Lack of mycothiol and ergothioneine induces different protective\nmechanisms in Mycobacterium smegmatis
Mycobacterium smegmatis contains the low molecular weight thiols, mycothiol (MSH) and ergothioneine (ESH). Examination of transposon mutants disrupted in mshC and egtA, involved in the biosynthesis of MSH and ESH respectively, demonstrated that both mutants were sensitive to oxidative, alkylating, and metal stress. However, the mshC mutant exhibited significantly more protein carbonylation and lipid peroxidation than wildtype, while the egtA mutant had less protein and lipid damage than wildtype. We further show that Ohr, KatN, and AhpC, involved in protection against oxidative stress, are upregulated in the egtA mutant. In the mshC mutant, an Usp and a putative thiol peroxidase are upregulated. In addition, mutants lacking MSH also contained higher levels of Coenzyme F420 as compared to wildtype and two Coenzyme F420 dependent enzymes were found to be upregulated. These results indicate that lack of MSH and ESH result in induction of different mechanisms for protecting against oxidative stress.
Introduction<!>Bacterial strains, culture conditions, and disk sensitivity assays<!>Creation and screening of a transposon mutant library and identification of\nthe disrupted gene<!>Determination of thiol levels and Coenzyme F420<!>Complementation of S24 (mshC mutant), R119 (egtA mutant)<!>Measurement of protein carbonylation, lipid peroxidation, and peroxidase\nactivity<!>Proteomic evaluation of wildtype and mutants, S24 and R119<!>Mycothiol, ergothioneine and coenzyme F420 levels in transposon mutants\nlacking MSH and ESH<!>Sensitivity of S24 and R119<!>Protein carbonylation and lipid oxidation in mutants lacking MSH and\nESH<!>Peroxidase activity in mutants lacking in MSH and ESH<!>2D- DIGE analysis of S24<!>2D-DIGE analysis of R119<!>Discussion<!>Conclusions<!>
<p>Mycothiol (MSH), the major low-molecular weight (LMW) thiol produced by
Actinomycetes, acts as a glutathione (GSH) analogue to maintain redox homeostasis,
and protects the cell against xenobiotic agents, notably in pathogens such as
Mycobacterium tuberculosis [1, 2]. MSH is present in mycobacteria at
millimolar levels [1] and is
synthesized in a five step pathway from glucose-6-phosphate with the enzymes MshA,
A2, B, C, D [2]. A second LMW
thiol, L-ergothioneine (ESH), is also synthesized by Actinomycetes but is present in
lesser amounts [3]. In M.
smegmatis, ESH biosynthesis is initiated by a methyltransferase (EgtD), and proceeds
through an additional five-step pathway involving the enzymes EgtA-E [4]. Unlike other LMW thiols such as MSH,
ESH is tautomeric [5] and
exists predominantly in the thione form in neutral aqueous solutions. In addition,
the redox potential of ESH is −60mV as compared to −250 mV of GSH
which may account for ESH's resistance to auto-oxidation [5].</p><p>While the role and function of MSH has been extensively studied, the role of
ESH in bacteria is not as well-described. MSH mutants are more susceptible than
wildtype to a number of antibiotics, oxidative stress, alkylating agents, and other
stresses but resistant to the pro-drugs, isoniazid and ethioniamide [6]. An M. smegmatis mutant disrupted in
egtD, which lacks ESH, and is more susceptible to the lipid peroxides, cumene
hydroperoxide (CHP) and tert-butyl hydroperoxide (tBOH), but not sensitive to the
antibiotics, rifampin, isoniazid, ethionamide, and ethambutol [3]. A double mutant disrupted in mshA
and egtD and lacking both ESH and MSH is significantly more sensitive to the
peroxides than either of the single mutants lacking either ESH or MSH.
Interestingly, levels of ESH are significantly higher in the mshA mutant indicating
that ESH may be able to compensate for the loss of MSH [7]. In addition, Ohr, an organic hydroperoxide
reductase, is substantially upregulated in both mshA and egtD mutants. Whether this
upregulation of Ohr is particular to mshA and egtD mutants or is common in mutants
disrupted in other genes in the biosynthetic pathway of MSH and ESH is unknown.</p><p>In a screen for genes involved in disulfide stress, a transposon mutant
library was screened for sensitivity to diamide, a thiol-oxidizing agent. Five
mutants lacking MSH and disrupted in mshC, S24, D43, D48, D41, R41, and a mutant
disrupted in egtA, R119, were identified as having increased sensitivity to diamide.
We demonstrate that the mshC mutant, S24, has significantly more protein
carbonylation and lipid peroxidation, measures of oxidative damage, while the egtA
mutant has less protein and lipid damage than wildtype. We further show that Ohr and
other enzymes involved in protection against oxidative stress are upregulated in the
egtA mutant, while in the mshC mutant, the levels of coenzyme F420, a coenzyme
involved in redox reactions, are increased.</p><!><p>M. smegmatis mc2155 and mutant strains were grown in
Middlebrook 7H9 broth (Difco) with 10% OADC and then diluted in
Middlebrook 7H9 broth with 0.05% Tween 80 and 1% glucose. The
strains were also grown on 7H11 solid medium with 0.5% glycerol with
1% glucose for disk sensitivity assays, which were performed as
previously described [6].
Antibiotics were added to the media for the transposon mutant (Kanamycin 25
Î¼g/ml) and complemented strains (Hygromycin 75 Î¼g/ml). Broth
cultures were grown in a shaking incubator and all cultures were grown at
37°C unless otherwise indicated.</p><!><p>An M. smegmatis transposon mutant library (EZ::TN <kan-2> Tnp
transposase and Tn5 kanamycin resistance marker) was constructed and screened
for diamide sensitive mutants as described by Rawat et al. (2002) [8]. To identify the site of
insertion, genomic DNA was digested with restriction enzymes Sa1I and PstI
followed by self-ligation and PCR amplification with primers complementary to
the transposon. The sequence of the amplified PCR product was compared to the M.
smegmatis genome sequence [8].</p><!><p>Thiols in cell extracts were labeled with monobromobimane (mBBr) as
previously described [6].
High-pressure liquid chromatographic (HPLC) analysis was carried out on a
HiChrom Ultrasphere Ion Pair column (5μm; 250 × 4.6 mm) with the
following gradient at 1ml/min of Buffer A, 0.25% glacial acetic acid, pH
4.0; and Buffer B, methanol (0 to 5 min, 10% A; 35 min, 18% A;
45 min 27% A; 47 min, 100% A; 49 min, 0%). HPLC analysis
for F420 content was performed as previously described [9] with the following modifications:
Shimadzu Prominence system (LC 20AD dual pumps, SIL-20A autosampler and SPD-M20A
Diodearray detector) was used for chromatographic separation and quantitative
analysis. Cell extracts were separated with a Vydac 218TP54 column (C18, 5
μm, 4.6 mm i.d. × 250 mm) having flow rate of 0.6 ml/min. A
linear gradient of sodium acetate buffer-acetonitrile solution was used and
elution was monitored at 400 nm. Identities of F420 peaks were
confirmed by their UV-visible spectrum collected by an in-line diode array
detector. Samples in quadruplicates were analyzed at least three times and
reported as averages ± SD. Student t test was performed with p ≤
0.05 regarded as significant.</p><!><p>M. tuberculosis mshC cloned into pSODIT[10] was introduced into S24 in order to
obtain the complemented strain, S24C. Primers, TEshAF, with a HindIII
restriction site (CTTAAGCTTCACCGACGAGGCCTGGAC) and TEshAR with a XhoI
restriction site (TGCCTCGAGTGAAGTCACGACGCCCCG ) were used to amplify M.
tuberculosis egtA (Rv3704c) and 297 base pairs upstream of the start codon,
which contained the promoter region of the gene. The 1609 bp amplicon and
complementation vector pHINT were digested with the HindIII and XhoI, ligated
and transformed into E. coli. After confirmation of cloning, the resulting
vector, pHINTEgtA, was electoporated into R119 competent cells. Kanamycin and
hygromycin resistant transformants were screened by PCR for M. tuberculosis egtA
and one colony, R119C, was characterized further.</p><!><p>Protein carbonylation and lipid peroxidation was measured using the
Protein Oxidation Detection Kit and TBARS Assay Kit, following the
manufacturer's instructions (Cayman Chemical). To assess protein
carbonylation, cells were grown until OD600 equaled one. Then one set
of 10 ml cultures in triplicate were treated with 5 mM
H2O2 and another set was left untreated for one hour.
The cultures were harvested and lysed using bead beating (Fast Prep FP120,
Thermo Electron company). The cell lysates were centrifuged at 13,000 rpm for 30
min at 4oC to get rid of cell debris and the protein concentration of the
supernatant was determined by Bio-Rad DC assay. Two mg/ml of protein was
incubated with 2,4-dinitrophenylhydrazine at room temperature in the dark for
one hour with frequent vortexing. The derivatized proteins were TCA precipitated
and the pellet was further extracted with ethanol/ethyl acetate (1:1) solution
three times. The resulting pellet was resuspended in guanidine hydrochloride.
After centrifugation, the supernatant containing the protein hydrrazones were
analyzed spectrophotometrically at 360 nm.</p><p>Lipid peroxidation was assessed by treating 1ml of cells in triplicate
at 1.0 OD600 in log phase with 30 mM FeSO4 and 5 mM
H2O2 for two hours. Cell lysates were prepared
similarly to the samples for protein carbonylation assay and to 100 Î¼l
of the cell lysate, 100 Î¼l of 10% SDS was added. A color reagent
was prepared by adding thiobarbaturic acid (TBA) to acetic acid followed by the
addition of sodium hydroxide to the solution. Four ml of the color reagent was
added to each sample and the samples were boiled for 1 hour and then transferred
to ice for 10 min to stop the reaction. After centrifugation at 1600 × g
for 10 min at 4°C, the supernatants were transferred to an ELISA plate
and A532 was determined in duplicate. The amount of lipid
peroxidation was calculated with reference to a standard curve of
malondialdehyde.</p><p>Peroxidase activity was determined for each strain in triplicate using
the Pierce Quantitative Peroxide Assay Kit, which detects levels of peroxides
based on the oxidation of ferrous to ferric ion at acidic pH. The cell free
lysate was prepared as described for the protein carbonylation assay. 300
Î¼g of protein was incubated with 45 Î¼M
H2O2, CHP, and TbOH separately for 30 min. The amount
of remaining peroxides was determined by addition of sulfuric acid solution
containing ferrous ion and xylenol orange and incubation at room temperature for
20 minutes. The ferric ion- xylenol orange purple complex was measured at 595
nm. The amount of peroxides was calculated with reference to a standard curve of
the relevant peroxide.</p><p>All the experiments were performed at least three times.</p><!><p>As described in Ta et al. (2011) [7], M. smegmatis strains were grown for 11 days on
Middlebrook 7H9 agar and 1.0% glucose at 23°C. Cells were
collected by centrifugation and resuspended in lysis buffer (50 mM Tris
containing 7M urea, 2M thiourea, and 4% 3-[(3-Cholamidopropyl)
dimethylammonio]-1-propanesulfonate (CHAPS)]. Two-dimensional
difference gel electrophoresis (2D-DIGE) analysis of the supernatant was
performed at Applied Biomics (Hayward, CA). Briefly, samples were fluorescently
labeled and subjected to isoelectric focusing (IEF) on a 13 cm pH gradient strip
(pH 4–6). The changes in abundance of proteins between the wildtype and
the mutants were identified using the ImageQuantTL and DeCyder softwares. Spots
were excised from the gel and identified by MALDI TOF/TOF (tandem mass
spectrometry MS/MS) on a 5800 mass spectrometer (AB Sciex). Resulting peptide
masses and associated fragmentation spectra were submitted to GPS Explorer
version 3.5 with the MASCOT search algorithm (Matrix science) and searched
against the National Center for Biotechnology Information non-redundant
database. Searches were performed without constraining protein molecular weight
or isoelectric point, with variable carbamidomethylation of cysteine and
oxidation of methionine residues, and with one missed cleavage allowed in the
search parameters. MASCOT searches were also performed to identify
phosphorylation in the proteins. Candidates with score of either protein C.I.
% or Ion C.I.% greater than 95 were considered significant.</p><!><p>Inverse PCR was performed to identify the disrupted genes in diamide
sensitive mutants, S24, D41, D43, D48, R41, and R119. Five of the mutants, S24,
D41, D43, D48, R41, are disrupted in mshC (MSMEG_4189) and did not contain any
MSH. In four of the five mutants disrupted in mshC, the transposon was inserted
at 589 nucleotides from the start codon. In the D48 mutant, the insertion site
was 599 nucleotides from the start codon. As the majority of the mutants had the
same transposon insertion site, we decided focus on only one of these mutants,
S24, further. The amount of ESH in S24, 0.37±0.04 μmol/g dry
cell weight, was higher than in wildtype (0.20 ± 0.01) and significantly
different. The increase in ESH was not as high as in the mshA mutant
(1.02±0.24) grown under the same growth conditions [7]. The complemented strain, S24C,
did not show a significant difference from wildtype in ESH levels (0.19
± 0.01) and MSH levels (wildtype: 6.21 ± 0.17, S24C:
5.90±0.66).</p><p>In the transposon mutant, R119, where the transposon is inserted 972
nucleotides after the start codon in egtA (MSMEG_6250), there was no ESH present
and MSH levels (6.10±0.37) were not statistically significantly
different than wildtype. The complemented strain, R119C, did not have
statistically significant different levels of MSH (5.88 ± 0.27) and ESH
from wildtype (0.24 ± 0.04).</p><p>Previously, we had demonstrated that mutants disrupted in fbiC, which is
involved in the biosynthesis of Coenzyme F420 (F420), a deazaflavin derivative
of FMN found in methanogenic Archaea and actinobacteria [11], are sensitive to oxidative stress
[12, 13]. We measured F420 levels to determine if
F420 can compensate for the lack of MSH or ESH. F420 levels were statistically
significantly higher in mshA (8.47±0.52 nmol/g wet cell weight) and mshC
(S24) mutants (8.05±0.24) as compared to wildtype (6.12±0.87).
Complementation of the mshA mutant showed partial restoration of wildtype F420
levels (7.16 ± 0.57) while complementation of S24 resulted in lower F420
levels (4.81±0.32) as compared to wildtype. Interestingly, R119 had
statistically significantly less F420 (4.21± 0.1) than the wildtype and
complementation (6.51±0.38) restored F420 to wildtype levels.</p><!><p>In liquid culture, there is no difference in growth between R119 and
wildtype under normal growth conditions (data not shown). However, R119, like
S24 is more susceptible to alkylating agents and the sensitivity is reversed by
complementation with the M. tuberculosis homolog (Table 1). In the case of oxidants, both mutants show
sensitivity to diamide and CHP, but only S24 demonstrates sensitivity to
H2O2.</p><p>As metal stress results in oxidative stress through by either the Fenton
reaction or by sequestering of LMW thiols, we also examined metal sensitivity.
Both mutants were sensitive to Cd2+, Cu2+,
SeO32−, TeO32−,
and CrO42−, but only S24 was more sensitive to
Fe2+, as compared to wildtype (Table 1). The sensitivity to oxidants, alkylating
agents, and metals was reversed in R119C and partially reversed in S24C.</p><!><p>Protein carbonylation, as an indicator for protein damage, was measured
in S24 and R119, with and without hydrogen peroxide. S24 demonstrated
significantly higher levels of protein carbonylation which was partially
reversed in the complemented strain, S24C. In contrast, R119 had lower levels of
protein carbonylation and the complemented strain, R119C, was not significantly
different from the wildtype. S24 also had significantly higher levels of lipid
peroxidation and R119 had lower lipid oxidation levels. The complemented strain,
R119C, was not significantly different from wildtype (Table 2). Thus, lack of MSH results in increased
damage while lack of ESH results in less damage to cellular proteins and
lipids.</p><!><p>To determine if MSH or ESH are involved as electron donors to a
peroxidase(s), the decomposition of H2O2, CHP, and tBOH
was measured using xylenol orange assay. The amount of peroxides was higher in
S24 than wildtype (Table 2) indicating
that MSH is able to either directly reduce the peroxides or act as an electron
donor to a peroxidase. In contrast, the amount of peroxides in R119 was the same
as wildtype (Table 2) indicating that ESH
is not directly involved in the reduction of peroxides or another mechanism is
induced in R119 that detoxifies peroxides. The peroxide levels in S24C were
similar to wildtype.</p><!><p>Since standard SDS-PAGE analysis of transposon mutants disrupted in mshA
and egtD demonstrated the overproduction of a band identified as Ohr
[3, 7], 2D-DIGE was performed on wildtype and
S24. In S24, 36 spots clearly demonstrated differences between S24 and wildtype
and of these 19 were conclusively identified (Table 3, SuppFig.
1). One of the upregulated proteins in the transposon mutant is
MSMEG_4283 (Spot 18), dihydrolipoamide acyltransferase (DlaT), which is the E2
component of pyruvate dehydrogenase complex, and contains a lipoyl binding site
[14]. M. tuberculosis
dlat mutant has severe growth defect in vitro, has reduced survival in bone
marrow-derived macrophages from c57bl/6 and iNOS −/− mice, and
is less able to grow in c57bl/6 mouse lungs, spleen and liver in vivo
[15]. Together with
lipoamide dehydrogenase (LpdC), AhpD, and AhpC, DlaT acts as an NADH dependent
peroxidase and peroxynitrite reductase to provide protection against oxidative
stress [14]. The
importance of this dihydrolipoamide system is highlighted by the discovery of
suppressor mutations in lipoamide dehydrogenase (lpdA) in E. coli. An E. coli
mutant lacking both glutathione reductase and thioredoxin reductase is not
viable but suppressor mutations in lpdA restore growth to this redox-defective
mutant by causing the accumulation of dihydrolipoamide, which reduces oxidized
glutaredoxins and circumvents the need for thioredoxin and glutathione in E.
coli [16].</p><p>MSMEG_4688, also upregulated, codes for a putative thiol peroxidase and
its M. tuberculosis ortholog, Rv2466c, has been shown to be involved in
activation of TP053, a thienopyrimidine compound considered to be a leading drug
target against tuberculosis [17]. Rv2466cis a member of the sigH regulon and is induced in
the Wayne model of dormancy [18]. The electron donor for this thiol peroxidase is unknown
although it is able to reduce peroxides using DTT as an electron donor
[17]. Interestingly,
two of the proteins up-regulated include coenzyme F420 dependent proteins:
MSMEG_1996 (Spot 16), a methyltetrahydromethanepterin reductase that reduces
oxidized coenzyme F420, and MSMEG_2027 (Spot 24), a quinone reductase, which
uses F420 as an electron donor or receptor to activate another new tuberculosis
drug, PAS-824 [13].
Another indication that S24 is under oxidative stress is the upregulation of
GAPDH, a marker of oxidative stress, which is present in two spots (11 and 12)
that represent different post-translational modifications in the form of
oxidation cysteine residues to sulfenic, sulfinic and sulfonic acid.</p><p>Several other proteins that are upregulated in S24 are associated with a
general stress response. Two Usps (universal stress proteins), MSMEG_4362 (spot
19) and MSMEG_3811 (Spots 22, 27, 28, 29), are differentially expressed.
MSMEG_3811 codes for an iron regulated single domain Usp and is the only Usp,
which has a homologue in M. leprae. The homolog in M. tuberculosis, Rv1636, is
the only Usp along with kdpD, which is not up-regulated during macrophage
infection [19]. Of the
spots representing MSMEG_3811, spots 22 and 28 are up-regulated in the wildtype,
and spots 27 and 29 are up-regulated in S24. A MASCOT search for
phosphorylations revealed that the wildtype MSMEG_3811 isoforms are
phosphorylated while the mshC isoforms are not.</p><p>Spot 13 is the 65 kDa heat shock protein, GroEl (MSMEG_0880), which
prevents misfolding and promotes the refolding and proper assembly of unfolded
polypeptides generated under stress conditions like high temperatures. Spot 26
is MSMEG_6431, the homologue of M. tuberculosis EspR (Rv3849), which regulates
ESX-1, required for secretion and virulence in mice [20]. RpsB (Spot 33), 30s ribosomal protein
S2, is involved in formation of the translation initiation complex and involved
in the metabolism of ppGpp, the mediator of the stringent response that
coordinates a variety of cellular activities in response to changes in
nutritional abundance. EspR, Tuf (Spot 8, 35) and the Usp, MSMEG_3811, are
regulated by iron levels, suggesting an interaction among iron levels, oxidative
stress, and LMW thiols.</p><p>Also upregulated in the mutant is nitrogen regulatory protein PII (Spot
30), which becomes uridylylated to pII-UMP by GlnD when the ratio of glutamine
to 2-ketoglutarate decreases. The pII-UMP allows the deadenylylation of
glutamine synthetase activating the enzyme. During nitrogen excess, p-II is
deuridylated and promotes the adenylation of glutamine synthetase, which was
identified in two spots, 3 and 9.</p><!><p>The DIGE protein profile of wildtype and R119 demonstrated that 98
proteins were differentially regulated (Table
4, SuppFig.
2). Of these 17 spots had a egtA−/wt ratio greater
than two and 18 spots had a egtA−/wt ratio less than
−2. Ohr is the most upregulated protein and was present in four spots
(Spots 88, 89, 90, 91). Oxidation of specific cysteines and perhaps
mycothiolation of other cysteines may be responsible for these multiple spots of
Ohr. In addition to Ohr, other proteins associated with protection against
oxidative stress, AhpC (Spot 72), another thiol peroxidase, and KatN (Spot 48),
a manganese containing catalase, are also up-regulated in R119. Another
up-regulated protein, MSMEG_4272 (Spot 95), is a member of an iron-sulfur
cluster biosynthesis protein family, which are dependent on LMW thiols such as
BSH for assembly [21]. In
addition, a MarR type regulator (Spot 82), upstream of a quinone reductase, is
up-regulated.</p><p>Proteins associated with metabolism are differentially expressed in R119
and wildtype. Lactate monooxygenase (Spot 36), which catalyzes the conversion of
lactate to acetate, is upregulated while glycerol kinase (Spot 22), which
catalyzes the transfer of a phosphate from ATP to glycerol dihydroxyacetone,
L-glyceraldehyde and D-glyceraldehyde during carbohydrate metabolism, is the
most downregulated protein. IlvC (Spot 40) associated with isoleucine and valine
metabolism is also downregulated. Other downregulated proteins include an
alcohol dehydrogenase (Spot 44), belonging to family of quinone reductases, a
flavin-containing monooxygenase (Spot 33), and MPT63 (Spot 19), an immunogenic
protein which has been implicated in virulence.</p><p>In contrast to S24, the Usp, MSMEG_3811, is down-regulated in R119.
GroEl, another general stress protein, is also down-regulated in R119 but
up-regulated in S24. Other protein markers for stress seen in S24 are not
observed in R119 thus does not appear to be in a "stressed"
state. 2D-DIGE of R119C demonstrated partial complementation by the M.
tuberculosis egtA gene. In particular, the R119C/WT ratios for spots
representing Ohr are significantly lower as compared to R119/WT. However, other
proteins are differentially expressed in both R119 and R119C but the R119C/WT
ratios are less than R119/WT ratios. As other phenotypic data for S24C
demonstrated that this strain was partially complemented by the M. tuberculosis
mshC, 2D-DIGE was not performed.</p><!><p>Herein, we confirm that both MSH and ESH protect mycobacterial cells against
a number of stresses including oxidative stress and alkylating and demonstrate that
these two LMW thiols are also involved in protection against metal stress. S24 is
extremely sensitive to oxidants and alkylating agents as previously reported for
other mutants lacking MSH [6]
while R119 is less sensitive (Table 1).
Similar to the M. smegmatis mutant disrupted in egtD, which is more susceptible to
the lipid peroxides, CHP and tert-butyl hydroperoxide (tBOH), R119 is sensitive to
CHP [3]. In contrast to the
Streptomyces coelicolor egtA mutant, R119 is not sensitive to hydrogen peroxide
[22].</p><p>Both mutants are also sensitive to metal stress. In vitro studies had
previously demonstrated that ESH is able to scavenge free radicals and metal ions.
In particular, ESH is known to form complexes with copper [23] in vitro but in vivo, copper sulfate had no
effect on conidial germination or hyphal growth in a mutant of Neurospora crassa
lacking ESH [24]. Other LMW
thiols like GSH are known to protect against cadmium toxicity in E. coli
[25] and mutants
disrupted in bacillithiol, which is structurally similar to MSH, are sensitive to
metal stress [26].</p><p>Recently, Servillio et al [27] reported that oxidation of ESH with hypochlorite, peroxynitrite
and hydrogen peroxide resulted in the sulfonated form (ESO3H), and
hercynine, the desulfurated form of ESH indicating that this thiol, like cysteine
and MSH, is oxidized irreversibly during oxidative stress. Moreover, Sao-Emani et al
(2013) reported that a double mutant lacking both ESH and MSH is significantly more
sensitive to the peroxides than either of the single mutants lacking either ESH or
MSH. Taken together, these results suggest that MSH and ESH may be able to partly
compensate for the loss of the other thiol. However, as the levels of ESH are
25–50 fold lower than MSH in M. smegmatis, ESH is less able to compensate
for MSH. In the mshA mutant, ESH levels are substantially higher but that is not the
in the mshC mutant. Instead, the levels of Coenzyme F420 are higher in
S24 but Coenzyme F420 still does not fully compensate for the lack of MSH
as S24 still displays a sensitivity to a number of stresses. As disruption of mshA
and eshD results in a dramatic overproduction of organic hydroperoxide resistance
protein, Ohr [3, 7], we sought to establish if the same was true
for mutants disrupted in mshC (S24) and eshA (R119). In S24, there is no
upregulation of Ohr; however, the upregulation of DlaT which is part of the system
that provides reducing equivalents to the thiol peroxidases, AhpC, and Ohr, and the
upregulation of the M. smegmatis homolog of the putative thiol peroxidase, Rv2466c,
implies that there is a major increase in oxidative stress in the mshC mutant.
Upregulation of an enzyme that likely serves as a F420-dependent quinone reductase,
preventing the formation of cytotoxic semiquinones, and upregulation of a putative
F420-dependent reductase implies that coenzyme F420, which has been shown to be
involved in protection against oxidative stress [12], may be able to partially substitute for MSH
in the mycobacterial cell.</p><p>Interestingly, multiple spots representing Usp (MSMEG_3811) are present in
the 2D- DIGE. These multiple spots are a result of different amounts of
phosphorylation of MSMEG_3811 in the wildtype. Intriguingly, Usp is not
phosphorylated in S24, implying a crosstalk between MSH and universal stress
proteins. Previously, we observed phosphorylation of another Usp, MSMEG_3940, during
stationary phase in wildtype M. smegmatis using a stain for phosphoproteins (data
not shown). Phosphorylation of Usps has also been demonstrated in Arabidopsis
challenged with biotic stress [28] although how this signal is translated into a protective
response is not clear. Lack of MSH alters the redox environment of the cell which
may cause oxidative modifications of kinases, either inhibiting or activating
them[29]. The resulting
phosphorylation events in turn would positively regulate some and negatively
regulate other signal transduction pathways [29]. In M. smegmatis wildtype, there would be presumably be less
mycothiolation of proteins as in C. glutamicum [30], including a kinase that phosphorylates Usp
or less mycothiolation of MSMEG_3811. This could result in phosphorylation or
autophosphorylation of MSMEG_3811, as in E. coli UspG [31]. In S24, mycothiolation of the kinase would
reduce activity and less phosphorylation or auto-phosphorylation. It is not clear
what the exact functions of Usps are but it is likely that post-translation
modifications of MSMEG_3811in S24 has far-reaching consequences involving changes in
metabolism and growth arrest.</p><p>Despite upregulation of genes involved in the stress response, S24 has
increased protein carbonylation and lipid peroxidation. Paradoxically, R119 has less
protein carbonylation and lipid oxidation than wildtype and when cell lysate of R119
is treated with inorganic and organic peroxides. This is in contrast to HeLA cells,
where disruption of the ESH transporter, ETT, results in an increase in protein and
lipid damage [32] and ETT
knockout fish, where there is an increase in levels of lipid peroxidation markers
after incubation with Pb2+ or Cu2 + and an
approximately four fold increase in DNA lesions in the skin [33]. The decreases in protein carbonylation,
lipid peroxidation, and wildtype levels of peroxides in R119 can be explained by the
dramatic upregulation of Ohr and other enzymes involved in reduction of oxidants,
such as the manganese dependent catalase, KatN, and AhpC. Intriguingly, neither Ohr
nor KatN are present in M. tuberculosis although there is a paralog of Ohr, OsmC
(Rv2923c). Recombinant OsmC from both M. smegmatis (MSMEG_2421) and M. tuberculosis
are able to reduce H2O2, CHP, and tBOH [34].</p><p>Multiple species of Ohr are present in R119, probably as a result of
mycothiolation or oxidation of exposed cysteine residues resulting in the formation
of sulfenic/sulfinic/sulfonic acids. Chi et al., 2013 [30] demonstrated that a number of proteins are
mycothiolated, including the antioxidant enzymes Tpx, Gpx, and MsrA, but not Ohr, in
MSH containing Corynebacterium glutamicum [30], although OhrR, the Ohr regulator, is known to be
bacillithiolated in Bacillus subtilis [35]. Whether "ergothiolation" serves a similar
protective role in mycobacteria remains to be seen.</p><!><p>Disruption of genes involved in biosynthesis of either MSH and ESH leads to
up-regulation of genes involved in oxidative stress, albeit the genes that are
induced are different in the two strains. The M. smegmatis MSH mutant displays
protein and lipid damage despite the up-regulation of enzymes involved in protection
against oxidative stress, and elevation of CoenzymeF420 levels. In
contrast, the ESH mutant demonstrates even less protein carbonylation and lipid
peroxidation than wildtype, likely due to the up-regulation of proteins that
specifically protect against oxidative stress, such as Ohr and KatN. Another key
difference is result is the up-regulation of general stress proteins, GroEl and an
Usp, which appears to be unphosphorylated in the MSH mutant. The lack of
phosphorylation of the Usp protein in the MSH mutant suggests crosstalk between the
two different signaling mechanisms in mycobacteria. Future studies are needed to
determine the interplay of these two post-translational modifications in the
signaling network in mycobacteria.</p><!><p>Fig 1: 2D DIGE gel of S24 and wildtype</p><p>Fig 2: 2D DIGE gel of R119, R119C and wildtype</p>