MECHANISMS OF RESISTANCE

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A Novel Mechanism of Inactivating Antibacterial Nitro

Compounds in the Human Pathogen Staphylococcus aureus by
Overexpression of a NADH-Dependent Flavin Nitroreductase
Ebaa M. El-Hossary,a,b,d Konrad U. Förstner,b Patrice François,c Damien Baud,c Karin Streker,b Jacques Schrenzel,c
Knut Ohlsen,b Ulrike Holzgrabea

a University of Würzburg, Institute of Pharmacy and Food Chemistry, Würzburg, Germany

b University of Würzburg, Institute for Molecular Infection Biology, Würzburg, Germany
c
University of Geneva Hospitals, Genomic Research Laboratory, Service of Infectious Diseases, Geneva,
Switzerland
d National Centre for Radiation Research & Technology, Egyptian Atomic Energy Authority, Cairo, Egypt

ABSTRACT Recently, the nitro-substituted bisquaternary bisnaphthalimides were re-

ported to have substantial anti-infective activity against Gram-positive bacteria, in-
cluding methicillin-resistant Staphylococcus aureus (MRSA). Here, we selected re-
sistant S. aureus clones by cultivation in increasing concentrations of the most active
compound, MT02. Interestingly, MT02-resistant variants induced a diffusible red color
of the broth. Chromatographic and spectroscopic investigations revealed a stepwise
reduction of the bisquaternary bisnaphthalimides’ nitro groups to amino groups. The
corresponding derivatives were completely inactive against staphylococci. RNA se-
quencing experiments revealed a strong overexpression of a novel oxidoreductase in
MT02-resistant strains. Deletion mutants of this enzyme did not produce the red
color and were not able to develop resistance against bisquaternary bisnaphthalim-
ides. Biochemical reactions confirmed an NADH-dependent deactivation of the nitro-
substituted compounds. Thus, this is the first report of a nitroreductase-based antibi-

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otic resistance mechanism in the human pathogen S. aureus.

KEYWORDS Staphylococcus aureus, antibacterial nitro compounds, bacterial

resistance, bacterial nitroreductases

I nfections due to antibiotic-resistant staphylococci are still a major threat for patients
in hospitals. Although the number of staphylococcal infections has slightly decreased
during recent years, multiresistant Staphylococcus aureus and S. epidermidis account for
Received 25 July 2017 Returned for
modification 11 August 2017 Accepted 28
more than 20% of all health care-associated infections, affecting more than 250,000 October 2017
Accepted manuscript posted online 13
patients in the United States and Europe each year (1). Moreover, methicillin-resistant
November 2017
S. aureus in the community (CA-MRSA) and in livestock (LA-MRSA) are on the rise, Citation El-Hossary EM, Förstner KU, François P,
creating a reservoir for the evolution of antibiotic-resistant clones (2). As a conse- Baud D, Streker K, Schrenzel J, Ohlsen K,
quence, the need for novel compounds with an alternative mode of action compared Holzgrabe U. 2018. A novel mechanism of
inactivating antibacterial nitro compounds in
to current antibiotics is of paramount importance. the human pathogen Staphylococcus aureus by
We have recently investigated the antibacterial activity of a novel nitro-active overexpression of a NADH-dependent flavin
compound called MT02 (3). MT02 was identified in an evaluation of bisquaternary nitroreductase. Antimicrob Agents Chemother
62:e01510-17. https://doi.org/10.1128/AAC
bisnaphthalimides exerting high antimicrobial activity against S. aureus and other .01510-17.
Gram-positive bacteria, such as S. epidermidis and Listeria monocytogenes, and moder- Copyright © 2018 American Society for
ate activity against streptococci. Mode-of-action studies-based global expression anal- Microbiology. All Rights Reserved.
ysis using DNA microarrays and radioactive labeling experiments generated evidence Address correspondence to Knut Ohlsen,
knut.ohlsen@uni-wuerzburg.de, or
that MT02 is a DNA-binding compound leading to inhibition of DNA replication. The
Ulrike Holzgrabe,
substance did not show cytotoxic activity in cell culture systems. The two permanent ulrike.holzgrabe@uni-wuerzburg.de.
positive charges of MT02 probably prevent penetration of the compound into eukary-

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El-Hossary et al. Antimicrobial Agents and Chemotherapy

otic cells. Moreover, MT02 is not active against Gram-negative bacteria owing to their
outer membrane, which acts as a penetration barrier.
Because of these properties, MT02 appears to be a promising candidate for the
development of a new antibacterial drug, and we were interested in the potential of S.
aureus to develop resistance against this compound. It is known that frequent exposure
of bacterial pathogens to subinhibitory (nonlethal) doses of antibiotics plays an impor-
tant role in the emergence of drug-resistant bacteria (4). Here, we report the selection
of MT02-resistant clones in S. aureus by subcultivation in stepwise increasing concen-
trations of MT02. By this process, resistant clones could be selected which, surprisingly,
produced a red color when adapted to higher concentrations of the compound. To
clarify the mechanism of resistance, we have identified the chemical nature of the red
color, which is an intermediate in the reduction of the nitro groups of MT02 into amino
groups. Furthermore, we found that this chemical process was initiated by overpro-
duction of an unknown oxidoreductase of S. aureus. Overall, we identified a novel
resistance mechanism against antibacterial compounds with active nitro groups.

RESULTS AND DISCUSSION

Selection of MT02-resistant S. aureus strains. The bisquaternary bisnaphthalimide
MT02 was previously shown to be active against several antibiotic-sensitive and
-resistant strains of S. aureus and S. epidermidis (3). To evaluate potential resistance
development, S. aureus strain MA12 was cultivated in the presence of stepwise increas-
ing concentrations of MT02, starting from subinhibitory concentrations up to 125 ␮M
(119.5 ␮g/ml). Surprisingly, when S. aureus MA12 was cultivated with 10 ␮M (9.5 ␮g/ml)
MT02, a red color appeared during the selection process (see Fig. S1 in the supple-
mental material). The red color intensified upon increasing concentrations of MT02.
Furthermore, the red color was produced under both aerobic and anaerobic conditions
and also by other genotypically different strains, such as 8325 (RN1), HG001, USA300,
LAC JE2, and Xen29, that produced the red color when selected for MT02 resistance.
Identification of the structures of colored compounds. To elucidate the structure
of the red compound, extracts were prepared from large-scale cultures of MT02-
resistant S. aureus MA12 cultivated with 125 ␮M (119.5 ␮g/ml) MT02 for 24 h. Harvest-
ing the bacterial cells resulted in a clear red supernatant, and a violet cell pellet became

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visible. The colored compounds were extracted with acetonitrile from the red super-
natant (extract 1) and from the violet cell pellet (extract 2), respectively. Interestingly,
prolongation of the incubation of the MT02-resistant strain with MT02 for 6 days
resulted in disappearance of the red color due to the formation of compounds 3 and
4 from compound 2. Harvesting the bacterial cells after 6 days of incubation afforded
a clear yellow supernatant and a dark brown cell pellet. Further extracts were obtained
from the yellow supernatant (extract 3) and from the brown cell pellet (extract 4).
Fractionation of the obtained extracts 1 to 4 was performed using semipreparative
reverse-phase high-performance liquid chromatography (RP-HPLC) with gradient elu-
tion. Compound 1 (colorless fraction) and compound 2 (red fraction) were isolated
from extract 1 or 2, while compounds 3 and 4 (yellow fractions) were purified from
extract 3 or 4 (Fig. S2).
Spectroscopic analysis of the purified compounds showed that compound 1 could
be assigned to the starting compound MT02, while compounds 2, 3, and 4 are MT02
derivatives with different substitutions on the 1,8-naphthalimide moiety. The mass
value of m/z 795.5 [M]⫹ of compound 2 indicated a hydride ion transfer to one of the
two nitro groups of MT02. Thus, it can be regarded as an intermediate on the reduction
pathway to nitroso compounds. Similar observations were reported by Christofferson
and Wilkie for a dinitrobenzamide compound (5). Due to its instability, compound 2
loses its red color when exposed to natural light at room temperature. Nuclear
magnetic resonance (NMR) spectra of the discolored compound 2 revealed the back
reaction to MT02.
1H-NMR data of the yellow compound 3 showed 10 signals, i.e., two sets of aromatic

protons (5 signals each) between ␦ of 7.30 and 9.48 ppm, suggesting an asymmetric

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FIG 1 Stepwise reduction of antibacterial agent MT02. The three MT02 derivatives, compounds 2 to 4, with

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different substitutions on the 1,8-naphthalimide moiety, were obtained by incubation of MT02-resistant S.
aureus MA12 strain with 125 ␮M (119.55 ␮g/ml) MT02 for 3 days. Compounds 2 to 4 were isolated from the
culture after 4, 24, and 72 h of incubation, respectively. The MIC values refer to strain S. aureus MA12. The
corresponding 1H-NMR spectra are indicated. The blue clamps indicate signals related to the protons of
the nitro-substituted 1,8-naphthalimide moiety, the red clamps signals related to the protons of the
amino-substituted 1,8-naphthalimide moiety.

structure. The signals between ␦ of 8 and 9.6 ppm are similar in multiplicity and
position to the nitro-substituted naphthalimide, whereas the signals between ␦ of 7.3
and 8.1 ppm can be assigned to an amino-substituted naphthalimide due to the huge
up-field shift. The asymmetric structure is supported by mass spectra showing m/z
382.3 [M]2⫹ and additional NH2 stretching vibrations at 3,356 and 3,229 cm⫺1 in the
infrared (IR) spectrum. Thus, a reduction of one nitro group must have taken place upon
incubation.
1H-NMR data of yellow compound 4 showed five signals for 10 aromatic protons at

␦ of 7.30 to 8.08 ppm, which are similar in multiplicity and position to the amino-
substituted naphthalimide moiety described for compound 3. Electrospray ionization-
mass spectrometry (ESI-MS) data of compound 4, showing m/z 367.1 [M]2⫹, confirmed
the symmetrical bisaminonaphthalimido structure of compound 4. The spectral data
are in accordance with corresponding data reported by Muth et al. (6).
Taken together, the aromatic nitro groups of MT02 (compound 1) were stepwise
reduced to amino groups upon incubation (Fig. 1). Since MT02 is stable in buffered
media and during contact with MT02-sensitive S. aureus, the reduction must have been
induced by an enzyme present in the resistant S. aureus strains.

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TABLE 1 In vitro antibacterial activity of compounds 1, 3, and 4

MIC or MBC (␮M) for compound:
1 (MT02)
Tested strains MIC MBC 3 (MIC) 4 (MIC)
Gram positive
Staphylococcus aureus 8325 2.5 5 ⬎40 ⬎40
S. aureus HG001 0.625 1.25 ⬎40 ⬎40
S. aureus MA12 1.25 2.5 ⬎40 ⬎40
S. aureus USA300 JE2 0.625 1.25 ⬎40 ⬎40
S. aureus Xen29 1.25 2.5 ⬎40 ⬎40
S. aureus NE118 0.625 1.25 NTa NT
S. aureus NE441 0.625 1.25 NT NT
S. aureus NE662 0.625 1.25 NT NT
S. aureus NE909 0.625 1.25 NT NT
Staphylococcus epidermidis RP62A 5 20 ⬎40 ⬎40
S. epidermidis 047 20 NT NT NT
S. epidermidis 195 10 20 NT NT
Enterococcus faecalis JH2-2 ⬎40 NT ⬎40 ⬎40
Enterococcus faecium 6413 ⬎40 NT ⬎40 ⬎40

Gram negative
Escherichia coli 536 ⬎40 NT ⬎40 ⬎40
Pseudomonas aeruginosa ⬎40 NT ⬎40 ⬎40
Yersinia pestis KUMA ⬎40 NT ⬎40 ⬎40
Yersinia pseudotuberculosis 252 01A ⬎40 NT ⬎40 ⬎40
aNT, not tested.

Antibacterial activity and cytotoxicity of the reduced compounds. We next were

interested if reduced compounds 3 and 4 possessed in vitro antibacterial activity
against selected clinically important Gram-positive and Gram-negative pathogenic
bacteria. MIC testing revealed that purified compounds 3 and 4 exhibited MIC values
against staphylococci higher than 40 ␮M (37.1 and 35.8 ␮g/ml, respectively), which are
considered to be inactive. For the other tested strains, all of the tested compounds
also were inactive (Table 1). The cytotoxicity of compounds 1 (MT02), 3, and 4 were

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measured in the macrophage cell line J774.1 using the alamarBlue-based cytotoxicity
test. None of the compounds showed any cytotoxic activity (50% inhibitory concen-
trations higher than 100 ␮M [95.6 ␮g/ml] after 48 h of incubation). We were not able
to test antibacterial activity and cytotoxicity of compound 2 due to its instability.
Expression of staphylococcal nitroreductases in the MT02-resistant strain. The
chemical analysis of the colored compounds 2 to 4, produced by MT02-resistant S.
aureus strains, suggested that bacterial nitroreductases confer the transformation of the
two nitro groups of MT02 into amino groups. So far, only two enzymes with confirmed
nitroreductase activity were described in S. aureus (7, 8). Previously, we could show that
NfrA (SAUSA300_0381) is a flavin mononucleotide-dependent NADPH oxidase involved
in oxidative stress response (8). The second putative nitroreductase, SAUSA300_0788, is
a bifunctional enzyme, and its inactivation increases the sensitivity of S. aureus to
S-nitrosoglutathione (GSNO) and augments resistance to nitrofurans (7). The expression of
the two genes, ntrA (SAUSA300_0788) and nfrA (SAUSA300_0381), was analyzed with
real-time reverse transcription-PCR (RT-PCR). In addition, we further analyzed the expression
of two other putative nitroreductase genes, SAUSA300_1986 and SAUSA300_2462, that we
identified by a domain search. All four genes were expressed in the S. aureus MT02-sensitive
as well as in the MT02-resistant strain at an optical density at 600 nm (OD600) of 1.0.
However, none of these four nitroreductases was found to be overexpressed in the
MT02-resistant S. aureus strains compared to their MT02-sensitive counterparts (data not
shown).
Furthermore, the inactivation of nfrA, ntrA, SAUSA300_1986, or SAUSA300_2462 in
MT02-sensitive S. aureus JE2 does not affect its ability to develop resistance against
MT02 or the production of the red color compound. These results indicate that these

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TABLE 2 Upregulated genes (top 10 and selected genes) in the MT02-resistant mutant
compared to the intermediately resistant strain
Gene Log2 fold
Locus name Function/role change
SAUSA300_0859 NADH-dependent flavin oxidoreductase 8.19
SAUSA300_1937 Phi77 ORF045-like protein 6.46
SAUSA300_1932 Phage-related hypothetical protein 6.36
SAUSA300_1938 Phi77 ORF006-like capsid protein 6.25
SAUSA300_1934 Phi77 ORF020-like protein, phage major tail protein 6.18
SAUSA300_1962 Phi77 ORF039-like protein 6.09
SAUSA300_1939 Phi77 ORF015-like protein protease 5.78
SAUSA300_1930 Phi77 ORF001-like protein 5.76
SAUSA300_1943 Phi77 ORF040-like protein 5.76
SAUSA300_1936 Phage-related hypothetical protein 5.75
SAUSA300_0860 rocD Ornithine oxo-acid transaminase 5.38
SAUSA300_01030 isdC Iron transport protein IsdC 3.35
SAUSA300_0117 sirA Iron compound ABC transporter SirA 3.77
SAUSA300_1715 ribD Riboflavin biosynthesis gene 3.27
SAUSA300_1713 ribA Riboflavin biosynthesis gene 3.11
SAUSA300_1714 ribB Riboflavin biosynthesis gene 3.06
SAUSA300_0504 pdxS Pyridoxal biosynthesis lyase PdxS 2.66
SAUSA300_0505 pdxT Glutamine amidotransferase subunit PdxT 2.55

four nitroreductases are not responsible for reduction of the nitro groups of MT02 and
subsequent bacterial resistance development.
Genome alterations are not responsible for MT02 resistance in S. aureus. In the
next step, whole-genome sequencing was performed to compare the genome of the
strain JE2, which was selected to grow in 5 ␮M (4.8 ␮g/ml) MT02 and does not produce
the red color when incubated with 5 ␮M (4.8 ␮g/ml) MT02, and the highly resistant
counterpart, which is resistant to 80 ␮M (76.5 ␮g/ml) MT02 and produces a dark red
color when incubated with 80 ␮M (76.5 ␮g/ml) MT02. Sequenced reads were assem-
bled into contigs exhibiting very high coverage values (950⫻ to 1,050⫻) and very
satisfactory assembly values: 17 contigs (⬎500 bp) with an N50 value between 550 and
630 kb, genome sizes of 2.851 Mbp, and a GC content of 32.60%. The maximum contig

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sizes approached 1 Mb (895 kb, 935 kb, 936 kb, and 936 kb). Pairwise comparison
showed no genotypic differences between the strains. Moreover, no single-nucleotide
polymorphisms, indels, or sequence recombinations were observed. Thus, no relevant
mutations were found that could be involved in the reduction of nitro groups,
suggesting further investigation by transcriptome analysis.
RNA-seq revealed massive overexpression of a novel putative nitroreductase
in MT02-resistant S. aureus. Since no genomic alterations were responsible for MT02
resistance and expression of known nitroreductases was not altered in MT02-resistant
strains, total RNA sequencing (RNA-seq) was applied to identify factors involved in the
resistance mechanism. Total RNA was isolated from mid-log cultures of strain JE2
passaged for growth in 5 ␮M (4.8 ␮g/ml) MT02 (no red color) and strain JE2 resistant
to MT02 grown in 80 ␮M MT02, producing a dark red color. The transcriptome was
determined using Illumina sequencing. Interestingly, the strongest deregulation, with
303-fold upregulation, was observed for SAUSA300_0859, which encodes a protein of
unknown function (Table 2 and Fig. S3). Further BLAST analysis revealed homology to
a family of NADH-dependent flavin oxidoreductases. Orthologs of SAUSA300_0859 are
highly conserved in all S. aureus strains; however, its biological function has not been
resolved yet. The gene rocD, downstream of SAUSA300_0859, was also highly upregu-
lated (Table 2 and Fig. 2; also see Fig. S4). The SAUSA300_0859 gene is located on the
bacterial chromosome in the vicinity of arginine/proline biosynthesis pathways, other
open reading frames (ORFs) encoding putative proteins with unknown function, and a
cation/proton antiporter complex (Fig. S3). In addition, several putative phage-related
ORFs also were upregulated (Table 2 and Table S1). In contrast, several toxin genes
encoding PSM-␤, Hld, alpha-toxin, and the two-component leukotoxin LukGH were

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FIG 2 Transcriptome profile of the genomic region within which SAUSA300_0859 is encoded. cDNA coverage from S.
aureus JE2 resistant to 80 ␮M MT02 (MT02_80; red line) and S. aureus JE2 resistant to 5 ␮M MT02 (MT02_5; blue line) are
shown.

strongly downregulated (Table 3). Overall, due to the putative oxidoreductase function,
SAUSA300_0859 appeared to be the top candidate to be involved in the reduction of
nitro groups of MT02 and, consequently, in MT02 resistance.
Expression of the putative nitroreductase gene SAUSA300_0859 in the MT02-
resistant strain. Based on the RNA sequencing results, which revealed overexpression
of the putative nitroreductase gene SAUSA300_0859, RT-PCR experiments were per-
formed to confirm RNA-seq results. We compared the expression of the SAUSA300_
0859 gene in the highly MT02-resistant S. aureus JE2 strain (resistant to 80 ␮M
[76.5 ␮g/ml] MT02) with the MT02-intermediate resistant strain (JE2 resistant to 5 ␮M

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[4.8 ␮g/ml] MT02) in logarithmic growth phase. A 160-fold overexpression of
SAUSA300_0859 in the MT02-resistant strain was observed, confirming the RNA-seq
results. In addition, we determined expression of the downstream genes SAUSA_0860
(rocD), which was also highly upregulated in RNA-seq, and SAUSA_0861, which was not
deregulated. RT-PCR results confirmed RNA-seq revealing 93-fold upregulation of
SAUSA_0860 (rocD) and no deregulation of SAUSA_0861 (ratio, 0.96).

TABLE 3 Downregulated genes (top 10 and selected genes) in the MT02-resistant mutant
compared to the intermediately resistant strain
Log2 fold
Locus Gene name Function/role change
SAUSA300_1068 psmß1 Cytolysin 7.52
SAUSA300_1067 psmß2 Cytolysin 7.36
SAUSA300_1988 hld Delta-hemolysin 5.34
SAUSA300_1058 Hla Alpha-hemolysin 3.94
SAUSA300_1974 lukG Two-component leukotoxin (LukGH) 3.80
SAUSA300_1975 lukH Two-component leukotoxin (LukGH) 3.66
SAUSA300_0151 adhE Acetaldehyde-CoA/alcohol dehydroge 3.41
SAUSA300_2154 lacB Galactose-6-phosphate isomerase subunit LacB 3.41
SAUSA300_2153 lacC Tagatose-6-phosphate kinase 3.31
SAUSA300_2155 lacA Galactose-6-phosphate isomerase subunit LacA 3.26
SAUSA300_1992 agrA Accessory gene regulator protein A 3.06
SAUSA300_1991 agrC Accessory gene regulator protein C 2.98
SAUSA300_1989 agrB Accessory gene regulator protein B 2.98
SAUSA300_1990 agrD Accessory gene regulator protein D 2.85

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SAUSA300_0859 mutation restored sensitivity in MT02-resistant strains. To

confirm the importance of SAUSA300_0859 in the development of resistance against
MT02, a transposon insertion mutant of the SAUSA300_0859 gene was obtained from
the Nebraska Transposon Mutant Library (9) in strain S. aureus JE2 and transduced to
other S. aureus strains that were either highly (80 ␮M [76.5 ␮g/ml] MT02) or interme-
diately (5 ␮M [4.8 ␮g/ml] MT02) resistant to MT02. In both situations, the intermediately
resistant strain (5 ␮M [4.8 ␮g/ml] MT02) and the highly MT02-resistant strain (80 ␮M
[76.5 ␮g/ml] MT02), the transposon insertion completely restored the MT02-sensitive
phenotype to wild-type levels (MIC of 0.625 ␮M [0.6 ␮g/ml] MT02).
The gene SAUSA300_0859 encodes the MT02-reducing enzyme. We biochemi-
cally analyzed the SAUSA300_0859-encoded protein by its expression as a His-tagged
fusion protein. After the protein purification procedure, the His tag was cut off as
described in Materials and Methods. The SAUSA300_0859-encoded protein is com-
posed of 375 amino acids and has a theoretical pI of 5.19 and a molecular mass of 42
kDa; the latter was confirmed on a Coomassie-stained polyacrylamide gel (data not
shown). The enzymatic reaction was carried out in a 96-well plate with constant
concentrations of MT02 (100 ␮M [95.6 ␮g/ml]) and NADH (60 ␮M). A 2-fold dilution
series of the SAUSA300_0859 protein was analyzed starting with 500 ␮g/ml to
0.48 ␮g/ml. Upon addition of the SAUSA300_0859 protein, the solution quickly turned
from light yellow to intense red, indicating the formation of compound 2. As a control,
we incubated the substrate MT02 with NADH alone or the protein with either MT02 or
NADH. Only a faint red color was observed when MT02 was incubated with NADH alone
due to transfer of a hydride ion from NADH to one of the two nitro groups forming
compound 2 (Fig. S5). Furthermore, at the highest SAUSA300_0859 protein concentra-
tion (500 ␮g/ml) the red color turned yellow after a longer incubation time of 24 h,
which indicates the subsequent reduction of the two nitro groups of MT02 to amino
groups. These results clearly show that SAUSA300_0859 is able to reduce MT02, being
the functional enzyme conferring MT02 resistance in S. aureus.
We can provide evidence that the novel oxidoreductase belongs to the class of
oxygen-insensitive nitroreductases (type I nitroreductases), as the red color associated
with MT02 resistance was also produced under anaerobic conditions. Oxygen-
insensitive nitroreductases (type I nitroreductases) are the key enzymes to degrade or

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transform nitroaromatic compounds by catalyzing the reduction of the aromatic ring or
reduction of the nitro groups to hydroxylamino and/or amino derivatives in a NAD(P)H-
dependent reaction (10). This result suggests that the nitroreductive process involves
an NAD(P)H-dependent hydride ion transfer that we have confirmed analytically and
biochemically. In recent years, type I nitroreductases have raised great interest due to
their application in prodrug activation for antibacterial drugs and chemotherapeutic
cancer treatments. Prominent examples of prodrug antibacterials are nitrofurans, such
as nitrofurantoin and metronidazole, which are activated by the corresponding reduc-
tion. Nitrofurans are broad-spectrum antimicrobial agents which are effective against
both Gram-positive and Gram-negative bacteria (11). Metronidazole, being one of the
few drugs of choice for the treatment of infections caused by Helicobacter pylori,
anaerobic bacteria, and protozoa, is intracellularly reduced by nitroreductases to its
active form. The resulting amino metabolites are highly active against DNA and inhibit
bacterial enzymes involved in the synthesis of DNA, RNA, and other metabolic enzymes
(12). In contrast to these prodrug compounds, the nitro group containing compound
MT02 used in this study exerts antibacterial activity without activation by nitroreduc-
tases. MT02-resistant strain JE2 showed no difference in sensitivity to the nitro group
containing the antibiotics nitrofurantoin and chloramphenicol, two antibiotics contain-
ing nitro groups, compared to the MT02-sensitive wild-type strain (data not shown).
This suggests specific mechanisms of nitro group reduction by S. aureus nitroreductases
depending on chemical properties. Interestingly, overexpression of a bacterial nitrore-
ductase plays a role in the inactivation of anti-infective drugs in mycobacteria (13).
Recently, antimycobacterial activity has been reported for a new class of antimycobac-

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El-Hossary et al. Antimicrobial Agents and Chemotherapy

terial agents, the 1,3-benzothiazin-4-ones (BTZs), which are characterized by an essen-

tial nitro group (14). In Mycobacterium smegmatis, resistance to BTZ043 was due to
overexpression of the nitroreductase NfnB, which leads to the inactivation of BTZ043 by
the reduction of the critical nitro group to an amino group (13). In that case, sponta-
neous mutations in the transcriptional repressor controlling expression of NfnB were
responsible for resistance development (13).
Concluding remarks. The molecular mechanism leading to overexpression of
SAUSA300_0859 is still unknown. Complete genome sequencing did not reveal any
genomic alterations, e.g., mutations in the promoter region that may be linked to
overexpression of the nitroreductase. Interestingly, the SAUSA300_0859 gene is located
in the vicinity of the arginine biosynthesis genes rocD, argG, and argH. Recently, it has
been shown that S. aureus is able to synthesize arginine via the urea cycle using proline
as the substrate (15). This pathway is under the control of the catabolite control protein
A (CcpA) and may be important for arginine synthesis under infection conditions.
However, it is not known if the nitroreductase SAUSA300_0859 is somehow involved in
the pathway of arginine biosynthesis. The downstream gene rocD encodes ornithine
aminotransferase, which catalyzes the formation of glutamate-5-semialdehyde from
ornithine or the reverse reaction. This enzyme is probably involved in arginine and
proline biosynthesis depending on substrate availability (15, 16). On the other hand, the
pronounced downregulation of toxin genes in the MT02 mutant remains to be ex-
plored. Strikingly, the six most downregulated genes belong to this class of virulence
factors. Since all genes of the agr quorum-sensing system, which positively regulates
toxin expression, are also strongly downregulated, resistance development against
MT02 is possibly linked to reduced agr expression (Table 3).
In conclusion, to the best of our knowledge, this is the first report of a
nitroreductase-based antibiotic resistance mechanism in the human pathogen S.
aureus. Further studies will be conducted to clarify the role of the nitroreductase
SAUSA300_0859 in metabolism, antibiotic resistance, and virulence of the human
pathogen S. aureus.

MATERIALS AND METHODS

Synthesis of MT02. MT02 was synthesized essentially as described before (17). The purity of

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synthesized MT02 was confirmed by high-performance liquid chromatography (HPLC).
MT02-resistant staphylococci selection. (i) Selection of MT02-resistant S. aureus in small
culture. Nine S. aureus strains, including HG001, MA12, 8325 (RN1), Xen29, LAC USA300 JE2, NE118,
NE441, NE662, and NE909, were used to select MT02-resistant clones by incubation in stepwise
increasing concentrations of MT02 from 0.5⫻ MIC to 200⫻ MIC. The MICs for all strains used are indicated
in Table 1. In the first step, 5 ml of Mueller-Hinton (MH) broth was inoculated with 50 ␮l of an overnight
culture of each MT02-sensitive S. aureus strain and incubated for 24 h at 37°C in a shaking incubator at
180 rpm. The next day, 5 ml of fresh MH medium containing a higher concentration of MT02 was
inoculated with 50 ␮l of the previous overnight cultures. At the beginning of the selection process,
subinhibitory concentrations of MT02 were used, starting with 0.3125 ␮M (0.3 ␮g/ml). This concentration
was increased in steps of 0.1⫻ MIC until reaching 2⫻ MIC, followed by increasing the concentration in
steps of 0.5⫻ MIC up to a total concentration of 10⫻ MIC. From this concentration, the increase was in
steps of 1⫻ MIC until reaching 25⫻ MIC and then in increments of 2.5⫻ MIC to a final concentration of
125 ␮M (200⫻ MIC, when the MIC was 0.625 ␮M [0.6 ␮g/ml]). The MT02-resistant S. aureus strain MA12
(resistant to 125 ␮M [95.5 ␮g/ml] MT02) obtained by the selection procedure was used for further
experiments to produce compounds 2 to 4.
(ii) Selection of MT02-resistant S. aureus JE2 in large cultures. Five hundred milliliters of MH broth
inoculated with 10 ml of an overnight culture of MT02-sensitive S. aureus JE2 was incubated for 24 h at
37°C in a shaking incubator at 180 rpm. The incubation was started in the presence of 0.625 ␮M (0.6
␮g/ml) MT02 (1⫻ MIC). The next day, 500 ml of fresh MH medium containing the next higher
concentration of MT02 (2⫻ MIC) was inoculated with 10 ml of the previous overnight cultures. The
concentration of MT02 was duplicated in each new cultivation every 24 h, until reaching 128⫻ MIC (80
␮M [76.5 ␮g/ml]). MT02-sensitive S. aureus JE2 and S. aureus JE2 strains resistant to 5, 10, and 80 ␮M (4.8,
9.6, and 76.5 ␮g/ml) MT02, respectively, were subjected to whole-genome sequencing, while S. aureus
JE2 strains resistant to 5 and 80 ␮M (4.8 and 76.5 ␮g/ml) MT02 were subjected to total RNA sequencing.
Incubation of MT02 with MT02-resistant S. aureus under anaerobic conditions. In a sealed jar
containing an AnaeroGen sachet (Thermo Fisher Scientific Inc., Schwerte, Germany), 5 ml of culture of MH
broth containing 125 ␮M MT02, inoculated with 50 ␮l of an overnight culture of MT02-resistant S. aureus
MA12 strain, was incubated for 24 h at 37°C in a shaking incubator at 180 to 200 rpm.

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Resistance to Antibacterial Nitro Compounds Antimicrobial Agents and Chemotherapy

Upscaled cultivation and extraction: production and extraction of compound 2 in MT02-

resistant S. aureus. The bacterial cultures were prepared by inoculation of 5-liter flasks containing 2
liters of MH broth with 40 ml of an overnight culture of MT02-resistant S. aureus MA12 and 12 ml of MT02
solution in dimethyl sulfoxide (DMSO) (10 mg/ml). The cultures then were cultivated with constant
shaking at 180 rpm for 24 h at 37°C. Following cultivation, the bacterial cells were harvested by
centrifugation for 10 min at 8,000 rpm at 4°C. After centrifugation, a clear red supernatant and a cell
pellet with a violet precipitate were obtained. The clear supernatant was collected and stored at ⫺20°C
for 48 h and then left to melt at 4°C. A violet precipitate again was formed and collected by another
centrifugation at 8,000 rpm for 10 min at 4°C. The obtained violet precipitates were dissolved in 5 ml
acetonitrile (extract 1). The cell pellet and the violet precipitate were resuspended in 10 ml acetonitrile
and centrifuged at 8,000 rpm for 10 min at 4°C. The obtained red clear supernatant was collected (extract
2). Extracts 1 and 2 were subjected to semipreparative HPLC and liquid chromatography-mass spec-
trometry (LC-MS) analysis.
Production and extraction of compounds 3 and 4 in MT02-resistant S. aureus. MT02-resistant S.
aureus MA12 was cultivated in 2 liters of MH broth with 60 mg/liter MT02 with constant shaking at 180
rpm at 37°C for 6 days. During the cultivation period, the red color was produced and disappeared
gradually. Following cultivation, the bacterial cells were harvested by centrifugation for 10 min at 8,000
rpm at 4°C. After centrifugation, a clear yellow supernatant and a cell pellet together with a brown
precipitate were obtained. The clear yellow supernatant was collected and stored at ⫺20°C for 48 h and
then left to melt at 4°C. A dark brown precipitate again was formed and collected by another
centrifugation at 8,000 rpm for 10 min at 4°C. The obtained dark brown precipitate was dissolved in 5
ml acetonitrile (extract 3). The cell pellet and the brown precipitate were resuspended again in 10 ml
acetonitrile and centrifuged at 8,000 rpm for 10 min at 4°C. The obtained yellow clear supernatant was
collected (extract 4). Extracts 3 and 4 were subjected to semipreparative HPLC and LC-MS analysis.
Semipreparative HPLC. All solvents used were of HPLC analytical grade and purchased from Aldrich
(Steinheim, Germany) and Merck (Darmstadt, Germany). An Agilent 1100 preparative HPLC (Agilent
Technologies, Böblingen, Germany) with a fraction collector and a multiple-wavelength detector was
used for fractionation of extracts 1 to 4. The extracts were fractionated using a semipreparative column
(Nucleodur Sphinx reverse phase; dimensions, 125 by 10 mm; particle size, 5 ␮m; Macherey-Nagel, Düren,
Germany), eluting with a mixture of H2O containing 0.1% (vol/vol) trifluoroacetic acid (TFA) (mobile
phase A) and acetonitrile containing 0.1% (vol/vol) TFA (mobile phase B), with gradients of 0 to 20 min
(5% B to 100% B) and 20 to 25 min (100% B to 5% B), flow rate of 4.4 ml/min, and an injection volume
of 150 ␮l. Pure compounds were found at retention times of 10.7, 9.5, 9.7, and 8.6 min for compounds
1 to 4, respectively.
LC-MS analysis. LC-MS was conducted on an Agilent 1100 analytical HPLC with diode array
detection and an Agilent LC/MSD trap. ESI-MS data were collected in both positive and negative mode.
Extracts 1 to 4 were subjected to LC-MS experiments using an analytical column (Nucleodur Sphinx
reverse phase; 150 by 4.6 mm; particle size, 5 ␮m; Macherey-Nagel), eluting with a mixture of H2O
containing 0.1% (vol/vol) TFA (mobile phase A) and acetonitrile containing 0.1% (vol/vol) TFA (mobile
phase B), with gradients of 0 to 25 min (5% B to 100% B), 25 to 30 min (100% B), and 30 to 35 min (100%
B to 5% B) and flow rate of 1 ml/min, with an injection volume of 20 ␮l for the extracts and 5 ␮l for

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compounds 1 to 4 purified by semipreparative HPLC. Pure compounds were found at retention times of
14.2, 12.4, 12.7, and 11.2 min for compounds 1 to 4, respectively. The following parameters were used
for MS detection: ESI nebulizer pressure, 50 lb/in2; drying gas, 10 liters/min; drying gas temperature,
350°C; capillary voltage, 3,500 V.
Spectroscopic analyses of compounds 1 to 4. IR spectra were acquired on a Jasco (Gross-Umstadt,
Germany) FT/IR-6100 Fourier transformation infrared spectrometer equipped with an attenuated total
reflection unit. 1H (400.132 MHz) and 13C (100.613 MHz) NMR spectra were recorded on a Bruker Avance
400 ultra shield spectrometer (Bruker Biospin, Ettlingen, Germany). As an internal standard, the signals
of the deuterated solvent were used (DMSO-d6; 1H 2.5 ppm, 13C 39.52 ppm). Abbreviations for data
quoted are the following: s, singlet; d, doublet; t, triplet, dd, doublet of doublet; and br, broad. UV spectra
were measured using an Agilent 1100 Series G1315B DAD UV detector (GenTech Scientific Inc., NY).
N1-(3-(5-amino-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-2,2-dimethylpropyl)-N6-(2,2-dimethyl-3-
(5-nitro-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)-N 1 ,N 1 ,N 6 ,N 6 -tetramethylhexane-1,6-
diaminium-dibromide (compound 3). IR [cm⫺1]: 786, 1198, 1540, 1623, 1665, 1709, 2963, 3059, 3250,
3349. 1H-NMR (DMSO-d6, ␦ [ppm]): 1.22 (s, 6H); 1.25 (s, 6H); 1.34 (br, 4H); 1.77 (br, 4H); 3.16 (s, 12); 3.41
(br, 4H); 3.51 (s, 2H); 3.48 (s, 2H); 4.11 (s, 2H); 4.14 (s, 2H); 7.26 (d, 1H); 7.60 (dd, 1H); 7.93 (d, 1H); 8.05 (m,
3H); 8.68 (d, 1H); 8.78 (d, 1H); 8.94 (s, 1H); 9.48 (s, 1H). 13C-NMR (DMSO-d6, ␦ [ppm]): 21.95, 25.34, 25.42,
48.56, 49.04, 52.01, 66.74, 71.62, 71.77, 111.69, 120.52, 121.76, 121.82, 122.54, 122.75, 122.83, 124.19,
125.55, 126.91, 129.22, 129.50, 129.61, 130.74, 131.47, 133.41, 133.98, 136.26, 145.74, 147.76, 163.51,
163.95, 164.68, 164.80. ESIMS (m/z): 877.4 [M⫹TFA]⫹, 382.3 [M]2⫹. UV (CH3CN/H2O, ␭max [nm]): 218.
N 1 ,N 6 -Bis(3-(5-amino-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-2,2-dimethylpropyl)-N 1 ,N 1 ,N 6 ,N 6 -
tetramethylhexane-1,6-diaminium-dibromide (compound 4). IR [cm⫺1]: 783, 1124, 1622, 1660, 1704,
2964, 3050, 3229, 3356. 1H-NMR (DMSO-d6, ␦ [ppm]): 1.22 (s, 12H), 1.32 (br, 4H), 1.75 (br, 4H), 3.15 (s, 12H),
3.39 (br, 4H), 3.46 (s, 4H), 4.11 (s, 4H), 7.30 (d, 2H), 7.62 (dd, 2H), 7.96 (d, 2H), 8.05 (d, 2H), 8.08 (d, 2H).
13C-NMR (DMSO-d , ␦ [ppm]): 22.00, 25.40, 25.42, 48.62, 51.94, 66.80, 71.77, 111.70, 120.58, 121.86, 122.63,
6
125.57, 126.94, 131.50, 133.47, 147.81, 164.72, 164.93. ESIMS (m/z): 847.5 [M⫹TFA]⫹, 367.1 [M]2⫹. UV
(CH3CN/H2O, ␭max [nm]): 238.
Determination of antibacterial activity. Compounds 1, 3, and 4 were dissolved in sufficient volume
of DMSO to make a final concentration of 20 mM. Bacterial strains (S. aureus NCTC 8325 [RN1], HG001,

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El-Hossary et al. Antimicrobial Agents and Chemotherapy

MA12, Xen29, and JE2; S. epidermidis RP62A, 047, and 195; Enterococcus faecalis JH2-2; Enterococcus
faecium 6413; Escherichia coli 536; Pseudomonas aeruginosa; Yersinia pestis KUMA; and Yersinia pseudo-
tuberculosis 252 01A) were cultivated overnight at 37°C (30°C for Yersinia) in LB (lysogeny broth; per liter,
10 g tryptone, 5 g yeast extract, 5 g NaCl) in a shaking incubator. The next day, the culture was diluted
1:100 in MH broth (23 g/liter) and again incubated until the cells reached the exponential growth phase.
Approximately 1 ⫻ 105 cells/ml were incubated with various concentrations of the compounds (40, 20,
10, 5, 2.5, 1.25, 0.625, and 0.3125 ␮M [38.3, 19.1, 9.6, 4.8, 2.4, 1.2, 0.6, and 0.3 ␮g/ml]) to make a final
volume of 200 ␮l in a 96-well plate at 37°C for 18 h (30°C for 48 h for Yersinia). The final concentration
of DMSO was 0.8% in each well. Following incubation, the optical densities of the cultures were
determined at 550 nm using an enzyme-linked immunosorbent assay (ELISA) microplate reader (Mut-
lisKan Ascent; Thermo Fisher Scientific, Waltham, MA, USA) with respect to the control without bacteria.
The lowest concentration of the compound where no bacterial growth is detectable was determined as
the MIC. The overnight cultures from the wells where no bacterial growth was detected were plated on
LB agar plates and incubated again overnight. The lowest compound concentration at which no growth
of the bacteria was detectable was determined as the minimal bactericidal concentration (MBC).
Cytotoxicity assay. The cytotoxicity of compounds 1, 3, and 4 was determined in the macrophage
cell line J774.1 using the alamarBlue-based cytotoxicity test (18). Macrophages were cultured in NaHCO3-
buffered RPMI medium containing 10% fetal calf serum (FCS), 2 mM glutamine, 10 mM HEPES, pH 7.2,
100 U/ml penicillin, 50 ␮g/ml gentamicin, and 50 ␮M 2-mercaptoethanol without phenol red in the
absence or presence of increasing concentrations of the compounds at a cell density of 1 ⫻ 105 cells/ml
for 24 h at 37°C, 5% CO2, and 95% humidity. Following the addition of 10% alamarBlue solution, the
plates were incubated for 24 and 48 h and the optical densities measured with a Multiscan Ascent ELISA
reader (Thermo Fisher Scientific) using a test wavelength of 540 nm and a reference wavelength of 630
nm. The final concentration of DMSO in the medium did not exceed 1% (vol/vol) and had no effect on
proliferation. Each compound was tested in duplicate.
RNA isolation. Bacterial strains (MT02-sensitive S. aureus MA12, MT02-resistant S. aureus MA12, and
S. aureus JE2 strains resistant to either 5 ␮M [4.8 ␮g/ml] or 80 ␮M [76.5 ␮g/ml] MT02, respectively) were
cultivated overnight at 37°C in LB medium in a shaking incubator at 200 rpm. The next day, the strains
were diluted in 100 ml MH broth (OD600 of 0.05) and again incubated until the cells reached the
exponential growth phase. The MT02-resistant S. aureus JE2 strain was incubated in the presence of 80
␮M (76.5 ␮g/ml) MT02 for RNA sequencing, while the MT02-resistant S. aureus MA12 strain was
incubated in the presence of 125 ␮M MT02 for RT-PCR experiments. RNA was isolated from the cells at
an OD600 of 1.0. At least 6 ml of the bacterial cultures was harvested by centrifugation for 10 min at
10,000 rpm, and the bacterial pellet was resuspended in 800 ␮l of RLT buffer (Qiagen, Hilden, Germany)
and mechanically disrupted with glass beads (2-ml lysing matrix tube E; MP Biochemicals GmbH,
Eschwege, Germany) in a Fastprep-24 (MP Biochemicals, Woburn, MA). The cell lysate was centrifuged for
2 min at 13,000 rpm, and the supernatant was used for RNA isolation. RNA was isolated with an RNeasy
minikit (Qiagen, Hilden, Germany) according to the instructions of the manufacturer. To remove the DNA
template, RNA was treated with RNase-free DNase I (New England, Biolabs Inc., Frankfurt am Main,
Germany). The same RNA isolation procedures were used for both real-time quantitative PCR experi-

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ments and RNA sequencing.
Real-time quantitative PCR. Two micrograms of the isolated RNA was reverse transcribed at 50°C
for 1 h by using Superscript III reverse transcriptase (Invitrogen, Karlsruhe, Germany). For the reverse
transcription, 200-ng portions of random hexamer primers (Amersham, Freiburg, Germany) were applied
in each reaction mixture. The reaction mixture was inactivated by incubating at 70°C for 15 min.
For amplification of nfrA, ntrA, SAUSA300_1986, SAUSA300_2462, and SAUSA300_0859 cDNAs, the
primers listed in Table S2 in the supplemental material were used. As a control, 16S rRNA primers were
used. For relative quantification of the expression of the four nitroreductases, RT-PCR was performed by
using a CFX96 real-time system device (C1000 Touch thermal cycler; Bio-Rad, Munich, Germany). The
amplification reaction was done with SsoAdvanced universal SYBR green supermix (Bio-Rad). Relative
quantification of the transcription of the four nitroreductases was performed as described by Pfaffl (19).
Chromosomal DNA isolation. Total bacterial DNA was extracted by using a DNeasy blood and tissue
kit (Qiagen) with the following modifications. Bacterial strains (MT02-sensitive S. aureus JE2 and S. aureus
JE2 strains resistant to 5, 10, and 80 ␮M [4.8, 9.6, and 76.5 ␮g/ml] MT02) were grown overnight at 37°C
in B medium supplemented with 1% glycine with constant shaking at 200 rpm. Bacteria were harvested
by centrifugation of 1.5 ml of the overnight culture for 8 min at 7,500 rpm. The cell pellet was
resuspended in 180 ␮l lysis buffer (0.23 M Tris-HCl, 0.23 M EDTA, and 0.025 M NaCl at pH 8). Lysostaphin
was added to the suspended cells at a final concentration of 100 ␮g/ml and incubated at 37°C for 1 h.
Every 10 min the mixture was inverted. The DNA isolation procedure was continued as described in the
manual of the extraction kit. In brief, proteinase K (25 ␮l) and 200 ␮l AL buffer were added to the mixture
and incubated for 30 min at 56°C. Afterwards, 200 ␮l ethanol (99.5%) was added and the mixture was
transferred to a column provided by the kit. The column was washed once with 500 ␮l of buffer AW1 and
once with 500 ␮l of buffer AW2. The DNA was eluted in 200 ␮l water. DNA was precipitated with ethanol.
Whole-genome sequencing. Purified genomic DNA from MT02-sensitive S. aureus JE2 and S. aureus
JE2 strains resistant to 5 (4.8 ␮g/ml) and 80 (76.5 ␮g/ml) ␮M MT02 was subjected to whole-genome
shotgun sequencing on a HiSeq2500 system (Illumina Inc., San Diego, CA). Following fragmentation, end
reparation, and sample tagging, the sequencer produced 20 to 22 million paired-end reads of 150 bases
in length. All strains were sequenced in a single multiplex run after barcoding according to the Nextera
XT kit by following the manufacturer’s recommendations. Read quality was assessed with FastQC by
Babraham Bioinformatics (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Genome assem-

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Resistance to Antibacterial Nitro Compounds Antimicrobial Agents and Chemotherapy

bly was performed using the Edena v3 assembler (20). Parameters were finely tuned to obtain the least
amount of contigs while observing the highest N50 value. Contigs with coverage of ⬍750⫻ were
removed in order to keep only highly reliable constructed sequences. Assembled contigs then were
carefully compared by simple pairwise alignment using BLAST (21) to determine the slightest genomic
variations between strains.
Total RNA sequencing. Total RNA of S. aureus JE2 strains resistant to 5 (4.8 ␮g/ml) and 80 (76.5
␮g/ml) ␮M MT02 was extracted with the RNeasy minikit (Qiagen). For high-throughput sequencing
experiments, total RNA was treated with the MICROBExpress kit (Thermo Fisher Scientific) to limit
contamination by multicopy structural rRNAs by following the manufacturer’s instructions, with slight
modifications. The maximal amount of total RNA per tube was limited to 4 to 5 ␮g, because previous
experiments showed important contamination levels when using 10 ␮g. Between each purification step,
RNA quality and quantity were determined by a Bioanalyzer (Agilent, Palo Alto, California, USA) using
RNA Nano chips and quantified using an ND-8000 (Thermo Fisher Scientific).
Fragmentation of messenger-enriched RNA was performed using zinc. Specific tags were ligated
before reverse transcription to conserve strand orientation. Single-stranded ligation of the 3= Illumina
adapter was followed by single-stranded ligation of 5= bar-coded adapter. Reverse transcription and PCR
amplification were performed to generate a cDNA colony template library, and gel purification with
selection of fragments of 20 to 300 nucleotides (nt) was performed. The libraries then were sequenced
in one channel in the Illumina genome analyzer GAII (Illumina Inc., San Diego, CA) for 50 cycles.
The raw RNA-Seq reads were quality trimmed (requiring a minimal Phred score of 20) and adapter
clipped with cutadapt (22), followed by read mapping against the genome of S. aureus USA300_FPR3757
(GenBank accession numbers NC_007793.1, NC_007790.1, NC_007791.1, and NC_007792.1), gene quan-
tification, and differential gene expression analysis with segemehl (23) and DESeq2 (24) by the READemp-
tion pipeline (25). Genes for which DESeq2 reported an adjusted P value below 0.05 were considered
differentially expressed. A Unix shell script that documents the complete data processing and analysis is
accessible at Zenodo at https://zenodo.org/record/159807.
Construction of SAUSA300_0859 mutant strain. A transposon insertion mutant in the
SAUSA300_0859 gene was acquired from the Nebraska Transposon Mutant Library (NTML). The library
was constructed in S. aureus LAC USA300 JE2 (ST8) (9). The transposon insertion mutation was transferred
to the MT02-resistant S. aureus LAC USA300 JE2 strain by phage transduction as described before (26).
Purification of SAUSA300_0859 protein and reduction of MT02. The SAUSA300_0859 gene was
expressed as a His-tagged fusion protein by using the pCDF vector (Novagen, Darmstadt, Germany). The
PCR product was amplified from chromosomal DNA by using Phusion DNA polymerase (New England
BioLabs, Frankfurt am Main, Germany) and cloned in the vector pCDF-trx3C with the restriction sites NcoI
and XhoI. For protein expression, the plasmid was transformed in the E. coli Rosetta strain. Cells were
grown in LB to an OD600 of 0.5. Isopropyl-␤-D-thiogalactoside (IPTG) was added to a final concentration
of 1 mM for induction. The culture was incubated for 24 h at 18°C in a shaking incubator at 200 rpm.
Bacteria were harvested by centrifugation for 15 min at 3,000 rpm at 4°C. The pellet was resuspended in
lysis buffer (50 mM Na2HPO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), and 0.5 mg/ml lysozyme was
added. Cell lysis was performed by sonication on ice for 3 min. The solution was centrifuged at 16,000

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rpm for 20 min at 4°C. The protein was purified from the supernatant by immobilized metal ion affinity
chromatography (IMAC) with an Ni-nitrilotriacetic acid resin spin column (Qiagen). The column was
equilibrated with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM
KH2PO4, pH 7.4) according to the instructions of the manufacturer. The supernatant was applied to the
column, and the column was washed with IMAC wash buffer (50 mM Na2HPO4, 300 mM NaCl, 5 mM
imidazole, pH 8.0). The protein was eluted from the column with elution buffer (250 mM imidazole, 50
mM Na2HPO4, 300 mM NaCl, pH 8.0). The protein was dialyzed against Tris-HCl buffer (100 mM Tris-HCl,
150 mM NaCl, pH 7.5) overnight at 4°C. To cut off the His tag from the protein, protease 3C (kindly
provided by Caroline Kisker, Rudolf-Virchow-Centre, Würzburg, Germany) was added to the dialysis tube.
To purify the tag-free protein, the solution again was applied to an Ni-nitrilotriacetic acid resin spin
column. The purified untagged protein is in the flowthrough, whereas uncleaved (His-tagged) protein
and the protease will stick to the column.
A 2-fold dilution series was prepared from the protein SAUSA300_0859, starting with a protein
concentration of 500 ␮g/ml. In a 96-well plate, 11 2-fold dilutions were analyzed (500 ␮g/ml, 250 ␮g/ml,
125 ␮g/ml, 62.5 ␮g/ml, 31.25 ␮g/ml, 15.6 ␮g/ml, 7.8 ␮g/ml, 3.9 ␮g/ml, 1.9 ␮g/ml, 0.975 ␮g/ml, and 0.48
␮g/ml). To each well 60 ␮M NADH and 100 ␮M (95.6 ␮g/ml) MT02 were added to a final volume of 100
␮l. As a negative control, the protein was incubated with either NADH or MT02. In an additional negative
control, NADH was incubated with MT02 without protein. The enzymatic reduction of MT02 was
visualized by the red color of the reduced MT02. The color was most intense after an incubation time of
2 h at room temperature.
Accession number(s). The RNA-seq data discussed in this publication have been deposited in NCBI’s
Gene Expression Omnibus (27) and are accessible through GEO Series accession number GSE87572.

SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/AAC
.01510-17.
SUPPLEMENTAL FILE 1, PDF file, 4.2 MB.
SUPPLEMENTAL FILE 2, XLSX file, 0.6 MB.

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El-Hossary et al.
ACKNOWLEDGMENT
We thank the German Research Foundation (DFG; Bonn, Germany) for financial
support (Sonderforschungsbereich 630, TR34) and the Alexander von Humboldt Foun-
dation for the postdoctoral fellowship given to E.M.E.H.
We also thank Liane Dreher (Institute for Molecular Infection Biology [IMIB], Univer-
sity of Würzburg) for the construction of SAUSA_0859 mutant strains, Caroline Richly
(former Bachelor student at IMIB) for protein purification, Svetlana Sologub (SFB 630
staff member, Z1, IMIB) for the cytotoxicity assay, and Eve-Julie Bonetti (University of
Geneva Hospitals, Genomic Research Laboratory) for RT-PCR experiments.
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