on demand redox buffering by H2S contributes to antibiotic resistance revealed by a bacteria-specific H2S donor

Article abstract of DOI:10.1039/c7sc00873b

Understanding the mechanisms of antimicrobial resistance (AMR) will help launch a counter-offensive against human pathogens that threaten our ability to effectively treat common infections. Herein, we report bis(4-nitrobenzyl)sulfanes, which are activated by a bacterial enzyme to produce hydrogen sulfide (H₂S) gas. We found that H₂S helps maintain redox homeostasis and protects bacteria against antibiotic-triggered oxidative stress on demand , through activation of alternate respiratory oxidases and cellular antioxidants. We discovered, a hitherto unknown role for this gas, that chemical inhibition of H₂S biosynthesis reversed antibiotic resistance in multidrug-resistant (MDR) uropathogenic Escherichia coli strains of clinical origin, whereas exposure to the H₂S donor restored drug tolerance. Together, our study provides a greater insight into the dynamic defence mechanisms of this gas, modes of antibiotic action as well as resistance while progressing towards new pharmacological targets to address AMR.

Full text of DOI:10.1039/c7sc00873b

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“On Demand” Redox Buffering by H2S Contributes to Antibiotic
Resistance Revealed by a Bacteria-Specific H2S Donor
Prashant Shukla,a,e† Vinayak S. Khodade,b† Mallojjala SharathChandra,b Preeti Chauhan,b Saurabh
Mishra,a Shivakumara Siddaramappa,c Bulagonda Eswarappa Pradeepd, Amit Singha,* and Harinath
Chakrapanib,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Understanding mechanisms of antimicrobial resistance (AMR) will help launch a counter-offensive against human
pathogens that threaten our ability to effectively treat common infections. Here, we report bis(4-nitrobenzyl)sulfanes,
which are activated by a bacterial enzyme to produce hydrogen sulfide (H2S) gas. We find that H2S helps maintain redox
homeostasis and protects bacteria against antibiotic-triggered oxidative stress “on demand” through activation of
alternate respiratory oxidases and cellular antioxidants. We discovered, a hitherto unknown role for this gas, that chemical
inhibition of H2S biosynthesis reversed antibiotic resistance in multidrug-resistant (MDR) uropathogenic bacteria of patient
origin, whereas exposure to the H2S donor restored drug tolerance. Together, our study provides greater insight into the
dynamic defence mechanisms of this gas, modes of antibiotic action as well as resistance while progressing towards new
pharmacological targets to address AMR.
Due to the dwindling arsenal of antibiotics, AMR is possibly the
biggest problem that this generation will face in the coming
decade. In order to address this complex socioeconomic public
health problem, multiple methodologies are necessary
including a better understanding of mechanisms of antibiotic
action and factors contributing to antibiotic resistance.7 Here,
we systematically investigated the dynamic effects of H2S in
protecting bacteria from antibiotic-induced stress and the role
of H2S in modulating AMR.
Introduction
Maintenance of redox homeostasis is fundamental to cellular
growth and survival. Induction of dysfunctional redox
environment is a common mechanism used against pathogens
by immune cells.1 In the past several decades, it has been well
established that gases such as hydrogen sulfide (H2S) and nitric
oxide (NO) affect cellular redox balance.2 Bacteria-derived H2S
through microbiota contribute significantly to repair
mechanisms and are vital for the health of the gastrointestinal
tract.3 Bacterial H2S is also implicated as a cytoprotective agent
against antibiotic-induced stress resulting in antibiotic
tolerance.4 Oxidant remediation by bacterial H2S is
operational, but precise mechanisms of protection remain to
be completely elucidated.5 Mapping out these cytoprotective
mechanisms will help progress towards new strategies to
combat the growing threat of antimicrobial resistance (AMR).6
Being a gaseous species, reliable detection8 as well as
controlled and site-specific generation of H2S within cells is
fundamental to understanding its biology.9 Numerous donors
of H2S3 are in development but none, to our knowledge,
distinguish one type of cells over others.10 Enzymes as
metabolic triggers for activation of donors offer distinct
advantages as they will facilitate localization of H2S. A H2S
generating functional group is tethered to a substrate for an
enzyme that is normally expressed in cells of interest (Figure
1a). Upon entry into cells, metabolism by the target enzyme
frees up the active H2S generator inside cells thus achieving
localized delivery. Recently, two esterase activated H2S donors
were reported with potential for wide applicability in cellular
studies (Figure 1b).11 However, generating H2S specifically in
certain cells over others might be problematic when using
esterase as a trigger. We chose E. coli nitroreductase (NTR), an
oxygen-insensitive bacterial enzyme that is frequently
expressed in bacteria but not in mammalian cells.12 Geminal
dithiols are reported to undergo hydrolysis in buffer to
produce H2S;13 4-nitroaryl groups are known substrates for
a. Department of Microbiology and Cell Biology & Centre for Infectious Disease and
Research, Indian Institute of Science, Bangalore 5600012, Karnataka, India. E-
mail: asingh@mcbl.iisc.ernet.in
b. Department of Chemistry, Indian Institute of Science Education and Research
Pune, Dr. Homi Bhabha Road, Pashan Pune 411 008, Maharashtra, India. E-mail:
harinath@iiserpune.ac.in
c. Institute of Bioinformatics and Applied Biotechnology Bengaluru 5600100,
Karnataka, India
d. Sri Sathya Sai Institute of Higher Learning, Vidyagiri, Prasanthi Nilayam, Andhra
Pradesh, India
e. International Centre for Genetic Engineering and Biotechnology, New Delhi, India
† These authors contributed equally
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
NTR. We hence designed
1b).
1
, a NTR-activated H2S donor (Figure
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incubated with NTR (Figure 2b). Next, NBD-FluorVeieswceAirntic,leaOnHli2nSe
sensitive dye was synthesized and wDaOs I:i1n0c.1u0b3a9t/Ced7SCi0n08t7h3eB
presence of 1c and NTR.16 Again, we find a distinct increase in
fluorescence attributable to H2S generation (Figure S1, ESI).
Thus, the formation of H2S was validated by several
independent assays suggesting that this compound is a reliable
donor of H2S.
The H2S donor 1c was able to maintain elevated levels of
H2S over 45 min (Figure 2c). The formation of a hydroxylamino-
or amino-aryl derivative, (Figure 1b, Scheme S2, ESI) which
self-immolates to generate a geminal dithiol is likely. This
geminal dithiol should hydrolyze to produce H2S and a
ketone.13 Accordingly, when 1e was incubated in buffer in the
presence of Zn and ammonium formate, acetophenone is
formed supporting the proposed mechanism (Figure S2, ESI).
Figure 1. (a) General design of an enzyme activated H2S donor. (b) Esterase
sensitive H2S donor; (b) Esterase activated H2S and COS/H2S donor; R1 can be a
non-steroidal anti-inflammatory drug (NSAID) while R2 is an aryl group or benzyl
(c) Bacterial enzyme nitroreductase (NTR) activated H2S donor and inset contains
compounds synthesized in this study.
Results and discussion
Synthesis of
1
is achieved by the reaction of variety of ketones
) (Table 1). A H2S-sensitive dye
(
2) with 4-nitrobenzyl thiol (3
BODIPY-azide 4a was employed to detect H2S. BODIPY-azide is
known to be reduced by H2S to produce a fluorescent amine
4b 14
Compounds 1a-1g were independently exposed to NTR
.
and all compounds were found to generate H2S under these
conditions (Figure 2a). The cyclopentyl derivative 1c was found
to be slightly better than the cohort of donors tested and this
compound was used for further studies.
Table 1. Synthesis of 1a-1g
Entry
R1
CH3
Et
Cyclopentyl
Cyclohexyl
Ph
R2
CH3
Et
Ketone
2a
2b
2c
2d
2e
2f
2g
Prod
1a
1b
1c
1d
1e
1f
Yield %
80
78
70
71
60
42
38
1
2
3
4
5
6
7
Figure 2. (a) Hydrogen sulfide generated during incubation of 1a
-1g (50 M) with
NTR in HEPES buffer pH 7.4 was estimated using a BODIPY-based sensor. (b) A
monobromobimane (6a) method for estimation of hydrogen sulfide. The
formation of thioether 6b supports the intermediacy of H2S. (c) Time course of
H2S generation during incubation of 1c in pH 7.4 buffer alone and in the presence
of NTR was assessed by BODIPY-based sensor 4a. (d) Flow cytometry analysis of
CH3
CH3
CH3
intrabacterial H2S generation in E. coli using the probe 4a. 1c was incubated for
20 minutes*** p-value=0.0001; NS = not significant. (e) Flow cytometry analysis
of intracellular H2S generation in THP-1 cells. 1c was incubated for 20 minutes
(100 M) and 4a was used for H2S detection NS = not significant.
4-FPh
Thiophene

1g
Having established that 1c generated H2S in cell-free
A monobromobimane (mBBr, 6a) assay (with some
modifications) was next used to confirm the production of
H2S.15 The electrophile mBBr reacts with sulfide anion to
produce a thioether which contains two bimane units (6b).
When 6a was treated with Na2S in pH 7.4 buffer, as expected,
6b was formed (Figure 2b). Under similar conditions, when 1c
was co-incubated with 6a and NTR, we found evidence for the
formation of 6b again supporting 1c as a source of H2S when
conditions in the presence of a bacterial enzyme, the ability of
this compound to permeate cells to be metabolized by NTR to
generate H2S was evaluated. First, a HPLC-based method was
used: E. coli cells were incubated with the H2S-sensitive dye 4a
and 1c. Cells were lysed and HPLC analysis of the lysate
revealed the formation of 4b supporting H2S generation
(Figure S3, ESI); a similar result was recorded for a variety of
bacteria supporting the broad utility of this donor. Next, flow
2 | J. Name., 2012, 00, 1-3
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cytometry analysis revealed the generation of H2S inside intact coli. In contrast, oxidative challenge with H2OV2,iewaArtriceleaOctnilvinee
bacterial cells when treated with 1c supporting the ability of oxygen species (ROS), rapidly increased tDhOeI:41005.1/043898/Cr7aStCi0o0,8a7n3dB
this donor to enhance H2S levels in live bacterial cells (Figure pre-treatment with 1c significantly reversed this response
2d). 4-Nitrobenzylbenzoate
5 (a likely competitive inhibitor) (Figure 3a). However, pre-treatment with either Na2S or 5 had
was next synthesized using a reported method.17 This no influence on H2O2-induced oxidative changes in biosensor
compound was by itself incapable of generating H2S in the response (Figure S6, ESI).
presence of NTR and also inhibited H2S generation from 1c
Importantly, protective influence of 1c on intrabacterial
(Figure 2a). The negative control was did not generate H2S redox potential translated into significantly higher resistance
5
within bacteria suggesting that the metabolism of the nitro displayed by E. coli against bactericidal concentrations of H2O2
group does not contribute to H2S production (Figure 2d). The (Figure 3b). Interestingly, H2S itself did not have any significant
H2S donor 1c was ineffective in generating H2S in E. coli strains effect on growth of E. coli. Thus, intervention by H2S occurs
lacking NTR (Figure S4, SI), confirming NTR-specificity in vivo. when other endogenous oxidant-remediation systems are
As NTR is predominantly produced in bacteria but not in overwhelmed. This property is consistent with the lower
mammalian cells, the H2S donor 1c must selectively enhance reduction potential of H2S when compared with major cellular
H2S in bacteria. Human monocytic cells (THP-1) were treated thiols such as glutathione19 and thus affords H2S a unique role
with 1c and H2S levels were assessed by flow cytometry. Here, in cellular redox chemistry. Furthermore, in contrast with
we find that while Na2S was capable of enhancing H2S levels other routinely used antioxidants in redox biology such as
within THP-1 cells, 1c remained completely ineffective (Figure thiourea and bipyridyl, 1c does not significantly affect bacterial
2e). Thus, 1c was selective in its ability to enhance H2S in growth (Figure 3b) suggesting that this tool would be
bacteria over mammalian cells (Figure S5, ESI). To our appropriate for studying H2S-mediated response to dynamic
knowledge, this is the first example of a H2S donor with species redox alterations during antibiotic-induced stress and lethality.
selectivity. Thus, this study lays the foundation for novel
The emerging model for antibiotic lethality involves the
methodologies for site-specific enhancement of H2S using this induction of complex redox and metabolic alterations as a
class of H2S donors. For example, selectively enhancing H2S in consequence of drugs' interaction with their specific targets.5,
20
microbiota to study effects of this gas on colorectal cancer and
Thus, it is important to understand the dynamic effects of
other similar pathophysiologies is possible.2
H2S in mitigating antibiotic-induced redox stress.21 To do this,
To begin understanding the mechanisms of H2S-mediated we exposed E. coli to clinically relevant concentrations of
oxidation remediation, we used non-invasive redox bactericidal antibiotic ampicillin (Amp; cell wall targeting) and
biosensor (roGFP2) and assessed dynamic changes in the an oxidative shift was recorded (Figure 3c).21a,
a
22
More
cytoplasmic redox potential of E. coli in response to oxidative importantly, pre-treatment with 1c reduced the degree of
stress.18 An increase in 405/488 nm excitation ratio of roGFP2 oxidative shift induced by Amp, resulting in significant
indicates oxidative stress, while reverse suggests reductive tolerance to antibiotics (Figure 3d). Amp-mediated increase in
changes.18b We first exposed E. coli expressing roGFP2 to 1c roGFP2 ratios emerged earlier than the time points at which
and measured the roGFP2 biosensor response and no significant killing was observed, indicating that oxidative stress
significant changes in 405/488 ratio was observed (Figure 3a). precedes death, and 1c-derived H2S protects bacteria by
Hence, H2S alone does not affect ambient redox-potential of E. maintaining cytoplasmic redox potential.
Figure 3. (a) Reduction-oxidation sensitive GFP (roGFP2) was used to measure dynamic changes in cytoplasmic redox potential of E. coli upon exposure to: H2O2, 1
mM; 1c, 100 M; (b) Time-kill analysis of E. coli treated with hydrogen peroxide (1 mM) and 1c (100 M) during 40 min. (c) Dynamic changes in cytoplasmic redox
potential of E. coli upon exposure to: Amp, 5
Dynamic changes in cytoplasmic redox potential of E. coli upon exposure to: Amik, 20
and 1c (100 M) during 120 min.

g/mL; 1c, 100

M; (d) Time-kill analysis of E. coli treated with Amp (5

g/mL) and 1c (100

M) during 120 min. (e)
g/mL)

g/mL; 1c, 100 M; (f) Time-kill analysis of E. coli treated with Amik (20 


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Similar results were recorded for Amikacin (Figure 3e-3f), a pretreatment resulted in a significant attenuation of Amp
translation inhibitor, and ciprofloxacin, a replication inhibitor lethality in the case of CyoA mutant (like WT strain), it was
(Figure S7, ESI). Altogether, data demonstrate that elevating completely ineffective in protecting cydB mutant (Figure 4b).
endogenous H2S levels can arrest antibiotics-triggered redox In LB medium, WT, cyoA, and cydB strains showed comparable
stress and killing.
growth profiles in the absence or presence of 1c, indicating the
Mechanisms of H2S-mediated protection from antibiotic- differences in Amp susceptibility is not a consequence of
induced lethality are poorly understood. It has been shown reduced growth rates. Sustenance of cydB expression in
that H2S elevates cellular antioxidant capacity and suppresses response to H2S-Amp combination, coupled with maintenance
iron load to mitigate antibiotic-linked ROS production.23 Since of H2S-mediated antibiotic tolerance in cyo mutant (where
sulfide is a potent ligand of copper and heme moieties, H2S aerobic respiration is mainly permitted by CydB) but not in
efficiently inhibits aerobic respiration by targeting copper- cydB mutant, suggest that H2S effect is likely dependent upon
heme containing cytochrome bo oxidase (CyoA).24 Under these cydB.24 Along with its role in respiration, CydB from E. coli has
conditions respiration becomes primarily dependent upon less been shown to reduce H2O2 by displaying catalase and quinol
energy efficient cytochrome bd oxidase (CydB).24 Interestingly, peroxidase activities.26 Therefore, maintenance of cydB
modulation of cytochrome oxidases expression is known to expression by H2S can potentiate antibiotic tolerance by
influence antibiotic toxicity.21b Therefore, we assessed bolstering the bacterial antioxidant capacity. Agreeing to this,
whether terminal oxidases are important contributory factors cydB mutant displayed heightened sensitivity to H2O2 as
in H2S-mediated antibiotic tolerance. First, quantitative compared to cyoA and WT strains (Figure S8, ESI).
reverse transcription-PCR (qRT-PCR) analysis of E. coli cells in
the absence or presence of 1c was conducted (see ESI). A
significant down-regulation of the genes encoding CyoA was
observed with 1c-treated bacteria (Figure 4a). The expression
of alternate oxidases was however either maintained
(cytochrome bd oxidase
I [cydB]) or highly induced
(cytochrome bd oxidase II [appY]) by H2S (Figure 4a). During
growth under low-O2 tension, E. coli down-regulates cyo
operon and upregulates cyd and app operons, indicating that
H2S triggers genetic and physiological changes comparable to
O2-limitation.25 Amp treatment reversed the influence of H2S
on the expression of cytochrome oxidases as cyoA and cydB
transcripts were significantly induced and repressed,
respectively, as compared to untreated cells (Figure 4a). The
appY transcript remained down-regulated in response to Amp.
Data suggest that Amp treatment promotes respiration via
energetically efficient CyoA, which is consistent with a recent
study demonstrating acceleration in aerobic respiration in
response to bactericidal antibiotics.21b
Having observed divergent effects of H2S and Amp on
cytochrome oxidases gene expression, we next examined the
outcome of H2S and Amp combination on transcription. qRT-
PCR analysis of E. coli pre-treated with 1c followed by
exposure to Amp showed severely down-regulated expression
cyoA, whereas expression of cydB and appY was robustly
maintained (Figure 4a) as compared to Amp alone. Thus,
maintenance of respiratory flux through cytochrome bd
oxidase I/II in response to H2S treatment may be a key trait
that permits adaptation upon subsequent exposure to
antibiotics. To examine this possibility, we assessed cell-killing
Figure 4. (a) qRT-PCR analysis of E. coli cells treated with 1c alone, Amp alone.
The fold-change is with respect to untreated control; 1c + Amp where fold-
change is with respect to Amp-treated cells. Fold change for each transcript was
calculated by normalizing expression with the housekeeping 16SrRNA transcript.
A 2-fold (p<0.05) increase or decrease in expression was considered significant in
all cases. (b) Growth curves of E. coli treated with Amp (5
g/mL) with and
without 1c (100
cydB mutant. (c) H2S levels measured using the H2S-sensitive dye 4a in the
presence of ASP, a biosynthesis inhibitor of H2S generation [d] Bacterial growth
measurements: Amp (100 g/mL); Asp (4 mM) and 1c (100 M) were used in this
study
M); Ctrl is untreated bacteria: Wild-type (WT); cyoA mutant;


In addition, consistent with previous results,4d we observed
that H2S is ineffective in protecting cells lacking cytoplasmic
catalase and peroxidase (KatA, KatE, ahpCF; HPX-) from Amp-
in respiratory mutants lacking either cyoA or cydB. While 1c
-
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induced lethality (Figure S9, ESI). Altogether, data implicate a inhibitors of 3-MST may find similar use in sensitizing resistant
View Article Online
central role for cytochrome bd oxidase and oxidant- pathogens.30 Our results provide a sound pharmacological
DOI: 10.1039/C7SC00873B
remediation mechanisms in diminishing the effectiveness of basis for design of inhibitors of biosynthesis of H2S as a
antibiotic by H2S (Figure 5).27
possible adjuvant.29-31 Furthermore, we identified critical
aspects of bacterial physiology that could be exploited as part
of new potentiation strategies. For examples, targeting
antioxidant enzymes and alternate respiratory complexes
(Cyd/App) is likely to enhance the killing potential of
antibiotics. A combination of molecules/drugs targeting H2S,
antioxidants, and respiration could have a remarkable impact
on drug-resistance and clinical outcomes.
Conclusions
In summary, we report a new H2S donor that reliably and
selectively enhances H2S within bacteria. An application of our
new tool clearly revealed that H2S is a key player in the
maintenance of intracellular redox balance of bacteria to
counteract lethal degree of oxidative stress induced by
antibiotics and the critical role that H2S played in modulating
drug resistance. Antibiotic resistance is emerging as possibly
the biggest global health challenge for this generation.
Therefore understanding pathogen defence mechanisms and
their consequences in drug resistance is critical. From the
evolutionary perspective, H2S generating enzymes are
prevalent in most sequenced bacterial genomes including
environmental bacteria, indicating a naturally conserved role
of H2S in ensuring survival. It is likely that H2S producing
capability is under the selective pressure in diverse
environmental bacteria owing to antimicrobials secreted by
other bacteria and fungi inhabiting the same niche. Our study
Figure 5. Evolving model for H2S mediated antibiotic tolerance. Bactericidal
antibiotics alters respiration and metabolism to elevate the production of
endogenous ROS. Based on our findings, we propose that bacterial H2S provides
tolerance to antibiotics by two mechanisms; (i) down-regulation of energy-
efficient cytochrome bo oxidase (Cyo) and induction of less-energy efficient
cytochrome bd oxidase I/II (CydAB/AppBC) to maintain respiratory flux and
redox balance, and (ii) augmentation of antioxidant capacity by elevating
catalase and superoxide dismutase (SOD) activities. NDH: NADH dehydrogenase
and SDH: Succinate Dehydrogenase.
Lastly, in order to examine if elevated endogenous H2S
levels are associated with drug resistance in the physiological
context of human infections, we measured the intracellular
H2S levels of several multidrug-resistant (MDR) E. coli strains
isolated from patients (Table S2, ESI for resistance profile)
suffering from urinary tract infections (UTI). The endogenous
H2S levels were considerably higher than WT indicating a
possible functional role for H2S in antibiotic resistance (Figure
presents significant advances towards
a
complete
understanding of antibiotic-induced stress and cytoprotective
mechanisms of H2S.
S10, SI).28 In the presence of
mercaptopyrvuate sulfurtransferase
a
well-established 3-
(3-MST) inhibitor
(aspartate, Asp), we find that H2S levels were significantly
diminished (Figure 4c).4d To understand the functional
relevance of endogenous H2S levels in drug resistance, we
monitored P14 strain’s resistance to Amp. We find that pre-
treatment with Asp efficiently inhibits the growth in response
to Amp (Figure 4d). More-importantly, co-treatment with Asp
and 1c, significantly restored resistance to Amp in the P14
strain (Figure 4d). Altogether, these findings revealed a
previously unknown contribution of H2S in cooperating with
genetic mechanisms of antibiotic resistance (Figure 5). Further
work is needed to examine H2S-mediated mechanisms
contributing to the emergence of drug-resistance in clinical
strains.5, 28 Among the major infectious diseases, UTI affects
millions and is further complicated by conditions such as
diabetes. E. coli has now become resistant to most major
classes of antibiotics and therefore there is an urgent need to
develop new therapeutics. Recently, Berkowitz and co-workers
have developed a CBS inhibitor that helps prevent the
deleterious effects of enhanced H2S such as neuronal cell
death during episodes of stroke.29 The inhibitors developed in
their study showed a marked diminution in neuronal cell death
when compared with untreated control. It is likely that
Acknowledgements
The authors thank the Department of Science and Technology
(DST, Grant number EMR/2015/000668), DBT-IISc program
(AS), and Council for Scientific and Industrial Research (CSIR)
for financial support. The authors thank James Imlay,
University of Illinois at Urbana-Champaign and Antonio Valle
Gallardo, Universidad de Cádiz for E. coli mutants.
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DOI: 10.1039/C7SC00873B
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