Chemical Modification and Detoxification of the Pseudomonas aeruginosa Toxin 2-Heptyl-4-hydroxyquinoline N-Oxide by Environmental and Pathogenic Bacteria

Article abstract of DOI:10.1021/acschembio.7b00345

2-Heptyl-4-hydroxyquinoline N-oxide (HQNO), a major secondary metabolite and virulence factor produced by the opportunistic pathogen Pseudomonas aeruginosa, acts as a potent inhibitor of respiratory electron transfer and thereby affects host cells as well as microorganisms. In this study, we demonstrate the previously unknown capability of environmental and pathogenic bacteria to transform and detoxify this compound. Strains of Arthrobacter and Rhodococcus spp. as well as Staphylococcus aureus introduced a hydroxyl group at C-3 of HQNO, whereas Mycobacterium abscessus, M. fortuitum, and M. smegmatis performed an O-methylation, forming 2-heptyl-1-methoxy-4-oxoquinoline as the initial metabolite. Bacillus spp. produced the glycosylated derivative 2-heptyl-1-(β-d-glucopyranosydyl)-4-oxoquinoline. Assaying the effects of these metabolites on cellular respiration and on quinol oxidase activity of membrane fractions revealed that their EC₅₀ values were up to 2 orders of magnitude higher than that of HQNO. Furthermore, cellular levels of reactive oxygen species were significantly lower in the presence of the metabolites than under the influence of HQNO. Therefore, the capacity to transform HQNO should lead to a competitive advantage against P. aeruginosa. Our findings contribute new insight into the metabolic diversity of bacteria and add another layer of complexity to the metabolic interactions which likely contribute to shaping polymicrobial communities comprising P. aeruginosa.

Full text of DOI:10.1021/acschembio.7b00345

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Article
Chemical Modification and Detoxification of the Pseudomonas
aeruginosa Toxin 2-Heptyl-4-hydroxyquinoline N-
oxide by Environmental and Pathogenic Bacteria
Sven Thierbach, Franziska S. Birmes, Matthias C Letzel, Ulrich Hennecke, and Susanne Fetzner
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00345 • Publication Date (Web): 14 Jul 2017
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Chemical Modification and Detoxification of the Pseudomonas aeruginosa
Toxin 2-Heptyl-4-hydroxyquinoline N-oxide by Environmental and
Pathogenic Bacteria
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Sven Thierbach†, Franziska S. Birmes†, Matthias C. Letzel‡, Ulrich Hennecke‡, and Susanne
Fetzner†*
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†Institute for Molecular Microbiology and Biotechnology, University of Münster, 48149 Münster,
Germany
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‡Organic Chemistry Institute, University of Münster, 48149 Münster, Germany
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Abstract
2ꢀHeptylꢀ4ꢀhydroxyquinoline Nꢀoxide (HQNO), a major secondary metabolite and virulence
factor produced by the opportunistic pathogen Pseudomonas aeruginosa, acts as potent inhibitor
of respiratory electron transfer and thereby affects host cells as well as microorganisms. In this
study, we demonstrate the previously unknown capability of environmental and pathogenic
bacteria to transform and detoxify this compound. Strains of Arthrobacter and Rhodococcus spp.
as well as Staphylococcus aureus introduced a hydroxyl group at Cꢀ3 of HQNO, whereas
Mycobacterium abscessus, M. fortuitum and M. smegmatis performed an Oꢀmethylation, forming
2ꢀheptylꢀ1ꢀmethoxyꢀ4ꢀoxoquinoline as initial metabolite. Bacillus spp. produced the glycosylated
derivative 2ꢀheptylꢀ1ꢀ(βꢀDꢀglucopyranosydyl)ꢀ4ꢀoxoquinoline. Assaying the effects of these
metabolites on cellular respiration and on quinol oxidase activity of membrane fractions revealed
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that their EC50 values were up to two orders of magnitude higher than that of HQNO.
Furthermore, cellular levels of reactive oxygen species were significantly lower in presence of
the metabolites than under influence of HQNO. Therefore, the capacity to transform HQNO
should lead to a competitive advantage against P. aeruginosa. Our findings contribute new
insight into the metabolic diversity of bacteria, and add another layer of complexity to the
metabolic interactions which likely contribute to shaping polymicrobial communities comprising
P. aeruginosa.
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Keywords:
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Pseudomonas aeruginosa, Mycobacterium, Staphylococcus, Bacillus, antibiotic, respiration,
alkylhydroxyquinoline Nꢀoxide, Oꢀmethylation, glucosylation, hydroxylation, biotransformation,
detoxification
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INTRODUCTION
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Pseudomonas aeruginosa is a ubiquitous Gramꢀnegative γꢀproteobacterium that inhabits soil and
aqueous environments. As one of the most important opportunistic human pathogens, it can
cause serious and lifeꢀthreatening infections in immunocompromised and cystic fibrosis (CF)
patients.1 Many CF patients experience an age dependent colonization pattern, with
Staphylococcus aureus being the prevalent pathogen in children and adolescents, which is
replaced by P. aeruginosa in adults, however, the CF lung microbiome can be highly diverse.2,3
CFꢀrelated lung disease also is a risk factor for chronic pulmonary infection with nontuberculous
mycobacteria (NTM). NTM are detected with increasing prevalence in the CF population, with
Mycobacterium abscessus being the most pathogenic and antibioticꢀresistant species.4ꢀ6
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P. aeruginosa produces an arsenal of virulence factors to colonize the host and to escape immune
defenses.1,7 Many of the extracellular factors, such as the redoxꢀactive pigment pyocyanin,
rhamnolipid surfactants, siderophores, and secondary metabolites deriving from the 2ꢀalkylꢀ
4(1H)ꢀquinolone biosynthetic pathway, not only affect the host but also have a negative impact
on other bacteria. Especially the 2ꢀalkylꢀ4(1H)ꢀquinolones (AQs), which differ in the length and
degree of saturation of the alkyl side chain, the substituent (–H or –OH) at the C3 position, and
the oxidation state of the quinoline nitrogen, exert various and often multiple biological
activities.8ꢀ15
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2ꢀHeptylꢀ4ꢀhydroxyquinoline Nꢀoxide (HQNO), one of the most abundant AQ compounds
synthesized by P. aeruginosa, reaches concentrations ranging from few µM up to 35 µM in
planktonic cultures16ꢀ18 and has been found in sputum, plasma and urine from CF patients
infected with P. aeruginosa.19,20 HQNO binds to the quinone reduction (Qi) site of the respiratory
menaquinol/ubiquinol:cytochrome c oxidoreductase (the cytochrome bc1 complex) and inhibits
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respiratory electron transfer.21 It also inhibits quinol oxidase activity of cytochrome bo3 and bd,
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and type II NADHꢀquinone oxidoreductase (NDHꢀ2) involved in bacterial respiratory
chains.24 Autoꢀpoisoning of the P. aeruginosa cytochrome bc1 complex by HQNO induces a
burst of reactive oxygen species (ROS) and affects membrane potential and permeability, finally
causing autolysis.25 Together with siderophores, HQNO is primarily responsible for P.
aeruginosaꢀmediated killing of S. aureus.26 Longꢀterm exposure of S. aureus to HQNO moreover
selects for smallꢀcolony variants (SCVs), which are well known for persistence in chronic
infections.20
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The complexity of polymicrobial infections in the CF lung raises the question of whether
competitors have evolved strategies to inactivate the toxic secondary metabolites produced by P.
aeruginosa. The same question applies to polymicrobial communities in environmental settings,
such as soil habitats. From a soil sample, we previously isolated a Rhodococcus erythropolis
strain with the ability to degrade the P. aeruginosa quorum sensing molecules 2ꢀheptylꢀ4ꢀ
hydroxyquinoline (HHQ) and 2ꢀheptylꢀ3ꢀhydroxyꢀ4(1H)ꢀquinolone (Pseudomonas quinolone
signal, PQS) to anthranilic acid.27 In this study, we tested a set of environmental and pathogenic
bacteria for their ability to convert HQNO, identified the major metabolites formed from HQNO
biotransformation, and assessed their effect on bacterial respiration, quinol oxidase activity, and
induction of ROS formation.
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RESULTS AND DISCUSSION
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Three different ways for transforming HQNO. To investigate whether bacteria are capable of
degrading the P. aeruginosa toxin HQNO, we used wellꢀknown model organisms
(Escherichia coli, Corynebacterium glutamicum, Bacillus spp.), representatives of bacteria
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commonly found in soil and water (Rhodococcus erythropolis, Arthrobacter spp., and members
of environmental NTM), and selected opportunistic pathogens coꢀexisting with P. aeruginosa in
clinical settings, e.g., in the lung of CF patients (S. aureus, M. abscessus).
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FIGURE 1.
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Cell suspensions of B. subtilis and B. licheniformis slowly consumed HQNO, with concomitant
transient accumulation of two metabolites (Figure 1a). MS analysis of the first compound
revealed an m/z of 422.217 ([C22H31NO7 + H+]), suggesting a hexosylꢀHQNO (Table 1 and
Supporting Figure S1a). When the growth medium was additionally supplemented with glucose
or galactose, the first step of HQNO conversion was accelerated, and hexosylꢀHQNO
accumulated in significantly higher amounts (data not shown). The metabolite was extracted from
galactose supplemented cultures of B. subtilis and purified by preparative HPLC for NMR
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analysis. Based on Jꢀcoupling constants J(H1’’ꢀH2’’) = 8.3 Hz, J(H2’’ꢀH3’’) = 9.0 Hz,
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the sugar moiety was identified as glucose. The βꢀconfiguration was deduced from the J(H1’’ꢀ
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H2’’) = 8.3 Hz coupling constant and the strong NOE between the H1’’ and H5’’ proton
indicating a 1,3ꢀdiaxial relationship. The regiochemistry of the substitution was deduced from the
strong NOE between the H8 proton of the quinoline system and the H1’’ proton of the sugar unit.
From these data, the metabolite was identified as 1ꢀ(βꢀglucopyranosydyl)ꢀ2ꢀheptylꢀ4ꢀ
hydroxyquinoline (GꢀHQNO; Table 1 and Supporting Figure S4).
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The m/z of 522.233 ([C26H35NO10 + H+]), observed for the second metabolite formed by the
Bacillus strains tested, is consistent with an acylated – possibly succinylated – derivative of
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glucosylꢀHQNO (SGꢀHQNO) (Supporting Figure S1b). Treatment of this biotransformation
intermediate with aqueous ammonia resulted in a deacylated but still glucosylated product with
HPLC elution behavior and molecular mass identical to that of the first intermediate (m/z of
422.217), indicating acylation of the glucosyl residue instead of a substituent of the aromatic ring.
MS/MS fragmentation of GꢀHQNO and SGꢀHQNO led to the same m/z of 260.166 with almost
100 % abundance (Supporting Figures S1c and S1d). When the metabolites were treated with
zinc/acetic acid to reduce any residual NꢀO bond, the same product was formed from both
compounds, which based on its HPLC retention time and UV spectrum was identified as HHQ.
This supports the hypothesis that HQNO modification by the two Bacillus spp. takes place at the
Nꢀoxide and involves Oꢀglucosylation and further acylation of the glucosyl residue. Other
metabolites besides Gꢀ and SGꢀHQNO were not detected in extracts of Bacillus cultures. Either,
further degradation proceeds without accumulation of intermediates, or intermediates formed
escape extraction and/or UV detection.
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Glycosylation and succinylation of natural products seem to be common strategies of bacilli. B.
subtilis natto as well as B. licheniformis have been reported to produce succinylated isoflavone
glycosides from soybean isoflavones.28 Furthermore, the UDPꢀglycosyltransferase YjiC (from B.
licheniformis) has been shown to glycosylate plant flavonoids, antibiotics such as mupirocin, and
macrolides such as epothilone A.29ꢀ32
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The three mycobacteria tested, M. abscessus, M. fortuitum and M. smegmatis, used another
strategy to modify HQNO. All three species were found to form 2ꢀheptylꢀ1ꢀmethoxyꢀ
4ꢀoxoquinoline (HMOQ) as a major transient metabolite (Figure 1b and Table 1). The identity of
HMOQ was established by comparison of its HPLC retention time and MS/MS fragmentation
pattern with an authentic standard obtained by chemical synthesis (Supporting Figure S2). HPLC
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analysis of samples taken after HMOQ had reached the peak concentration suggested formation
of a multitude of metabolites at low concentrations which show UV spectra resembling that of
HMOQ. Therefore, we tentatively assume that the compounds are structurally closely related,
with modifications possibly introduced mainly to the alkyl chain.
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Already decades ago, mycobacteria were observed to modify phenolic and catecholic compounds
by methylation. M. fortuitum CGꢀ2, an isolate from soil, is capable of Oꢀmethylating a plethora of
chlorinated phenols, guaiacols, syringols, and hydroquinones, with a preference for orthoꢀ
chlorinated substrates.33 M. vanbaalenii PYRꢀ1 neutralizes the toxic effect of catechols by Oꢀ
methylation34, generating less reactive intermediates which in contrast to catechol do not promote
formation of hydroxyl radicals by the Fenton reaction.35
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Cell suspensions of Arthrobacter sp. Rue61a, Paenarthrobacter aurescens TC1, R. erythropolis
BG43, R. erythropolis PR4, S. aureus Newman, and S. aureus USAꢀ300 were also capable of
converting HQNO (Figure 1c). MS analysis of the transiently accumulating metabolite revealed
an m/z of 276.159 (for [C16H21NO3 + H+]), consistent with a hydroxylated form of HQNO (Table
1 and Supporting Figures S3a,b). The MS/MS fragmentation pattern as well as the 1HꢀNMR data
suggest the hydroxyl group to be located at the C3ꢀposition of the heteroaromatic ring
(Supporting Figures S3c, S6). This is supported by the observation that zinc/acetic acid reduction
of the Nꢀoxide group of this compound yielded PQS, which was identified by HPLC and its
characteristic UV spectrum. Hence, we propose that these Gramꢀpositive bacteria hydroxylate
HQNO to 2ꢀheptylꢀ3,4ꢀdihydroxyquinolineꢀNꢀoxide (“PQSꢀNꢀoxide”, PQSꢀNO). The observed
gradual decrease in PQSꢀNO levels in the S. aureus cultures (Figure 1c) may in parts be due to its
spontaneous decomposition to PQS (halfꢀlife in sterile LB at 30 °C is 2.5 hours).
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Formation of Gꢀ and SGꢀHQNO, HMOQ, and PQSꢀNO was organismꢀspecific under the
conditions of the biotransformation experiments. We did not detect GꢀHQNO or SGꢀHQNO in
cultures other than Bacillus spp., or HMOQ in cultures other than Mycobacterium spp., and we
also could not detect PQSꢀNO in Bacillus or Mycobacterium cultures.
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For C. glutamicum, E. coli BL21, and P. putida KT2440, we could not observe any HQNO
conversion under the conditions tested. The strains were still able to grow in presence of 10 µM
HQNO.
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Inhibitory effects of HQNO and its metabolites on cellular respiration and menaquinol
oxidase activity. The antimicrobial activity of HQNO has been reported to be mainly due to
inhibition of respiratory electron transfer. To find out whether the chemical modifications of
HQNO performed by the bacteria tested correlate with detoxification, we compared the
respiration activity of cell suspensions, as well as menaquinol oxidase activity of membrane
preparations, in response to HQNO and to the major metabolite formed by the respective strain.
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FIGURE 2.
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Respiration of B. subtilis 168 cells was highly sensitive to HQNO which showed an EC50 value of
0.4 µM. Correspondingly, growth of freshly inoculated cultures was inhibited by 10 µM HQNO
(Supporting Figure S7a). The menaquinol oxidase activity of membrane fractions prepared from
B. subtilis was drastically inhibited by HQNO, with the EC50 value of 1.6 µM being slightly
higher than that observed for cellular respiration (Figure 2a,d). In contrast, the EC50 of GꢀHQNO
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on cellular respiration was more than two orders of magnitude higher, with a value of 84.4 µM
being far above the range of physiological concentrations (Figure 2a). In line with the in vivo
data, the menaquinol oxidase activity of membrane fractions was only weakly inhibited by Gꢀ
HQNO, which under the conditions of the assay had an EC50 value of 128.4 µM (Figure 2d).
Conjugation of HQNO with the bulky and polar glucose residue may efficiently prevent binding
to the quinone/quinol binding sites of menaquinol oxidases. Additional acylation – possibly a
succinylation – of GꢀHQNO by Bacillus could introduce a negative charge, which should support
release of the compound from the membrane.
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Cells of M. abscessus DSM 44196 as well as M. smegmatis mc2 155 (data not shown) showed
only little response to both HQNO and the metabolite HMOQ. At 100 µM of HQNO or HMOQ,
the residual relative respiratory activities of M. abscessus were still 81% and 94%, respectively
(Figure 2b). On the one hand, the complex cell wall of mycobacteria may protect them against
toxic compounds. On the other hand, in order to maintain ATP levels the mycobacteria might
reroute electron transfer by rapidly switching between terminal oxidases, thereby increasing O2
consumption, as shown recently for M. tuberculosis.36 In line with the insensitivity of respiratory
activity of M. abscessus cells to HQNO, the growth curve of M. abscessus was unaffected by 10
µM HQNO (Supporting Figure S7b). However, in vitro experiments using membrane fractions of
M. abscessus revealed significant inhibition of menaquinol oxidase activity by HQNO, with an
EC50 value of 4 µM (Figure 2e), but much less sensitivity toward HMOQ. The estimated EC50
value for HMOQ of 189 µM suggests that methylation of the Nꢀoxide is a highly efficient way to
counteract poisoning of menaquinol oxidases.
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S. aureus is well known to be very sensitive to HQNO.20,26,37,38 This is reflected by inhibition of
growth in presence of 10 µM HQNO, however, the cells resumed growth after having decreased
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the HQNO levels by biotransformation (Supporting Figure S7c). For S. aureus, the EC50 value of
HQNO on cellular respiration was determined as 3.2 µM. The hydroxylated metabolite, PQSꢀNO,
showed only a slightly reduced inhibitory effect with an EC50 value of 12.3 µM (Figure 2c). The
results of the menaquinol oxidase assays were similar, with HQNO acting as highly efficient
inhibitor and PQSꢀNO also exerting a substantial inhibitory effect (Figure 2f), with estimated
EC50 values for HQNO and PQSꢀNO of 0.2 µM and 1.8 µM, respectively.
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Whereas HQNO hydroxylation to PQSꢀNO led to only moderate detoxification for S. aureus, the
metabolites formed by M. abscessus and B. subtilis had EC50 values which were increased by
more than one order of magnitude compared to that of HQNO. Therefore, modifying the Nꢀoxide
functional group appears to be an efficient strategy to detoxify HQNO as respiratory inhibitor.
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Cellular ROS levels in presence of HQNO and its metabolites. Inhibition of quinone reduction
at the Qi site of the cytochrome bc1 complex, using, e.g., antimycin A, has been reported to
significantly increase ROS production at the Q0 site.39 To test for cellular ROS, we measured
fluorescence intensities of 2′,7′ꢀdichlorodihydrofluorescein (DCF), a fluorescent probe for
cellular oxidant stress.40
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FIGURE 3.
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When cell suspensions were incubated with HQNO, a 2ꢀfold and even 4ꢀfold increase of DCF
fluorescence was observed for B. subtilis 168 and M. abscessus, respectively, indicating elevated
oxidative stress (Figure 3). Even though the presence of 100 µM GꢀHQNO or HMOQ, which still
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showed some inhibition in the quinol oxidase assay, resulted in slightly higher DCF fluorescence
increases compared to the DMSO controls, the ROSꢀinducing effect of the metabolites was
significantly lower than that of HQNO.
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Mycobacterial growth under aerobic conditions mainly relies on the cytochrome bc1ꢀaa3
branch.41 Thus, HQNO inhibition of cytochrome bc1, which was reported to mediate ROS
production when inhibited at the Qi site39, may account for the observed strong induction of ROS
formation in M. abscessus. In contrast, B. subtilis uses a branched respiratory chain with
cytochrome aa3 and bd as terminal quinol oxidases, with cytochrome aa3 as the major terminal
oxidase during exponential growth.42 Homologs of these quinol oxidases from other organisms as
well as cytochrome aa3 (Qox) of B. subtilis have been shown to be sensitive towards HQNO, but
it has not been reported whether inhibition leads to ROS formation.22,43,44 However, since
electrons from the menaquinol pool of B. subtilis can also enter the cytochrome oxidase branch
via the cytochrome bc complex42, the observed moderate increase in ROS production in HQNOꢀ
treated cells of B. subtilis might be due to inhibition of cytochrome bc.
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Surprisingly, S. aureus Newman did not show an altered DCF fluorescence increase in the
presence of HQNO compared to the control (Figure 3). This may be due to the fact that S. aureus
in contrast to the other model organisms tested completely lacks the bc1 complex, and only
possesses aa3ꢀ and bdꢀtype terminal oxidases.45,46 Remarkably, PQSꢀNO even reduced the DCF
fluorescence increase to almost 0.4ꢀfold of the control, suggesting antioxidant capacity of this
metabolite.
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Conclusions. In this study, we demonstrate that bacteria have the capability to transform the
respiratory inhibitor HQNO, a major toxin produced by the opportunistic pathogen P.
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aeruginosa. Among the bacteria tested, we identified three different ways to covalently modify
and detoxify the respiratory inhibitor HQNO, illustrated in Figure 4. Bacillus spp. initially
transformed HQNO to the glucosylated derivative GꢀHQNO, which was significantly less
inhibitory on respiration and menaquinol oxidase activity than HQNO, and induced less oxidative
stress. The three Mycobacterium spp. tested catalyzed an initial Oꢀmethylation of the Nꢀoxide,
which also resulted in a substantial quenching of the inhibitory activity on quinol oxidase.
Oxidative stress in mycobacteria was drastically increased by HQNO, whereas HMOQ showed
an almost negligible effect. S. aureus as well as Arthrobacter and Rhodococcus spp. were found
to introduce a hydroxyl substituent at C3 of HQNO, instead of modifying the Nꢀoxide group. The
resulting PQSꢀNO exhibited only slightly less respiratory inhibition than HQNO, but it quenched
oxidative stress in S. aureus. The molecular mechanisms underlying the observed physiological
effects of HQNO modification remain to be elucidated. However, our discovery of HQNO
biotransformation and detoxification adds another layer of complexity to the multifaceted
interactions within microbial communities. Microorganisms capable of detoxifying HQNO
clearly should gain a competitive advantage in mixedꢀspecies microbial communities with P.
aeruginosa. Thus, future studies should evaluate the significance of HQNO modification and
degradation in the context of coꢀculture and coꢀinfection. HQNO does not function as a quorum
sensing signal molecule,47 and the HQNO derivatives seem not to affect biofilm formation or to
interfere with P. aeruginosa quorum sensing, as suggested by absence of effects on PQS and
pyocyanin production (Supporting Figure S8). However, autoꢀpoisoning of P. aeruginosa might
be decreased in coꢀcultures,25 and the HQNO metabolites might exert other biological activities.
Thus, another key question is whether the metabolites influence interspecies behavior by
affecting the phenotypes not only of P. aeruginosa, but also of other bacteria, fungi, and host
cells. From a more pharmacological point of view, the selective chemical modification of the
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heterocyclic Nꢀoxide may also open up perspectives for the development of new bioactive
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compounds.
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METHODS
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Chemicals and chemical synthesis. Chemicals were purchased from Aldrich, Acros, TCI, or
Merck, and used as received. HHQ and HQNO were prepared according to literature
procedures.48,49 A detailed description of the synthesis of 2ꢀheptylꢀ1ꢀmethoxyꢀ4ꢀoxoquinoline
(HMOQ) is provided in the Supporting Information.
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N-oxide reduction. HQNO metabolites were dissolved in 100% acetic acid and a few grains of
zinc powder were added to reduce hydroxyquinoline Nꢀoxides to the corresponding
hydroxyquinolines. After incubation for 15 min at room temperature the suspension was
centrifuged to pellet undissolved particles. The supernatant was dried to completion and the
residual sample was redissolved in methanol for HPLC analysis.
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Analytical methods. Detailed information about analytical and preparative HPLC, MS analyses
and NMR analyses is given in the Supporting Information.
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Bacterial strains and growth conditions. R. erythropolis BG43 (DSM 46869), R. erythropolis
PR4 (NBRC 100887), Arthrobacter sp. Rue 61a (DSM 24942), Paenarthrobacter (formerly:
Arthrobacter) aurescens TC1 (ATCC BAAꢀ1386), Corynebacterium glutamicum (DSM 20300),
Bacillus subtilis 168 (DSM 402), Bacillus licheniformis (DSM 13), Pseudomonas putida KT2440
(DSM 6125), Staphylococcus aureus Newman (NCTC 8178), S. aureus USA30050 and
Escherichia coli BL21 were grown in LB (lysogeny broth; 5 g/L yeast extract, 10 g/L peptone,
tryptic digested, 10 g/L NaCl, pH 7.3) at 37°C (E. coli, B. subtilis, S. aureus) or 30°C (all other
strains). Mycobacterium abscessus (DSM 44196), M. fortuitum (DSM 46621) and M. smegmatis
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strain mc2 155 (ATCC 700084) were cultivated at 37 °C in DSM 219 medium (per L: yeast
extract 2.0 g; proteose peptone 2.0 g, casein peptone, tryptic digested, 2.0 g, Na2HPO4 × 12 H2O
2.5 g, KH2PO4 1.0 g, Naꢀcitrate 1.5 g, MgSO4 × 7 H2O 0.6 g, glycerol 50.0 ml, Tween 0.5 g, pH
7.0).
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Biotransformation of HQNO and organic extraction. For biotransformation experiments, cells
were pelleted by centrifugation (7,000 × g, 5 min) and resuspended in fresh medium adjusting an
optical density (OD600nm) of 2. HQNO (Enzo Life Sciences, dissolved in methanol) was added to
each culture to a final concentration of 10 µM, and cultures were incubated under vigorous
shaking at 37°C. At appropriate time intervals 1 mL samples were withdrawn and extracted three
times with 0.5 mL ethyl acetate (acidified with 1 mL/L acetic acid) as described previously.27 The
organic phase was dried to completion and 200 µL methanol was added to redissolve the sample
for HPLC analysis. For the preparation of larger amounts of the metabolites by biotransformation
and preparative HPLC, cultures were fed with the synthesized HQNO.
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Cellular respiration and quinol oxidase assay. To determine the effect of HQNO and HQNO
metabolites on cellular respiration, exponentially growing cells were adjusted to an OD600 of 0.5.
1 mL cell suspension was mixed with 5 µL of the compound to be tested (dissolved in DMSO)
and shaken vigorously for 10 min at room temperature prior to monitoring cellular O2
consumption. The protocol used for preparation of cell membranes is provided in the Supporting
Information. Inhibition of quinol oxidase activity of membrane fractions was measured using a
coupled enzyme assay. The reaction was carried out in airꢀsaturated 50 mM potassium phosphate
buffer, pH 6.5, at room temperature. The quinol substrate 2,3ꢀdimethylꢀ1,4ꢀnaphthoquinol
(DMNH2) was formed from 2,3ꢀdimethylꢀ1,4ꢀnaphtoquinone (DMN, purchased from
RareChemicals, Kiel, Germany) and NADH by DTꢀdiaphorase (Sigma). The reaction mixture
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(final volume of 500 µL) contained 250 µM DMN, 20 µg/mL DTꢀdiaphorase, varying
concentrations of HQNO or its metabolites, and 10ꢀ40 µL of the membrane fraction. The assay
was started by the addition of 400 µM NADH. Autoxidation of reduced DMN was measured
separately in the absence of the membrane fraction. For measuring cellular respiration as well as
for the quinol oxidase assay, oxygen consumption was monitored with a Clarkꢀtype oxygen
electrode equipped with a magnetically stirred chamber. Reference samples without inhibitors
contained the same amount of DMSO. EC50 values were estimated from fitting the data to a dose
response curve (Hill equation).
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Cellular ROS detection. The cellular production of ROS in the presence of HQNO or its
metabolites was measured using the cell permeable dye 2’,7’ꢀdichlorodihydrofluorescein
diacetate (H2DCFꢀDA, purchased from Sigma).40 B. subtilis, M. abscessus and S. aureus were
grown as described above. Cells were pelleted by centrifugation, resuspended in fresh medium
adjusting an OD600 of 1, supplemented with 50 µM of H2DCFꢀDA, and shaken vigorously at
37 °C for 30 min. Prior to the addition of 100 µM of HQNO or its metabolite, cells were washed
once with phosphate buffered saline (PBS, pH 7.5) to remove residual extracellular dye, and
resuspended in PBS. Suspensions of mycobacteria were additionally supplemented with 0.05%
(w/v) Tween80. Aliquots of the cell suspensions were transferred to a 96ꢀwell microtiter plate
(200 µL per well) and the fluorescence increase (Ex/Em of 490/510ꢀ570 nm) over 30 min was
measured using the GloMax®ꢀMulti+ microtiter plate reader (Promega). Results represent the
mean of three independent experiments, each measured in three technical replicates.
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AUTHOR INFORMATION
Corresponding Author
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*Eꢀmail: fetzner@uniꢀmuenster.de. Tel. 049 (251) 8339824. Fax +49 (251) 8338388.
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Author contributions
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S.F. conceived the study. S.F. and S.T. coordinated the study. F.S.B. performed
biotransformations and biofilm and pyocyanin assays. S.T. performed all other physiological and
biochemical experiments. U.H. performed all chemical syntheses, NMR analyses, and together
with M.C.L. performed MS analyses. All authors contributed to interpretation of data. S.F. and
S.T. wrote the manuscript. S.T. and U.H. prepared the figures. All authors contributed to and
approved the final version of the manuscript.
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ACKNOWLEDGEMENTS
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Research reported in this publication was supported by the Deutsche Forschungsgemeinschaft
(grant FE 383/25ꢀ1 to S.F., and Heisenberg grant HE 6020/3ꢀ1 to U.H.). F.B. thanks the
Studienstiftung des Deutschen Volkes for a PhD scholarship and support. We thank T. Martins
and P. Fricke for help with biotransformation experiments and respiratory measurements, B. Kahl
for providing S. aureus strains, and B. Philipp for access to the electrospray ionization ion trap
mass spectrometer (DFG grant INST 211/646ꢀ1) which was used for initial MS analyses.
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ASSOCIATED CONTENT
Supporting Information
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Description of chemical syntheses, analytical procedures, membrane preparation protocols, and
biofilm and pyocyanin assays; Figures S1ꢀS6 providing supporting MS and NMR data; Figure S7
providing supporting information on the effect of HQNO on bacterial growth; Figure S8
providing supporting data on the effect of HQNO metabolites on P. aeruginosa (PDF file).
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FIGURE LEGENDS
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Figure 1. Biotransformation of HQNO by cell suspensions (initial OD600 nm of 2.0) of B.
subtilis 168, M. abscessus DSM 44196, and S. aureus Newman. Conversion of HQNO (black
squares) led to transient formation of: (a) GꢀHQNO (blue filled squares) and SGꢀHQNO (blue
open squares) by B. subtilis 168; (b) HMOQ (red squares) by M. abscessus DSM 44196; (c)
PQSꢀNO (green squares) by S. aureus Newman. Symbols and error bars represent the means and
standard errors of three biological replicates. Culture media or spent media did not mediate
conversion of HQNO.
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Figure 2. Inhibitory effect of HQNO and key metabolites on cellular respiration and
menaquinol oxidase. The effect of HQNO (black), GꢀHQNO (blue), HMOQ (red), and PQSꢀNO
(green) on oxygen consumption by cell suspensions is shown in (a, b, c), while effects on the
menaquinol oxidase activity of membrane fractions are shown in (d, e, f). Panels (a, d): B.
subtilis 168; (b, e): M. abscessus DSM 44196; (c, f): S. aureus Newman. Data were normalized to
cellular/enzymatic O2 consumption rates (cOCR/eOCR) of the respective reference measurement
(without inhibitor) which was set to 100 %. Symbols and error bars represent the means and
standard errors of three biological replicates. EC50 values (see text) were estimated by fitting the
data to dose response curves (solid lines).
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Figure 3. Relative increase of the fluorescence intensity of DCF, a probe for cellular ROS,
in the presence of HQNO and key metabolites. H2DCFꢀDA treated cells of B. subtilis 168, M.
abscessus DSM 44196 and S. aureus Newman were incubated for 30 min with 100 µM of HQNO
(dark bars), and 100 µM of GꢀHQNO (blue bar), HMOQ (red bar), and PQSꢀNO (green bar),
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respectively. Each bar, which is normalized to the fluorescence increase of the respective control
containing only DMSO, and error bar represent the mean ± SE of three independent experiments,
each measured in three technical replicates. Oneꢀway ANOVA was used for statistical analysis, *
P<0.02, ** P<0.01 and ***P<0.0001.
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Figure 4. Overview of the effects of HQNO and its metabolites on the respective bacterial
species. Inhibition of quinol oxidase activity, as indicated by EC50 values, is reduced by HQNO
conversion. Metabolites in comparison to HQNO induced less cellular oxidant stress, as indicated
by fold changes in cellular ROS production (ROS levels in DMSO controls set to 1).
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TABLE
Table 1. Key metabolites of HQNO conversion.
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Organisms
HQNO Metabolites
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Fragment
Ions b
RT a
m/z [M + H+] b
Structure c
B. subtilis d
13.9 min
422.217
260.16391
B. licheniformis
M. abscessus d
M. fortuitum
M. smegmatis
159.06853
186.09225
172.076511
25.2 min
23.5 min
274.1801
276.159
S. aureus
175.06265
178.04974
188.07055
R. erythropolis
A. sp. Rue61a d
P. aurescens
560
a HPLC retention time.
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b MS and MS/MS data of the key metabolites. See also Supporting Information, Figure S1ꢀS3.
c Structure proposal based on MS and NMR data. See also Supporting Information, Figure S4ꢀS6.
d denotes the organism cultivated for preparative isolation of the respective metabolite.
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