i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 6 ( 2 0 1 9 ) 2 0 e2 5

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indian-journal-of-tuberculosis/

Review article

Efflux pump as alternate mechanism for drug

resistance in Mycobacterium tuberculosis

Akbar Kanji, Rumina Hasan, Zahra Hasan*

Department of Pathology and Laboratory Medicine, The Aga Khan University, Karachi, Pakistan

article info abstract

Article history: Tuberculosis (TB) remains an important global public health issue with an approximate
Received 20 February 2018 prevalence of 10 million people with TB worldwide in 2015. Since antibiotic treatment is
Accepted 26 July 2018 one of the foremost tools for TB control, knowledge of Mycobacterium tuberculosis (MTB) drug
Available online 1 August 2018 resistance is an important component for disease control. Although gene mutations in
specific loci of the MTB genomes are reported as the primary basis for drug resistance,
Keywords: additional mechanisms conferring resistance to MTB are thought to exist. Efflux is a
Drug efflux pumps ubiquitous mechanism responsible for innate and acquired drug resistance in prokaryotic
Drug resistance and eukaryotic cells. MTB presents a large number of putative drug efflux pumps compared
Mycobacterium tuberculosis to its genome size. Bioinformatics-based evidence has shown an association between drug
ABC transporters efflux and innate or acquired resistance in MTB. This review describes the recent under-
MFS transporters standing of drug efflux in MTB.
© 2018 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.

Patients affected with MDRTB are treated following the

1. Introduction recommendations of the WHO according to defined parame-
ters.1 There are no official specific recommendations for the
The high mortality and morbidity associated with tubercu- treatment of patients with XDR-TB, although positive experi-
losis (TB), especially in developing countries, is one of the ences have been reported.3 Patients affected with XDR-TB
important features characterizing TB as a major public health remain with fewer options for treatment and risk higher
concern globally. In the absence of a more effective vaccine, mortalities, especially in human immunodeficiency virus
chemotherapy is one of the main TB control tools. There has (HIV) co-infected persons as has been reported earlier.4 The
been an increase in the prevalence of TB cases with multi- innate drug resistance in MTB has been recognized to the
drug- and extensively-drug resistance in several settings.1 combination of a highly impermeable mycolic acid-containing
Multi drug resistant TB (MDR) is caused by strains of MTB cell wall and an active drug efflux mechanism.5 As MTB does
that are resistant to at least rifampicin and isoniazid, two not contain plasmids, no horizontal gene transfer occurs,
important drugs in the treatment of the disease. Extensively however spontaneous mutations in specific target causes MTB
drug resistant TB (XDR) is caused by strains of MTB that in to be resistant to a given drug.6
addition to being MDR are also resistant to any fluo- The knowledge of drug resistance in MTB has increased
roquinolones and to one of the three injectable second-line substantially in the last twenty years, gene mutations in several
drugs: kanamycin, capreomycin or amikacin.2

* Corresponding author. Tel.: þ92 300 8247815.

E-mail address: [email protected] (Z. Hasan).
https://doi.org/10.1016/j.ijtb.2018.07.008
0019-5707/© 2018 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.
 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 6 ( 2 0 1 9 ) 2 0 e2 5 21

loci have been characterized as the main basis for drug resis- efflux pump genes have been identified which include:
tance.7 However, in a certain proportion of clinical MTB strains, Rv0194, Rv1218c-Rv1217c, drrA-drrB-drrC, Rv1273c-Rv1272c,
drug resistance cannot be explained by the presence of gene Rv2688c-Rv2687c-Rv2686c, Rv1348-Rv1349, Rv1456c-Rv1457c-
mutations. It has been previously shown in 30% of isoniazid Rv1458c, Rv1473, Rv1667c-Rv1668c, Rv1686c-Rv1687c, Rv1819,
resistant and 5% of rifampicin resistant MTB clinical strains, Rv2477.22 They use the energy from ATP hydrolysis to export
this suggests that alternate mechanisms of drug resistance the substrate.22 The members of the ABC superfamily use
must be present.8,9 Amongst these, efflux has been anticipated Adenosine tri phosphate (ATP) as the energy source and are
as the basis for drug resistance in clinical strains which do not considered as primary transporters.23 Usually, the common
have gene mutations described earlier.10,11 feature of all ABC transporters is consisting of two hydro-
Drug efflux has also been associated with pathogenicity, phobic membrane spanning domains (MSDs) associated with
virulence, biofilm formation and coordinate gene expression two cytoplasmic nucleotide binding domains (NBDs).24 Whilst
according to the density of bacterial population (quorum the MFS, SMR, RND, and MATE members are secondary
sensing).12 However, not all currently available anti- transporters, characteristically energized by the proton
tuberculosis drugs, are considered as substrates of efflux motive force (Hþ or Naþ).
mechanisms.13 Analysis of the available bacterial genomes has
shown that putative drug efflux pumps (EPs) constitute 6%e18% 1.4. Association between ABC efflux pump expression
of all transporters found in any given bacterial cell. MTB pre- and drug sensitivity of MTB
sents one of the largest numbers of putative EPs compared to its
genome size.14 The mechanisms for the expression and regu- Efflux pumps belonging to the ABC class, encoded by Rv1218c,
lation of these efflux pumps are not yet fully understood. and the SMR class, encoded by Rv3065, have been shown to
Moreover, their role in drug resistance needs to be understood.15 play important roles in mediating the efflux of different
chemical classes and antibiotics in M. smegmatis.25 Efflux
1.1. Drug efflux in the genus Mycobacterium pumps encoded by Rv0849 and Tap (Rv1258c) also help in the
efflux of these compounds, but to a lesser extent in MTB.25
In Mycobacterium smegmatis as well as in other mycobacteria, the
cell wall, rich in mycolic acids, functions as an efficient barrier 1.5. Rv1217c (MT1255)e1218c (MT1256): tetronasin-
preventing the access of several molecules including antibi- transport membrane protein ABC transporter
otics. However, this is not enough for explaining the innate drug
resistance of these microorganisms.16 Although efflux mecha- This efflux pump has been shown to be composed of two
nisms have been studied in several mycobacteria17 M. smeg- copies of the Rv1218c, active as the NBD, and one copy of the
matis has been used as the model system for expressing Rv1217c, which was fused by two MSDs.24 Previous studies
heterologous putative efflux pump genes and to study the efflux showed that Rv1218c is responsible for the efflux of a wide
mechanism.18 The first efflux pump described in mycobacteria variety of drugs. The Minimum Inhibitory Concentrations
was the LfrA; a Major Facilitator Super-family (MFS) present in (MICs) of these drugs decreased 4e8-fold in the Rv1218c
M. smegmatis that expressed in multicopy plasmids confers low- mutant compared to those of the wild-type strain.26 A previ-
level resistance to fluoroquinolones, ethidium bromide, acri- ous study has shown that Rv1218c, a major ABC transporter in
dine and some quaternary ammonium compounds.19 Other MTB, was found to be responsible for the efflux of a wide va-
Efflux pumps initially characterized in mycobacteria were TetV riety of substrates including novobiocins, pyrazolones, biar-
conferring resistance to tetracycline20 and Tap (Rv1258c) has ylpiperazines, bisanilinopyrimidines, pyrroles, and pyridones.
been shown to confer low-level resistance to aminoglycosides
and tetracycline when overexpressed in M. smegmatis.21 1.5.1. Rv2686c-Rv2687c-Rv2688c
The reports mentioned below can be considered as pre- The MTB operon encoded by Rv2686c-Rv2687c-Rv2688c region
cursors of the different studies of drug efflux in MTB. conferred resistance to fluoroquinolones, when overex-
pressed in M. smegmatis showed 8- fold increase in the MIC for
1.2. Putative efflux pumps in MTB M. smegmatis strains. However, in the presence of an efflux
inhibitor it showed reduced ciprofloxacin resistance to the
Putative efflux pumps also called transporters are categorized same level as that of control which do not contain this
in five superfamilies: ATP-binding cassette (ABC), Major operon.27
Facilitator Super-family (MFS), Resistance Nodulation Division
(RND), Small Multidrug Resistance (SMR) and Multidrug and 1.6. Daunorubicin-dim-transport ATP-binding protein
Toxic-Compound Extrusion (MATE). ABC transporter drrA (Rv2936) and drrB (Rv2937)

1.3. ABC efflux pumps The drrA and drrB, encode an ATP binding cassette (ABC) type
transporter, drrA encodes the nucleotide binding domain,
The ABC transporters have been found both in prokaryotes while the drrB is encoded by the membrane integral compo-
and eukaryotes and constitute a large superfamily of multi- nent.16 The drr operon has been shown to play a significant
subunit permeases that transport amino acids, antibiotics role in virulence of MTB,17 this operon has been shown to be
and polysaccharides across biological membranes.10 ABC an important drug target, as it exports a surface antigenic lipid
class is the largest known super-family which encodes 2.5% of known as phthiocerol dimycocerosates (DIMs) to the cell
the genome of MTB. By sequence analysis at least 12 putative surface.18
22 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 6 ( 2 0 1 9 ) 2 0 e2 5

The Rv0194, an ABC multidrug efflux pump was identified either the hypothetical transcriptional regulator, or in its pu-
in MTB while investigating the molecular mechanisms for tative promoter or operator region of the Rv0678 gene.34
resistance to beta-lactam antibiotics.28 The low level expres-
sion of Rv0194 increased resistance of Mycobacterium bovis BCG 2.2. Overexpression of efflux pump genes and
to several other antibiotics.28 antimicrobial drug resistance profile
Based on bioinformatics predictions (http://www.
membranetransport.org/) up to 20 potential efflux pump Increased resistance to several drugs has been reported using
genes belonging to the MFS drug transporters have been M. smegmatis or M. bovis as expression hosts and plasmids
identified in the MTB genome.29 carrying genes coding for putative efflux pumps in MTB. Using
this method, Rv1258c and Rv1410 (P55) encoding MFS trans-
porters, produced resistance to tetracycline and aminoglyco-
2. Major Facilitator Super-family (MFS) sides. In the same way, Rv1634 gene, another member of the
MFS family has been shown to be overexpressed and confer
2.1. Association between ABC efflux pump expression resistance to fluoroquinolones.30
and drug sensitivity of MTB The stp gene (Rv2333c) from MTB has been shown to confer
resistance to spectinomycin and tetracycline when expressed
According to the sequence and motif similarity to efflux in M. bovis BCG.17 Overexpression studies have also shown
pumps identified in numerous microorganisms, at least 16 that an ABC-type efflux pump gene, Rv0194 conferred multi-
putative efflux pump genes of the MFS family have been drug resistance in M. bovis BCG and M. smegmatis, and also
identified in MTB which include: Rv0037c, Rv0191, Rv0783c, reduced the accumulation of ethidium bromide in the latter.28
Rv0849, Rv1250, Rv1258c, Rv1410c, Rv1634, Rv1877, Rv2333c, Using the recombinant avirulent strains of MTB H37Ra, the
Rv2456c, Rv2459, Rv2846c (efpA), Rv28994, Rv3239c and overexpression of Rv2459 (jef A) led to an increase in minimum
Rv3728.30 The Tap (Rv1258c) protein and LfrA mentioned inhibitory concentrations (MICs) of anti-tuberculosis first line
before are efflux pumps belonging to the MFS family which drugs such as isoniazid and ethambutol. These MIC values
has been shown to confer resistance to antibiotics such as were again decreased when growing the bacteria in the pres-
tetracycline and fluoroquinolones, respectively.21 The P55 ence of the efflux pump inhibitors carbonyl cyanide m-chlor-
protein encoded by the Rv1410c gene was classified as a ophenyl hydrazone (CCCP) and verapamil. Bioinformatics
multidrug efflux pump of the MFS family in MTB and M. bovis analyses have also shown a close association of the JefA protein
conferring resistance to streptomycin and tetracycline.31 P55 with drug efflux pumps in other clinically relevant bacteria.35
has been previously shown to require the MTB cell surface
lipoprotein, LprG (Rv1411c) to function properly.32 Both the P55 2.3. Drug-induced efflux pumps
and LprG lipoproteins have been shown to play an important
role in the persistence of MTB during infection.33 Exploring drug-induced alterations in gene expression has
The MTB Rv3065 Multidrug resistance protein (mmr) has associated a number of efflux pumps with different drugs.
been identified as a multidrug efflux pump of the SMR family Using this approach Siddiqi et al. reported a MDR MTB clinical
that confers resistance to acriflavine, ethidium bromide and isolate that when grown in the presence of sub-inhibitory
erythromycin in M. smegmatis when expressed in a multicopy concentrations of rifampicin and ofloxacin showed
vector.20 A previous study has shown that an efflux pump of increased transcription of the gene Rv1258c, that encodes a
the RND family has also been found in gram negative bacteria. tap-like efflux pump. Although drug resistance-associated
Moreover, the conserved transmembrane transport protein, mutations were detected in gyrA and rpoB, it was assumed
mmpL7 gene that encodes a putative RND transporter confers that the high level of resistance to rifampicin and ofloxacin
a high-level resistance to isoniazid, when overexpressed in M. could reflect an additional overexpression of the gene
smegmatis.27 This resistance was reversed and MTB strains Rv1258c.36 The overexpression of efflux pump genes were also
were shown to become sensitive in the presence of efflux found in another study with a clinical MDR isolate in which
pump inhibitors. the expression of Rv1258c and a multidrug efflux pump P55
The MTB genome encodes 15 putative transmembrane (Rv1410c) was significantly increased in the presence of
proteins, predicted to belong to the RND family (http:// rifampicin or isoniazid.37 Sharma et al also reported the as-
genolist.pasteur.fr/TubercuList/). Since these proteins sociation of Rv1258c as a putative efflux pump in MTB by
showed some characteristics only associated with mycobac- showing that piperine, a transetrans isomer of 1-piperoyl-
teria they were designated MmpL (mycobacterial membrane piperidine, inhibited the clinically overexpressed Rv1258c
proteins).16 Transporters belonging to the MATE family have gene.38 A genome-wide expression analysis using microarray
not been reported in mycobacteria being more common in hybridization and induced gene expression in the presence of
Escherichia coli and Vibrio sp.16 Previously, it was also found an antibiotic was reported by Wilson et al, it was found that
that there was an increased transcription of mmpS5-mmpL5 isoniazid and ethionamide increased the expression of efpA, a
genes and econazole drug efflux, encodes a hypothetical member of the MFS family of efflux pumps.39
efflux pump of the RND family characterizing several azole A study by Gupta et al, using a DNA microarray with 25
resistant spontaneous mutants of MTB and M. bovis BCG. drug efflux pump genes of MTB found overexpression of ten
In addition, it has been established that up-regulation of genes after exposure to various anti-TB drugs. These included
mmpS5-mmpL5 genes were associated with mutations in Rv3065 and Rv2938 already reported as active MTB drug efflux
pumps genes in previous studies and another eight efflux
 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 6 ( 2 0 1 9 ) 2 0 e2 5 23

pump genes reported for the first time (Rv1819, Rv2209, deficiency causes this attenuation of infection in mice, but it
Rv2459, Rv2477c, Rv2688, Rv2846, Rv2994, and Rv3728).40 At the has been reported that in B. abortus bacA mutant induce larger
same time, overexpression of the efflux pump genes Rv2459, amounts of pro-inflammatory cytokines compared to the B.
Rv3728, and Rv3065 was associated with resistance to the abortus wild type strain.47
combination of first line anti-TB drugs, isoniazid and etham-
butol along with streptomycin, were identified to group
together, indicating their possible significance in the devel- 3. Conclusion
opment of multidrug resistance in MTB. An interesting study
has reported the induction of high-level resistance to isoni- Bioinformatics and experimental data showing the relation-
azid (up to 20 mg/ml) in MTB strains, which could be reduced ship between efflux mechanisms and drug resistance in MTB
100-fold by sub-inhibitory concentrations of reserpine, sup- have emphasized the need to further our understanding of the
porting the argument that induced drug resistance may be mechanisms of drug resistance in TB. It is necessary to better
due to an drug efflux pump mechanism.41 identify the correlation between efflux pump gene expression
In MTB, an antibiotic inhibitory system similar to the and their inducers, both under environmental conditions and
multidrug-resistance system present in Actinobacter sps has in the presence of drugs. Whilst, the efflux pumps described in
been described. This system is dependent on the whiB7 gene, MTB show a small number of substrates, point mutations
which is induced by sub-inhibitory concentrations of antibiotics. could significantly alter the spectrum of substrates as has
The expression of whiB7 was found to be increased five-fold in been described in other microorganisms.48 Molecular analysis
the late logarithmic and early stationary phases with conse- using multicopy plasmids or gene knock-outs has the
quent reduction to control levels in the late stationary phase of advantage of identifying the accurate position or locus
growth. Gene expression analysis has shown that transcription involved in efflux-mediated resistance; however, its limitation
of whiB7 determined drug resistance by activating the expres- being the limited approach on the complex interaction be-
sion of a regulon that includes Rv1258c and Rv1473.42 In MTB, it tween the efflux pumps, the regulatory proteins and several
was observed that low concentrations of salicylate induced inducers.
resistance to different anti-TB drugs such as rifampicin, isoni- On the other hand, the phenotypic methods have the benefit
azid, ethambutol and streptomycin.43 Although salicylate can of identifying vital efflux mechanisms as a result of the broad
also induce antibiotic resistance through a Mar-independent interaction of several genes responsible for efflux, but they are
pathway, it is possible to infer the presence of a transcriptional unable to determine the exact efflux pump involved. Whilst the
activator like multi antibiotic resistance locus, MarA in MTB.44 role of efflux in adaptive and innate drug resistance is not
In the MTB genome, it has been shown that the Rv1931c, completely explained, it is probable to consider this mecha-
Rv3736 and Rv3833 genes show 30% of identity with MarA and nism as contributing factor to clinical drug resistance working
could be related to an efflux pump gene overexpression in combination with other mechanisms of resistance such as
(http://genolist.pasteur.fr/TubercuList/). It remains to be impermeability of the cell wall and drug resistance mutations.
investigated how these mechanisms of transcriptional regu- Clinically, the detection of drug efflux remains an impor-
lation function and if they are responsible for clinically rele- tant goal since it allows identifying mechanisms that could be
vant drug resistance in MTB. related to an increase in drug resistance. For this reason it
would be important to develop precise and simple diagnostic
2.4. Drug efflux pumps and MTB virulence methods that could identify and characterize efflux events.
Along this line, although not yet used consistently, minimum
It has been shown that there is an association between efflux inhibitory concentration determination in the presence or
pumps and virulence. In some bacteria efflux pumps can also absence of known efflux inhibitors or assessment of substrate
export virulence determinants such as adhesins, toxins or accumulation using radiolabeled substrates or instrument-
other proteins that are important for colonization and free methods have been proposed.41
persistence in human and animal cells.45 The survival of MTB The recent understanding on efflux pump shows that
within the macrophage depends on its capacity to inhibit the efflux inhibitors could become candidate tools to treat infec-
normal maturation of the phagosome. The isolation of tious diseases.49 Certain compounds already in use in clinical
defective MTB mutants unable to arrest phagosome matura- practice for other purposes, such as verapamil, reserpine and
tion showed that some affected genes were homologues of omeprazole are capable of inhibiting efflux mechanisms in
putative efflux pumps and lipid synthesis enzymes. One such several eukaryotic and prokaryotic cells. However, they have
mutant had a knockout in Rv1819c gene, which is character- been found to be mostly active at concentrations higher than
ized as a putative efflux pump of the ABC family. However, it is clinically used.50
interesting to note that an increasing number of MDR ABC In conclusion, several compounds that inhibit efflux ac-
transporters have been shown to transport lipids. Thus, it is tivity have been synthesized or obtained from natural sources,
likely that Rv1819c might also be responsible for the transport but none of them are currently used to treat infectious
of lipids to the exterior of the bacterium.46 diseases.50
It has been shown by Domenech et al that the ABC trans- Since the numbers of antimicrobials available for the treat-
porter encoded by Rv1819c, which shares 39% similarity with ment of TB are limited and the unquestionable association be-
the BacA protein of Brucella abortus, plays an important role in tween efflux and drug resistance in MTB, it is crucial to extend
the maintenance of extended chronic TB infection in mice.47 our knowledge on drug efflux as well as to develop compounds
However, it is not known by which mechanism the BacA that could counterbalance this resistance mechanism.
24 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 6 ( 2 0 1 9 ) 2 0 e2 5

16. Li XZ, Zhang L, Nikaido H. Efflux pump-mediated intrinsic

Conflicts of interest drug resistance in Mycobacterium smegmatis. Antimicrob
Agents Chemother. 2004;48(7):2415e2423.
The authors have none to declare. 17. Ramon-Garcia S, Martin C, Thompson CJ, Ainsa JA. Role of the
Mycobacterium tuberculosis P55 efflux pump in intrinsic drug
resistance, oxidative stress responses, and growth. Antimicrob
Agents Chemother. 2009;53(9):3675e3682.
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