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Free Radical Biology and Medicine 53 (2012) 1625–1641
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Free Radical Biology and Medicine
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Review Article
The mechanism of redox sensing in Mycobacterium tuberculosis
Shabir Ahmad Bhat, Nisha Singh, Abhishek Trivedi, Pallavi Kansal, Pawan Gupta, Ashwani Kumar n
Council of Scientific and Industrial Research, Institute of Microbial Technology, Chandigarh 160036, India
a r t i c l e i n f o
abstract
Article history:
Received 12 April 2012
Received in revised form
3 August 2012
Accepted 3 August 2012
Available online 11 August 2012
Tuberculosis epidemics have defied constraint despite the availability of effective treatment for the past
half-century. Mycobacterium tuberculosis, the causative agent of TB, is continually exposed to a number
of redox stressors during its pathogenic cycle. The mechanisms used by Mtb to sense redox stress and
to maintain redox homeostasis are central to the success of Mtb as a pathogen. Careful analysis of the
Mtb genome has revealed that Mtb lacks classical redox sensors such as FNR, FixL, and OxyR. Recent
studies, however, have established that Mtb is equipped with various sophisticated redox sensors that
can detect diverse types of redox stress, including hypoxia, nitric oxide, carbon monoxide, and the
intracellular redox environment. Some of these sensors, such as heme-based DosS and DosT, are unique
to mycobacteria, whereas others, such as the WhiB proteins and anti-s factor RsrA, are unique to
actinobacteria. This article provides a comprehensive review of the literature on these redox-sensory
modules in the context of TB pathogenesis.
& 2012 Elsevier Inc. All rights reserved.
Keywords:
Redox homeostasis
Redox sensing
Virulence
DosS
DosT
DosR
WhiB3
Anti-s factors
Serine–threonine kinases
Metabolic flexibility
Free radicals
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1626
The role of redox stress in tuberculosis pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1626
The role of ROS in tuberculosis pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1626
The role of hypoxia in latency and reactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1626
The role of nitric oxide (NO) in tuberculosis pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627
The role of acidic pH stress in tuberculosis pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1628
Mtb lacks classical redox sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1628
DosS and DosT: heme-based sensors of redox, hypoxia, NO, and CO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1629
The Dos regulon and the mycobacterial response to hypoxia, NO, and CO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1629
The molecular mechanism of redox and gas sensing by DosS and DosT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1631
WhiB proteins as iron–sulfur cluster-based sensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1632
WhiBs as iron–sulfur cluster proteins with protein disulfide reductase activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1632
WhiBs as redox-responsive transcriptional factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634
WhiB proteins as virulence and stress response factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634
Redox-regulated s factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634
The role of serine–threonine kinases in redox homeostasis of mycobacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1636
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1637
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1637
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1637
n
Corresponding author. Fax: þ91 172 2690585.
E-mail address: [email protected] (A. Kumar).
0891-5849/$ - see front matter & 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.freeradbiomed.2012.08.008
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Introduction
Infectious diseases lead to the deaths of 15 million people
annually and continue to be the primary cause of morbidity and
mortality in underdeveloped and developing nations [1]. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis
(TB), has latently infected one-third of the world’s population and
remains the leading cause of death by a single infectious agent
(1.7 million deaths annually). Of those infected with Mtb, only
5–10% develop active TB. The remaining individuals can control
but not eliminate the infection. These infected individuals have a
10% lifetime risk of developing TB, but suppression of the immune
system by aging, HIV infection, or immunosuppressive therapy
may increase their risk to 10% annually. The genetic and mechanistic details of how Mtb switches from the persistent state, which
can last for decades, to the replicative form are not well characterized and represent an important gap in our understanding of
the biology of TB pathogenesis.
Mtb faces a number of host-generated oxidoreductive stresses
during its cycle of infection. These stresses are believed to be
important for the transition between latency and reactivation.
Mtb senses redox stress through diverse mechanisms and then
exploits its metabolic flexibility to survive the stress and to
initiate the genetic program for entering and establishing a
dormant state. A better understanding of Mtb physiology, including the mechanistic details of its sensory pathways, is important
for the development of better diagnostic tools, effective vaccines,
and potent drugs. The role of redox homeostasis in TB pathogenesis
has recently been reviewed elsewhere [2,3]; hence this review
focuses on the redox-sensing mechanisms used by mycobacteria.
The role of redox stress in TB pathogenesis
During infection, mycobacteria are exposed to a number of
redox stresses, such as reactive oxygen species (ROS), reactive
nitrogen species (RNS), acidic pH, nutrient starvation, and
hypoxia. Exposure of mycobacteria to redox stress triggers
changes in metabolism and physiology that not only help it to
survive inside the host, but also allow it to express virulence
factors that ultimately lead to disease pathogenesis. The level of
redox stress in the microenvironment surrounding Mtb plays an
important role in determining whether the bacilli enter the
nonreplicating persistent state or the replicative state.
The role of ROS in TB pathogenesis
The primary type of cells encountered by Mtb upon infection is
the alveolar macrophage. Human alveolar macrophages employ a
battery of enzymes to generate oxidative stress in an effort to
eliminate the pathogen. Examples of these enzymes include
NADPH oxidase (NOX), myeloperoxidase, catalase, and hydrolases. NOX is a multiprotein enzyme system with core components such as p40phox, p47phox, p67phox, p22phox, and gp91phox, as
well as a number of regulatory components. NOX extracts
electrons from NADPH and transfers them to oxygen to generate
superoxide radicals using a low-potential b-type cytochrome [4].
Superoxide radicals leads to generation of other ROS such as
H2O2, hypochlorite, and hydroxyl radicals. A number of mycobacterial species, including Mtb [5], are susceptible to the cidal
and growth-inhibitory activities of ROS. ROS have the potential to
damage a number of cellular components, including lipids,
proteins, and DNA. The toxic effect of ROS is further compounded
in Mtb by the absence of important DNA repair pathways such as
mismatch repair [6,7]. Mtb is equipped, however, with a number
of protective mechanisms and enzymes, including superoxide
dismutase, catalase (KatG), alkyl hydroperoxidase (AhpC), and
peroxiredoxins, to neutralize the redox stress generated by the
macrophage cells [2]. In addition, the mycolic acid-rich cell wall
of Mtb also protects against ROS. Mtb maintains its intracellular
redox potential using mycothiol as an intracellular redox buffer [8]. Mycothiol is a conjugate of N-acetylcysteine with a
pseudo-disaccharide of glucosamine and myoinositol [9]. Using
these protective mechanisms in a regulated manner, Mtb can
survive physiologically relevant levels of ROS [2].
The role of ROS in TB pathogenesis is highlighted by the
observation that alveolar macrophages and blood monocytes
obtained from active TB patients produce significantly reduced
levels of ROS upon Mtb infection compared with cells from
healthy individuals [10,11]. This decrease in ROS is related to
the decreased activities of NADPH oxidase and the enzymes of the
hexose monophosphate shunt [10]. Because only a small percentage of infected individuals develop active TB, it is possible that
the extent of ROS production by alveolar macrophages upon Mtb
infection could dictate the outcome of infection. Further evidence
for a role of ROS in TB pathogenesis comes from the observation
that NOX2 is essential for Toll-like receptor 2-dependent inflammatory responses and 1,25-dihydroxyvitamin D3-mediated antimicrobial activity against Mtb via cathelicidin expression [12].
These observations suggest that ROS not only have potential
mycobactericidal activity but also are important for activation
of the inflammatory/antimicrobial response of macrophages.
Furthermore, ROS are important for modulation of apoptosis,
which is emerging as a major pathway of Mtb clearance in vivo
[13]. ROS induce apoptosis through activation of apoptosisregulating signal kinase 1. In line with this, the p47phox pathway
of ROS generation is responsible for the induction of proinflammatory responses during TB [14,15]. Finally, the importance of
ROS in human TB is indicated by the fact that patients with
chronic granulomatous disease (with a genetic defect in ROS
production) are susceptible to infection from various species of
Mycobacterium [16–19]. These studies strongly suggest that levels
of ROS in the host immune system play an important role in TB
pathogenesis.
The role of hypoxia in latency and reactivation
TB primarily affects the lungs. The oxygen-rich environment of
the lungs is important for the growth of bacteria and the
establishment of productive infection. Oxygen is also required
for luxuriant growth of Mtb in vitro. On the other hand, gradual
depletion of oxygen helps Mtb transition into a nonreplicative,
drug-unresponsive, persistent state, wherein the bacteria can
survive for decades and could resume an active replicative state
upon exposure to ambient levels of oxygen [20,21]. Exposure to
hypoxia leads to a number of changes in the physiology of
mycobacterial cells, including inhibition of DNA, RNA, and protein
synthesis; variability in acid fastness; thickening of the cell wall;
and unresponsiveness to anti-TB drugs such as isoniazid [21].
These changes are similar to changes in the metabolism of bacilli
recovered from infected tissues [22,23] and suggest that hypoxia
is a physiologically relevant stress that modulates the outcome of
the infection.
Interestingly, during the preantibiotics era, treatment of TB
included patient care at sanatoriums, therapeutic pneumothorax,
thoracoplasty, and surgical removal of infected tissue. Many of
these methods involved reduction of oxygen tension in the lung,
thereby potentially initiating dormancy in Mtb and relieving the
symptoms of TB. As an example, treatment of TB was primarily
done at the sanatoriums. Most of the sanatoriums were constructed at high altitudes, where oxygen tension is low [24].
S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641
The low oxygen tension at high altitude could be one of the
underlying causes of relatively infrequent occurrence of active TB
among people living at high altitudes [25–27]. In the preantibiotics era, pneumothorax was used as one of the ways to cure TB. In
therapeutic pneumothorax, air is artificially introduced into the
pleural cavity, causing partial collapse of a lung and leading to
reduced oxygen tension in the surrounding region [28]. Moreover,
therapeutic pneumothorax often leads to fibrosis around the
diseased tissue and thus containment of the infection, as seen
with closed tubercular cavities potentially harboring dormant
Mtb. Similar to therapeutic pneumothorax, thoracoplasty also
involves surgery of the thorax for TB treatment [29]. In thoracoplasty, ribs from one side of the thorax are bent or removed to
collapse the infected portion of lung permanently. Therapeutic
pneumothorax and thoracoplasty were recently used successfully
for treatment of multidrug-resistant and extensively drugresistant cases of TB, further emphasizing the role of oxygen
tension in mycobacterial growth and latency [30–32]. More
evidence that oxygen tension is involved in latency comes from
the observations that postprimary TB is mainly associated with
the upper lobe of the lung (the most oxygen-rich tissue of the
body) [33,34] and that actively replicating bacteria are recovered
from the lesions exposed to air, whereas less bacterial load is
observed in lesions lacking direct contact with oxygen [35]. In
addition, human granulomas are often avascular, indicating that
Mtb faces hypoxia in humans [36]. Consistent with this hypothesis, granulomas of nonprimates, rabbits, and guinea pigs are also
hypoxic [37]. These observations strongly suggest that the levels
of oxygen in the granuloma dictate whether the bacteria will
actively replicate to cause active TB disease or enter a state of
nonreplicative persistence (Fig. 1). Because the transition from
replicative state into nonreplicative state requires synchronized
regulation of Mtb metabolism, Mtb must employ a battery of
versatile sensors to continuously monitor the levels of oxygen in
its microenvironment.
The role of nitric oxide (NO) in TB pathogenesis
Similar to hypoxia, exposure to NO is believed to be a
physiologically relevant signal for initiating mycobacterial persistence. Low concentrations of NO inhibit Mtb respiration [38],
leading to inhibition of anabolic processes such as DNA, RNA, and
protein synthesis and thus inhibiting Mtb growth and promoting
the drug-unresponsive persistent state [38]. Moderate and high
concentrations of NO have bacteriostatic and bactericidal effects
on Mtb, respectively [39,40]. Furthermore, exposure of Mtb to NO
induces a genetic response similar to the one initiated by hypoxia
[38,41,42].
A significant body of evidence demonstrating the importance
of NO in TB pathogenesis has emerged from the murine model of
TB. Murine macrophages produce bactericidal levels of NO upon
activation with appropriate stimuli and are important for the
control of TB [43]. The inhibition of exponential replication of
mycobacteria in the murine model depends on the presence of
inducible NO synthase (iNOS) [44] and inhibition of iNOS during
the chronic phase of infection leads to exacerbation of the
infection and death of the infected animal. In addition, iNOSdeficient mice are more susceptible to intravenous [44] and
aerogenic infection [45]. Intriguingly, the presence of NO is
essential for maintaining a latent TB infection in the murine
model [46], suggesting a critical role for NO in TB dormancy.
Infection
Active
Tuberculosis
Latent
Tuberculosis
Hy
pox
i
a/ E
fficie
nt imm
un
1627
S
e response/ ROS/ RN
/ An
s
rug
Bd
ti-T
B-cells
T-cells
Extracellular mycobacteria
Foamy macrophages
Infected macrophages
Ruptured macrophages
Intracellular mycobacteria
Fibroblast
Collagen fibres
Fig. 1. TB pathogenesis. Upon infection with bacilli residing in aerosols, 5–10% of individuals develop active tuberculosis disease, whereas 90–95% remain latently
infected. The hallmark of tuberculosis disease is granuloma formation. The outcome of the Mtb infection is guided by the prevailing redox state of the environment in the
granuloma. The structure of granulomas in active TB patients and in latently infected individuals is depicted. The latent infection is favored by hypoxia, efficient immune
response, anti-TB drugs, ROS/RNS, and also by surgical interventions such as therapeutic pneumothorax or thoracoplasty. The reactivation of latent infection is assisted by
immunosuppression through HIV infection or administration of immunosuppressive drugs, nutrient starvation, and hyperoxia.
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S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641
The precise role of NO in human pulmonary TB remains
elusive, because of a lack of vital resources and reagents such as
a human alveolar macrophage cell line. However, significant
evidence points to the importance of NO in human TB. Mtb
infection of peripheral blood-derived monocytes or human monocyte cell lines leads to induction of iNOS and thus increased NO;
this increase in NO is important for inhibition of Mtb growth [47].
More significantly, pulmonary macrophages from healthy individuals produce inhibitory concentrations of NO upon mycobacterial infection [48–50]. iNOS, endothelial NOS, and nitrotyrosine are
detected in surgically resected lung tissue from patients with
active pulmonary TB, suggesting a role for iNOS in human
pulmonary TB [51]. Furthermore, TB patients exhale higher levels
of NO than healthy control subjects do and their alveolar macrophages exhibit higher levels of iNOS activity [52]. Exhaled NO has
recently been proposed as a potential diagnostic marker of TB
[53]. In mice, iNOS is induced by interferon-g (IFN-g). In humans,
mutations in the IFN-g receptor [54] and polymorphisms of the
IFN-g promoter [55] are manifested as susceptibility to mycobacterial infection, further emphasizing the role of NO in TB pathogenesis. Arginine is administered as a dietary supplement that
promotes increased NO and thus has the potential to improve the
clinical outcome of active TB [56]. Furthermore, 1,25-dihydroxyvitamin D3 in the presence of IFN-g leads to induction of iNOS
and reduces the growth of mycobacteria in human cells [57,58].
In addition to NO being mycobactericidal, it is also a secondary
messenger that can modulate the adaptive immune system and
function of phagocytic cells to affect the outcome of infection.
This aspect of NO’s role in TB pathogenesis, however, has not been
well studied. It was shown recently that NO induces apoptosis of
macrophage cells to restrict the growth of Mtb [59]. Furthermore,
NO produced by mesenchymal stem cells recruited at the site of
infection can suppress the T cell response [60]. Together, these
results suggest that iNOS-generated NO at higher concentrations
could kill mycobacterial cells, whereas at the lower concentration
it promotes the nonreplicative persistent state of Mtb by induction of the Dos regulon. Thus sensing, adaptation, and escape from
the bactericidal activity of NO play a crucial role in TB pathogenesis. However, the molecular mechanisms of NO sensing and
adaptation remain poorly understood.
The role of acidic pH stress in tuberculosis pathogenesis
Mtb is an intracellular pathogen that resides primarily in the
phagosomes of alveolar macrophages. The pH of phagosomes in
naı̈ve macrophages ranges from 6.3 to 6.5 [61], whereas the pH of
activated macrophages ranges from 4.5 to 4.8 [62]. To establish a
productive or latent infection, Mtb must survive the acid stress
manifested by macrophages. To survive inside phagosomes and
phagolysosomes, Mtb inhibits the acidification of phagosomes by
producing and secreting ammonia [63] or by inhibiting vacuolar
ATPase through secretory factors [64]. Mtb must also adjust its
metabolism to maintain its internal pH despite fluctuations of the
pH in its microenvironment [65]. A coordinated transcriptional
response is observed when Mtb is exposed to pH values encountered inside naı̈ve or activated macrophages [66]. A number of
in vitro and in vivo studies indicate that Mtb can resist significant
levels of acidic pH stress and that its pH response is not similar to
the classical acid tolerance phenotype of enteric bacteria [67]. In the
classical acid tolerance phenotype, bacteria can resist exposure to
highly acidic conditions after being primed with mildly acidic
conditions [67]. The hypothesis that Mtb is exposed to acidic pH
in human pulmonary TB is also supported by the fact that the
frontline anti-TB drug pyrazinamide is activated only under acidic
conditions [68]. These observations suggest that acidic pH plays an
important role in the TB pathogenesis; however, the genetic
mechanisms of pH sensing and tolerance are not known.
Mtb lacks classical redox sensors
The above-cited literature suggests that Mtb is exposed to a
number of redox stresses such as ROS, hypoxia, NO, and acidic pH
during its cycle of infection. The success of Mtb as a human
pathogen indicates that it can perceive these stresses in the
surrounding environment and modulate its metabolism to maintain its intracellular redox state. A number of classical sensors are
employed by various species of bacteria to sense redox stress and
hypoxia. These sensors include OxyR of Salmonella; FixL of
Rhizobium; SoxR, fumarate/nitrate reduction regulator (FNR),
and ArcB of Escherichia coli; and RexA of Streptomyces.
OxyR regulates the transcriptional response of bacteria to
peroxide stress. The OxyR regulon includes several of the oxidative stress enzymes such as KatG, alkyl hydroperoxide reductase,
thioredoxins, glutaredoxins, and the enzymes of the iron–sulfur
cluster biosynthetic pathway [69,70]. OxyR harbors six Cys
residues, of which Cys199 and Cys208 are redox reactive [71].
Under nonoxidizing conditions, Cys199 remains buried in a hydrophobic cavity and interacts with neighboring Arg266. This interaction renders Cys199 oxidation sensitive. In the presence of
oxidative stress, Cys199 is oxidized to sulfenic acid and comes
close to Cys208, thus facilitating disulfide bond formation [72]. The
tetrameric form of oxidized OxyR can activate the transcription of
the OxyR regulon. Upon establishment of a reducing intracellular
environment, the oxidized OxyR can be reduced back to the
transcriptionally inactive form by glutaredoxins [71].
The other classical sensors include the heme-based oxygen
sensor FixL, iron–sulfur cluster-based sensors SoxR and FNR, and
the NADH/NAD þ sensor RexA. FixL is a heme-based sensor of
Rhizobium meliloti that regulates the expression of genes involved
in nitrogen fixation in response to the presence of oxygen [73].
The binding of O2 to the heme of FixL inhibits the kinase activity
of FixL, whereas in the absence of O2, the heme remains in the
deoxy form, which possesses increased kinase activity [74]. SoxR
is an iron–sulfur cluster-based sensor that regulates the transcriptional response of E. coli to the superoxide radical. SoxR
contains a [2Fe–2S] cluster [75]. Under normoxic conditions, the
SoxR iron–sulfur cluster is maintained in the [2Fe–2S]1 þ state;
upon exposure to superoxide radicals, it is oxidized to [2Fe–2S]2 þ
[76]. This transition initiates transcription by SoxR [77]. FNR is
also an iron–sulfur cluster-based redox sensor. However, it
regulates components of an alternative electron transport chain
through a different mechanism. Under anaerobic conditions, the
FNR iron–sulfur cluster is cubic, [4Fe–4S]2 þ , and upon exposure
to oxygen, it is converted into the planar [2Fe–2S]1 þ [78]. This
switch of oxidation states is associated with conversion of
transcriptionally active, dimeric FNR into inactive, monomeric
FNR [79].
RexA, in Streptomyces coelicolor, is another unique sensor of
intracellular redox status. RexA harbors a Rossmann fold capable
of binding pyridine nucleotides at the C terminus, whereas the N
terminus acts as a DNA-binding domain. Both NAD þ and NADH
can bind to the C-terminal sensory fold of Rex, but only NADH
binding decreases the affinity of RexA for the promoters of the
cydABCD and hemACD operons and leads to reduced expression
[80]. The decreased flow of electrons in the electron transport
chain (ETC) caused by the unavailability of oxygen during hypoxia
leads to accumulation of NADH. On the other hand, ArcB monitors
the redox state of the cell by sensing the levels of reduced
ubiquinones in the ETC [81]. Under hypoxic conditions, inhibition
of respiration leads to accumulation of the reduced form of
S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641
ubiquinones. Accumulated ubiquinol initiates the formation of
interchain disulfide bridges in the ArcB dimer. The disulfide bond
formation leads to inhibition of the kinase activity of ArcB [82].
Bacteria are persistently exposed to organic hydroperoxides
(OHPs) resulting from oxidation of unsaturated fatty acids by
molecular oxygen or ROS. Bacteria employ a family of proteins
known as organic hydroperoxide resistance protein (Ohr) to
detoxify the OHPs [88]. The expression of Ohr is under the control
of an organic hydroperoxide sensor, OhrR (organic peroxide
resistance regulator) [89]. OhrR acts as a repressor of ohr. OhrR
harbors a redox-active conserved cysteine that reacts with the
OHP to generate a sulfenic acid intermediate. This sulfenic acid
derivative of OhrR could react with the low-molecular-weight
thiol leading to formation of a mixed-disulfide species of protein
that is incapable of binding to DNA. This mixed-disulfide species
could be reconverted to the thiol form upon reduction through a
thiol–disulfide exchange reaction. This reduction leads to activation of DNA binding activity of OhrR [90–92]. A homolog of Ohr
has been identified and characterized in Mycobacterium smegmatis
(MSMEG_0447) by exploiting the transposon mutagenesis of a
strain lacking mycothiol (MSH) [93]. This strain overproduced Ohr
to compensate for the lack of MSH [93]. However, a homolog of
ohr could not be detected in pathogenic mycobacteria [93]. The
absence of Ohr in the Mtb genome could be one of the underlying
reasons for its increased sensitivity toward the loss of MSH and
organic hydroperoxides compared to M. smegmatis [93]. Although
Mtb lacks the ohr gene, it possesses another protein (Rv2923c) of
the OsmC protein subfamily previously known for the response to
osmotic stress. This protein has a higher degree of similarity with
Ohr proteins. Rv2923c and its homolog in M. smegmatis
(MSMEG_2421) have been shown to possess organic hydroperoxide reductase activity. Unlike Ohr proteins, OsmC proteins can
reduce hydrogen peroxide as well [94]. Identifying a regulator of
osmC could lead to the identification of a peroxide sensor in
mycobacteria.
MgrA is another sensor protein belonging to the MarR family of
proteins possessing a helix–turn–helix (HTH) domain and which
bind to DNA. This regulator protein regulates autolysis, virulence
genes, and efflux pumps and is also involved in biofilm regulation
[95–97]. Similar to OhrR, MgrA harbors a redox-active cysteine that
regulates its DNA-binding activity [98,99]. This conserved cysteine
could be oxidized by various species of ROS including hydroperoxide
and organic peroxide. This oxidation leads to conformational
changes in the structure of MgrA, decreasing its affinity for DNA
[98,99]. Interestingly, PknB of Mtb has significant similarity to MgrA,
but its function as a redox sensor has not been analyzed.
In E. coli and Salmonella, pretreatment with mild oxidative
stress provides protection from stringent oxidative stress. This
protective response comes from the presence of redox sensors
such as OxyR. A similar protective response is detected only in the
saprophytic species of mycobacteria, such as M. smegmatis, and is
absent from the pathogenic, slow-growing species of mycobacteria, including Mtb [83], suggesting that Mtb lacks the classical
sensors of oxidative stress. A more detailed analysis suggests that
the genetic locus coding for divergent expression of OxyR and
AhpC is conserved in all species of mycobacteria, but the OxyR of
pathogenic, slow-growing mycobacterial species is inactivated by
multiple mutations [84]. Sequencing of the Mtb genome revealed
a putative cyclic AMP receptor protein (Crp)/FNR homolog
encoded by open reading frame Rv3676. This protein shares 32%
sequence identity with E. coli Crp and is devoid of cationic metal
ion [85]. It is a transcription regulator that is regulated by
cellular cAMP levels [86]. It plays an important role in bacterial
survival in the mouse TB model and regulates the expression of
resuscitation-promoting factor [87]. Despite the homology to
FNR, Mtb lacks a true [Fe–S] cluster-based FNR.
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Using the published genome and proteome sequences of Mtb,
other homologs of classical redox sensors could not be detected.
Recently published work by Voskuil et al. suggests that exposure
to different levels of oxidative stress mediated by different
oxidizing agents such as H2O2 and NO results in different
transcriptional responses [100]. A number of uncharacterized
putative redox regulators were also induced as a part of the
transcriptional response to oxidative stress. However, the redoxsensing/regulating mechanism for these and many other uncharacterized sensors/regulators in the biology of Mycobacterium
remains to be analyzed. The protective response conferred by
Mtb despite the absence of classical redox sensors suggests that
Mtb could utilize novel sensors to perceive the surrounding
microenvironment. The discovery of mycobacterial redox sensors
and their characterization was begun only recently and is
believed to be an area of scientific interest in which coming years
will see the characterization of more redox sensors and their
importance in TB biology. A number of recently published elegant
studies have established an important role for some of the
recently characterized redox regulators in the virulence and
pathogenesis of tuberculosis. A recently published review has
described the role of redox signaling in human pathogens [99];
hence a detailed description of the current understanding of
mycobacterial redox sensors is presented in the following
sections.
DosS and DosT: heme-based sensors of redox, hypoxia, NO, and
CO
Although Mtb lacks classical redox sensors such as FixL, OxyR,
and FNR, it possesses novel heme-based sensors in the form of
DosS and DosT. DosS and DosT, along with DosR, constitute the
DosRST two-component system, wherein DosS and DosT act as
sensor histidine kinases that sense the presence or absence of
ligands (that may also act as an oxidant) and relay it to the
response regulator DosR (Fig. 2). The Dos regulon is believed to
play an important role in the transition of Mtb from the actively
replicating state to the nonreplicating, persistent state. The
following sections describe the molecular mechanism of redox
and gas sensing by DosS and DosT and the role of the Dos regulon
in Mtb physiology.
The Dos regulon and the mycobacterial response to hypoxia, NO,
and CO
The potential role of hypoxia in human TB and Mtb latency has
prompted researchers to invest significant effort toward understanding the effect of hypoxia on the physiology of Mtb. Mtb is
aerobic and sudden depletion of oxygen is lethal [101], but
gradual depletion of oxygen initiates a phenotypic switch in
Mtb that enables it to survive for decades [20,21]. Upon exposure
to hypoxia in vitro, Mtb becomes elongated, its cell wall becomes
thick, the acid-fast character of Mtb is lost, and it becomes
unresponsive to anti-TB drugs [21,102]. These phenotypic characteristics are very similar to the distinctive attributes of latent
Mtb, suggesting that hypoxia plays an important role in Mtb
latency. In general, hypoxia reduces the flow of electrons in the
ETC because of the unavailability of oxygen as a terminal electron
acceptor. This decreased flow of electrons leads to an accumulation of NADH, reduced ubiquinones, and reduced cytochromes in
the bacteria. Survival of bacteria during hypoxia depends on
induced expression of an alternative cytochrome oxidase (cytochrome BD oxidase) with higher affinity for oxygen, changeover
from proton-pumping NADH dehydrogenase I to non-protonpumping NADH dehydrogenase II, and a number of other important
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Fig. 2. Sensing mechanism of DosS/DosT. (A) The role of the Dos regulon. DosR regulates 50 genes that are collectively known as the Dos regulon. The Dos regulon is activated
under hypoxic and reductive conditions and in the presence of CO and NO. The Dos regulon includes genes encoding the nitrate–nitrite antiporter (NarK2) and nitrate reductase
(NarX). These proteins facilitate the survival of Mtb in hypoxia and acidic pH and its in vivo persistence. DosR also regulates expression of the fumarate reductases (frdABCD),
ferridoxin (fdxA), and formate-hydrogen lyase systems that act as alternate electron transfer systems in the absence of oxygen. Furthermore DosR regulates other physiological
responses such as DNA biosynthesis, triacylglycerol (TAG) biosynthesis, and growth of Mtb by regulating the expression of ribonucleoside diphosphate reductase (nrdZ),
triacylglycerol synthase (tgs1), and Rv2633, respectively. (B) Mechanism of redox/gas sensing by DosS. The kinase activity of DosS is regulated by the ligation/redox state of the heme
iron. Under hypoxic conditions the heme iron of DosS is in the deoxy ferrous (Fe2 þ ) form and the DosS protein is kinetically active. Binding of NO or CO to heme iron does not inhibit
the kinase activity of DosS. However, under normoxic conditions the DosS heme iron either binds oxygen or becomes oxidized. This binding of oxygen or oxidation of heme iron
leads to inhibition of kinase activity. The redox sensing model proposes another level of regulation by unknown factors that could reduce the Fe3 þ heme iron into the Fe2 þ form.
(C) Mechanism of gas sensing by DosT. Unlike the redox sensing mechanism of DosS, DosT heme iron binds oxygen in a concentration-dependent manner. This binding of oxygen
leads to inhibition of the kinase activity of DosT. However, similar to DosS, the binding of NO and CO could lock DosT in the active conformation.
S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641
metabolic changes. These alterations help the bacteria to minimize the reductive stress generated from the accumulation of
reduced carriers of electrons.
Bacteria have evolved sensors such as ArcB of E. coli to sense
the depletion of oxidized ubiquinones and RexA of S. coelicolor to
sense NADH/NAD þ . To understand Mtb’s response to hypoxia,
Sherman et al. exposed Mtb cells to defined hypoxia and analyzed
the transcriptome using microarray technology [103]. This study
demonstrated that, similar to E. coli and other bacteria, Mtb
responds to hypoxia by inhibiting overall biosynthesis of RNA
and protein. Approximately 50 genes were upregulated in
response to hypoxia (Fig. 2A). This transcriptional response was
termed the Dos (dormancy) regulon [104]. The Dos regulon
contains a paired two-component system called DosRS, which,
via phosphorylation and activation of DosR by DosS, regulates the
DosR regulon [42,103]. DosT (Rv2027c), an orphan histidine
kinase and a homolog of DosS, can also specifically phosphorylate
DosR [105,106]. Unlike DosS, which is under the control of DosR,
DosT is constitutively expressed and is not regulated by DosR. As
with exposure to hypoxia, exposure of Mtb to NO inhibits the
respiration of Mtb, restricts its growth, and specifically induces
the Dos regulon [38]. Furthermore, we and others [107,108] have
demonstrated that exposure to CO also induces the Dos regulon.
Interestingly, Mtb infection of naı̈ve macrophages induces the
expression of heme oxygenase 1 (HO-1), independent of IFN-g or
iNOS [107]. HO-1 catalyzes the oxidative degradation of heme
and leads to formation of biliverdin, molecular iron, and CO. Using
bone marrow-derived macrophages from HO-1 knockout mice,
we [107] and others [108] have demonstrated that physiological
levels of CO lead to the induction of the Dos regulon in naı̈ve
macrophages.
Under hypoxic or anoxic conditions, bacteria must use alternate electron acceptors such as nitrate or fumarate for continued
production of ATP through the ETC. For example, E. coli reduces
nitrate to nitrite through the nitrate reductase complex using
electron flux from the ETC. In E. coli, this system requires a
nitrate–nitrite antiporter that is involved in the uptake of nitrate
and the export of nitrite that results from nitrate respiration
[109]. The level of nitrate reductase complex is not transcriptionally regulated; instead, the expression of the transporter is
regulated and is induced upon hypoxia [110]. Similar to E. coli,
Mtb can use alternate electron acceptors in the absence of
oxygen. A number of enzyme systems required for continuing
the ETC in the absence of oxygen are under the control of the Dos
regulon. These systems include nitrate reductase, fumarate reductase, formate-hydrogen lyase, and ferridoxins. DosR regulates
the expression of the nitrate–nitrite antiporter narK2 along with
the fused nitrate reductase narX. This system is important for the
survival of Mtb in hypoxia [111] and acidic pH [112] and for
in vivo persistence [113]. These observations suggest that DosRregulated nitrate respiration is used by Mtb for survival in vivo
and could be important for transition into latency. The other
preferred alternate terminal electron acceptor is fumarate. Fumarate can be reduced to succinate by fumarate reductase using the
flux of electrons from the etc. Indeed, increased levels of succinate
are secreted by Mtb exposed to hypoxia [114]. The expression of
fumarate reductase (frdABCD) is controlled by DosR. Furthermore,
DosR regulates the expression of fdxA, an alternate electron
transfer protein that may play an important role in electron
transfer reactions during adaptation to hypoxia. Interestingly,
DosR also regulates the expression of enzymes encoding the
formate-hydrogen lyase (FLH) system. The FLH system converts
formate into H2 and CO2 using two enzymes: formate
dehydrogenase-H and hydrogenase-3 [115]. This system functions in the absence of oxygen and other alternate terminal
electron acceptors.
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In addition to regulating alternate electron acceptors, DosR also
regulates a number of important pathways that help Mtb adapt
during stress conditions. In a series of elegant experiments, Leistikow et al. demonstrated that DosR is essential for maintaining
energy levels and redox balance during the downshift to the
nonreplicating state upon anaerobiosis [116]. Additionally, the Dos
regulon is critical for resumption of growth upon the return of
aerobic conditions. DosR governs DNA biosynthesis, triacylglycerol
biosynthesis, and general stress response upon exposure to hypoxia
and NO [116]. It regulates the expression of ribonucleosidediphosphate reductase (nrdZ), which catalyzes the biosynthesis of
deoxyribonucleotides from the corresponding ribonucleotides using
the reductive energy provided by thioredoxins. DosR also regulates
tgs1, which encodes triacylglycerol synthase 1 (Tgs1), one of the
most active of 10 triacylglycerol synthases encoded by the Mtb
genome. It was recently demonstrated that triacylglycerol biosynthesis plays an important role in reducing the growth rate of Mtb by
redirecting cellular carbon fluxes away from the tricarboxylic acid
cycle [117]. tgs1 mutants cannot arrest their growth, they consume
oxygen faster than wild-type Mtb, and they show inefficient
prolonged survival in the absence of oxygen. DosR regulates
approximately eight universal stress proteins, including Rv2623.
Rv2623 is believed to regulate TB latency; an Rv2623-deficient
mutant fails to restrict growth in response to the adaptive immune
response and thus cannot establish chronic infection in guinea pig
and mouse models of TB [118]. Because of unrestricted growth,
Rv2623-deficient Mtb strains exhibit a hypervirulent phenotype.
These observations suggest that DosR plays an important role in
Mtb’s adaptation to hypoxia.
The molecular mechanism of redox and gas sensing by DosS and DosT
DosS and DosT are sensor histidine kinases that relay signals to
the response regulator DosR. They are equipped with a sensory
domain and an activator/transmitter domain. The sensory
domains contain two tandem GAF domains, so-named because
of their characteristic presence in sensor proteins, namely, cGMPspecific phosphodiesterases, adenylyl cyclases, and FhlA. The
transmitter domain of both sensors has a histidine kinase domain
and an ATPase domain. Sardiwal et al. have demonstrated that the
first GAF domain of DosS (GAF-A) covalently binds heme at His149
[119]; these proteins are the first examples of GAF-based proteins
capable of binding heme. The unique coordination/redox chemistry of this heme allows DosS and DosT to modulate their kinase
activity in response to variable levels of O2, NO, and CO. In 2007,
the biochemical properties of DosS and DosT were independently
characterized by us and others [120–122]. These studies showed
that DosS and DosT bind heme type B. When oxygen binds to the
heme of the DosS and DosT sensory domains, a signal is relayed to
the activator/transmitter domain that results in the inhibition of
the kinase activity of DosS and DosT (Fig. 2B and C). NO and CO
binding do not inhibit the kinase activity; in contrast, these
studies suggest that NO and CO can form complexes with the
heme that could potentially lock DosS and DosT in their active
states and lead to activation of the Dos regulon even in the
presence of O2. A distal tyrosine (Tyr171) and the second GAF
domain have been implicated in ligand discrimination [123,124].
Despite remarkable similarities between DosS and DosT, there
are also a number of critical biochemical differences. For example,
DosS and DosT have different binding affinities for gaseous ligands
[122]. Furthermore, DosT is extremely resistant to oxidation
[121,122], whereas DosS either is readily oxidized by O2 [121]
or forms an unstable oxy complex [120,122,123]. The rate of
DosS oxidation and whether oxygen makes a stable complex with
the ferrous form of heme iron remain controversial (Fig. 2B). Our
group has used absorbance spectroscopy and electron paramagnetic
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resonance (EPR) spectroscopy to demonstrate that in the absence of
O2, the heme iron of DosS exists in the deoxy ferrous form (Fe2 þ ).
Upon exposure to O2, the heme iron is rapidly oxidized to the ‘‘met’’
(Fe3 þ ) form [121]. We have also shown that the met form of the
protein is kinetically inactive. In addition, oxidizing agents other
than O2, such as ferricyanide, can convert the active ferrous heme of
DosS into the inactive met form [121]. Thus, DosS can be classified
as a redox sensor.
A different interpretation of kinase activity of DosS comes from
Sausa et al. [122], Yukl et al. [123], and Ioanaviciu et al. [120]. These
groups performed elegant experiments using absorbance spectroscopy and resonance Raman spectroscopy to demonstrate that DosS
can form a relatively stable oxy form (Fe2 þ –O2). Moreover, they also
demonstrated that the oxy form of DosS has NO dioxygenase
activity similar to NO-detoxifying bacterial flavohemoglobins,
further emphasizing the existence of a stable oxy–heme complex
of DosS [125]. These results have led them to conclude that DosS is
an oxygen sensor rather than a redox sensor.
Our model of DosS as a redox sensor is supported by Cho et al.
[126], who solved the crystal structures of oxidized, reduced, and
reduced-then-air-oxidized complexes of the DosS GAF-A domain.
They demonstrated that the met form of the GAF-A domain
harbors a hexacoordinated heme ligated to His149 at the proximal
end and a water molecule at the distal end. The reduced protein
has a pentacoordinated heme that forms the met complex upon
exposure to oxygen. Furthermore, this met heme in the GAF-A
domain can be reduced by FADH, suggesting the possibility that
an FADH-binding protein may control the rate of DosS reduction.
Cho et al. further observed that heme inside the GAF-A domain
has a restricted environment that does not permit a stable oxy–
heme complex. Instead, a prevalent network of hydrogen bonds
involving multiple water molecules and the side chains of several
amino acids facilitates transfer of electrons from oxygen to heme
[126]. These findings were further extended by a comparison of
the crystal structures of the DosS and DosT GAF-A domains [127].
Whereas DosT has a wide-open channel that facilitates formation
of the oxy–heme complex, the DosS GAF-A channel is narrow and
closed by the carboxylate group of Glu87, which obstructs the
access of O2 to the heme iron. These observations were further
supported by the experiments in which replacement of Glu87 in
DosS with Ala or Gly resulted in a GAF-A domain structure that
allowed stable oxy–heme complex formation. In contrast, a
conversion of glycine to glutamate at position 87 in DosT GAF-A
rendered DosT oxidation sensitive [127].
The redox sensor model of DosS was further supported by
elegant in vivo studies performed by Honaker et al. [128]. This
group showed that a reduced electron transport system in the
presence of ambient oxygen leads to upregulation of the Dos
regulon through DosS; these results confirm that DosS is a redox
sensor. They also observed that DosT does not respond to the
reduced electron transport system; these results further confirmed that DosT is an oxygen sensor. The ETC was more reduced
during hypoxic conditions; they reasoned that the reduced ETC is
responsible for hypoxic induction of the Dos regulon in the DosT
mutant. To further investigate, they inhibited oxidoreductases or
the synthesis of menaquinone and observed that in both cases,
the extent of Dos regulon induction was diminished [128].
The above discussion details the controversy surrounding the
identity of DosS as a redox sensor or as an oxygen sensor. The
underlying causes of these divergent results are unclear and
indicate the difficulties associated with working on these
oxygen-sensitive proteins. Despite controversy over the mechanism of action, the above studies agree that Mtb DosS senses the
presence of oxygen. The conclusive elucidation of the mechanism
of redox and hypoxia sensing by DosS and DosT in vivo is an area
of interest that requires more research.
WhiB proteins as iron–sulfur cluster-based sensors
Actinobacteria employ a number of mechanisms and pathways
for survival during oxidative stress, such as the thioredoxin
system and mycothiol pathways. In addition to these important
thiol-specific antioxidant systems, another family of proteins has
been recently shown to possess properties similar to those of
thioredoxins and mycoredoxins. Interestingly, the proteins of this
family, called WhiB proteins, harbor an Fe–S cluster and can bind
DNA. WhiB proteins were first reported in 1992 by Davis and
Chater [129] as products of a screen for genes involved in
Streptomyces sporulation. This study demonstrated that the
S. coelicolor whiB mutants formed white spores instead of grey
spores (thus the name), suggesting a potential role in the regulation of sporulation [129]. Although the sporulation phenomenon
has not been reported for Mtb, seven WhiB-like proteins (WhiB1–
WhiB7) are present in Mtb and these proteins are in fact
conserved throughout the Actinomycetes [6,130]. Sequence alignment of WhiB proteins revealed the presence of a C-terminal HTH
motif, four cysteine residues, and a conserved CXXC motif formed
by the middle two cysteines, with the exception of WhiB5, which
has a CXXXC motif. To begin deciphering the physiological
function of WhiB proteins in mycobacteria, Gomez and Bishai
tried to create a knockout mutant of whmD (now known as whiB2)
[131]. However, unlike nonessential Streptomyces whiB genes,
whiB2 is essential and can be disrupted only through complementation in trans. The conditional knockout of whiB2 exhibits
filamentous branched growth resulting from aberrations in septum formation and placement, implicating WhiB2 in the regulation of mycobacterial septum formation and cell division [131].
Other whiB genes (such as whiB3 and whiB7) can be disrupted
without the need for complementation in trans [132,133] and
WhiB proteins have been reported to be involved in a variety of
physiological processes, from thioredoxin-like activity [134] to
antioxidant response [135] and transcriptional regulatory activity
[136]. The following section describes the roles of WhiB proteins
in redox sensing, in detoxifying thiol-specific oxidative stress, and
as transcriptional regulators of virulence (Fig. 3).
WhiBs as iron–sulfur cluster proteins with protein disulfide reductase
activity
Pioneering studies by Buttner and co-workers first demonstrated the ability of WhiD of S. coelicolor to bind a [4Fe–4S]2 þ
cluster through the four invariant cysteines [137]. This [4Fe–
4S]2 þ cluster is oxygen sensitive and can be converted to a [2Fe–
2S]2 þ cluster or completely destroyed [137] upon exposure to O2.
These properties are similar to those of the oxygen sensor FNR of
E. coli [78]. Interestingly, the four cysteine residues that harbor
the iron–sulfur cluster are essential for the function of WhiD.
Along similar lines, Steyn and co-workers have reconstituted and
characterized the iron–sulfur cluster of WhiB3. They suggested
the potential role of WhiB3 in sensing NO and O2 [138]. Their
elegant study identified IscS as a cysteine desulfurase and found it
to be the main protein involved in the in vitro assembly of
WhiB3’s iron–sulfur cluster. Using 35S-labeled cysteines, they
developed a traceable, in vitro reconstitution system for the
iron–sulfur cluster in WhiB3. This group also established the
requirement for all four cysteines in the coordination of the iron–
sulfur cluster. Using EPR spectroscopy, they demonstrated that
exposure to O2 leads to conversion of [4Fe–4S]2 þ into [3Fe–4S] þ 1,
followed by complete destruction of the iron–sulfur cluster,
suggesting that WhiB3 is an oxygen sensor similar to FNR of
E. coli. Using EPR studies, they further demonstrated that the iron–
sulfur cluster of WhiB3 reacts to NO and leads to formation of a
dinitrosyl iron complex (DNIC) and thus could potentially be used
S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641
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Fig. 3. Model depicting redox regulation of WhiB proteins. Under hypoxic condition, the WhiB3 protein harbors a [4Feþ 4S]2 þ cluster synthesized by the iron–sulfur
cluster-generating machinery consisting of NifS/SufS/IscS. Upon exposure to normoxic conditions the [4Feþ 4S]2 þ cluster of WhiB3 is oxidized to [3Fe þ4S]1 þ , releasing
one Fe2 þ , and continual exposure to oxygen leads to further oxidation of the Fe–S cluster to [2Fe þ2S]2 þ . This oxidation-assisted destruction of the Fe–S cluster leads to
conformational changes in WhiB proteins that enhance the DNA-binding activity of WhiB3. The activation of WhiB protein could also be achieved through oxidation of
CXXC (as in the case of WhiB1) at the cost of reduction of its substrate GlgB or other proteins (not yet identified) to form a disulfide bond. The [4Fe þ4S]2 þ cluster of the
WhiB protein could also react with NO to form DNIC. This interaction with NO also activates some WhiB proteins. The active form of WhiB protein has DNA-binding
activity and regulates transcription of pks2, pks3, antibacterial resistance genes, and the erm gene.
by Mtb to sense NO. Interestingly, the mutant of WhiB3 displayed
diminished rugosity in the colony, suggesting an important role
for WhiB3 in the regulation of colony morphology. Furthermore,
DWhiB3 was attenuated for growth on medium containing
carbohydrates as the carbon source, and in contrast the mutant
grew significantly better in medium that contained fatty acids as
the sole source of carbon. Because the growth on fatty acids is
associated with increased reductive stress, these results suggested that WhiB3 could be a sensor of reductive stress, counter
to the in vitro experiments suggesting that its iron–sulfur cluster
could act as a nano-switch for sensing the presence of O2 and NO
[138]. Further scrutiny of the composition of lipids of the whiB3
mutant suggested that WhiB3 regulated the lipid production in
Mtb [136]. It was found that WhiB3 regulates the production of
the virulence lipids polyacyltrehaloses (PAT), diacyltrehaloses
(DAT), sulfolipids (SL-1), trehalose monomycolates, and trehalose
dimycolates [136]. Further examination demonstrated that the
WhiB3 protein binds to the promoters of pks2 (required for SL-1
biosynthesis) and pks3 (required for PAT/DAT synthesis) and this
binding is dictated by the redox state of conserved cysteines.
Under oxidizing conditions the binding is enhanced, whereas
reducing conditions disrupt the DNA-binding activity, suggesting
that WhiB3 is a sensor of oxidative stress rather than reductive
stress [136]. Furthermore, this O2/NO-sensing ability of WhiB in
mycobacteria is associated with the upregulation of Pks1/Pks3
during macrophage infection and during persistence [66]. The
role of WhiB proteins as an Fe–S cluster-based redox sensor was
also supported by a recent study that demonstrated that WhiB4 of
Mtb contains an oxygen- and NO-sensitive Fe–S cluster. Interestingly, the WhiB4 mutant displays an altered redox balance with
accumulating NADH levels in cytoplasm and is resistant to
oxidative stress in vitro and in vivo. This mutant was also found
to display hypervirulence in the lungs of guinea pigs [139].
The above-cited literature suggests that the WhiB proteins use
their Fe–S cluster and act as redox-sensing proteins; however,
Agrawal and co-workers [140,134] have attributed a thiol-specific
antioxidant property to the WhiB proteins. They cleverly demonstrated that WhiB1 and WhiB4 proteins possess protein disulfide
reductase activity and that this activity is regulated through an
iron–sulfur cluster. No protein disulfide activity was seen in the
case of WhiD of S. coelicolor [141]. This property of reducing
disulfide bonds of proteins was attributed to the CXXC motif and
was established using the insulin disulfide reductase assay, in
which reduction of the disulfide bond between the two chains of
insulin leads to precipitation of the b chain and a subsequent
increase in OD650. It is likely that these proteins also act as protein
disulfide reductases in vivo. If they do, the following question
arises: after WhiB1 and WhiB4 reduce the disulfide bonds of their
substrates, how are the oxidized WhiB proteins converted back to
their reduced form? The Agrawal group showed that the existing
thioredoxin reductase cannot reduce WhiB4 [140], but they
suggested that the WhiB proteins could function as ‘‘mycoredoxins,’’ using the reductive energy from the NADPH channeled
through mycothiol. This hypothesis that WhiBs are mycoredoxins
capable of binding iron–sulfur clusters was supported by the
discovery that some glutaredoxins can also bind iron–sulfur
clusters [142]. Mycoredoxins were recently identified and characterized in Corynebacterium glutamicum by Messens and coworkers [143]. The WhiB proteins, if acting as mycoredoxins,
would depend on MSH, and the phenotype of MSH-deficient
mycobacteria should be the same as that of the whiB mutant.
Contrary to this, no such phenotype has been reported for the
MSH-deficient mutant, suggesting that some other uncharacterized proteins could function as mycoredoxins [143]. Recently,
Agrawal and co-workers used a yeast two-hybrid screen to
identify the a-(1,4)-glucan branching enzyme (GlgB) as the
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interacting partner of WhiB1 [144], but more research is needed
to find other interacting partners and to establish the mechanism
by which WhiB obtains reducing equivalents. Furthermore, a
recent study has suggested that WhiB2 has chaperone-like
activity, adding a new dimension to the function of WhiB proteins
and raising the possibility that WhiB proteins could be multifunctional proteins [145].
WhiBs as redox-responsive transcriptional factors
Since their discovery in actinobacteria, WhiB proteins have
been postulated to be DNA-binding proteins. WhiB3 was known
to physically interact with the principle s factor SigA (RpoV)
[133], but the first evidence of its DNA-binding activity was
provided only recently by Singh et al. [136], who conclusively
demonstrated that WhiB3 can bind specifically to the putative
promoter sequences of the lipid biosynthesis genes pks2 and pks3.
They also established that oxidized apo-WhiB3 possesses a
stronger affinity for DNA than holo-WhiB3 or reduced apo-WhiB3,
suggesting an important redox-sensitive switch for DNA binding
and transcriptional regulation [136]. Earlier work by the same
group established that WhiB3 is involved in O2 sensing. They used
EPR spectroscopy to demonstrate that oxygen catalyzes the
oxidation of the redox-responsive [4Fe–4S]2 þ cluster to form a
[3Fe–4S] þ species and that for WhiB3 to function as a DNAbinding protein, the iron–sulfur cluster is ultimately destroyed
[138]. Another recent report by Smith et al. [146] identified a 37bp region of protection by WhiB1 protein in DNase I footprinting
assays. This group demonstrated that the iron–sulfur cluster of
WhiB1 is oxygen tolerant and reacts rapidly with NO to form
dinuclear dinitrosyl-iron thiol complexes. They also showed that
holo-WhiB1 (with [4Fe–4S]2 þ ) has lower affinity for its specific
DNA than apo-WhiB1 (lacking the iron–sulfur cluster) or holoWhiB1 treated with NO [146]. Furthermore, DNA-binding activity
has been detected in WhiB2 and WhiBTM4 (a WhiB homolog) of
mycobacteriophage TM4 [147]. Another recent study demonstrated that the WhiB4 of Mtb is capable of nonspecifically
binding to GC-rich regions of DNA and this binding leads to
repression of transcription. It was further demonstrated that the
oxidizing conditions that convert the holo-form of WhiB4 into the
apo-form enhance the binding of WhiB4 with DNA and hence
induce the repression of transcription [139]. Taken as a whole, the
above-cited literature suggests that DNA-binding activity is a
common feature among WhiB proteins, but more research is
needed to establish that WhiB proteins are iron–sulfur clusterbased nano-sensors of O2 and NO.
WhiB proteins as virulence and stress response factors
The first evidence of the importance of WhiB3 in Mtb virulence
was provided by Steyn et al. in 2002 [133]. Using a yeast twohybrid screen, this group identified WhiB3 as a virulence factor
that interacts with RpoV from the virulent strains of Mtb but not
with the mutated (Arg515His) allele from the avirulent strain. The
interaction of WhiB3 with s factors was suggestive that WhiB3
could function either as a transcription factor or an anti-s factor.
It was later demonstrated that WhiB3 harbors an O2- and NOsensitive iron–sulfur cluster that can regulate the expression of
stress- and virulence-associated genes in response to redox stress
[136,138]. Importantly, a whiB3 mutant of H37Rv shows no
growth defect in the murine and guinea pig models of TB,
whereas the whiB3 mutant of Mycobacterium bovis is attenuated
in both models. These observations suggest that the role of WhiB3
is distinct in different species of pathogenic mycobacteria, in
particular Mtb and M. bovis [133]. The expression of WhiB2 and
WhiB3 of Mycobacterium avium is upregulated during oxidative
stress and pH stress, suggesting a role for these proteins in
sensing and responding to these stresses [148]. Moreover, in
mouse lungs and macrophages, increased expression of WhiB3
is observed during early infection. The WhiB3 expression profile is
dependent on cell density and indicates its regulation by ‘‘quorum
sensing’’ [149].
WhiB7 is involved in regulating many antibacterial resistance
genes in mycobacteria; it is induced by erythromycin, tetracycline, capreomycin, streptomycin, and kanamycin. In contrast, the
whiB7 mutant is hypersensitive to these antimicrobial agents,
suggesting that WhiB7 could be a potent chemotherapeutic target
[132,150,151]. A recent study by Thompson and co-workers
reported that WhiB7 responds to the level of MSH/MSSM, is
induced in a reducing environment, and thus is directly involved
in the regulation of redox homeostasis of the cell [153]. This study
also demonstrated that the macrolide resistance mediated by
erythromycin ribosome methyltransferase (ERM) [152] is actually
mediated by WhiB7 acting as a transcription factor for the erm
gene [153]. Further investigation of the regulatory roles of WhiB
proteins in transcription and redox detoxification will provide us
a better understanding of their physiological function.
Redox-regulated r factors
Sigma (s) factors are the primary regulators of bacterial gene
expression. The Mtb genome encodes 13 members of the s70
family [6]. SigA, SigB, and SigC belong to groups 1, 2, and 3,
respectively, and the rest belong to group 4. Group 4 s factors are
involved in sensing extracytoplasmic signals; these proteins are
also called extracytoplasmic function s factors [154]. s factors
regulate transcriptional responses for numerous cellular processes in prokaryotes, including stress response and growth.
Normally, s factors can recognize specific promoter sequences
and interact with components of RNA polymerase, but they
cannot directly sense relevant specific signals. Instead, they are
guided by anti-s factors, anti-anti-s factors, and a variety of
sensors and transcription regulators to synchronize the appropriate transcriptional response to stress. SigH, SigE, SigL, and SigF
play important roles in the survival of Mtb against redox stress;
their mechanism of redox sensing is described below (Fig. 4).
The role of SigH in oxidative stress was first demonstrated by
Fernandes et al. using M. smegmatis sigH mutants [155]. These
mutants are extremely susceptible to cumene hydroperoxide,
suggesting that SigH is important for the regulation of mycobacterial response to oxidative stress. The same group later demonstrated that SigH is a homolog of SigR of Streptomyces and plays
an important role in the protection of Mtb from oxidative thiol
stress and ROS [156,157]. It was shown that SigH protects against
oxidative stress by regulating the expression of thioredoxins
(trxB1 and trxC), thioredoxin reductase, and stress-responsive s
factor SigE (Fig. 4A). SigE and SigH regulate the expression of
stress-responsive s factor SigB. Further evidence for the protective role of SigH comes from the observation that it is upregulated
upon infection of macrophages [158]. Interestingly, infection of
mice with a sigH mutant of Mtb [159] produces an organ burden
similar to that of mice infected with wild-type Mtb. However, the
immunopathology of animals infected with sigH mutant strains is
nominal compared with that of mice infected with wild-type Mtb,
suggesting that SigH regulates the immunopathogenic virulence
factors of Mtb [159]. Additionally, ingenious studies performed by
Song et al. have demonstrated that Rv3221a, a gene in the same
operon as sigH, codes for a bona fide anti-s factor against SigH
[160], named RshA. RshA interacts specifically with SigH in a 1:1
stoichiometry in vitro and in vivo [160] and the interaction of RshA
with SigH leads to inhibition of SigH-dependent transcription
S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641
1635
Fig. 4. Response of s factors and anti-s factors to redox stress. The activity of s factors is regulated by an intricate network of anti-s factors and anti-anti-s factors. The
binding of an anti-s factor inhibits the activity of s and is regulated as shown in (A) and (B). (A) Redox regulation of DNA-binding activity of SigH and SigE. SigH regulates
the expression thioredoxins (trxB, trxC) and thioredoxin reductase (trxR), which constitutes a vital response to oxidative stress. Under stress-free conditions, anti-s factor
RshA binds SigH and inhibits the transcription of thioredoxins, thioredoxin reductase, and sigE. The release of SigH from anti-s factor RshA is achieved under oxidative
stress by two mechanisms: (i) under oxidative stress the Zn2 þ bound to the cysteines and histidine of RshA is released to form a disulfide bond, resulting in conformational
change in RshA leading to release of SigH. (ii) Phosphorylation of RshA by PknB renders it inactive and activates SigH. In addition to activation by SigH, the sigE
transcriptional activation is also regulated by polyphosphate (poly-P) stress. Poly-P activates the MprAB two-component system, which regulates transcription of SigE.
However, the activity of SigE is also regulated posttranslationally through anti-s factor RseE. SigE is released from RseA by two mechanisms: (i) under oxidative stress the
redox-reactive cysteines of RseA undergo oxidation to form a disulfide bond and induce conformational changes guiding the release of SigE. (ii) SigE leads to transcriptional
activation of relA, clgR, and sigB. ClgR upregulates the ClpC1P2 system. This ClpC1P2 binds to phosphorylated RseA (phosphorylated by PknB) and RseA is cleaved by these
proteases, activating SigE. SigE also regulates its own transcription. (B) Redox-mediated regulation of SigF. Antibiotics, stationary phase, anaerobiosis, and oxidative stress
lead to activation of sigF and anti-s factor usfX transcription. SigF is involved in persistence and possesses a complex control mechanism involving anti-anti-s factors RsfA
and RsfB. One mechanism involves the binding of RsfA to UsfX under reductive stress in the stoichiometry 1:2. This binding of RsfA to UsfX releases and activates SigF. The
other mechanism involves the anti-anti-s factor RsfB, which becomes phosphorylated by an unknown kinase and releases SigF to activate the genes involved in
persistence of the bacteria under oxidative stress.
in vitro. The peptides involved in RshA–SigH interactions were
identified through a phage display screen. These peptides
inhibited SigH by mimicking RshA [161]. More importantly,
the interaction was sensitive to oxidative stress and heat
shock [160]. Similar to RsrA of Streptomyces, RshA harbors an
HXXXCXXC motif. This motif, called the ZAS motif, is found
in a number of redox-sensitive, zinc-associated anti-s factors.
The cysteine thiols of the ZAS motif bind Zn2 þ under reducing
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S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641
conditions; under oxidizing conditions, these redox-active
cysteines form a disulfide bond and release the zinc [162,163].
When the cysteines of the HXXXCXXC motif in RshA were
mutated to alanine, the inhibitory effect of RshA on transcription
by SigH was abolished. Furthermore, oxidation of these conserved
cysteine residues inhibited the binding of RshA with SigH [160],
thus ensuring induction of a protective response during oxidative
stress.
Another important s factor that regulates mycobacterial
response to oxidative stress is SigE, as evident from the observation
that sigE knockout strains of M. smegmatis are more susceptible than
wild-type strains to oxidative stress and to several membranedisrupting agents [164]. Further experiments showed that sigE
expression is induced upon phagocytosis of macrophages [165]
and that Mtb mutants of sigE are defective in survival against
oxidative stress and inside the macrophages [166]. Additionally,
SigE is important in Mtb virulence in the mouse model of TB
[167,168]. The two-component system MprAB, which responds to
polyphosphate stress [169] and is implicated in the persistence of
Mtb in vivo [170], was shown to regulate the expression of SigE.
Interestingly, SigE regulates its own expression and directly regulates the level of RelA and MprAB, thereby creating a feedback loop.
RelA is important for long-term survival of mycobacteria under
starvation conditions [171]. SigE also regulates the expression of
clgR, sigB, and a number of other genes involved in the survival of
Mtb under stress conditions [166]. Importantly, the function of SigE
is regulated by the anti-s factor RseA, which has a ZAS motif
(Fig. 4A). RseA interacts with and inhibits the function of SigE under
reducing conditions. This interaction requires Cys70 and Cys73 of the
ZAS motif and is disrupted by oxidizing conditions, suggesting that
the disulfide bond formation between the two cysteines acts as the
regulatory step of the interaction [172]. Another level of complexity
in the regulation of SigE function is introduced by phosphorylation
of RseA on Thr39 by PknB in response to surface stress-imparting
agents such as vancomycin. This phosphorylation renders RseA
susceptible to proteolysis by the SigE-regulated ClpC1P2 proteolytic
machinery. This signaling constitutes an envelope stress-induced
positive feedback loop for SigE-dependent transcription [172].
Another redox-regulated s factor is SigL of Mtb. SigL is
cotranscribed with and binds specifically to RslA (Rv0736). RslA
is predicted to be a membrane protein, wherein the ZAS motif on
the N terminus of the protein faces the exterior of the cell and the
C-terminal anti-s factor domain resides in the cytosol. SigL
regulates a number of cell envelope proteins, suggesting its
importance in TB pathogenesis. This idea is supported by the
observation that mice infected with a sigL mutant of Mtb survive
longer than mice infected with wild-type Mtb [173]. Elegant
studies performed by Thakur et al. have elucidated the mechanism of regulation of SigL through the redox sensor RslA [174]. This
group solved the crystal structure of the SigL–RslA complex and
observed that under reducing conditions, the C-terminal anti-s
factor domain of RslA binds SigL and the N-terminal domain
coordinates Zn2 þ through His25, His50, Cys54, and Cys57. RslA
inhibits transcription by SigL through the occlusion of the s4
domain, which recognizes the 35 region of SigL-specific promoters. Oxidative stress assists the release of Zn2 þ through
formation of a disulfide bond between the cysteine residues of
the ZAS motif. This drives a conformational change in the anti-s
factor domain of RslA and reduces its affinity for SigL, thus
permitting transcription activation by SigL [174].
Recently obtained evidence links SigF with redox stress. SigF is
a homolog of bacillus s factor, which regulates sporulation in
response to nutrient and amino acid starvation. SigF is induced
upon antibiotic exposure, in the stationary phase of growth, upon
anaerobiosis, and in response to oxidative stress [175], suggesting
a role for SigF in Mtb redox homeostasis. Importantly, the SigF
mutant is attenuated for persistence in the murine model of TB
[176]. SigF regulates a number of genes involved in stability of the
cell envelope, including sulfolipids [176]. SigF itself is regulated
by the anti-s factor UsfX (Rv3287c). usfX is located upstream of
sigF in the Mtb genome and is transcribed as an operon under the
control of SigF [177] (Fig. 4B). Well-executed studies by Beaucher
et al. demonstrated that UsfX specifically binds and inhibits
transcription by SigF [178]. This study further identified two
anti-anti-s factors: RsfA (Rv1365c) and RsfB (Rv3687c). RsfA
binds specifically to UsfX and relieves SigF inhibition under
reducing conditions. This binding depends on Cys73 and Cys109.
RsfB, however, seems to be regulated through phosphorylation of
Ser61. Interestingly, Rv1364c also shares significant sequence
similarity with the anti-anti-s factors of SigF, but formal evidence
that Rv1364c regulates SigF is lacking. Importantly, Rv1364c has a
PAS domain with very high affinity for fatty acids (particularly
palmitoleic acid), suggesting that it may regulate SigF in response
to fatty acids [179]. In summary, a complex network of s factors
and their regulatory protein partners ensures survival of Mtb
during physiological redox stress.
The role of serine–threonine kinases in redox homeostasis of
mycobacterium
In most eukaryotes, signal is perceived by a sensor and then
relayed through an intricate network of kinases and phosphatases to
generate a synchronized response. The Mycobacterium genome
encodes 11 serine/threonine kinases (STKs) (PknA to PknL, except
PknC), one serine/threonine phosphatase (PstP), a tyrosine kinase
(PtkA), and two tyrosine phosphatases (PtpA and PtpB). These kinases
and phosphatases, along with two-component systems and onecomponent proteins, constitute the signal sensing and transducing
machinery of Mtb [180]. Knowledge of the molecular basis of this
signal transduction network lies at the heart of our ability to create
novel therapeutic tools; our current understanding of the roles of
kinases in the redox homeostasis of Mtb is described here (Fig. 5).
PknA and PknB are encoded by an operon located near the origin
of replication. This operon also codes for PstP, RodA (a protein
involved in determination of cell shape), and PbpA (a protein
involved in peptidoglycan biosynthesis). PknA and PknB are essential genes that are involved in the regulation of cell growth and
morphology. PknA can phosphorylate PknB, Wag31, FipA, FtsZ, and
FtsQ [174,181]. Wag31, FipA, FtsZ, and FtsQ are involved in cell
division. Interestingly, phosphorylation of FipA on Thr77 and FtsZ on
Thr343 by PknA is required for cell growth under oxidative stress
[181], suggesting an important role for PknA in sensing oxidative
stress and regulating oxidative stress-responsive growth. PknA is
sufficiently different from human kinases and thus is an attractive
drug target. With the aim of discovering novel anti-TB drugs,
Magnet et al. [182] have devised a screen for identifying PknA
inhibitors [164]. Of the 12,000 compounds that were screened, three
potential hits with a minimum inhibitory concentration below
10 mM were identified [182].
PknB phosphorylates the oxidative response s factor SigH and its
anti-s factor RshA. Phosphorylation of RshA by PknB leads to
disruption of the interaction between SigH and RshA and thus
regulates the induction of oxidative stress response by mycobacteria
[183]. Similar to PknB, PknD indirectly regulates the transcriptional
response of SigF through phosphorylation of the anti-anti-s factor
Rv0516c. However, this phosphorylation differs from classical phosphorylation on a conserved internal threonine and is located on the
second amino acid at the N terminus (Thr2) [184].
Two of the STKs (PknG and PknE) harbor redox-sensitive
domains, suggesting that these STKs could be important for
sensing the redox environment of mycobacteria. Of these, PknG
S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641
1637
Fig. 5. Protein kinases as redox sensors. Top left: PknA phosphorylates PknB, Wag31, FtsQ, FipA, and FtsZ. They are involved in the control of cell growth and morphology;
among these proteins FipA and FtsZ are very important as they are involved in cell growth under oxidative stress. Phosphorylated PknB also activates SigH by
phosphorylating the anti-s factor RshA. Top right: PknG activates a-ketoglutarate decarboxylase and glutamate dehydrogenase by phosphorylation of inhibitory protein
GarA. Bottom: PknD is involved in activation of SigF by phosphorylating and inhibiting the binding of anti-s factor Rv0516 to SigF.
is of special interest and is studied vigorously. PknG inhibits the
phagosome–lysosome fusion of infected macrophages and thus
plays an important role in mycobacterial virulence [185]. The
inhibition of PknG’s kinase activity by chemical inhibitors leads to
increased clearance of mycobacterial infection, suggesting that
PknG could be exploited as a potential drug target [185]. PknG has
an N-terminal rubredoxin domain, a central kinase domain, and a
C-terminal tetratricopeptide repeat domain. Rubredoxin domains
are small domains that bind iron tetrahedrally through four
cysteine residues that reside in two CXXCG motifs. They are
usually found in electron carrier proteins, but the rubredoxin
domain in PknG, a sensor STK, is probably a redox sensor. The
rubredoxin domain is important for regulating the function of
PknG [186,187]. The kinase activity of PknG is induced in
response to oxidative stress and the cysteines of the CXXCG motif
are closely involved in regulating that kinase activity [187].
Additionally, PknG leads to phosphorylation of GarA (glycogen
accumulation regulator) in Mtb [188]. Unphosphorylated GarA
binds and inhibits a-ketoglutarate decarboxylase and glutamate
dehydrogenase and thus regulates metabolic flux in response to
nitrogen availability [188].
PknE harbors a thioredoxin fold. This fold is often present in
redox sensors, indicating that PknE might act as a redox sensor.
Interestingly, expression of PknE is responsive to NO and deletion
of PknE leads to increased resistance to NO and increased
sensitivity to reducing agents [189]. Similar to PknE, deletion of
PknH in Mtb also leads to increased resistance to acidified NO,
implying that PknH could also act as a redox sensor. Recently, AvGay and co-workers demonstrated that PknH phosphorylates the
NO-responsive DosR [190] on Thr198 and Thr205. These two
phosphorylations and the phosphorylation by DosS and DosT on
Asp act cooperatively to enhance the transcription activation by
DosR in response to NO [190]. In summary, the STKs of Mtb are
important for sensing redox stress and orchestrating an ordered
response to the stress.
in vitro leads to induction of specific transcriptional responses that
result in networked regulation of Mtb’s metabolism facilitating the
maintenance of intracellular redox state and survival. The induction
of specific transcriptional responses despite a lack of classical redox
sensors suggests that Mtb possesses a battery of redox sensors to
steadily monitor the extracellular and intracellular redox status.
Some of these sensors have been recently discovered; however,
many more redox sensors are yet to be discovered. The important
redox sensors of Mtb include the heme based sensors DosS and
DosT, Fe–S cluster-based WhiB proteins, redox-responsive thiolbased anti-s factors/anti-anti-s factors, and thioredoxin-,
rubredoxin-fold-dependent redox-sensitive kinases. The DosS and
DosT proteins monitor changes in the levels of oxygen, redox, NO,
and CO to facilitate the transition from an actively multiplying state
to a nonreplicative state. The WhiB proteins sense changes in the
NO, oxygen, and intracellular redox state to regulate the metabolism
of virulence lipids and response to antibiotics. The anti-s/anti-antis factors and serine–threonine kinases monitor the levels of ROS.
However, we still do not know the identity of the sensors involved
or the genetic pathways involved in reactivation of Mtb upon reaeration/dampened immune system. Thus many more sensors and
regulatory pathways await discovery. Given the unique and prolonged latency of Mtb, these pathways and sensory modules would
be unique to mycobacteria and could be exploited for discovering
drugs that may target the bacteria in a persistent/latent infection.
Acknowledgments
We thank Dr. Girish Sahni for his help and support. We
gratefully acknowledge IMTECH (a constituent laboratory of the
CSIR) for providing the infrastructural facilities and financial
support. A.K. is supported by funding from the CSIR (OLP-70
and SIP10) and DBT (BT/PR15097 and BT/PR15086/GBD/27/307/
2011) India. S.A.B., N.S., and P.K. acknowledge the fellowships
from CSIR and A.T. is grateful to UGC for a fellowship.
Concluding remarks
References
Mtb is persistently exposed to numerous redox stresses during
its pathogenic cycle. These stresses include host-generated ROS,
acidic pH, RNS, and hypoxia. Exposure of Mtb to these stresses
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