflux systems, including the RND pumps
AcrD,23,24 AcrEF,24 MdsAB25 and MdtABC,26 as well as for the
MFS systems EmrAB and EmrKY,27 and also the ABC drug
transporter MacAB.28 Similarly, AcrA has been shown to associate with AcrD29,30 and AcrF,30,31 as well as with AcrB and
TolC. In contrast, AcrB is not known to cooperate with other
efflux pump components, but in E. coli it has been shown
recently to have a role in contact-dependent growth inhibition
(CDI), possibly as a downstream target for the CDI signal,
which is independent of both AcrA and TolC.32 The amino acid
sequence of AcrAB-TolC of E. coli is very similar to that of
Salmonella, with 94%, 97% and 94% similarity for AcrA, AcrB
and TolC, respectively, so it is thought that they are also functionally similar.9
In addition to an established role in antimicrobial resistance,
MDR efflux pumps, including AcrAB-TolC, have been shown to
have a role in pathogenicity in a variety of organisms.14,25,33 – 38
In Salmonella Typhimurium, disruption of either acrB or tolC
led not only to hypersusceptibility to a range of antimicrobial
compounds, but also to a significantly reduced ability to adhere
to, or invade, human intestinal epithelial cells (INT-407) or
mouse macrophages (RAW 264.7) in vitro. Inactivation of either
acrB or tolC also led to reduced colonization and persistence in
poultry14 and reduced virulence in the mouse model of invasive
Salmonella infection.25 The effect seen in vivo can be partially
explained by the finding that many MDR efflux pumps, or
certain components thereof, are also responsible for exporting
host-derived substrates, such as bile salts and fatty acids.
Deletion of acrAB in E. coli led to increased susceptibility to
bile salts and decanoate (a fatty acid), and the expression of
these genes was elevated in the presence of decanoate.39 These
data support the hypothesis that AcrAB-TolC exports these compounds and therefore has a physiological role in enabling these
enteric bacteria to survive within the hostile environment of the
host.40 However, this does not explain the effect seen in vitro in
tissue culture.
In previous work, only the transporter (e.g. AcrB), the outer
membrane protein (e.g. TolC) or AcrA with AcrB have been
investigated for their contribution to innate MDR and pathogenicity. However, we wished to investigate the contribution of the
PAP, AcrA, along with the hypothesis that AcrA has a role in
the MDR and pathogenicity of S. enterica serovar Typhimurium.
Materials and methods
Bacterial strains and construction of mutants
S. enterica serovar Typhimurium strain SL1344 was used throughout
this study.41 Strains L109 (tolC::aph) and L110 (acrB::aph) were
constructed during a previous study and are the P22 transductants of
L108 and L643, respectively.14 The acrA gene was inactivated in
Salmonella Typhimurium SL1344 by insertion of the aph gene
between nucleotides 99 and 1088 as described previously,12,42 using
the primers shown in Table 1. Sequences were taken from xbase
(http://xbase.bham.ac.uk/colibase/) and primers designed using
PRIMER v.2.00 software (Scientific and Educational Software, UK)
and produced by Invitrogen (Invitrogen Ltd, UK). PCR and sequencing was used to confirm that the aph gene had inserted in the correct
position. The acrA::aph construct was transduced into SL1344 using
P22 phage, with selection for kanamycin resistance. Putative transductants were selected on LB agar containing kanamycin, and allelic
replacement was confirmed by PCR and sequencing. The aph gene
was removed using the pCP20 plasmid, encoding the FLP recombinase gene.42 Removal was confirmed by the loss of kanamycin
resistance, followed by PCR and sequencing. The acrA mutant
(L884) was complemented by cloning wild-type acrA, along with the
native promoter, into the low copy number vector pWKS3043 after
digestion of both the plasmid and insert with BamHI and HindIII.
The resulting plasmid ( pWKS30acrA) was sequenced to confirm
insert identity.
The growth kinetics of parent and mutant strains was determined
using a FLUOstar OPTIMA (BMG Labtech, Aylesbury, UK).
Overnight cultures were diluted 1:100 in LB broth and grown at
378C with shaking at 150 rpm. The optical density (at 600 nm) was
measured every 10 min for 24 h.
RT – PCR
Bacterial strains were grown in minimal medium (Teknova, USA) at
378C with aeration until mid-logarithmic phase. A 95% ethanol/5%
phenol mixture was added and incubated on ice for 30 min. After
centrifugation, the supernatant was discarded and the pellets resuspended in TE buffer with 50 mg/L lysozyme and incubated at room
temperature for 5 min. RNA was then isolated using the Promega
SV Total RNA Purification Kit (Promega Corporation, UK, Z3100),
according to the manufacturer’s instructions. Residual DNA was
removed by digestion with Promega RNase-free DNase (Promega
Corporation, UK, M6101), according to the manufacturer’s instructions. cDNA was synthesized using Invitrogen Superscript II
Reverse Transcriptase (Invitrogen, UK). PCRs for acrA, acrB and
16s RNA were set up using the cDNA as a template and the internal
primers shown in Table 1. For the 16s RNA PCR, cDNA was used
at a 1:1000 dilution, while for both acrA and acrB PCRs the cDNA
was used at a dilution of 1:5.12 The PCR amplimers were quantified
using dHPLC, as described previously.12
Western blotting
Bacterial strains were grown in LB broth (Sigma) at 378C with aeration until mid-logarithmic phase. Total cellular protein was obtained
following sonication and samples were run on 12% Bis-Tris gels
(Invitrogen) along with the Magic MarkTM XP molecular weight
966
Role of AcrA in Salmonella
Table 1. Primer sequences
Description
acrA knockout primer forward
acrA knockout primer reverse
Upstream acrA
Downstream acrA
Internal acrA forward
Internal acrA reverse
Internal 16s forward
Internal 16s reverse
Internal acrB forward
Internal acrB reverse
Internal aph forward
Internal aph reverse
acrA complementation forward
acrA complementation reverse
a
Primer sequence
AGC GCT AAC AGG ATG TGA CGA CAA ACA GGA CCA
GCA AGG CGT GTA GGC TGG AGC TGC TTCa
CTG TAC TTT AAC CTG TGC GCC AGG ACG TAC TTT
TTG CAG CGG GAA TTA GCC ATG GTC CATa
ACA TCC AGG ATG TGT TGT CG
CAA TCG TCG GAT ATT GCG CT
GCA GTA CAT CAG TAA GCA GG
CCT TGC GTT ACG GAT GAC TT
CCT CAG CAC ATT GAC GTT AC
TTC CTC CAG ATC TCT ACG CA
CGT GTT ATG ACG GAA GAA GG
GCC ATA CCG ACG ACG ATA AT
CGG TGC CCT GAA TGA ACT GC
CGG CCA CAG TCG ATG AAT CC
TCA AGC TTA CAT CCA GGA TGT GTT GTC G
TCG GAT CCA TCG TCG GAT ATT GCG CTA C
Underlined sequences correspond to the 20 bp homology to the aph gene encoding kanamycin resistance.
marker (Invitrogen). For subsequent immunoblotting, proteins were
transferred to a PVDF membrane (Amersham) by electrophoresis
for 3 h at 48C. The membrane was blocked with 5% non-fat milk
solution. After overnight incubation with either AcrA or AcrB
primary antibody [kindly provided by Helen Zgurskaya and Elena
Tikhonova (University of Oklahoma) and Eva and Vassilis
Koronakis (University of Cambridge), respectively], the proteins
were visualized using an HRP-linked anti-rabbit secondary antibody
raised in donkey (GE Healthcare) and an ECL western blotting
detection system (GE Healthcare).
Determination of the antimicrobial susceptibility
to antibiotics, dyes and detergents
The MICs of a range of antibiotics, dyes and detergents were determined using the standardized agar doubling dilution method as
described by the BSAC.13 Each value is the modal value from at
least three independent experiments and the inherent error of the
doubling dilution method is + one dilution.13
(Sigma). To measure association of bacterial cells with either
INT-407 or RAW 264.7 cells, the washed monolayers were inoculated with 5107 cfu bacteria and incubated for 2 h at 378C in 5%
CO2. The infected monolayers were then washed six times with
HBSS and disrupted by adding 1% Triton X-100 and stirring with a
magnetic flea for 10 min. The number of cfu/mL was determined by
serial dilution onto LB agar and incubation for 16 h.
Invasion assays were performed in parallel with the association
assays. After the initial 2 h incubation as described, the infected
monolayers were washed six times with HBSS before addition of
either MEM or DMEM containing 100 mg/L gentamicin to kill all
external bacteria. Plates were incubated for a further 2 h and monolayers disrupted by adding 1% Triton and stirring with a magnetic
flea for 10 min, and the cfu/mL determined by serial dilution onto
LB agar.
Adhesion was calculated as the number of bacteria associated
with the eukaryotic cells minus the number that had invaded. Each
assay was repeated a minimum of three times, with each repeat
including four technical replicates per bacterial strain. The results
were analysed using Student’s t-test and P values of 0.05 were
taken as significant.
Adhesion and invasion assays
The ability of the strains to adhere to, and invade, INT-407 (human
embryonic intestine cells) and RAW 264.7 (mouse macrophages)
was determined as previously described.14,44 Briefly, INT-407 cells
were maintained in minimal essential medium (MEM), while RAW
264.7 cells required Dulbecco’s modified Eagle’s medium
(DMEM), both supplemented with 10% heat-inactivated fetal calf
serum (Invitrogen), 1% non-essential amino acids (NEAAs)
(Sigma), 1% glutamine (Sigma) and 50 mg/L gentamicin (Sigma).
Plates (24-well) were seeded with either INT-407 or RAW 264.7
cells and incubated for 48 h at 378C and 5% CO2 until confluent.
Confluent monolayers contained 4–6105 cells per well. Prior to
the assays, the monolayers were washed three times in Hanks’
balanced salt solution (HBSS) (Sigma). For tissue culture assays,
bacterial cultures were grown overnight in LB broth at 378C, with
agitation and washed in PBS before being diluted in MEM or
DMEM supplemented with 1% NEAAs (Sigma) and 1% glutamine
Accumulation of Hoeschst H33342 (bis-benzamide)
Hoechst H33342 (bis-benzamide) fluoresces when bound to DNA
and therefore its accumulation can be used to assess the relative
level of active efflux in the mutants.45,46 Bacteria were grown overnight in LB broth, pelleted and resuspended in PBS to an OD600 of
0.1. Hoechst H33342 was added to a final concentration of 2.5 mM.
Fluorescence intensity was measured over 30 min at excitation and
emission wavelengths of 350 and 461 nm, respectively, in a
FLUOstar OPTIMA (BMG Labtech). The experiments were performed in duplicate on three separate occasions (i.e. three biological
replicates each with two technical replicates). Student’s t-tests of the
mean values accumulated after 50 min were performed to compare
the accumulation of Hoescht H33342 by each strain with that of the
parental wild-type strain, SL1344; P values of ,0.05 were taken to
be significant.
967
Blair et al.
Table 2. Salmonella Typhimurium strains used in this study
Strain
SL1344
L109a
L110a
L823
L884
L976
L979
Mean generation time (min)+SD
P value
Reference
32.0+2.65
28.5+1.29
28.6+0.57
—
0.07
0.09
30.5+1.22
0.26
41
14
12
this study
this study
this study
this study
tolC::aph
acrB::aph
acrA::aph
acrA::aph transduced in SL1344
DacrA
acrA::aph, pWKS30acrA
L109 and L110 are the P22 transductants of L108 and L643, respectively.14
L979
(acrA::aph, pWKS30acrA)
Results
Inactivation of acrA alone leads to hypersusceptibility to a
range of antibiotics, dyes and detergents, and reduced efflux
Salmonella Typhimurium SL1344 was susceptible to all 13
agents tested (Table 3). The acrA mutants L884 (acrA::aph) and
L979 (acrA::aph, pWKS30acrA) showed the expected increase
in kanamycin resistance to .256 mg/L due to the incorporation
of the aph gene and increased susceptibility to almost all other
agents. MIC values for the acrB and tolC mutants were all
within one dilution of previously published data from this laboratory.12,14 The susceptibility profile of the acrA mutant (L884)
L884 (acrA::aph)
L976 (ΔacrA)
L110 (acrB::aph)
(b) AcrB
L109 (tolC::aph)
(a) AcrA
SL1344 (WT)
The acrA gene was inactivated by insertion of the aph gene conferring kanamycin resistance. The inserted sequence also
includes a ribosomal binding site and an inframe start codon to
allow expression of downstream acrB. The mutant was designated L823 (Table 2). The disrupted region was transduced into
the wild-type SL1344 background using P22 phage to eliminate
non-specific mutations caused by the l red recombinase used in
the creation of the mutant. The transduced strain was designated
L884 (Table 2). The aph gene was removed using the pCP20
plasmid as previously described42 to ensure there were no polar
effects from the insertion and this kanamycin susceptible mutant
was named L976 (Table 2). All constructs were verified by PCR
and sequencing. Mutants containing acrA::aph or DacrA were
complemented with wild-type acrA cloned into the low copy
number vector pWKS30 (Table 2).43 There was no significant
difference between the generation times or the optical density at
which stationary phase occurred in minimal medium for SL1344
or any of the constructs (Table 2).
RT –PCR was performed to confirm that acrB was transcribed in L884 (acrA::aph). This was necessary because acrA
and acrB are found in a single operon, so to determine the role
of AcrA alone it is crucial that transcription of acrB is not
affected by the disruption of the acrA region. RT – PCR showed
that acrA was expressed by SL1344 and L979 (acrA::aph,
pWKS30acrA), but was not expressed by L884 (acrA::aph). The
acrB gene was expressed by all strains tested. Western blotting
with anti-AcrB antibody confirmed the expression of the AcrB
protein in all three strains, while blotting with anti-AcrA antibody confirmed that AcrA expression had been abolished in the
acrA mutants (Figure 1).
SL1344
Construction of a mutant with inactivation of acrA alone
L884 (acrA::aph)
a
Genotype
Figure 1. Western Blotting for AcrA and AcrB. (a) AcrA expression was
eliminated after disruption of acrA in L884 (acrA::aph) or L976 (DacrA),
but expression was detected in the complemented strain L979 (acrA::aph,
pWKS30acrA). (b) AcrB expression was eliminated by disruption of acrB
(L110), but expression was not affected by disruption of either acrA (L884)
or tolC (L109).
was distinct from that of the acrB (L110) and tolC (L109)
inactivated strains (Table 3). While L884 (acrA::aph) was as
susceptible (+1 dilution) as L110 (acrB::aph) to ampicillin,
nalidixic acid, chloramphenicol, tetracycline, ethidium bromide,
bile, SDS and Triton, it was more susceptible than the acrB
mutant to triclosan, acriflavine and fusidic acid. The acrA mutant
(L884) was as susceptible as the tolC mutant (+1 dilution) to
ampicillin, nalidixic acid, chloramphenicol, tetracycline, acriflavine and fusidic acid, while L109 (tolC::aph) was more susceptible to ethidium bromide, bile, SDS and Triton. The acrA
mutant was more susceptible than the tolC mutant to ciprofloxacin and triclosan. Complementation of both acrA::aph and
DacrA restored antibiotic susceptibility to that of the parental
strain, SL1344 (Table 3).
In order to compare the levels of active efflux in the acrA
mutant, accumulation of Hoechst H33342 was determined and
compared with that in SL1344 and the acrB and tolC mutants.
Accumulation of Hoechst H33342 was 2-fold higher following
inactivation of acrA compared with the wild-type strain SL1344
968
Role of AcrA in Salmonella
Table 3. MICs of antibiotics, dyes and detergents
MIC (mg/L)
Strain
Genotype
SL1344
L823
L884
L976
L979
L110
L109
KAN
AMP
8
1
acrA::aph
.256
0.25
acrA::aph
.256
0.25
DacrA
8
0.25
acrA::aph, pWKS30acrA .256 .128
acrB::aph
.256
0.25
tolC::aph
.256
0.25
NAL
CIP
4
0.5
0.5
0.5
4
1
1
0.015
0.002
0.002
0.002
0.015
0.015
0.015
CHL TET TRIC EtBr ACR FUS
4
0.5
0.5
0.5
4
1
1
1
0.5
0.5
0.5
1
1
0.5
bile
SDS Triton X-100
0.06 .256 256 .512 .1024 .512
0.008
64 16
4
2048
256
0.008
64 16
4
2048
256
0.008
64 16
4
2048
256
0.06 .256 256
512 .1024
512
0.03
64 64
16
1024
128
0.015
16 16
4
256
64
.20000
.20000
.20000
.20000
.20000
.20000
1200
(a) 3500
Inactivation of acrA alone attenuates virulence in vitro
Arbitrary fluorescence units
KAN, kanamycin; AMP, ampicillin; NAL, nalidixic acid; CIP, ciprofloxacin; CHL, chloramphenicol; TET, tetracycline; TRIC, triclosan; EtBr, ethidium
bromide; ACR, acriflavine; FUS, fusidic acid.
Bold formatting indicates significantly increased susceptibility (MIC changed 2-fold).
Triton X-100 was used in all the tissue culture assays to disrupt
the eukaryotic cell monolayer, allowing the number of bacteria
to be determined. It has been shown previously that this concentration of the detergent does not affect viability of either the
acrB or tolC mutants (L110 and L109, respectively) during the
timespan of the assay.14 Susceptibility to Triton X-100 was not
affected by inactivation of acrA or acrB (Table 3). To ensure
that Triton X-100 was not affecting the viability of the acrA
mutant during the tissue culture assays, bacterial recovery after
exposure to Triton X-100 and water were compared. As shown
previously for the other strains,14 there was no significant difference in the viable count over the time period studied (data not
shown). In addition, growth kinetics of the bacterial strains in
the continuous presence of 1% Triton X-100 and with 1% Triton
X-100 added after 2 h (logarithmic growth phase) were
measured. There was no significant difference between the generation times of the wild-type parental strain, SL1344, and the
mutants when in the continuous presence of 1% Triton X-100 or
with Triton X-100 added during logarithmic growth phase (data
not shown).
The ability of Salmonella Typhimurium L884 (acrA::aph)
and L979 (acrA::aph, pWKS30acrA) to adhere to, and invade,
human embryonic intestine cells (INT-407) in vitro was determined and compared with that of SL1344, L109 (tolC::aph) and
L110 (acrB::aph) (Figure 3). As previously published,14 the
ability of L109 (tolC::aph) to adhere to INT-407 cells was significantly decreased compared with SL1344 (P ¼ 0.0002), while
L110 (acrB::aph) and L884 (acrA::aph) were unaffected
(P ¼ 0.07 and 0.34, respectively). All the mutants, L109
(tolC::aph), L110 (acrB::aph) and L884 (acrA::aph), had a significantly reduced ability to invade the INT-407 cell line compared with the parental strain, SL1344 (0.003%, 1.00% and
5.77% of wild-type invasion, respectively; P values ranging
from 0.00019 to 0.00021). When the acrA mutation was complemented with pWKS30acrA, the mutant phenotype was abolished
(Figure 3). A similar pattern was seen with a mouse macrophage
cell line (RAW 264.7); however, in this case the ability of all
mutants to adhere to, and invade, the eukaryotic cells was significantly impaired and L109 (tolC::aph) completely lost the
ability to invade (data not shown).
3000
2500
2000
1500
1000
SL1344
500
L884 (acrA::aph)
0
20
0
40
0
60
0
80
0
10
00
12
00
14
00
16
0
18 0
00
20
00
22
00
24
00
26
00
28
00
30
00
0
Time (s)
Fold change
(b)
*
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
*
*
SL1344
L884
L979 (L884 +
L110
L109
(acrA::aph) pWKS30acrA) (acrB::aph) (tolC::aph)
Figure 2. Hoechst H33342 efflux. (a) The accumulation of Hoechst H33342
in L884 (acrA::aph) and SL1344 over time. (b) The mean fold change in
accumulation of Hoechst H33342 after 50 min in SL1344, the acrA mutant
(L884), the acrA-complemented strain (L979), the acrB mutant (L110) and
the tolC mutant (L109). Student’s t-tests were performed to compare the
accumulation of each strain with that of the wild-type, SL1344, returning
P values of 0.04, 0.21, 0.005 and 0.003, respectively. P values of ,0.05 are
indicated by asterisks.
(P ¼ 0.04), indicating a lower level of efflux. This phenotype
was abolished in the complemented strain (L979). The acrB
mutant accumulated 2.9-fold more Hoechst H33342 (P ¼ 0.005),
while the tolC mutant accumulated 3.6-fold more (P ¼ 0.003)
(Figure 2).
969
Blair et al.
1.0E + 07
*
1.0E + 06
*
cfu/mL
1.0E + 05
*
1.0E + 04
1.0E + 03
*
1.0E + 02
1.0E + 01
SL1344
L884
L979
L110
L109
(acrA::aph) (L884 + (acrB::aph) (tolC::aph)
pWKS30acrA)
Adhesion
Invasion
Figure 3. Ability of Salmonella Typhimurium SL1344 and mutants thereof
to adhere to, and invade, human intestinal cells (INT-407). Adhesion of the
acrA::aph mutant strain to the INT cell line was not significantly different
from the parental strain. However, the mutant had a significantly reduced
ability to invade. This phenotype was abolished when the acrA mutation was
complemented with pWKS30acrA. P values of ,0.05 are indicated by
asterisks.
Discussion
Inactivation of acrA, as found previously with mutants lacking
AcrB or TolC, led to increased susceptibility to a range of antibiotics, dyes and detergents compared with the parental strain
Salmonella Typhimurium SL1344; however, subtle differences
were seen in phenotype, dependent upon which gene (acrA,
acrB or tolC) was inactivated. The susceptibility of L884
(acrA::aph) to antimicrobials, although distinct, was generally
similar to, and in some cases greater than, that of L110
(acrB::aph), which suggests an important role for the periplasmic adaptor protein, AcrA, in efficient efflux. These data can be
partially explained by the reported promiscuity of the AcrA
protein with other efflux proteins of the RND family of efflux
pumps, including AcrD and AcrF.29 – 31 The greatest increase in
susceptibility was seen with the inactivation of tolC, particularly
to fusidic acid, nalidixic acid and Triton X-100, as previously
reported for both Salmonella and E. coli.14,24,25 TolC also acts
as the outer membrane component for many efflux systems,
including other RND pumps,23 – 26 as well members of the
MFS27 and ABC drug transporter families;28 therefore, disruption of TolC will not only affect the ability of the AcrAB pump
to function, but will interfere with other export pathways.
Interestingly, the acrA mutant was more susceptible to ciprofloxacin and triclosan than the tolC mutant. This may be because
the AcrA protein is also promiscuous and may associate with
efflux pumps of other families, which can also transport ciprofloxacin and triclosan.
Accumulation of the fluorescent dye Hoechst H33342 was
increased 2-fold in the acrA mutant (L884 acrA::aph), showing
a significant decrease in efflux. These data suggest that the PAP
is an integral component of the efflux process and that without it
efficient function is diminished. This supports the hypothesis
that the PAP has an active role in the pump function.47
The observed decrease in efflux goes some way to explain
the increase in susceptibility of strains lacking AcrAB-TolC to
the antimicrobials tested. The decreased efflux means that toxic
compounds are not extruded as efficiently and, therefore,
accumulate inside the cell and are lethal at a lower total concentration. However, the effect is substrate-specific, as the acrA
mutant is the most susceptible to triclosan while it does not have
the most attenuated efflux.
As previously published,14 inactivation of either acrB or tolC
led to a reduced ability to invade INT-407 cells compared with
the parental strain and inactivation of tolC also affected adherence. In addition, we have now shown that inactivation of acrA
also affects the ability to invade host cells. The involvement of
RND efflux pump proteins, including AcrB and TolC and homologues thereof in other bacteria, in pathogenicity is now well
established.14,25,33 – 38,40,48 This study extends these findings from
this, and from other laboratories, to include the role of the periplasmic adaptor protein, AcrA, in this process.
Loss of functional acrA significantly affected the efflux
activity, antibiotic susceptibility and also the virulence of the
mutant. This suggests a key role for AcrA in the efficient operation of this pump, which is separate and distinct from that of
AcrB (or other protein partners) or TolC. This is complementary
to the growing body of structural data showing that AcrA physically interacts with both AcrB and TolC,17,18,49 and that the
conformational flexibility seen in E. coli AcrA could mediate
the opening of the TolC channel.16,50 The extent of the change
in phenotype is probably augmented by the promiscuity of
AcrA, such that disruption of acrA will also cause the function
of other RND pumps to be impaired.23,24,29 – 31
Acknowledgements
We are grateful to Helen Zgurskaya and Elena Tikhonova for
their gifts of the anti-AcrA antibodies and technical advice with
regard to western blotting. We are grateful to Eva and Vassilis
Koronakis for the gift of anti-AcrB antibodies. We thank Sarah
Coleman for her technical assistance.
Funding
The study was funded by the Medical Research Council doctoral
training account to the University of Birmingham Medical
School for J. M. A. B.
Transparency declarations
None to declare.
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