ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2009, p. 3675–3682 Vol. 53, No.

9
0066-4804/09/$08.00⫹0 doi:10.1128/AAC.00550-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Role of the Mycobacterium tuberculosis P55 Efflux Pump in Intrinsic

Drug Resistance, Oxidative Stress Responses, and Growth䌤
Santiago Ramón-García,1,2* Carlos Martín,1 Charles J. Thompson,2† and José A. Aínsa1†
Departamento de Microbiología, Medicina Preventiva y Salud Pública, Universidad de Zaragoza, Zaragoza 50009, and
CIBER Enfermedades Respiratorias, Spain1; and Department of Microbiology and Immunology,
Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, Vancouver,
British Columbia V6T 1Z3, Canada2
Received 23 April 2009/Returned for modification 10 June 2009/Accepted 18 June 2009

Bacterial efflux pumps have traditionally been studied as low-level drug resistance determinants. Recent
insights have suggested that efflux systems are often involved with fundamental cellular physiological pro-
cesses, suggesting that drug extrusion may be a secondary function. In Mycobacterium tuberculosis, little is
known about the physiological or drug resistance roles of efflux pumps. Using Mycobacterium bovis BCG as a
model system, we showed that deletion of the Rv1410c gene encoding the P55 efflux pump made the strain more
susceptible to a range of toxic compounds, including rifampin (rifampicin) and clofazimine, which are first-
and second-line antituberculosis drugs. The efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone
(CCCP) and valinomycin inhibited the P55-determined drug resistance, suggesting the active export of the
compounds by use of the transmembrane proton and electrochemical gradients as sources of energy. In
addition, the P55 efflux pump mutant was more susceptible to redox compounds and displayed increased
intracellular redox potential, suggesting an essential role of the efflux pump in detoxification processes coupled
to oxidative balance within the cell. Finally, cells that lacked the p55 gene displayed smaller colony sizes and
had a growth defect in liquid culture. This, together with an increased susceptibility to the cell wall-targeting
compounds bacitracin and vancomycin, suggested that P55 is needed for proper cell wall assembly and normal
growth in vitro. Thus, P55 plays a fundamental role in oxidative stress responses and in vitro cell growth, in
addition to contributing to intrinsic antibiotic resistance. Inhibitors of the P55 efflux pump could help to
improve current treatments for tuberculosis.

Tuberculosis (TB) continues to be one of the main causes of While drug resistance in Mycobacterium tuberculosis clinical
morbidity and mortality worldwide. The most recent WHO isolates is often due to the acquisition of mutations in genes
report estimates that there are 9.2 million new cases of TB and encoding drug targets or enzymes activating prodrugs, such
1.7 million deaths per year (41), significant numbers of which mutations are not found in many low-level-drug-resistant iso-
occur among human immunodeficiency virus-positive patients. lates, suggesting the contribution of efflux pumps (11). In fact,
Along with human immunodeficiency virus coinfection, multi- many M. tuberculosis efflux pumps contribute to drug resistance
drug-resistant TB (MDR-TB) and extensively drug-resistant under laboratory conditions, and there are reports describing
TB (XDR-TB) pose major threats that challenge TB control, increases in the levels of expression of efflux pumps in various
especially in those regions of the world with the highest burden drug-resistant M. tuberculosis isolates (11, 15, 17, 32, 33, 38). In
of TB. addition, the inactivation of certain efflux pumps attenuates M.
Efflux pumps are membrane proteins that export substrates tuberculosis, indicating that, like other bacterial pathogens, ef-
from bacterial and eukaryotic cells. They confer resistance to flux pumps also contribute to virulence (10).
anticancer drugs in tumor cells and to antibiotics in bacteria, We have previously characterized the contribution of the M.
often providing low levels of intrinsic multidrug resistance (25). tuberculosis P55 efflux pump to drug resistance in Mycobacte-
Their activities allow better tolerance of drugs and thus may rium smegmatis (39). The gene encoding the P55 efflux pump,
potentiate the acquisition of chromosomal mutations that pro- Rv1410c, forms an operon with Rv1411c (5), which encodes the
vide higher levels of resistance (29, 30). In recent years, it has lipoprotein LprG. Both genes are predicted to support the
become evident that efflux pumps have important functions in growth of M. tuberculosis in vivo (6, 37). A recent report has
many other cellular processes, such as physiological homeosta- demonstrated that this operon is required for survival in the
sis, resistance to stress conditions, lipid transport, and viru- presence of ethidium bromide and for maintenance of a nor-
lence (24). mal cell surface composition in M. smegmatis (13).
In the study described here, we characterized P55 in the TB
vaccine strain, M. bovis BCG. Our results demonstrate that P55
* Corresponding author. Mailing address: Department of Microbi- plays a role in at least three important processes: it extrudes
ology and Immunology, Life Science Centre, University of British and thus provides resistance to several drugs (including ri-
Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia fampin [rifampicin], one of the most important frontline TB
V6T 1Z3, Canada. Phone: (604) 822-8094. Fax: (604) 822-6041. E-mail:
[email protected].
drugs), it is part of the oxidative stress response, and it is
† These authors contributed equally to this work. needed to maintain normal growth characteristics both on
䌤
Published ahead of print on 29 June 2009. solid medium and in liquid medium.

3675
3676 RAMÓN-GARCÍA ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1. Strains and plasmids used in this study electroporation with a Gene Pulser (Bio-Rad Laboratories Inc., Richmond, CA)
(26).
Reference or Strain construction. The plasmids used for the overexpression and inactiva-
Strain or plasmid Description
source tion of the p55 gene in M. bovis BCG are listed in Table 1.
M. bovis BCG (i) Overexpression. The p27-p55 operon, expressed under the control of its
Pasteur 1173 Wild type Laboratory own promoter, was cloned into the pSUM41 vector, yielding plasmid pPAZ23
collection (39). Plasmid pPAZ23 was electroporated into M. bovis BCG, resulting in M.
KOP55 Pasteur p55::⍀hyg This study bovis BCG PAZ23 (Table 1).
KOP55 PAZ23 Strain KOP55 containing pPAZ23 This study (ii) Inactivation and complementation. A suicide delivery plasmid containing
PAZ23 Strain Pasteur 1173 containing This study the p55 gene interrupted by the insertion of a hygromycin resistance cassette
pPAZ23 (⍀hyg) was constructed (pILI12). Initially, a 2.1-kb PCR product from M. tuber-
culosis H37Rv genomic DNA (with an engineered EcoRI site on one side)
Plasmids containing the p27-p55 operon was cloned into the pGEM-T Easy vector. The
pGEM-T Easy E. coli cloning vector, ampicillin Promega fragment was then removed by EcoRI digestion and cloned into the EcoRI-
resistance digested vector pIJ2925, resulting in plasmid pSAN6. A 2.1-kb BglII-Asp718I
pIJ2925 Cloning vector, pUC18 derivative 16 fragment from pSAN6 was transferred into BamHI-Asp718I-digested vector
with BglII sites flanking MCS,a p2NIL, resulting in plasmid pILI10. p55 was then interrupted by the insertion of
ampicillin resistance a ⍀hyg cassette (derived from BamHI-digested plasmid pHP45-⍀hyg) into its
pSUM series E. coli-mycobacteria shuttle 2 unique Bsp120I restriction site, resulting in plasmid pILI11. A cassette contain-
vectors, nontranscribed MCS, ing the lacZ and sacB genes from pGOAL17 was then cloned into the single PacI
kanamycin resistance site of pILI11 to generate the suicide delivery vector pILI12. pILI12 was used to
p2NIL Gene manipulation mycobacterial 27 transform M. bovis BCG. Single-crossover (SXO) and double-crossover (DXO)
suicide vector, kanamycin transformants were selected as described elsewhere (27). Candidates for SXO
resistance and DXO were analyzed by PCR with primers specific for the p55 gene flanking
pGOAL17 PAg85-lacZ Phsp60-sacB PacI 27 the ⍀hyg insertion point and primers specific for the kanamycin resistance gene
cassette vector, ampicillin of the vector. Candidates that had the expected PCR patterns were streaked onto
resistance plates with 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside and either kana-
pHP45-⍀hyg ⍀hyg, ampicillin resistance 31 mycin or hygromycin for phenotypic analysis. Finally, Southern blotting analysis
pSAN6 p27-p55 cloned into pIJ2925 This study was done with PvuII- and PstI-digested DNA (Fig. 1 and data not shown,
pPAZ23 Pp27-p55-p27-p55 cloned into 39 respectively) to confirm SXO and DXO. The M. bovis BCG strain with the
pSUM41 inactivated p55 gene was named M. bovis BCG KOP55 (Table 1). M. bovis BCG
pILI10 p27-p55 cloned into p2NIL This study KOP55 was complemented by introducing pPAZ23 containing the p27-p55
pILI11 p27-p55::⍀hyg cloned into p2NIL This study operon, resulting in M. bovis BCG KOP55 PAZ23.
pILI12 p27-p55::⍀hyg cloned into p2NIL This study Drug susceptibility assays. Two methods were used to determine the drug
with pGOAL17 PacI cassette susceptibilities of the M. bovis BCG strains. First, a conventional disc assay was
used to test the redox compounds. Briefly, a suspension containing 106 cells/ml
a
MCS, multicloning site. was spread on Middlebrook 7H10 agar plates, and discs containing the redox
compounds were placed onto the lawn. Halos were recorded after 14 days at
37°C. Second, MICs were determined by using serial twofold dilutions of the
compounds in both liquid and solid media. For determination of the MICs in
liquid medium, the resazurin assay was carried out essentially as described
MATERIALS AND METHODS previously (32), except that the plates were incubated for 8 days at 37°C and for
Gene nomenclature. M. tuberculosis and M. bovis BCG have a DNA sequence an additional 2 days after the addition of the redox indicator (resazurin). For
identity of greater than 99.9% (3). The nucleotide sequences of the M. tubercu- agar-based determinations, log-phase cultures were diluted to 105 cells/ml and 10
losis Rv1410c and Rv1411c genes (http://genolist.pasteur.fr/TubercuList/) are ␮l was spotted onto Middlebrook 7H10 agar plates containing serial twofold
identical to those of the BCG1471c and BCG1472c genes, respectively, from M. antibiotic dilutions. Visible growth was scored after 21 days of incubation at
bovis BCG Pasteur 1173 P2 (http://genolist.pasteur.fr/BCGList/). In this report, 37°C. The experiments were carried out in triplicate and were repeated at least
both Rv1410c and BCG1471c are referred to as p55, and both Rv1411c and three times.
BCG1472c are referred to as p27. MTT assays. The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
Bacterial strains, growth conditions, and chemicals. The strains used in this razolium bromide (MTT) was used to estimate the intracellular redox potential,
study are listed in Table 1. The sequences of the oligonucleotides used are essentially as described previously (7), with some modifications. Briefly, M. bovis
available upon request. M. bovis BCG was cultivated at 37°C in Middlebrook 7H9 BCG cultures at an optical density (OD) at 600 nm of 0.2 were treated at 37°C
broth (Difco) supplemented with 10% Middlebrook albumin-dextrose-catalase with drug or solvent alone for 1 h (100 ␮l/well in Honeycomb 2 plates in
(Difco) and 0.05% (vol/vol) Tween 80 or on Middlebrook 7H10 agar quadruplicate) before the addition of 25 ␮l of 2 mg/ml MTT and were then
plates (Difco) supplemented with 10% (vol/vol) oleic acid albumin-dextrose- incubated for an additional 3 h. The absorbance at 600 nm was recorded after the
catalase (Difco). Escherichia coli was grown at 37°C in Luria-Bertani (LB) broth formazan precipitate was solubilized by the addition of 25 ␮l of 10% sodium
or on LB agar plates. For the selection of resistance markers in mycobacteria, dodecyl sulfate and subsequent shaking for 30 min in a BioScreen C plate reader
hygromycin or kanamycin was added to the cultures at final concentrations of 10 (Oy Growth Curves Ab Ltd., Helsinki, Finland). All drugs except menadione and
mg/liter and 20 mg/liter, respectively. Plasmids were maintained in E. coli with diamide were added at a concentration of 10-fold the MIC for the wild-type
appropriate antibiotics for selection (100 mg/liter of ampicillin, 20 mg/liter of strain. Menadione was added at eightfold the MIC, and diamide was added at the
kanamycin). Acriflavine, amikacin, bacitracin, carbonyl cyanide m-chlorophenyl- MIC. The assay was repeated at least three independent times.
hydrazone (CCCP), cerulenin, D-cycloserine, ethidium bromide, ciprofloxacin, Growth kinetics. Experiments were carried out in a BioScreen C plate reader
clofazimine, chloramphenicol, chlorpromazine, diamide, DL-dithiothreitol, econ- (Oy Growth Curves Ab Ltd.) at 37°C with continuous shaking. The cells were
azole, ethambutol, ethionamide, fluconazole, gentamicin, hydrogen peroxide, grown to stationary phase, and quadruplicate Honeycomb 2 plate wells were
imipenem, isoniazid, L-glutathione (reduced), menadione, monobromobimane inoculated with 250 ␮l of 105 cells/ml. The absorbance at 600 nm was recorded
(mBBr), novobiocin, ofloxacin, p-aminosalicylic acid, PA-824, reserpine, ri- every 12 h for 18 days.
fampin, streptomycin, spectinomycin, tetracycline, triclosan, valinomycin, and
vancomycin were used in the mycobacterial antimicrobial susceptibility tests.
DNA manipulations. DNA manipulations were carried out by standard tech- RESULTS
niques (35). Mycobacterial genomic DNA was isolated as described previously
(26). Southern blotting analysis was done with an ECL direct nucleic acid label-
P55 efflux pump drug resistance phenotypes are dependent
ing and detection system (Amersham Biosciences), according to the manufac- on genetic background. A M. bovis BCG Pasteur derivative in
turer’s instructions. Both E. coli and the mycobacteria were transformed by which the gene encoding the P55 efflux pump (Rv1410c) was
VOL. 53, 2009 ROLE OF THE M. TUBERCULOSIS P55 EFFLUX PUMP 3677

FIG. 1. Southern blot analysis for p55 gene inactivation. Genomic DNA isolated from the M. bovis BCG wild-type (WT), M. bovis BCG
single-crossover (SXO), and M. bovis BCG double-crossover (DXO) strains was digested with PvuII and hybridized to a probe corresponding to
a 0.9-kb p55 internal region. The DXO strain carrying the inactivated p55 gene lost the wild-type 5-kb band and gained a 7.3-kb band, reflecting
the insertion of the 2.3-kb hygromycin resistance cassette (cf. lane WT and lane DXO). The SXO strain showed two bands (9.3 and 7.2 kb) due
to the incorporation of the suicide vector in the genome. The diagram showing the expected PvuII digestion fragment is not shown to scale. ⍀hyg,
cassette containing the hygromycin resistance gene; pGOAL17, cassette containing the lacZ and sacB genes; Km, kanamycin resistance gene from
p2NIL vector.

inactivated (strain KOP55) was constructed by the insertion of wall lipid (40). Other compounds induced only either p27 or
a cassette containing the hygromycin resistance gene (hyg) and p55. The level of expression of p55 was increased by treatment
a transcriptional terminator. Previous studies have shown a with clofazimine (a second-line anti-TB drug), which disrupts
decrease in susceptibility to tetracycline and aminoglycoside the membrane potential; triclosan, which inhibits fatty acid
when the p55 gene is overexpressed in M. smegmatis (39) and biosynthesis in bacteria; and thioridazine, another phenothia-
that deletion of the chromosomal p55 ortholog in M. smegmatis zine which also affects respiration. Menadione, which is both
results in increased susceptibility to ethidium bromide (13). an inhibitor of respiration and a generator of superoxide, in-
However, in M. bovis BCG, the similar p55 overexpression or creased the expression of only p27. Significant decreases in the
inactivation constructions did not alter resistance to these levels of expression of these genes were observed only after
drugs (data not shown). The genetic divergence between M. treatment with valinomycin. This drug, which facilitates the
smegmatis (6.98-megabase genome, 67.4% G⫹C content) and transfer of potassium ions across cell membranes and which
M. bovis BCG (4.37-megabase genome, 65.6% G⫹C content), thus dissipates the transmembrane potential, decreased the
which have an average of 68% nucleotide identity between levels of expression of both genes. Together, these data pro-
orthologous genes, could explain this difference in their phe- vide general concepts and specific compounds for analysis in
notypes. Paralogous transporters in different species may be the context of the P55 phenotypes.
controlled by different regulatory systems and/or may have Inactivation of the p55 gene in M. bovis BCG resulted in
different substrate specificities. In addition, comparative ge- increased susceptibility to rifampin and other drugs. Drug
netic studies may be complicated by the presence of transport-
ers with overlapping substrate specificities. These results rein-
force the observation that efflux pump substrate specificity and TABLE 2. Analysis of Boshoff databasea for p27 and p55 genes
from Mycobacterium tuberculosis
the phenotypes of null mutants or overexpressing strains are
determined by the genetic background of the bacterial host Fold induction
(24). Treatment b
Up (⬎2-fold) Down (⬍0.5-fold)
Microarray data analysis reveals possible P55 efflux pump
p27 p55 p27 p55
substrates. Studies of compounds that induce P55 expression
might give important insights into its substrates. To investigate Chlorpromazine x x
this possibility, a microarray database (generated by Boshoff Clofazimine x
et al. [7]) which recorded the transcriptional responses of M. Menadione x
Triclosan x
tuberculosis to a series of 75 different drugs and physiological Thioridazine x
conditions was analyzed. Eight conditions which increased or Valinomycin x x
decreased p27 and p55 gene expression at least twofold in at Novobiocin x x
least three independent experiments were selected (Table 2). PA-824 x xc
The levels of expression of both genes increased after treat- a
The database of Boshoff et al. (7).
b
ments with chlorpromazine, which is a phenothiazine predicted Treatments for which p27 and p55 gene expression was found to be up- or
downregulated at least twofold in three or more independent experiments.
to affect respiration; novobiocin, which is a gyrase inhibitor; c
Microarray analyses for which p55 gene expression was upregulated at least
and PA-824, which inhibits the synthesis of protein and cell twofold only in two independent experiments.
3678 RAMÓN-GARCÍA ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 3. Antimicrobial susceptibilities of M. bovis Expression of p55 complements the mutant phenotype.
BCG P55 strains Complementation tests were carried out to confirm that the
MIC (mg/liter) for M. bovis BCG straina: sensitivity phenotype observed in KOP55 was due to inactiva-
Compound tion of the p55 gene rather than suppressor mutations at other
Wild type KOP55b KOP55 PAZ23c PAZ23d
locations. Plasmid pPAZ23 was introduced into the KOP55
Bacitracin ⬎256 16 ⬎256 NDf mutant to express the p27 and p55 genes under the control of
Bacitracin ⫹ CCCPe ⬎256 16 ⬎256
CCCP 4 2 ND ND the native operon promoter (5). When mutant KOP55 was
Clofazimine 0.1 0.025 0.1 0.1 complemented with the wild-type genes, the levels of resis-
Chlorpromazine 4 4 ND ND
Econazole 6.4 1.6 6.4 12.8 tance to bacitracin, clofazimine, econazole, novobiocin, PA-
Ethambutol 4 2 ND ND 824, rifampin, valinomycin, and vancomycin returned to the
Novobiocin 16 2 16 ND
Novobiocin ⫹ CCCPe 4 1 4 wild-type levels (Table 3). The introduction of pPAZ23 into
Novobiocin ⫹ VALe 8 0.5 8 the parental wild-type strain resulted in a 2-fold increase in the
Novobiocin ⫹ RPe 16–8 1 16–8
PA-824 0.25 0.125 0.25 ND MICs of econazole and vancomycin (whereas inactivation of
Reserpine 2 2 2 ND the p55 gene resulted in 4- and 16-fold decreases in the MICs
Rifampin 0.0032 0.0004 0.0032 0.0032
Rifampin ⫹ CCCPe 0.0008 0.0002 0.0008 of these compounds, respectively) and did not alter its suscep-
Rifampin ⫹ VALe 0.0016 0.0002 0.0016 tibility to clofazimine and rifampin (Table 3). In summary,
Rifampin ⫹ RPe 0.0032–0.0016 0.0004 0.0032–0.0016
Valinomycin 0.4 0.2 0.4 ND these results indicate that the P55 efflux pump provides low-
Vancomycin 4 0.25 4 8 level intrinsic resistance to a range of different antimicrobial
Vancomycin ⫹ CCCPe 4 0.25 4
Triclosan 8 8 ND ND
compounds in M. bovis BCG.
The P55 efflux pump uses the proton and electrochemical
a
MICs were assayed over a range of twofold dilutions of antibiotics. The MICs gradients across the membrane as sources of energy to extrude
of clofazimine, chlorpromazine, econazole, and triclosan were determined on
agar plates. The MICs of bacitracin, CCCP, ethambutol, novobiocin, reserpine, compounds from the cell. To elucidate whether the P55 efflux
rifampin, valinomycin, and vancomycin were determined by the resazurin assay. pump provides intrinsic drug resistance by actively extruding
No difference in susceptibility between wild-type and KOP55 strains was ob-
served for the following compounds: acriflavine, amikacin, cerulenin, D-cy-
the compounds or by an indirect effect, the drug susceptibilities
closerine, ethidium bromide, ciprofloxacin, chloramphenicol, ethionamide, flu- of the different P55-related strains were tested in the presence
conazole, gentamicin, imipenem, isoniazid, ofloxacin, p-aminosalicylic acid, of subinhibitory concentrations of standard efflux pump inhib-
streptomycin, spectinomycin, and tetracycline (data not shown).
b
KOP55 is M. bovis BCG with the p55 gene inactivated. itors. The inhibitors tested included CCCP, a proton uncou-
c
KOP55 PAZ23 is complemented M. bovis BCG KOP55 with plasmid pler; valinomycin, a K⫹-specific transporter that dissipates the
pPAZ23 (p27-p55 operon cloned into pSUM41). electrochemical potential across the membrane; and reserpine,
d
PAZ23 is M. bovis BCG containing plasmid pPAZ23 (p27-p55 operon in
pSUM36). a general inhibitor of bacterial efflux pumps.
e
CCCP, valinomycin (VAL), and reserpine (RP) were present at concentra- Subinhibitory concentrations of CCCP increased the suscep-
tions of 1, 0.1, and 0.5 mg/liter, respectively (fourfold the sub-MIC for the M.
bovis BCG wild type).
tibility of the wild-type strain to novobiocin and rifampin (four-
f
ND, not determined. fold). In addition, the novobiocin and rifampin resistance of
KOP55 restored by pPAZ23 (expressing p27-p55) was abol-
ished in the presence of CCCP. These results suggest that this
susceptibility assays were carried out to determine whether the proton uncoupler has a direct effect on the function of P55
M. bovis BCG KOP55 mutant had become sensitive to a range (Table 3) and reconfirmed that rifampin and novobiocin are
of different compounds (Table 3). To explore the possibility substrates of the P55 efflux pump. In contrast, at the same
that the regulatory responses predict functional properties, concentration, CCCP did not affect the MICs of bacitracin and
compounds in the database of Boshoff et al. (7) that apparently vancomycin, suggesting that intrinsic P55-dependent resistance
altered the expression of p55 were included in the study. In- to bacitracin and vancomycin was mediated by a mechanism
deed, the KOP55 mutant was more susceptible to some com- that is not dependent on active transport. Valinomycin in-
pounds that alter p55 expression, including clofazimine, novo- creased the susceptibility of the wild-type strain to rifampin
biocin, PA-824, and valinomycin. However, compound-induced and novobiocin by twofold (Table 3). These data suggest that
expression was apparently not essential for P55-dependent resis- P55 uses the electrochemical gradient to extrude these antibi-
tance. While rifampin and econazole treatment did not result in otics. Subinhibitory concentrations of reserpine had little or no
detectable changes in p55 gene expression, the susceptibility to effect on the sensitivity to rifampin and novobiocin of the M.
these compounds increased in the mutant strain (for rifampin, bovis BCG wild-type, mutant, and complemented strains. Re-
eightfold; for econazole, fourfold) (Table 3). On the other serpine is apparently not as efficient as CCCP at inhibiting the
hand, susceptibility to chlorpromazine and triclosan, which function of P55 in M. bovis BCG.
increased the level of p55 expression according to the infor- Together, these data indicated that rifampin and novobiocin,
mation from the database of Boshoff et al. (7), was unchanged. but probably not bacitracin and vancomycin, are extruded from
Finally, since the P55 efflux pump may participate in the trans- the cell by P55 and that the efflux pump is using the proton and
port of lipids across the membrane (13), a P55 inhibitor may electrochemical gradients across the membrane as sources of
have an effect on cell envelope biosynthesis, making the bac- energy.
teria more sensitive to compounds that target the correspond- The P55 efflux pump is involved in oxidative stress-related
ing assembly step. In fact, the KOP55 mutant was more sen- processes within the cell. Analysis of the database of Boshoff
sitive than the wild type to some cell wall-targeting compounds, et al. (7) revealed p27- or p55-inducing compounds that either
such as ethambutol, vancomycin, and bacitracin (Table 3), but generate reactive oxygen species or alter redox potential (Ta-
not to cerulenin, D-cycloserine, or imipenem (data not shown). ble 2). On the basis of these results, the susceptibility of
VOL. 53, 2009 ROLE OF THE M. TUBERCULOSIS P55 EFFLUX PUMP 3679

TABLE 4. Susceptibilities of M. bovis BCG P55 strains to the cytoplasmic redox potential (7). The most dramatic obser-
redox compounds vation was a threefold increase in the MTT reduction activity
Inhibition zoneb (mm) for of KOP55 compared to the activities of the wild type and the
Disc load M. bovis BCG strains complemented mutant strain. MTT could be a P55 efflux pump
Redox compounda
(␮mol)
Wild type KOP55c substrate. To rule out the possibility that a strain lacking the
P55 efflux pump might be impaired in its ability to export MTT
Hydrogen peroxide 80 23 23
DL-Dithiothreitol 5 0 30d and thus accumulates more formazan than the wild-type strain,
L-Glutathione reduced 10 0 0 CCCP was used in control experiments. The observed propor-
Diamide 5 0 30 tional declines in the intracellular redox potentials of all three
Menadione 0.125 20 16 strains indicated that the assay measured the increased intra-
mBBre 0.5 0.125–0.25 cellular reductive power of KOP55 and did not quantify the
a MTT P55-dependent transport activity (Fig. 2). These results
Dimethyl sulfoxide, which was used as the solvent for some of these com-
pounds, had no effect on the growth of either strain (data not shown). suggest an increased intracellular redox potential in KOP55
b
Halos were recorded after 14 days at 37°C. compared to the redox potentials of the wild-type and comple-
c
KOP55 is M. bovis BCG with the p55 gene inactivated.
d
Delayed growth inside the halo. mented mutant strains under normal growth conditions.
e
Susceptibility to monobromobimane (mBBr) was determined by the Resaz- The effects of drugs on the intracellular redox potentials in
urin assay, and the concentrations are shown as the MICs (mM) for M. bovis the wild type and the KOP55 mutant were compared by the
BCG strains.
MTT assay (Fig. 2). Rifampin, econazole, and vancomycin
treatment induced no significant changes (data not shown).
KOP55 to various redox compounds was tested (Table 4). The Diamide decreased the abilities of the mutant and wild-type
mutant strain was not altered in its sensitivity to hydrogen strains to reduce MTT (by approximately 35 and 25%, respec-
peroxide (neither gene was induced by H2O2). However, it was tively), while the ability of the complemented strain to reduce
more resistant to menadione, a respiratory inhibitor and su- MTT remained unchanged. This suggested that the increased
peroxide-generating compound (Table 3). Compounds directly expression of the p27-p55 operon cloned into the low-copy-
involved in the reduction of disulfide bonds were also tested. number plasmid (the complemented strain) might protect
The mutant strain was more sensitive to the strong reducing against the oxidative effects caused by diamide. KOP55 under-
agent DL-dithiothreitol but not to glutathione, a weaker thiol went an ⬃15% reduction in its intracellular reductive power
reductant. KOP55 was also more susceptible to diamide, a after treatment with clofazimine, while no effect was observed
thiol-oxidizing agent. Mycothiol, the primary reducing agent in in either the wild-type or the complemented strain. In contrast,
the mycobacterial cytosol (34), can be specifically alkylated by under these conditions, menadione dramatically increased the
mBBr; KOP55 was more sensitive to mBBr. These results intracellular redox potential of the wild-type and comple-
demonstrate that the inactivation of p55 alters the sensitivity of mented strains (⬃3.5-fold) but had a minimal effect on the
M. bovis BCG to superoxides and some agents that disrupt mutant (⬃1.3-fold), almost abolishing the difference observed
systems needed to maintain the thiol redox balance in the among the untreated mutant, complemented, and wild-type
cytoplasm. strains. In summary, these results suggest that the P55 efflux
To further investigate the role of the P55 efflux pump in the pump plays a role in the maintenance of the oxidative balance
maintenance of the general redox balance, MTT assays were within the cell.
performed (Fig. 2). The rate of MTT reduction required to Cell growth is defective in M. bovis BCG KOP55. The
generate the colored product formazan is commonly inter- KOP55 mutant displayed two growth defects in liquid cultures:
preted as a reflection of the levels of NADH, which determines it had an increased lag phase and reached stationary phase at

FIG. 2. Intracellular redox potential of M. bovis BCG P55 strains. Cell cultures in quadruplicate wells were treated with drugs for 1 h before
the addition of MTT. The reduced formazan product was measured at 600 nm after an additional 3 h of incubation. The dimethyl sulfoxide solvent
had no effect compared to no treatment (data not shown). The results shown represent data from one of three independent experiments. Abs600,
absorbance at 600 nm.
3680 RAMÓN-GARCÍA ET AL. ANTIMICROB. AGENTS CHEMOTHER.

suggest that efflux systems are primarily involved with funda-

mental cellular physiological processes and that drug extrusion
is a nonspecific, secondary role (19, 20, 30). Thus, physiological
regulatory systems may determine the levels of drug resistance.
The expression database of Boshoff et al. (7) contains infor-
mation from 430 microarray profiles and has records of the
transcriptional regulation of M. tuberculosis genes in response
to 75 conditions (including drugs) that elicit defined metabolic
or functional changes (7). Our analysis of the results of drug
resistance assays in combination with the regulatory data gen-
erated by Boshoff et al. (7) allowed us to identify additional
P55 efflux pump substrates as well as to elucidate the involve-
FIG. 3. Growth kinetics of M. bovis BCG wild-type (squares), ment of P55 in physiological processes. Interestingly, the level
KOP55 (triangles), and KOP55 complemented (circles) strains. of p55 gene expression increased in response to clofazimine,
Growth curves are representative of those from two similar indepen- triclosan, and the respiration inhibitors phenothiazines. How-
dent experiments done in quadruplicate. Error bars indicate standard
deviations. OD600, OD at 600 nm. ever, only clofazimine was shown to be a P55 substrate. Boshoff
et al. (7) reported a general increased level of expression of
potential drug detoxification and efflux mechanisms in re-
a lower OD. The wild-type strain reached stationary phase with sponse to treatment with the compounds mentioned above,
an OD of 1.3, while the KOP55 mutant was able to reach a suggesting the presence of generalized, perhaps redundant reg-
stationary-phase OD of only 1.0 (Fig. 3). In addition, strain ulatory systems. In this regard, the P55 efflux pump would play
KOP55 initiated similar growth rates, but only after a 48-h a role in the detoxification systems linked to respiratory pro-
delay, apparently reflecting a prolonged recovery from station- cesses and maintenance of the redox balance within the cell.
ary-phase metabolism. Interestingly, the delayed outgrowth This is supported by the increased sensitivity of the P55 mutant
phenotype could be partially complemented by the introduc- to various redox compounds, including clofazimine, DL-dithio-
tion of the p27-p55 operon. Complementation reduced the lag threitol, and diamide, as well as the thiol alkylating agent
phase from 48 to 24 h; however, it did not allow the strain to mBBr. In the cytoplasm, mBBr specifically binds to mycothiol,
reach the same stationary-phase OD value (OD ⫽ 1.0) as the the thiol reducing agent that is involved in the primary detox-
wild type (OD ⫽ 1.3). The instability of the complementing ification pathway in mycobacteria. Although the products of
plasmid or the reduced level of expression of the cloned gene this pathway are exported, the nature of the corresponding
during this growth phase might explain this incomplete transport systems is unknown (23). Our data suggest a possible
complementation phenotype. In addition, when it was grown in role for the P55 efflux pump in eliminating the toxic products
liquid cultures without Tween, KOP55 displayed increased generated by a thiol reductant (including mycothiol) in Myco-
clumping compared with that of either the wild-type or the bacterium.
complemented strains, suggesting an altered cell wall compo- The MTT assay allowed further analysis of P55’s role in
sition. This phenotype was abolished in the presence of Tween general redox processes. Tetrazolium salts are widely used to
(data not shown). The results of these experiments demon- measure metabolic activity, as reflected by NADH levels. Since
strate that KOP55 has a growth defect in both the early and the NADH is the primary metabolite catalyzing the reduction of
final stages of cell growth in liquid culture. MTT to formazan (4), the rate of MTT reduction can be
To observe bacterial colonial growth, strains were plated on interpreted as a direct reflection of the cellular NADH con-
Middlebrook 7H10 agar, and the diameters of 100 colonies tent. The mutant strain displayed a large (threefold) increase
were measured. KOP55 mutant colonies were roughly half the in the level of formazan accumulation, suggesting a major
size (diameter, 1.52 ⫾ 0.12 mm) of colonies of the wild-type change in the intracellular reductive potential, most likely due
strain (diameter, 2.7 ⫾ 0.15 mm). However, the morphologies to increased NADH levels. This change was fully reversed by
of the mutant and wild-type colonies appeared to be similar genetic complementation by use of the p27-p55 operon (Fig.
when they were observed under an inverted microscope (⫻100 2). On the other hand, KOP55 was less susceptible to mena-
magnification), and no differences in the sizes or the morphol- dione (Table 4). During 4 h of exposure, menadione caused an
ogies of single bacilli were observed under a fluorescence mi- overall increase in the amount of MTT reduction products in
croscope after auramine-rhodamine staining (data not shown). all strains, probably by increasing the transfer of electrons from
Together, these data suggest that the P55 efflux pump plays NADH to MTT (4). Thus, menadione promotes net NADH
an important role in bacterial growth, even though it does not synthesis or accumulation, which is reflected in higher levels of
affect the cellular or the colonial morphology, which is proba- MTT reduction. After menadione treatment, the threefold dif-
bly related to its putative role as a transporter of lipids across ference in the reductive power of MTT displayed by the
the membrane. KOP55 mutant strain was almost abolished, suggesting that
menadione may in some way mimic the effect of the p55 mu-
tation. These data provide additional evidence that the P55
DISCUSSION
efflux pump serves as a key detoxification system that maintains
Efflux pumps have traditionally been linked to their multi- within the cell an oxidative balance that is probably related to
drug extrusion capabilities, which confer clinically significant respiratory processes. In fact, the respiratory modulators chlor-
levels of antibiotic resistance (29). However, recent insights promazine and triclosan increased the level of p55 expression
VOL. 53, 2009 ROLE OF THE M. TUBERCULOSIS P55 EFFLUX PUMP 3681

(7), even though they are not P55 substrates, as shown by our sion-like transporter MmpL7 extrudes isoniazid when the
susceptibility data. mmpL7 gene from M. tuberculosis is expressed in M. smegma-
Farrow and Rubin (13) showed that deletion of the p27-p55 tis. However, this is not observed in M. tuberculosis, in which
operon leads to morphological changes in M. smegmatis and the natural substrate of MmpL7, the lipid phthiocerol dimyco-
speculated that this was due to an inability to transport cell wall cerosate, competes with isoniazid. The findings of that study
lipids. Thus, any deficiency in lipid transport across the cyto- are in agreement with our own data showing that the substrate
plasmic membrane would affect the integrity of the cell wall, specificity of the P55 efflux pump is dependent on the genetic
making the mutant more sensitive to drugs targeting the cell background, and it is a clear example of the relevance of the
envelope and also altering its growth characteristics. In M. bacterial host when the activities of efflux pumps are analyzed.
bovis, two findings support these speculations. First, the mu- Other authors have also reported the importance of the iniA
tant strain has a growth defect. This clearly shows that bacteria gene product for the activity of an efflux pump that confers
lacking P55 have a physiological perturbation that affects their isoniazid and ethambutol tolerance (9).
fitness under in vitro growth conditions. Second, the P55 mu- To our knowledge, this is the first report of rifampin trans-
tant strain is highly sensitive to bacitracin and vancomycin, port by an efflux pump in mycobacteria. Other studies reported
which both target the peptidoglycan layer of the cell envelope, increases in the levels of expression of efflux pumps, including
suggesting the possible lack of an important substrate needed P55, after the treatment of clinical M. tuberculosis strains with
for the integrity of the cell wall. It is tempting to suggest that rifampin and isoniazid (15, 17, 38). However, those studies did
P55 could actively transport these antibiotics away from the not show a direct correlation between expression of the P55
peptidoglycan layer, perhaps in combination with a periplasmic efflux pump and intrinsic resistance to rifampin in mycobacte-
adaptor and TolC-like proteins, as has been described for ria. A decrease in the intracellular levels of rifampin would
other efflux pumps (21). However, CCCP was unable to inhibit allow the bacillus to better adapt to the presence of rifampin by
P55-mediated bacitracin and vancomycin resistance, whereas it acquiring mutations in the rpoB gene. This is of particular
could inhibit P55-mediated rifampin and novobiocin resis- importance, since the p55 gene is present in all M. tuberculosis
tance. This suggests that P55 contributes to the intrinsic strains whose genomes have been sequenced, such as the lab-
resistance to bacitracin and vancomycin, perhaps by contribut- oratory strain M. tuberculosis H37Rv, as well as clinical isolates
ing to the impermeability of the cell wall, but it might not be M. tuberculosis CDC1551, F11, Haarlem, C, KZN 4207, KZN
actively transporting both drugs. 1435 (an MDR-TB strain), and KZN 605 (an XDR-TB strain)
In general, the overexpression of an efflux pump decreases (http://www.broad.mit.edu/). In addition, PCR experiments
the susceptibility of bacteria to substrates of the pump, and have detected p55 in 10 of 10 DNA samples from our collec-
susceptibility increases when the gene encoding the efflux tion of M. tuberculosis complex clinical isolates, which in-
pump is inactivated. The introduction of pPAZ23 (which con- cludes 4 rifampin-resistant strains (the XDR strain MBZ
tains the p27 and p55 genes under the control of the operon [36] and 3 MDR-TB strains) and 6 rifampin-susceptible
promoter) into the KOP55 mutant restored drug susceptibility isolates of M. tuberculosis from different geographic origins
levels, suggesting that p55 was being expressed. However, when (data not shown).
additional copies of the operon were introduced (pPAZ23) P55 is a transporter belonging to the drug/H⫹ antiporter
into the wild-type strain, little or no decrease in drug suscep- 14-transmembrane-domain (DHA14) family whose members
tibility was observed, suggesting the presence of autoregulatory are thought to export cationic molecules by proton motive
control mechanisms. Indeed, we have obtained preliminary force (12). We demonstrated that this is the main source of
evidence that the p55 gene is also expressed independently energy used by the P55 efflux pump to extrude drugs. On the
under the control of its own promoter. It is noteworthy that the other hand, the fact that valinomycin also exerted an effect on
level of p55 transcription from this promoter increased fivefold drug transport suggests that the electrochemical potential
after exposure to rifampin and vancomycin, both of which are across the membrane can also be used as a source of energy.
substrates of the efflux pump (S. Ramón-García and J. A. Surprisingly, reserpine had very little inhibitory effect on the
Aínsa, unpublished data). The induction of genes encoding expelling activity of P55 in M. bovis BCG since it inhibits
efflux pumps by their own substrates has been described pre- tetracycline efflux by another mycobacterial DHA14 family
viously (1, 8, 14, 18). In this regard, we propose a model in efflux pump (32), as well as the transport of ethidium bromide
which p55 can be expressed selectively under its own promoter by the homologue of P55 in M. smegmatis (13).
or under the control of the operon promoter (in combination In most bacterial species, efflux pump inhibitors can reduce
with p27), depending on the particular conditions that the resistance levels both in wild-type strains and in those with
bacteria confront, such as antibiotics, a need for detoxification, acquired target mutations (22). In this regard, an inhibitor of
oxidative stress, or other physiological conditions. We are car- the P55 efflux pump would render the bacillus more susceptible
rying out studies to test this hypothesis. to the first- and second-line drugs rifampin and clofazimine (or
As efflux pump-mediated drug extrusion in bacteria is of other drugs transported by P55), in either susceptible or MDR-
clinical significance (29), determination of the substrate pro- and XDR-TB strains. In a scenario in which few or no effective
files of these transporters could allow the design of new ther- drugs are available for the treatment of patients infected with
apies against bacterial infections. In mycobacteria, many efflux MDR or XDR M. tuberculosis strains, the possibility that ri-
pumps may contribute to intrinsic drug resistance (11). How- fampin may be able to be reintroduced is very appealing. In
ever, only a few of them are known to provide resistance to addition, it would also reduce the emergence of rifampin-
anti-TB drugs and, in particular, to first-line anti-TB drugs. resistant strains, and since a p55 mutant of M. tuberculosis has
Pasca et al. (28) showed that the resistance-nodulation-divi- been predicted to be attenuated (37), a putative P55 efflux
3682 RAMÓN-GARCÍA ET AL. ANTIMICROB. AGENTS CHEMOTHER.

pump inhibitor could also reduce the virulence of the tubercle 16. Janssen, G. R., and M. J. Bibb. 1993. Derivatives of pUC18 that have BglII
sites flanking a modified multiple cloning site and that retain the ability to
bacillus. Thus, this inhibitor would help to control the spread identify recombinant clones by visual screening of Escherichia coli colonies.
of this devastating disease. We currently are performing high- Gene 124:133–134.
throughput chemical screens to explore this concept. 17. Jiang, X., W. Zhang, Y. Zhang, F. Gao, C. Lu, X. Zhang, and H. Wang. 2008.
Assessment of efflux pump gene expression in a clinical isolate Mycobacte-
ACKNOWLEDGMENTS rium tuberculosis by real-time reverse transcription PCR. Microb. Drug Re-
sist. 14:7–11.
We gratefully acknowledge Helena Boshoff and Clifton E. Barry III 18. Kaatz, G. W., and S. M. Seo. 1995. Inducible NorA-mediated multidrug
for the kind gift of PA-824. We are also grateful to Charles Howes for resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 39:
his help analyzing the database of Boshoff et al. (7) and to Cristina 2650–2655.
Villellas and Carmen Lafoz for technical assistance with the clinical 19. Krulwich, T. A., O. Lewinson, E. Padan, and E. Bibi. 2005. Do physiological
roles foster persistence of drug/multidrug-efflux transporters? A case study.
distribution studies of the p55 gene and Southern blot analysis. Nat. Rev. Microbiol. 3:566–572.
This work was supported by European Union grant QLK2-CT-2000- 20. Lewinson, O., E. Padan, and E. Bibi. 2004. Alkali tolerance: a biological
017610 (to C.M.), Spanish Ministry of Science and Education grants function for a multidrug transporter in pH homeostasis. Proc. Natl. Acad.
BIO2002-01297 and BIO2005-01810 (to J.A.A.), and Canadian Insti- Sci. USA 101:14073–14078.
tute of Health Research grants MOP-82745 and MOP-82855 (to 21. Li, X. Z., and H. Nikaido. 2004. Efflux-mediated drug resistance in bacteria.
C.J.T.). S.R.-G. held grants from the Spanish Ministry of Science Drugs 64:159–204.
and Education (AP2001-1114) and the Fundación Alfonso Martin 22. Lomovskaya, O., M. S. Warren, A. Lee, J. Galazzo, R. Fronko, M. Lee, J.
Escudero (Spain). Blais, D. Cho, S. Chamberland, T. Renau, R. Leger, S. Hecker, W. Watkins,
K. Hoshino, H. Ishida, and V. J. Lee. 2001. Identification and characteriza-
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