A small-molecule modulator of metal homeostasis in gram-positive pathogens

Article abstract of DOI:10.1128/mBio.02555-20

Metals are essential nutrients that all living organisms acquire from their environment. While metals are necessary for life, excess metal uptake can be toxic; therefore, intracellular metal levels are tightly regulated in bacterial cells. Staphylococcus aureus, a Gram-positive bacterium, relies on metal uptake and metabolism to colonize vertebrates. Thus, we hypothesized that an expanded understanding of metal homeostasis in S. aureus will lead to the discovery of pathways that can be targeted with future antimicrobials. We sought to identify small molecules that inhibit S. aureus growth in a metal-dependent manner as a strategy to uncover pathways that maintain metal homeostasis. Here, we demonstrate that VU0026921 kills S. aureus through disruption of metal homeostasis. VU0026921 activity was characterized through cell culture assays, transcriptional sequencing, compound structure-activity relationship, reactive oxygen species (ROS) generation assays, metal binding assays, and metal level analyses. VU0026921 disrupts metal homeostasis in S. aureus, increasing intracellular accumulation of metals and leading to toxicity through mismetalation of enzymes, generation of reactive oxygen species, or disruption of other cellular processes. Antioxidants partially protect S. aureus from VU0026921 killing, emphasizing the role of reactive oxygen species in the mechanism of killing, but VU0026921 also kills S. aureus anaerobically, indicating that the observed toxicity is not solely oxygen dependent. VU0026921 disrupts metal homeostasis in multiple Gram-positive bacteria, leading to increased reactive oxygen species and cell death, demonstrating the broad applicability of these findings. Further, this study validates VU0026921 as a probe to further decipher mechanisms required to maintain metal homeostasis in Gram-positive bacteria. IMPORTANCE Staphylococcus aureus is a leading agent of antibiotic-resistant bacterial infections in the world. S. aureus tightly controls metal homeostasis during infection, and disruption of metal uptake systems impairs staphylococcal virulence. We identified small molecules that interfere with metal handling in S. aureus to develop chemical probes to investigate metallobiology in this organism. Compound VU0026921 was identified as a small molecule that kills S. aureus both aerobically and anaerobically. The activity of VU0026921 is modulated by metal supplementation, is enhanced by genetic inactivation of Mn homeostasis genes, and correlates with increased cellular reactive oxygen species. Treatment with VU0026921 causes accumulation of multiple metals within S. aureus cells and concomitant upregulation of genes involved in metal detoxification. This work defines a small-molecule probe for further defining the role of metal toxicity in S. aureus and validates future antibiotic development targeting metal toxicity pathways.

Full text of DOI:10.1128/mBio.02555-20

RESEARCH ARTICLE
Host-Microbe Biology
crossm
A Small-Molecule Modulator of Metal Homeostasis in Gram-
Positive Pathogens
Lillian J. Juttukonda,a,g* William N. Beavers,a,g Daisy Unsihuay,a,g* Kwangho Kim,b Gleb Pishchany,c Kyle J. Horning,d
Andy Weiss,a,g Hassan Al-Tameemi,e Jeffrey M. Boyd,e Gary A. Sulikowski,a,b,f Aaron B. Bowman,d* Eric P. Skaara,g
aVanderbilt Institute for Infection, Immunology and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, USA
bChemical Synthesis Core, Vanderbilt University, Nashville, Tennessee, USA
cDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA
dVanderbilt Brain Institute, Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
eDepartment of Biochemistry and Microbiology, Rutgers, the State University of New Jersey, New Brunswick, New Jersey, USA
fDepartment of Chemistry, Vanderbilt University, Nashville, Tennessee, USA
gDepartment of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
Lillian J. Juttukonda and William N. Beavers contributed equally to this work. Author order was determined in order of increasing seniority.
ABSTRACT Metals are essential nutrients that all living organisms acquire from
their environment. While metals are necessary for life, excess metal uptake can be
toxic; therefore, intracellular metal levels are tightly regulated in bacterial cells.
Staphylococcus aureus, a Gram-positive bacterium, relies on metal uptake and metab-
olism to colonize vertebrates. Thus, we hypothesized that an expanded understand-
ing of metal homeostasis in S. aureus will lead to the discovery of pathways that can
be targeted with future antimicrobials. We sought to identify small molecules that
inhibit S. aureus growth in a metal-dependent manner as a strategy to uncover path-
ways that maintain metal homeostasis. Here, we demonstrate that VU0026921 kills S. au-
reus through disruption of metal homeostasis. VU0026921 activity was characterized
through cell culture assays, transcriptional sequencing, compound structure-activity re-
lationship, reactive oxygen species (ROS) generation assays, metal binding assays,
and metal level analyses. VU0026921 disrupts metal homeostasis in S. aureus, in-
Citation Juttukonda LJ, Beavers WN, Unsihuay
D, Kim K, Pishchany G, Horning KJ, Weiss A,
Al-Tameemi H, Boyd JM, Sulikowski GA,
Bowman AB, Skaar EP. 2020. A small-molecule
modulator of metal homeostasis in Gram-
positive pathogens. mBio 11:e02555-20.
https://doi.org/10.1128/mBio.02555-20.
creasing intracellular accumulation of metals and leading to toxicity through mis-
metalation of enzymes, generation of reactive oxygen species, or disruption of other
cellular processes. Antioxidants partially protect S. aureus from VU0026921 killing,
emphasizing the role of reactive oxygen species in the mechanism of killing, but
VU0026921 also kills S. aureus anaerobically, indicating that the observed toxicity is
not solely oxygen dependent. VU0026921 disrupts metal homeostasis in multiple
Gram-positive bacteria, leading to increased reactive oxygen species and cell death,
demonstrating the broad applicability of these findings. Further, this study validates
VU0026921 as a probe to further decipher mechanisms required to maintain metal
homeostasis in Gram-positive bacteria.
Editor Paul Dunman, University of Rochester
Copyright © 2020 Juttukonda et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Eric P. Skaar,
eric.skaar@vumc.org.
IMPORTANCE Staphylococcus aureus is a leading agent of antibiotic-resistant bacte-
rial infections in the world. S. aureus tightly controls metal homeostasis during infec-
tion, and disruption of metal uptake systems impairs staphylococcal virulence. We iden-
tified small molecules that interfere with metal handling in S. aureus to develop
chemical probes to investigate metallobiology in this organism. Compound VU0026921
was identified as a small molecule that kills S. aureus both aerobically and anaerobi-
cally. The activity of VU0026921 is modulated by metal supplementation, is enhanced
by genetic inactivation of Mn homeostasis genes, and correlates with increased cellular
reactive oxygen species. Treatment with VU0026921 causes accumulation of multiple
metals within S. aureus cells and concomitant upregulation of genes involved in metal
* Present address: Lillian J. Juttukonda, Boston
Combined Residency Program, Boston
Children’s Hospital, Boston, Massachusetts,
USA; Daisy Unsihuay, Department of Chemistry,
Purdue University, West Lafayette, Indiana, USA;
Aaron B. Bowman, School of Health Sciences,
Purdue Institute for Integrative Neuroscience,
Purdue University, West Lafayette, Indiana, USA.
Received 9 September 2020
Accepted 29 September 2020
Published 27 October 2020
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detoxification. This work defines a small-molecule probe for further defining the role of
metal toxicity in S. aureus and validates future antibiotic development targeting metal
toxicity pathways.
KEYWORDS MRSA, Staphylococcus aureus, antibiotics, cobalt, copper, manganese,
metalloregulation
acterial infections remain a significant cause of morbidity and mortality worldwide,
B
and antibiotic resistance has become commonplace amid a decades-long drought
in antibiotic development (1, 2). Most antibiotics brought to market are derived from
existing antibiotic classes and may be rendered obsolete through shared antibiotic
resistance mechanisms (3). Therefore, therapeutic development for antibiotic-resistant
bacterial pathogens has been named a priority by numerous public health organiza-
tions (4, 5).
Staphylococcus aureus is a major threat to human health and one of the most
common causes of bacterial infection in the world (6). Effective antibiotic treatment
prevents morbidity and mortality due to S. aureus infections. However, methicillin-
resistant S. aureus (MRSA) strains are resistant to multiple classes of beta-lactam
antibiotics (7). MRSA surgical site wounds and central line-associated bloodstream
infections in the United States alone cost more than $1.3 billion per year (8). The
Centers for Disease Control and Prevention labeled MRSA a “serious threat,” and the
World Health Organization categorized the need for new antibiotics against MRSA as a
“high priority” (4, 5). Investigations into processes essential for S. aureus fitness may
reveal fruitful avenues for the development of novel antibiotics.
The transition metals iron (Fe), manganese (Mn), and zinc (Zn) serve as structural
elements within macromolecules and as essential cofactors for enzymes and are thus
necessary for S. aureus survival. However, transition metals are toxic when present in
excess as they compete with cognate metals for protein binding pockets or transport
(9, 10). Furthermore, redox-active metals can generate reactive oxygen species (ROS)
through Fenton chemistry (11, 12). Consequently, transition metal homeostasis is
imperative for pathogen success during infection. Vertebrate hosts limit bacterial
access to essential metals in a process termed “nutritional immunity,” and a parallel
process exists for intoxicating invaders with highly reactive metals (13–16). Bacteria
strictly control metal levels such that metals are available to support biological pro-
cesses while preventing toxicity. Metal levels are maintained through a balance of
import and efflux systems as well as metal-sequestering molecules within the cell (9).
During infection, maintaining appropriate metal ratios is essential for bacterial survival
and virulence. We predict that metal homeostasis is a process that can be exploited by
targeted antimicrobial therapy during infection.
Given the importance of metal acquisition and homeostasis in bacterial physiology,
several studies have investigated the potential of developing antimicrobials that act by
disrupting metal homeostasis (17). These approaches have included gallium-based
antimicrobials, which disrupt Fe import and homeostasis (18, 19); gallium porphyrin
molecules that disrupt heme-iron import and homeostasis (20); and metal chelators (21,
22). However, these strategies have heretofore relied on imposing metal starvation,
whereas exploitation of metal toxicity is less common.
Here, we present the identification of a small molecule that causes metal accumu-
lation and toxicity in S. aureus. A library of compounds previously identified to alter Mn
transport in mammalian cells (23) was screened for the ability to differentially inhibit
growth of S. aureus lacking high-affinity Mn uptake machinery. Compound VU0026921
(‘921) decreases S. aureus viability, exhibiting enhanced activity in an S. aureus strain
lacking high-affinity Mn import systems. Supplementation with divalent transition
metals modulates the antimicrobial activity of VU0026921, and cellular ROS levels are
inversely correlated with the ability of each metal-VU0026921 combination to kill S.
aureus. However, the activity of VU0026921 is not mediated solely through ROS
generation because antioxidants provide only partial protection from VU0026921
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killing, and the molecule kills S. aureus both aerobically and anaerobically. RNA se-
quencing revealed that VU0026921 causes upregulation of multiple metal exporters
and metal homeostasis machinery, suggesting that VU0026921 induces a metal toxicity
response in S. aureus. Indeed, VU0026921 binds cobalt (Co), copper (Cu), Fe, Mn, and Zn,
leading to increases in cellular Zn, Fe, and Cu following compound exposure as
uncovered by elemental mass spectrometry. Cotreatment with Mn and VU0026921 led
to increases in cellular Mn and abrogated accumulation of other metals, suggesting
that intracellular Mn maintains cellular viability. Additionally, the exogenous addition of
Cu exacerbates the killing of S. aureus by VU0026921, while the exogenous addition of
Co is more protective than Mn. VU0026921 has low toxicity in Gram-negative species
but kills all of the S. aureus strains and other Gram-positive pathogens tested. Taken
together, this work reports the discovery of a small molecule that dysregulates metal
homeostasis in S. aureus and other Gram-positive pathogens, causing substantial
toxicity to this organism, and facilitates future elucidation of metal dysregulation as an
antistaphylococcal treatment.
RESULTS
Identification of a small molecule that kills S. aureus. We sought to identify small
molecules that disrupt S. aureus growth in an Mn-dependent manner with the goal of
identifying chemical probes for understanding S. aureus metallobiology. We tested a
collection of 39 compounds that alter the net uptake of Mn into mammalian cell lines
without causing toxicity (23). The compounds were tested in S. aureus Newman and
Newman ΔmntH/C, a strain that is Mn starved due to deletion of two Mn import
systems (24). This strain does not have a growth defect in rich medium but grows
poorly under Mn-limited conditions and is hypersusceptible to oxidative stress because
of impaired superoxide dismutase activity (24, 25). This strategy was chosen to identify
compounds that imposed stress on S. aureus with impaired Mn homeostasis while also
selecting for small molecules that did not target the high-affinity Mn import systems.
Any compounds discovered that differentially inhibit S. aureus Newman and ΔmntH/C
presumably are altering intracellular Mn in S. aureus. Eight compounds were identified
that are antimicrobial at concentrations of 10 ␮M or lower, and 7 of those demon-
strated growth inhibition that can be reversed by the addition of MnCl2 to the medium
in stoichiometric ratios, suggesting that they inhibit growth by chelating Mn in the
medium (Fig. 1A and also Table S1B in the supplemental material). Compound
VU0026921 was selected for further study because it exhibits differential growth
inhibition of the ΔmntH/C strain relative to the wild type (WT) (Fig. 1B), and reversal of
growth inhibition by Mn is not complete and requires 50-fold excess Mn (Fig. 1C). These
data suggest that VU0026921 interferes with S. aureus Mn homeostasis or disrupts a
process that can be rescued by excess Mn. Moreover, these results imply that the
activity of VU0026921 does not occur simply through extracellular Mn limitation, as a
substantial excess of Mn is required to reverse the phenotype.
Aerobic antimicrobial activity of VU0026921 is modulated by Mn. Compound
VU0026921 inhibits S. aureus growth in a manner modulated by Mn (Fig. 1C). The S.
aureus lag phase is prolonged by treatment with 5 ␮M VU0026921, and the addition of
Mn partially rescues this phenotype (Fig. S1A). To determine whether VU0026921
inhibits growth or decreases cell viability, S. aureus CFU were measured every 2 h in the
presence of VU0026921 (Fig. 1D). While CFU increase in the vehicle-treated group, CFU
decrease following treatment with VU0026921, and cotreatment of Mn with VU0026921
protects S. aureus from killing by VU0026921. These data demonstrate that excess Mn
protects S. aureus from VU0026921-mediated killing. To determine if metal restriction
enhances S. aureus susceptibility to VU0026921-mediated killing, S. aureus mid-
exponential-phase cultures were cotreated with VU0026921, Mn, and the metal chela-
tor EDTA. Cotreatment with EDTA and VU0026921 decreases S. aureus CFU more than
treatment with VU0026921 alone, and the decrease in CFU could be reversed by the
addition of Mn (Fig. 1E). Together, these data are consistent with a model in which
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FIG 1 Identification of a small molecule that is antimicrobial to S. aureus. (A) Workflow for identifying
compounds of interest. (B) Percent growth inhibition by treatment with the indicated concentrations of
compound ‘921 relative to vehicle control for S. aureus Newman or ΔmntH/C strain. Data are mean Ϯ standard
deviation from duplicate measurements. Statistical significance was determined by t test where * ϭ P Ͻ 0.05
and ** ϭ P Ͻ 0.01. (C) Relative growth at 12 h with 5 ␮M ‘921 Ϯ MnCl2 is graphed as % of growth without
compound or MnCl2. Data are mean Ϯ standard deviation from triplicate measurements acquired on two
separate days and combined (n ϭ 6). Statistical significance was determined by one-way analysis of variance
(ANOVA) with Dunnett’s multiple-comparison test comparing each concentration of Mn to no-Mn control
where *** ϭ P Ͻ 0.001. (D) CFU recovered at 2-h intervals from growth curve of S. aureus Newman treated with
vehicle, 5 ␮M ‘921, or 5 ␮M ‘921 ϩ 1 mM MnCl2 over time. Data are mean Ϯ standard deviation from
quadruplicate measurements. (E) CFU recovered following 24-h treatment of mid-exponential-phase S. aureus
Newman cultures with the indicated combinations of vehicle, 1 mM MnCl2, 100 ␮M ‘921, and 1 mM EDTA. Data
are mean Ϯ standard deviation from triplicate measurements acquired on two separate days combined
(n ϭ 6). Statistical significance was determined by one-way ANOVA with Sidak’s multiple-comparison test
comparing each concentration of Mn to no-Mn control where * ϭ P Ͻ 0.05 and *** ϭ P Ͻ 0.001. Experiments
in panels F and G were performed in an anaerobic chamber. (F) Growth of S. aureus Newman in the presence
of vehicle, 1 mM MnCl2, 5 ␮M ‘921, or 5 ␮M ‘921 ϩ 1 mM MnCl2. Data are mean Ϯ standard deviation for
triplicate measurements. (G) CFU recovered following treatment of mid-exponential-phase cultures of S. aureus
with vehicle, 1 mM or 100 ␮M MnCl2, 100 ␮M ‘921, or combinations of MnCl2 ϩ ‘921 for 24 h. Data are mean Ϯ
standard deviation for triplicate measurements.
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VU0026921 kills S. aureus through a process that is altered by the availability of Mn in
the medium.
We considered the possibility that Mn may rescue S. aureus from VU0026921-
mediated killing by detoxifying oxygen radicals. Some antimicrobial molecules cause
the generation of ROS, and S. aureus uses Mn to detoxify superoxide in both superoxide
dismutase (SOD)-dependent and SOD-independent mechanisms (25–29). VU0026921
inhibits S. aureus growth under anaerobic conditions, demonstrating that oxygen is not
required for VU0026921 toxicity (Fig. 1F and G). In contrast to the phenotype seen
under aerobic conditions, the addition of Mn does not decrease the anaerobic toxicity
of VU0026921 (Fig. 1F and G). These results suggest that VU0026921 may have multiple
cellular targets, which is commonly observed for biologically active small molecules
(30).
Isolation of VU0026921-resistant S. aureus strains. Multiple strategies were
employed to attempt to generate resistant strains to identify cellular targets of
VU0026921 toxicity. First, selection was attempted by plating S. aureus on solid medium
impregnated with VU0026921. Despite multiple iterations using various concentrations,
growth conditions, and media, single colonies were never observed; growth occurred
in a lawn, no growth occurred, or groups of colonies occurred on the edges of plates.
Bacteria isolated from VU0026921-impregnated plates did not have enhanced resis-
tance in the presence of VU0026921 under solid or liquid growth conditions. The
second selection strategy employed was serial passage in escalating concentrations of
VU0026921 in liquid medium. While growth was observed after incubation in the
presence of low concentrations of compound, bacterial isolates from these experiments
did not exhibit either transient or permanent enhanced resistance in the presence of
VU0026921 under solid or liquid growth conditions. Finally, a third strategy involved
screening transposon mutants for enhanced growth in liquid medium containing
VU0026921; a pilot screen of approximately 200 known MRSA transposon mutants did
not identify any strains with significantly enhanced growth that was reproducible
across multiple experimental days, and this strategy was not further pursued. No
attempts were made to isolate resistant mutants using chemical mutagenesis or pooled
transposon libraries. The fact that VU0026921-resistant strains could not be readily
isolated is consistent with the possibility of multiple cellular targets of the compound.
VU0026921 induces a transcriptional response indicative of metal toxicity.
Seeking insight into cellular processes affected by VU0026921, we performed RNA
sequencing on aerobically grown exponential-phase S. aureus Newman following
30-min treatment with 100 ␮M VU0026921. Exposure to VU0026921 resulted in 298
upregulated and 220 downregulated genes at a cutoff of log2|FC| ϭ 2 (Table S2A),
demonstrating that VU0026921 dramatically alters the transcriptional profile of S.
aureus. A Cluster of Orthologous Groups of proteins (COG) analysis for the up- and
downregulated transcripts following VU0026921 treatment (Table S2B) demonstrates
that the major upregulated COGs are related to toxin production, posttranslational
modification/protein turnover/chaperone functions, and translation while energy pro-
duction is the major downregulated COG, consistent with the stress that S. aureus
experiences upon VU0026921 treatment (31–33). Since cotreatment with Mn rescues S.
aureus from VU0026921-mediated toxicity, we also analyzed RNA sequencing results for
S. aureus cotreated with VU0026921 and Mn to identify Mn-dependent transcriptional
changes. Two hundred sixteen genes are upregulated and 147 genes are downregu-
lated by cotreatment with VU0026921 and Mn compared to treatment with compound
alone (Table S3). These changes are VU0026921 dependent, as treatment with MnCl2
without compound only downregulates the high-affinity mntABC operon, known to be
repressed by Mn, and upregulates the Mn exporter mntE (34, 35) (Table S4).
Accordingly, the global transcriptional profiles of vehicle-treated and Mn-treated
biological replicates cluster together on a multidimensional scaling (MDS) plot, whereas
both of the VU0026921-treated groups cluster independently (Fig. 2A). The COG
pathways containing the greatest number of Mn-dependent transcriptional changes
between VU0026921 treatment and cotreatment with VU0026921 and Mn are toxin
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FIG 2 VU0026921 induces a metal toxicity transcriptional response. RNA sequencing was performed on mid-
exponential S. aureus Newman cultures exposed to vehicle, 1 mM MnCl2, 100 ␮M ‘921, or 100 ␮M ‘921 ϩ 1 mM
MnCl2 for 30 min. (A) Multidimensional scaling visualization of RNA sequencing biological replicates. The x and y
axes are dimensionless units. The closeness of symbols to each other on the 2-dimensional plot represents the
relatedness of the transcriptional profiles of the represented data sets. (B) Categories of genes whose transcription
was significantly different between 100 ␮M ‘921 treatment alone and cotreatment with 1 mM MnCl2. (C) Fold
change (log2) of transcript abundance for all S. aureus genes following treatment with VU0026921 compared to
vehicle-treated controls. (D) Fold change (log2) of transcript abundance for all S. aureus genes following treatment
with VU0026921 and 1 mM MnCl2 compared to ‘921 alone. For panels C and D, genes involved in metal
homeostasis and reactive oxygen species (ROS) detoxification are highlighted. Dashed lines indicate genes with
greater than 2-fold change and a false-discovery rate of less than 0.01.
production, amino acid metabolism and transport, and genes involved in transcrip-
tion (Fig. 2B). This suggests that the addition of Mn does not simply inactivate
VU0026921 or reverse VU0026921 activity but instead alters the physiology of the
S. aureus cell to promote survival. Similar transcriptional changes, such as upregu-
lation of superantigen-like genes NWMN_0390-91 and NWMN_1076-77, were observed
in other unrelated studies and may be a marker of cellular stress (31–33). The global
transcriptional profile of S. aureus treated with VU0026921 indicates that the cellular
stress induced by this compound causes changes to S. aureus physiology.
We hypothesized that VU0026921 targets Mn uptake or utilization and anticipated
that VU0026921 may alter the transcription of genes involved in metal homeostasis.
Interestingly, genes encoding transition metal import systems, including Mn, Fe, and Zn
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import systems, are downregulated or unchanged by VU0026921 treatment (Fig. 2C
and Table S5). In contrast, genes encoding exporters for the highly reactive metal Cu
and the reactive Fe-containing tetrapyrrole heme are highly upregulated by VU0026921
(Fig. 2C and Table S5). Moreover, genes thought to function in intracellular metal
buffering are increased by treatment with VU0026921 (Fig. 2C and Table S5). This
transcriptional pattern suggests that VU0026921 induces a metal toxicity response in S.
aureus.
Metals alone and the mismetalation of enzymes can generate ROS, which damage
cellular macromolecules (36, 37). Genes involved in detoxification of ROS are upregu-
lated following treatment with VU0026921 (Fig. 2C and Table S5). The transcriptional
upregulation of metal export and ROS detoxification systems coupled with unchanged
or downregulated genes encoding metal uptake and metal-binding proteins suggests
that VU0026921 causes S. aureus to experience metal toxicity. The addition of Mn,
which reverses the antimicrobial activity of VU0026921 toward S. aureus, leads to
downregulation of metal export genes and ROS-detoxification systems (Fig. 2D). Taken
together, these transcriptional changes implicate intracellular metal homeostasis as a
target of VU0026921 toxicity.
VU0026921 causes intracellular metal accumulation in S. aureus. To determine
the impact of VU0026921 treatment on S. aureus metal levels, inductively coupled
plasma mass spectrometry (ICP-MS) was performed on S. aureus cells exposed to
VU0026921 for 30 min. Cu, Fe, and Zn concentrations increase in cells treated with
VU0026921, whereas there is no difference in the concentration of Co, and Mn
decreases slightly upon VU0026921 treatment (Fig. 3A to E). This decrease in intracel-
lular Mn may explain why VU0026921 inhibits S. aureus ΔmntH/C more than the
wild-type strain (Fig. 1B), as this mutant is Mn starved at baseline. In contrast, cells
treated with VU0026921 and 1 mM Mn demonstrate significantly higher Mn concen-
trations than treatment with either compound or Mn alone (Fig. 3A). Additionally,
VU0026921- and Mn-cotreated samples have lower Fe and Zn levels than samples
treated with compound alone (Fig. 3D and E). Cu levels are not changed when
comparing VU0026921 treatment to VU0026921 plus Mn treatment.
Cotreatment with EDTA decreases the intracellular concentrations of Mn, Cu, Fe, and
Zn compared to VU0026921 treatment alone, suggesting that EDTA competes with
VU0026921 for binding of these metals and further implicating metal uptake as the
mechanism of action by VU0026921 (Fig. S2A and C to E). S. aureus is Mn depleted when
VU0026921 and EDTA are used as cotreatment (Fig. S2A), explaining why the combi-
nation treatment of VU0026921 and EDTA inhibits S. aureus growth so well (Fig. 1E). The
combination of VU0026921 and EDTA is more effective at preventing Zn uptake
(Fig. S2D) than Fe uptake (Fig. S2E). Presumably, this is due to the formation constant
for the EDTA ϩ Zn complex being 158-fold greater than the formation constant for
EDTA ϩ Fe (38). Co levels are not affected by VU0026921 or EDTA (Fig. 3B and Fig. S2B).
Therefore, VU0026921 alters metal homeostasis and leads to accumulation of Zn, Fe,
and Cu in the cell, but Mn accumulation is dependent on excess Mn being present in
the medium.
Mn is used by S. aureus as both a SOD-dependent and SOD-independent antioxi-
dant; therefore, the hypothesis that VU0026921 induces ROS in S. aureus was tested.
VU0026921-induced ROS production was quantified using the compound dihydrorho-
damine 123 (DHR123), which is nonfluorescent until it is oxidized by cellular oxidants.
VU0026921 induces increased ROS in S. aureus and cotreatment with Mn decreases the
amount of ROS generated (Fig. 3F). These data correlate S. aureus ROS levels with
VU0026921 killing of S. aureus and rescue of killing by Mn as observed in Fig. 1D.
Genes involved in Mn import and ROS detoxification protect against
VU0026921 toxicity. Based on the transcriptional profile of S. aureus and the increase
in ROS following treatment with VU0026921, we hypothesized that proteins involved in
maintaining metal homeostasis may contribute to VU0026921 toxicity by modulating
intracellular metal availability. S. aureus strains with mutations in genes related to
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FIG 3 VU0026921 causes metal accumulation and oxidative stress in S. aureus. Metal isotopes 55Mn (A), 59Co (B), 63Cu (C),
56Fe (D), and 66Zn (E) were measured by ICP-MS in S. aureus Newman treated for 30 min with vehicle, 1 mM MnCl2, 100 ␮M
‘921, or 100 ␮M ‘921 ϩ 1 mM MnCl2. Data are mean Ϯ standard deviation normalized to 34S to account for differences in
growth of four biological replicates. Statistical significance was determined by one-way ANOVA where ns ϭ P Ͼ 0.05, * ϭ
P Ͻ 0.05, ** ϭ P Ͻ 0.01, *** ϭ P Ͻ 0.001, and **** ϭ P Ͻ 0.0001. (F) ROS levels in S. aureus cultures treated with vehicle,
50 ␮M ‘921, 1 mM MnCl2, or 50 ␮M ‘921 ϩ 1 mM MnCl2 for 6 h. Data are mean Ϯ standard deviation for six biological
replicates. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test where ** ϭ
P Ͻ 0.01 and **** ϭ P Ͻ 0.0001. (G) CFU recovered following 24-h treatment of mid-exponential-phase cultures of S. aureus
Newman, ΔmntH/C, or ΔsodA/sodM with vehicle, 1 mM MnCl2, 100 ␮M ‘921, or 1 mM Mn ϩ 100 ␮M ‘921. Data are mean Ϯ
standard deviation combined from three independent triplicate experiments, performed on separate days (n ϭ 9).
Statistical significance was determined by one-way ANOVA with Dunnett’s multiple-comparison test where ### ϭ
P Ͻ 0.001 compared to WT vehicle-treated bacteria and ** ϭ P Ͻ 0.01 and *** ϭ P Ͻ 0.001 for the comparisons indicated
by the bars. (H) S. aureus Newman was treated with vehicle, 50 ␮M ‘921, 80 ␮M MitoTEMPO, or 50 ␮M ‘921 ϩ 80 ␮M
MitoTEMPO, and growth was monitored by optical density at 600 nm for 24 h. Data are mean Ϯ standard deviation for six
biological replicates.
antioxidant defenses and Mn import were tested for sensitivity to compound
VU0026921 in a 24-h killing assay. The deletion of genes encoding the ROS-detoxifying
enzymes SodA and SodM enhances susceptibility to VU0026921 in a manner reversed
by excess Mn (Fig. 3G). To further test the role of ROS in VU0026921 killing of S. aureus,
the superoxide scavenger MitoTEMPO (39) was used to deplete VU0026921-generated
ROS. As seen in Fig. 3H, the cotreatment of MitoTEMPO with VU0026921 protects S.
aureus from VU0026921 killing but does not completely rescue the phenotype, indi-
cating that VU0026921 does not kill S. aureus solely through ROS generation, which is
consistent with Fig. 1E, where VU0026921 can kill S. aureus independently of oxygen.
These findings suggest that VU0026921 may act, in part, through generation of
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superoxide and the resulting downstream oxidants, either through direct action by
VU0026921 or through mismetalation that occurs during VU0026921 treatment. More-
over, genetic inactivation of Mn importers MntH and MntC enhances susceptibility to
VU0026921, and this susceptibility is reversed by the addition of excess Mn to the
growth medium (Fig. 3G). These results suggest that an increased ratio of intracellular
Mn relative to other metals protects against VU0026921 toxicity.
Co and Mn protect S. aureus from VU0026921 killing, while Cu exacerbates
killing. VU0026921 increases the uptake of transition metals (Fig. 3A to E), so the
hypothesis that metals added to the medium will alter the killing of S. aureus by
VU0026921 was tested. Neither Fe nor Zn modulates VU0026921 killing (Fig. 4A and B).
Consistent with previous experiments, Mn added to the media protects S. aureus from
VU0026921 killing (Fig. 4C). Co also protects S. aureus from VU0026921-mediated killing,
is more protective than Mn (Fig. 4D), and protects at a much lower molar ratio to
VU0026921 than Mn (Fig. S2F). Co protection, similarly to what was seen with Mn,
decreases intracellular ROS induced by VU0026921 (Fig. 4E), and the protective effects
of Co are not mediated through vitamin B12 (Fig. S2G).
In contrast to Co and Mn, cotreatment with Cu exacerbates S. aureus killing by
VU0026921 (Fig. 4F), which corresponds with the increase in intracellular Cu observed
in VU0026921-treated S. aureus (Fig. 3C). To understand whether Cu export protects
against VU0026921 toxicity, we used an MRSA strain of S. aureus with defined Cu export
systems. USA300 LAC (40) accounts for most MRSA infections in the United States (41).
This S. aureus strain has two systems that protect against Cu toxicity, CopAZ and CopBL
(42). Figure 4H demonstrates that VU0026921 is also growth inhibitory to USA300 LAC
and that deletion of the Cu export genes (Δcop) increases VU0026921 toxicity in the
presence of 10 ␮M Cu, which is not toxic without VU0026921. The Cu in tryptic soy
broth (TSB) alone, approximately 100 nM Cu, is not sufficient to kill the Δcop mutant
when treated with VU0026921. These data suggest that VU0026921-facilitated Cu
uptake is toxic to S. aureus (Fig. 4F). These data may have implications for future
therapies that combine small molecules that induce metal uptake and Cu. Since Cu has
strong mismetalation properties based on the Irving-Williams series and can participate
in Fenton chemistry, the hypothesis that VU0026921-mediated Cu uptake increases
ROS-dependent killing of S. aureus was tested. Cotreatment of Cu with VU0026921
increases ROS compared to VU0026921 treatment alone, correlating with the increased
killing seen under these same conditions (Fig. 4G). Taken together, these data confirm
that VU0026921 alters S. aureus metal homeostasis and imply that its antimicrobial
mechanism relates to the accumulation of intracellular metals and generation of ROS.
Metal binding stabilizes VU0026921. We hypothesized that VU0026921 causes
accumulation of cellular metals by directly binding and promoting metal transport into
the cell. VU0026921 absorbs light between 300 and 400 nm (Fig. 5A). The absorbance
intensity of VU0026921 decreases over time, indicating that the compound is changing
or degrading during the 120 min of this experiment (Fig. 5A). The absorbance spectrum
of VU0026921 shifts to longer wavelengths in the presence of Co, Cu, Fe, Mn, and Zn
(Fig. 5B to F), suggesting that VU0026921 binds these metals. When Cu and VU0026921
are combined, the absorbance shift occurs faster than the plate reader is able to acquire
the initial absorbance scan (Fig. 5C). Additionally, the formation constant for the
EDTA ϩ Cu complex is over 30,000-fold greater than the formation constant for EDTA ϩ
Fe (38), but the decrease in intracellular Cu (Fig. S2C) levels is similar to Fe (Fig. S2D)
when comparing VU0026921 ϩ EDTA to VU0026921, further emphasizing that
VU0026921 and Cu have rapid and strong binding kinetics (Fig. 5C). The binding
kinetics with Mn are considerably slower, and VU0026921 is not completely bound to
Mn at the end of the 120-min experiment (Fig. 5E). Slow binding kinetics may explain
why so much Mn is needed to protect S. aureus from VU0026921 toxicity. The rapid
binding of Cu and the slow binding of Mn with VU0026921 may also explain why
treatment with VU0026921 and Mn does not reduce the amount of Cu (Fig. 3C) taken
up by the cells as it does for Fe and Zn (Fig. 3D and E). Metals that bind VU0026921
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FIG 4 Mn and Co protect S. aureus against VU0026921 toxicity, while Cu exacerbates it. (A to D and F) CFU recovered
following 4-h treatment of S. aureus Newman with vehicle, 100 ␮M ‘921, 100 ␮M FeSO4 (A), ZnCl2 (B), MnCl2 (C), CoCl2
(D), and CuSO4 (F), or 10 ␮M ‘921 ϩ 100 ␮M metal. Data presented are mean Ϯ standard deviation from triplicate
measurements. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test
where ns ϭ P Ͼ 0.05, *** ϭ P Ͻ 0.001, and **** ϭ P Ͻ 0.0001. (E and G) ROS levels in S. aureus cultures treated with
vehicle, 50 ␮M ‘921, 100 ␮M CoCl2 (E) or CuSO4 (G), and 50 ␮M ‘921 ϩ 100 ␮M metal for 6 h. Data are mean Ϯ
standard deviation for six biological replicates. Statistical significance was determined for each condition compared
to ‘921 treatment by one-way ANOVA with Tukey’s multiple-comparison test where *** ϭ P Ͻ 0.001 and **** ϭ
P Ͻ 0.0001. (H) USA300, an MRSA strain, and USA300 ΔcopAZ ΔcopBL (Δcop) were treated with vehicle, 5 ␮M ‘921,
10 ␮M CuSO4, or 5 ␮M ‘921 ϩ 10 ␮M CuSO4, and growth was monitored by optical density at 600 nm for 24 h. Data
are mean Ϯ standard deviation for six biological replicates.
appear to stabilize the molecule, so that the decrease in absorbance intensity seen in
VU0026921 alone is decreased. The divalent cations Ca and Mg, which do not appear
to bind VU0026921, also do not have the stabilizing effects on the molecule seen for
the other metals (Fig. 5G and H). The binding of VU0026921 to these metals as well as
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FIG 5 Binding of divalent transition metals stabilized VU0026921. (A to H) Absorbance was measured
from 280 nm to 600 nm in 10-nm increments of 0 or 100 ␮M ‘921 combined with vehicle (A) or 500 ␮M
CoCl2 (B), CuSO4 (C), FeSO4 (D), MnCl2 (E), ZnCl2 (F), CaCl2 (G), or MgCl2 (H). Absorbance measurements
were taken at 0, 5, 15, 30, 60, or 120 min after the addition of metals. The final absorbance spectra are
‘921 ϩ metal with spectra for metal alone subtracted. Spectra displayed are representative of a single
experiment that was performed three times. The spectrum of ‘921 at 0 min was included in each panel
as a reference of the compound absorbance alone.
the increase in intracellular metal concentrations (Fig. 3A to E) supports the hypothesis
that VU0026921 promotes metal transport into the cell.
Preliminary structure-activity relationship study of VU0026921 reveals func-
tional groups required for toxicity. Five small molecules were synthesized based on
the VU0026921 backbone to determine what structural features are required to support
metal-dependent killing activity and to identify where the molecule can be modified
with affinity tags for target identification and future probe improvement (Fig. 6A).
Additionally, VU0026921 is not stable in TSB (Fig. 5A and Fig. S1B and C), so an active
compound with more medium stability will aid in future studies of small-molecule-
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FIG 6 Chemical features of VU0026921 required for toxicity. (A) Chemical structures of VU0026921 and analogs.
(B) Relative potency of 100 ␮M (each) analog—VU0026921 (‘921), VU0849731 (‘731), VU0849732 (‘732), VU0849730
(‘730), or VU0849729 (‘729)—measured as growth of S. aureus as a percentage of untreated cells at 4 h for each
compound tested at the indicated concentrations. Data are mean Ϯ standard deviation from triplicate measure-
ments. (C) S. aureus Newman was treated with vehicle, 100 ␮M ‘921, or 100 ␮M ‘921 analog, and growth was
monitored by optical density at 600 nm for 24 h. Data are mean Ϯ standard deviation from triplicate measure-
ments. (D) ROS levels in S. aureus cultures treated with vehicle, 50 ␮M ‘921, or 50 ␮M ‘921-2 for 6 h. Data are
mean Ϯ standard deviation for six biological replicates. Statistical significance was determined by one-way ANOVA
with Tukey’s multiple-comparison test where ns ϭ P Ͼ 0.05 and **** ϭ P Ͻ 0.0001.
mediated metal uptake. Trifluoromethyl ketones readily hydrate and serve as Zn-
binding hydroxamic acid replacements in histone deacetylase (HDAC) inhibitors (43);
therefore, we first assessed a methyl ketone replacement for the trifluoromethyl group
of VU0026921 (VU0026921-2). As anticipated, methyl ketone VU0026921 does not
inhibit S. aureus growth (Fig. 6B and C). Additionally, the increase in ROS observed
following the treatment of S. aureus with VU0026921 does not occur in cells treated
with VU0026921-2, correlating ROS levels with the bactericidal ability of these com-
pounds in S. aureus (Fig. 6D). Likewise, substitution of the acyl hydrazide linker for a
hydrazine linker (VU0849731) decreases the antimicrobial activity (Fig. 6B and C),
suggesting that metal binding and/or NH acidity may be important for antimicrobial
activity. The importance of the latter property is further supported by the finding that
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FIG 7 VU0026921 S. aureus killing is not through fatty acid biosynthesis inhibition. (A and B) MIC of
irgasan (A), a FabI inhibitor that halts fatty acid biosynthesis and inhibits S. aureus strain RN4220, and
VU0026921 (B). Irgasan has an increased MIC in S. aureus RN4220 mutants with fatty acid biosynthesis
disabled when cotreated with oleic acid, while ‘921 (B) does not, indicating that ‘921 does not inhibit
fatty acid biosynthesis. Note: no growth is observed for accD mutants in the absence of oleic acid. (C and
D) Platensimycin (C), a FabF inhibitor, has an increased MIC in E. faecalis when cotreated with oleic acid,
while ‘921 (D) does not, suggesting that ‘921 does not inhibit fatty acid biosynthesis in E. faecalis. All data
are mean Ϯ standard deviation for triplicate measurements. Note: 10 ␮g/ml was the highest concen-
tration of platensimycin tested. The actual MIC of platensimycin against E. faecalis in the presence of oleic
acid may be higher.
replacement of the para-bromo group with less electron-withdrawing groups such as
para-amino (VU0849730) or N-acetamido (VU0849729) results in loss of activity (Fig. 6B
and C). In sharp contrast, replacement of the bromo group with an electron-
withdrawing para-nitro group (VU0849732) only mildly impacts antimicrobial activity
(Fig. 6B and C). Together, these data suggest that electron-withdrawing trifluoromethyl
ketone and acyl hydrazide groups across the compound contribute to activity.
VU0026921 does not inhibit fatty acid biosynthesis. Due to structural similarities
between VU0026921 and isoniazid, the hypothesis that the VU0026921 mechanism of
action is through the inhibition of fatty acid biosynthesis was tested. Irgasan, a FabI
inhibitor, was used as a positive-control compound in these studies. In wild-type S.
aureus, inhibition of fatty acid biosynthesis is not reversible by the addition of external
fatty acids (44). However, a mutant in acetyl coenzyme A carboxylase accD both is a
fatty acid auxotroph and is resistant to inhibitors of fatty acid biosynthesis (45) (Fig. 7A).
This increase in MIC is not observed in accD mutants when VU0026921 is cotreated with
oleic acid (Fig. 7B), indicating that VU0026921 does not inhibit S. aureus through the
inhibition of fatty acid biosynthesis. In contrast to S. aureus, fatty acid biosynthesis
inhibition in wild-type Lactobacillales is reversible with fatty acids (45). Addition of oleic
acid reversed the activity of a FabF inhibitor, platensimycin, in Enterococcus faecalis,
increasing its MIC by at least 100-fold (Fig. 7C). However, no MIC increase is observed
when oleic acid is added in combination with VU0026921 (Fig. 7D), indicating that
VU0026921 does not inhibit fatty acid biosynthesis in E. faecalis. In total these data
demonstrate that VU0026921, despite having structural similarities to isoniazid, does
not target the same cellular process as isoniazid.
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FIG 8 VU0026921 is growth inhibitory toward Gram-positive bacteria. (A to F) S. aureus MW2 (A), UAMS1 (B), HG003
(C), 8325-4 (D), RN6390 (E), and SH1000 (F) were treated with vehicle or 5 ␮M or 20 ␮M ‘921, and growth was
monitored by optical density at 600 nm for 24 h. Data are mean Ϯ standard deviation from triplicate measure-
ments. (G and H) The Gram-positive organisms Bacillus anthracis (G) and Micrococcus luteus (H) were treated with
vehicle or 5 ␮M ‘921, and growth was monitored by optical density at 600 nm for 16 h. Data are mean Ϯ standard
deviation from quadruplicate measurements.
VU0026921 kills Gram-positive bacteria. As proof-of-concept that the process of
generating metal toxicity has potential as an antibiotic strategy, we assessed the
antimicrobial activity of VU0026921 across a range of bacterial pathogens. In addition
to inhibiting the growth of methicillin-sensitive S. aureus (Fig. 1), VU0026921 inhibits
the growth of MRSA (Fig. 4H) as well as S. aureus strains MW2 (Fig. 8A), UAMS1 (Fig. 8B),
HG003 (Fig. 8C), 8325-4 (Fig. 8D), RN6390 (Fig. 8E), and SH1000 (Fig. 8F). VU0026921
also inhibits the growth of the Gram-positive organisms E. faecalis (Fig. 7D), Bacillus
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anthracis (Fig. 8G), and Micrococcus luteus (Fig. 8H). B. anthracis is a highly virulent
organism with potential for use as a bioweapon (46) and a rapidly increasing infection
rate resulting from intravenous, illicit drug use (47). M. luteus is often used to screen
compounds for antimicrobial activity (48, 49). VU0026921 does not inhibit the growth
of the Gram-negative species Escherichia coli DH5␣ (Fig. S3A), E. coli MG1655 (Fig. S3C),
Pseudomonas aeruginosa PAO1 (Fig. S3E), P. aeruginosa PA14 (Fig. S3F), and Klebsiella
pneumoniae TOP2 (Fig. S3G). The Gram-negative species E. coli SLO1B (Fig. S3B), E. coli
BL21 (Fig. S3D), and Acinetobacter baumannii 17978 (Fig. S3H) are all slightly inhibited
by VU0026921, although at concentrations more than 10-fold higher than what is
needed to inhibit growth of Gram-positive species. Together, these data suggest that
VU0026921 is far more antimicrobial against Gram-positive than Gram-negative organ-
isms. These results provide preliminary evidence that small molecules can be devel-
oped that target metal toxicity in Gram-positive pathogens.
DISCUSSION
There is significant need to develop antimicrobials to combat drug-resistant bacte-
ria. Here, we demonstrate that intracellular metal homeostasis can be targeted by small
molecules and that perturbation of metal homeostasis by small molecules has antimi-
crobial effects. We propose targeting metal homeostasis as it represents an avenue
toward additional novel antibiotic discovery. We identify VU0026921 as a small mole-
cule toxic to S. aureus and present evidence that VU0026921 disrupts metal homeo-
stasis. Divalent cations modulate the antimicrobial activity and stability in solution of
VU0026921. Exposure of S. aureus to VU0026921 upregulates metal and ROS detoxifi-
cation systems, and ROS levels correlate with VU0026921 toxicity. S. aureus accumulates
metals following incubation with VU0026921. Finally, inactivation of genes involved in
Mn import and utilization enhances S. aureus susceptibility to VU0026921. VU0026921
has antimicrobial activity against all S. aureus strains tested as well as other Gram-
positive organisms. Together, these results establish VU0026921 as a probe for studying
Gram-positive microbial metal metabolism and support the possibility of developing
small-molecule antimicrobials that target metal uptake.
VU0026921 exerts its antimicrobial activity by targeting metal import and homeo-
stasis rather than by acting as an extracellular metal chelator and sequestering metals
from S. aureus. While VU0026921 binds to divalent transition metals and inhibits
bacterial growth in a manner both reversed and exacerbated by the addition of
different metals, properties that would be expected for a metal chelator, it does so in
a nonstoichiometric manner. Furthermore, an extracellular metal chelator would be
expected to inhibit Gram-negative as well as Gram-positive bacteria, but VU0026921
slightly inhibits the growth of some Gram-negative organisms at concentrations more than
10-fold higher than what is needed to inhibit Gram-positive organisms. This result suggests
that penetration into the cell or a specific cellular target is required for the antimicrobial
activity of VU0026921. Finally, ICP-MS results demonstrate that VU0026921 increases cel-
lular metal levels in S. aureus, whereas an extracellular chelator would be expected to
decrease cellular metal levels.
While the exact mechanism of action for VU0026921 remains unknown, we propose
a model of activity wherein VU0026921 causes an influx of metal, leading to metal-
dependent toxicity. This model is supported by the increase in cellular metal levels
following treatment with VU0026921. VU0026921 may potentiate metal influx by
interacting with metal transporters, such as by locking these proteins into an open
conformation. A second possibility is that VU0026921 is a metallophore that binds
metals in growth medium and facilitates transport of these metals into S. aureus. Both
of these models are consistent with the observation that metals present in growth
medium become concentrated in the S. aureus cell following treatment with
VU0026921. Notably, Mn does not accumulate when cells are treated with VU0026921
in TSB alone, potentially due to the slow kinetics of binding between VU0026921 and
Mn compared to other metals tested. The proposed model of VU0026921-mediated
metal influx is also supported by transcriptomic analyses, which reveal that treatment
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with compound leads to upregulation of metal efflux genes and downregulation of
genes encoding metal-binding proteins, the transcriptional pattern expected to restore
metal equilibrium. The additional transcriptional changes observed in the RNA se-
quencing results are most likely secondary effects of the stress experienced by the cell
following dramatic changes in metal levels. We were unable to identify a nonessential
gene required for VU0026921 toxicity in S. aureus or isolate a VU0026921-resistant S.
aureus strain. These findings suggest that VU0026921 has multiple targets within the
cell or has an antimicrobial activity that does not require a specific cellular target, such
as generation of ROS, which directly correlates with killing by VU0026921 and metals.
The question of how intracellular metal accumulation causes cellular toxicity in
VU0026921-treated cells remains unanswered. While this study does not elucidate the
cellular targets of metal-dependent toxicity, one possible mechanism is metal catalyz-
ing the production of ROS, such as through the Fenton and Haber-Weiss reactions (50).
Deletion of sodA and sodM enhanced VU0026921 toxicity, and cotreatment with
MitoTEMPO, a superoxide dismutase mimetic, is partially protective, implicating ROS in
VU0026921 toxicity. One metal that can be toxic to cells through all of the above
mechanisms is Cu. VU0026921 treatment induces Cu uptake by S. aureus and increased
cellular ROS. Inactivation of the S. aureus Cu detoxification systems exacerbates the
toxicity of VU0026921 cotreated with nontoxic Cu levels, indicating a mechanism of Cu
toxicity that proceeds though the accumulation of intracellular Cu.
Mn rescues VU0026921 aerobically, and Mn has been previously described as an
antioxidant and may protect S. aureus from ROS by forming antioxidant small mole-
cules (26, 51, 52), serving as a cofactor for superoxide dismutase (25, 28), or replacing
oxidized Fe in mononuclear enzymes (53). Mn does not protect S. aureus from anaer-
obic VU0026921 toxicity, demonstrating that Mn protects S. aureus from oxygen-
dependent toxicity, such as ROS. A second possible mechanism of metal toxicity is
replacement of cognate metals in metalloenzymes (9, 54, 55). This mechanism may
explain VU0026921-mediated toxicity in anaerobically grown cells and why antioxi-
dants do not completely protect S. aureus. Cotreatment with Mn and VU0026921 causes
massive accumulation of Mn within the S. aureus cell, suggesting that Mn may protect
S. aureus from VU0026921 toxicity by blocking the import of other, more toxic metals.
This is further supported by the decrease in cellular Fe and Zn levels in S. aureus
cotreated with VU0026921 and Mn compared to cells treated with VU0026921 alone.
Exogenous Co protects S. aureus from VU0026921 killing but is not accumulated under
any of the conditions tested by ICP-MS even though Co is present in TSB at similar
levels as Cu. Cellular Cu levels, which are increased upon VU0026921 treatment, do not
decrease like Fe and Zn when 1 mM Mn is cotreated with VU0026921. The addition of
exogenous Mn represents 10,000-fold more Mn than Cu in the medium, which is
insufficient to compete with Cu uptake, which correlates with the rapid binding kinetics
of Cu and slow binding kinetics of Mn with VU0026921.
Co, in contrast to Mn, completely protects S. aureus from VU0026921 killing through
a mechanism that eliminates VU0026921-induced cellular ROS and is independent of
vitamin B12, the major Co-containing molecule in cells. Co also maximally protects S.
aureus from VU0026921 killing at much lower metal/VU0026921 molar ratios. A Co/
VU0026921 molar ratio of 2 gives complete protection, while it takes an Mn/VU0026921
ratio of 50 to get 80% rescue. The near-unity stoichiometry of protection by Co is
consistent with Co binding to VU0026921, preventing other metals from binding.
However, further experiments are necessary to completely understand the role of Co in
protecting S. aureus from VU0026921 killing.
Recent studies emphasizing the importance of metal homeostasis in bacterial
pathogenesis have garnered enthusiasm for developing metal-dependent antimicro-
bials (17–22). Most previous studies emphasized the development of antimicrobials to
restrict metal acquisition by pathogens rather than antimicrobials that cause metal
accumulation and toxicity. However, bacterial pathogens also must balance metal
toxicity during infection. Cefiderocol, a siderophore cephalosporin that increases cel-
lular Fe levels in bacteria, was recently approved for the treatment of Gram-negative
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urinary tract infections, emphasizing that metal uptake can be exploited for antimicro-
bial development (56). There are tissue-specific fitness defects for S. aureus and
Streptococcus pneumoniae strains lacking an Mn efflux system (34, 57); a Helicobacter
pylori strain lacking a Cu, Cd, and Ni efflux pump (58); and several bacterial pathogens
lacking Cu detoxification systems (59). An intriguing possibility raised by this study is
the concept of targeting specific infectious niches where metal toxicity occurs. How-
ever, several key challenges will have to be overcome for VU0026921 derivatives to be
used as effective antimicrobials. First, VU0026921 appears to be unstable in aqueous
medium and rapidly loses activity in solution, and future compound development will
require identifying active analogs with improved stability. Another caveat is that
VU0026921 increases Mn levels in mammalian cells in the presence of high extracellular
Mn levels (23), and any clinical studies must closely investigate the potential for causing
deleterious side effects in the host. Therefore, VU0026921 is best suited as a chemical
probe used to expand our understanding of Gram-positive metal homeostasis, and this
initial work with VU0026921 provides proof-of-concept evidence that metal toxicity can
be exploited as an antimicrobial strategy against Gram-positive pathogens through the
activity of a small molecule.
MATERIALS AND METHODS
Bacterial strains and reagents. The strains used in this study are described in Table S6 in the
supplemental material. S. aureus strains were grown in tryptic soy broth (TSB); M. luteus, E. coli, P.
aeruginosa, A. baumannii, and K. pneumoniae strains were grown in lysogeny broth (LB); and B. anthracis
was grown in brain heart infusion broth (BHI). All cultures were grown aerobically at 37°C with 180-rpm
shaking. Solid medium contained 1.5% agar. For all assays, bacterial strains were streaked from Ϫ80°C
stocks 2 days prior to the assay. Overnight cultures were grown for 12 to 15 h in 5 ml medium in 15-ml
conical tubes.
Creation of USA300 ⌬copAZ ⌬copBL. JMB1100 chromosomal DNA was used as a template for PCRs.
Escherichia coli PX5␣ (Protein Express) was used as a cloning host and for plasmid propagation. Plasmids
were isolated and transformed into S. aureus strain RN4220 (60) using a standard protocol (61), and
assays were conducted using phage 80␣ (62). All strains were verified by PCR and/or sequencing.
Construction of plasmids for mutant generation was done using yeast homologous recombination
cloning as previously outlined (63, 64). The following primer pairs were used to create the amplicons
necessary to make pJB38_ΔcopAZ: Ycc Pjb38 for and YCC CopAZ rev; CopAZ Up for and CopAZ up rev;
CopAZ dwn for and pJB38 CopAZ rev. The amplified PCR fragments were combined with EcoRI-linearized
pJB38 plasmid and transformed into Saccharomyces cerevisiae FY2. Mutant strains were constructed
using the pJB38 allelic exchange as described previously (65). The ΔcopAZ ΔcopBL strain (cop-) was
created using the ΔcopBL mutant (JMB7901) (ΔSAUSA300_0078-0079) (42) and pJB38_ΔcopAZ.
Compound acquisition and storage. The compounds in the 39-compound toolbox were purchased
from Chemical Diversity (San Diego, CA) and stored as 10 mM stocks in dimethyl sulfoxide (DMSO) at
Ϫ80°C. Compound VU0026921 was repurchased three times and confirmed to exert similar activity in
each batch. Additionally, the structural integrity of purchased VU0026921 was confirmed by nuclear
magnetic resonance (NMR). Finally, VU0026921 and VU0026921 analogs were resynthesized by the
Vanderbilt Chemical Synthesis Core and used to confirm antimicrobial activity and Mn rescue.
Compound preparation. (i) 4-Bromo-N=-(2-(2,2,2-trifluoroacetyl)cyclopent-1-en-1-yl)benzo-
hydrazide (VU0026921). To
a solution of 2-(2,2,2-trifluoroacetyl)cyclopentan-1-one (0.562 ml,
4.65 mmol) in ethanol (10 ml) was added a solution of 4-bromobenzohydrazide (1.0 g, 4.65 mmol) in
ethanol (12 ml) dropwise. The reaction mixture was stirred at room temperature for 1 h, solvent was
removed in vacuo, and the residue was recrystallized (ethanol) to afford 792 mg (74%) of the derived
hydrazone as a white solid: 1H NMR (deuterated methanol [MeOD], 400 MHz) ␦ (ppm) 7.71 (d, J ϭ 8.4 Hz,
2H), 7.59 (d, J ϭ 8.4 Hz, 2H), 2.79 (t, J ϭ 7.2 Hz, 2H), 2.70 (t, J ϭ 7.6 Hz, 2H), 1.92 (t, J ϭ 7.2 Hz, 2H); liquid
chromatography-mass spectrometry (LC-MS) (electrospray ionization [ESI]) tR: 1.09 min (Ͼ99%, evapo-
rative light scattering detection [ELSD]), m/z: 377.0 [MϩH]ϩ.
(ii) 2,2,2-Trifluoro-1-(2-(2-(4-nitrophenyl)hydrazinyl)cyclopent-1-en-1-yl)ethan-1-one (VU0849731).
1H NMR (MeOD, 400 MHz) ␦ (ppm) 8.19 (d, J ϭ 9.2 Hz, 2H), 6.96 (d, J ϭ 9.2 Hz, 2H), 2.82 to 2.72 (m, 4H), 2.04
to 1.94 (m, 2H).
(iii) 4-Nitro-N=-(2-(2,2,2-trifluoroacetyl)cyclopent-1-en-1-yl)benzohydrazide (VU0849732). 1H
NMR (MeOD, 400 MHz) ␦ (ppm) 8.38 (d, J ϭ 8.8 Hz, 2H), 8.11 (d, J ϭ 8.8 Hz, 2H), 2.85 to 2.78 (m, 4H), 1.99
(q, J ϭ 7.6 Hz, 2H); LC-MS (ESI) tR: 1.01 min (Ͼ99%, ELSD), m/z: 344.0 [MϩH]ϩ.
(iv) 4-Amino-N=-(2-(2,2,2-trifluoroacetyl)cyclopent-1-en-1-yl)benzohydrazide (VU0849730). To
a solution of 4-nitro-N=-(2-(2,2,2-trifluoroacetyl)cyclopent-1-en-1-yl)benzohydrazide (50 mg, 0.15 mmol)
in ethyl acetate (EtOAc), followed by tin chloride (142 mg, 0.75 mmol). After stirring for 4 h at 80°C,
reaction mixture was poured into NaOH solution (2 ml, 2 M), yellow product was recrystallized from
methanol (MeOH) (33 mg, 70%). 1H NMR (DMSO, 400 MHz) ␦ (ppm) 7.67 (d, J ϭ 8.8 Hz, 2H), 6.53 (d,
J ϭ 8.8 Hz, 2H), 5.87 (s, -NH2), 2.81 to 2.76 (m, 2H), 1.99 to 1.91 (m, 2H), 1.28 to 1.11 (m, 2H); LC-MS (ESI)
tR: 0.83 min (Ͼ99%, ELSD), m/z: 314.1 [MϩH]ϩ.
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Juttukonda et al.
(v) N-(4-(2-(2-(2,2,2-Trifluoroacetyl)cyclopent-1-en-1-yl)hydrazine-1-carbonyl)phenyl) acetamide
(VU0849729). To
a
solution of 4-amino-N=-(2-(2,2,2-trifluoroacetyl)cyclopent-1-en-1-yl)benzohydrazide
(55 mg, 0.176 mmol) at 0°C in dichloromethane (10 ml) was added acetic anhydride (33 ␮l, 0.35 mmol). The
reaction mixture was stirred at room temperature for 16 h, quenched with saturated NaHCO3, and extracted
with dichloromethane (3 ϫ 20 ml). Organic extracts were combined, dried over MgSO4, and concentrated in
vacuo. The residue was purified by column chromatography using dichloromethane-methanol (Combi-flash
Rf, 0% to 10% MeOH gradient) to afford yellow solid (44 mg, 72%). 1H NMR (DMSO, 400 MHz) ␦ (ppm) 10.22
(s, -NH), 7.94 (d, J ϭ 8.8 Hz, 2H), 7.67 (d, J ϭ 8.8 Hz, 2H), 2.85 to 2.80 (m, 2H), 2.60 to 2.45 (m, 2H), 2.08 (s, 3H),
2.01 (t, J ϭ 8.0 Hz, 2H); LC-MS (ESI) tR: 0.87 min (Ͼ99%, ELSD), m/z: 356.1 [MϩH]ϩ.
Antimicrobial growth assays. All growth curves were carried out in the rich growth medium
described for each strain above. For each growth curve, bacterial strains were streaked from Ϫ80°C
stocks 2 days prior to the assay. Overnight cultures were treated at stationary phase or grown for 12 to
15 h in 5 ml of medium in 15-ml conical tubes, and 1 ␮l of overnight culture was added to 100 ␮l of
medium in a round-bottom 96-well plate (Corning, Corning, NY). MnCl2 (Thermo Fisher, Waltham, MA, or
Sigma Millipore, St. Louis, MO) was prepared as a 100 mM stock in deionized water, sterile filtered, and
stored at room temperature. Individual components of the growth assay were added to the 96-well plate
in the following order: growth medium, growth medium containing additional Mn, and growth medium
containing compound. Immediately after adding compound, cultures were added and optical density
absorbance at 600 nm (OD600) at the zero-time point was established. Ninety-six-well plates were grown
at 37°C in a shaking incubator and removed periodically for OD600 measurements with a plate reader
(BioTek, Winooski, VT). Alternatively, kinetic reads were carried out in the plate reader at 37°C with linear
shaking.
VU0026921 compound stability in medium. VU0026921 was diluted to 50 ␮M or 3 ␮M in TSB and
incubated shaking at 37°C for 0, 4, 8, or 24 h. After incubation in TSB, kinetic growth curves were
performed in S. aureus Newman as described above.
Growth curve VU0026921 killing assays. Overnight cultures of S. aureus strain Newman were
diluted 1:100 into 96-well plates containing TSB, MnCl2, and VU0026921 at the indicated concentrations
and grown at 37°C in a shaking incubator. At 0, 2, 4, 6, and 8 h postinoculation, 5 ␮l of culture was
removed, serially diluted in sterile phosphate-buffered saline (PBS; Corning, Corning, NY), and spot plated
onto tryptic soy agar (TSA). CFU were calculated by dilution factor per 1 ml of culture.
Bacterial killing assays. Overnight cultures of S. aureus were treated at stationary phase or diluted
1:100 into 5 ml TSB in 15-ml conical tubes and incubated at 37°C in a shaking incubator until cultures
reached OD600 of 0.4. Mid-exponential cultures were pelleted at 3,220 ϫ g for 10 min, and pellets were
resuspended in 2.5 ml TSB. TSB, 25 ␮l of resuspended culture, MnCl2, and VU0026921 were added to a
final volume of 100 ␮l and final concentrations of 1 mM for MnCl2 and 100 ␮M for ‘921. For Fig. 1E, EDTA
(Millipore Sigma, St. Louis, MO) was added to a final concentration of 0 or 1 mM. For Fig. 4A to E, MnCl2,
ZnCl2 (Millipore Sigma, St. Louis, MO), CoCl2 (Thermo Fisher, Waltham, MA), CuSO4 (Millipore Sigma, St.
Louis, MO), or FeSO4 (Millipore Sigma, St. Louis, MO) was added to a final concentration of 100 ␮M. After
incubation at 37°C for the indicated lengths of time, 10 ␮l of culture was removed, serially diluted in
sterile PBS, and spot plated onto TSA for CFU enumeration.
Anaerobic growth curves and killing assays. For experiments performed under anaerobic condi-
tions, an anaerobic chamber was employed (Coy Laboratory Products, Grass Lake, MI). S. aureus Newman
WT was streaked onto TSA and grown aerobically for 24 h at 37°C. Cultures were started from single
colonies in 5 ml of anaerobic TSB and grown at 37°C for 15 h. For growth curves, overnight cultures were
subcultured 1:100 into 100 ␮l of anaerobic TSB containing 0 or 1 mM MnCl2 and 0, 1.25, 5, 10, or 20 ␮M
VU0026921 in a round-bottom 96-well plate (Corning, Corning, NY) covered with a Breathe-Easy
gas-permeable seal (Sigma, St. Louis, MO). Growth was measured by optical density over time in a BioTek
Synergy H1. For killing assays, overnight cultures were diluted 1:100 into 100 ␮l anaerobic TSB in a
round-bottom 96-well plate and incubated at 37°C with shaking until cultures reached OD600 of 0.35.
Sterile water or MnCl2 to a final concentration of 100 ␮M or 1 mM was added as well as sterile DMSO or
VU0026921 to a final concentration of 100 ␮M. After anaerobic incubation at 37°C with shaking for 24 h,
5 ␮l of culture was removed, serially diluted in sterile PBS, and spot plated onto TSA for CFU
enumeration.
Growth for transcriptome sequencing and RNA isolation. Overnight cultures of S. aureus strain
Newman were diluted 1:100 into 5 ml TSB in 15-ml conical tubes and grown to mid-exponential
phase (OD600 ϭ 0.4). Cells were pelleted at 3,220 ϫ g for 10 min at room temperature, supernatants
were discarded, and cells were resuspended in 2.5 ml TSB. TSB, VU0026921 or vehicle control, Mn or
vehicle control, and 2 ml of resuspended bacterial culture were added to a 15-ml conical tube to a
final volume of 8 ml and final concentration of 0 or 100 ␮M VU0026921 and 0 or 1 mM MnCl2.
Following incubation at 37°C with 180-rpm shaking for 30 min, 10-␮l aliquots were removed for CFU
plating and cells were harvested by centrifugation at 6,000 ϫ g for 10 min at 4°C. Pellets were
resuspended in LETS buffer (0.1 M LiCl, 10 mM EDTA, 10 mM Tris HCl, pH 7.4, 1% SDS), homogenized in
a bead beater (Fastprep-24; MP Biomedical, Santa Ana, CA) with Lysing Matrix B beads (MP Biomedical,
Santa Ana, CA) at a speed of 6 m/s for 45 s, heated at 55°C for 5 min, and centrifuged for 10 min at
15,000 rpm. The upper phase was collected, mixed with 1 ml TRI Reagent (Sigma, St. Louis, MO), and
incubated for 5 min at room temperature. Chloroform (0.2 ml, Acros Organics, Waltham, MA) was added,
samples were vigorously shaken for 15 s, and samples were incubated at room temperature for 2 min.
Following centrifugation at 4°C for 15 min, 600 ␮l of the upper aqueous phase was collected, and RNA
was precipitated with 1 ml isopropanol (Sigma, St. Louis, MO). RNA was washed with 70% ethanol (Sigma,
St. Louis, MO) and resuspended in 100 ␮l DNase-free, RNase-free water (Thermo Fisher, Waltham, MA).
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DNA contamination was removed by treatment with 8 ␮l RQ1 enzyme (Promega, Madison, WI), 12 ␮l
10ϫ RQ1 buffer, and 2 ␮l RiboLock RNase inhibitor (Thermo Fisher, Waltham, MA) for 2 h at 37°C. DNase
was removed, samples were further purified by the RNeasy kit (Qiagen, Hilden, Germany) using the
manufacturer’s instructions, and RNA was stored long-term at Ϫ80°C.
RNA sequencing. RNA sequencing was performed by Vanderbilt Technologies for Advanced Genom-
ics Core Facility (VANTAGE). RNA sequencing was performed on three biological replicates per condition.
Total RNA quality was assessed using the 2100 Bioanalyzer (Agilent). At least 200 ng of DNase-treated
total RNA with an RNA integrity number greater than 6 was used for rRNA depletion and generation of
cDNA libraries via the ScriptSeq complete (bacterial) kit (Illumina/Epicentre) following the manufacturer’s
protocol. Library quality was assessed using the 2100 Bioanalyzer (Agilent), and libraries were quantitated
using KAPA library quantification kits (KAPA Biosystems). Pooled libraries were subjected to 75-bp
paired-end sequencing according to the manufacturer’s protocol (Illumina HiSeq3000). Bcl2fastq2 con-
version software (Illumina) was used to generate demultiplexed Fastq files.
RNA sequencing analysis. RNA sequencing data were trimmed to remove all bases below Q3 and
had adapter sequence removed, using FaQCs (66). Trimmed sequences were aligned to the Staphylo-
coccus aureus subsp. aureus strain Newman reference genome (accession number GCF_000010465.1)
using BWA (67) and default alignment options. Read counts were generated using HT-Seq v0.6.1p1 (68)
and default options. The EdgeR (69) package for R was used to analyze count files to identify differential
gene expression (DGE). Read counts per gene were also analyzed and visualized using the Degust public
server, Version 2.1, degust.erc.monash.edu. Raw read CSV files were uploaded to the server, analysis
performed with the built-in Voom/Limma settings, the FDR cutoff modified to Ͻ 0.01, and the log fold
change cutoff changed to 2. COG designations for individual genes were manually curated from
microbesonline.org.
Inductively coupled plasma mass spectrometry. Four biological replicate overnight cultures of S.
aureus Newman were diluted 1:100 into 5 ml TSB in 15 ml conical tubes and grown to mid-
exponential phase for 3 h at 37°C with 180-rpm shaking. Cells were pelleted at 3,220 ϫ g for 10 min
at room temperature, supernatants were discarded, and cells were resuspended in 1 ml TSB
containing 100 ␮M VU0026921, 1 mM MnCl2, 100 ␮M EDTA, 100 ␮M VU0026921 ϩ 1 mM MnCl2, or
100 ␮M VU0026921 ϩ 100 ␮M EDTA. Following incubation at 37°C with 180-rpm shaking for 30 min, cells
were harvested by centrifugation at 3,220 ϫ g for 10 min at 4°C and washed with 10 ml PBS (Corning,
Corning, NY). Pellets were digested in 200 ␮l HNO3 (Optima grade metal-free; Fisher, Waltham, MA) and
50 ␮l hydrogen peroxide (ultratrace grade; Sigma) at 65°C overnight. Before analysis, 2 ml of UltraPure
water was added to each sample. Elemental quantification of acid-digested samples was performed
using an Agilent 7700 inductively coupled plasma mass spectrometer (Agilent, Santa Clara, CA) attached
to a Teledyne CETAC Technologies ASX-560 autosampler (Teledyne CETAC Technologies, Omaha, NE).
The following settings were fixed for the analysis: cell entrance ϭ Ϫ40 V, cell exit ϭ Ϫ60 V, plate bias ϭ
Ϫ60 V, OctP bias ϭ Ϫ18 V, and collision cell helium flow ϭ 4.5 ml/min. Optimal voltages for Extract 2,
Omega Bias, Omega Lens, OctP RF, and Deflect were determined empirically before each sample set was
analyzed. Element calibration curves were generated using Aristar ICP standard mix (VWR, Radnor, PA).
Samples were introduced by peristaltic pump with 0.5-mm-internal-diameter tubing through a MicroMist
borosilicate glass nebulizer (Agilent). Samples were initially up taken at 0.5 revolutions per second (rps)
for 30 s followed by 30 s at 0.1 rps to stabilize the signal. Samples were analyzed in spectrum mode at
0.1 rps, collecting three points across each peak and performing three replicates of 100 sweeps for each
element analyzed. Sampling probe and tubing were rinsed for 20 s at 0.5 rps with 2% nitric acid between
every sample. Levels of 34S, 59Co, 63Cu, 66Zn, 55Mn, and 56Fe were measured, concentrations were
determined utilizing a standard curve for each metal, and results were normalized by the concentration
of 34S. Data were acquired and analyzed using the Agilent Mass Hunter workstation software version
A.01.02.
Reactive oxygen species quantification with dihydrorhodamine 123. S. aureus Newman over-
night cultures were diluted 10-fold into 150 ␮l TSB in black-walled 96-well plates containing (Fig. 3F)
50 ␮M DHR123 (Thermo Fisher, Waltham, MA) and vehicle, 50 ␮M ‘921, 1 mM MnCl2, or 50 ␮M
‘921 ϩ 1 mM MnCl2 or (Fig. 4E and G) vehicle, 50 ␮M ‘921, 100 ␮M CoCl2 or CuSO4, or 50 ␮M
‘921 ϩ 100 ␮M CoCl2 or CuSO4. Plates were incubated with shaking linearly at 37°C for 8 h on a BioTek
Cytation 5 (BioTek, Winooski, VT). Every 30 min, the optical density was measured at 600 nm to determine
bacterial growth and DHR123 fluorescence (excitation ϭ 507 nm, emission ϭ 529 nm) was measured to
determine cellular ROS levels. ROS levels were normalized to OD600 to account for differences in S. aureus
viability.
Spectrophotometric determination of VU0026921 metal binding. Zero or 100 ␮M ‘921 was
combined with 0 or 500 ␮M CoCl2, CuSO4, MgCl2, CaCl2, MnCl2, FeSO4, or ZnCl2 in 200 ␮l water at 25°C.
Absorbance spectra were taken from 280 nm to 600 nm in 10-nm increments on a BioTek Cytation 5 at
0, 5, 15, 30, 60, and 120 min following plating. Final spectra are metal spectra alone subtracted from
‘921 ϩ metal spectra.
Fatty acid biosynthesis inhibition experiments. Approximately 105 CFU of indicated bacteria were
inoculated into 1 ml liquid medium (TSB) containing 2-fold dilutions of indicated antibiotics with or
without oleic acid (18:1) supplemented at 40 ␮g/ml (S. aureus) or 10 ␮g/ml (E. faecalis) and incubated 20
h with shaking overnight in 13- by 100-mm glass test tubes at 37°C. The lowest dilution of antibiotics
preventing visible growth of S. aureus was recorded as the MIC.
Statistical analyses. All raw numerical data were saved in Excel files and imported into GraphPad
Prism for statistical analysis. All graphs are the result of a single representative experiment performed in
biological triplicate, and all experiments were repeated to confirm results.
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Data availability. The data received from RNA sequencing experiments have been deposited into
the NCBI Gene Expression Omnibus (accession no. GSE128062). The portion of the data set which
compares S. aureus treated with and without 1 mM MnCl2 has been previously published (34) and is also
accessible under GEO accession number GSE124285.
SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
FIG S1, TIF file, 2.7 MB.
FIG S2, TIF file, 2.7 MB.
FIG S3, TIF file, 2.7 MB.
TABLE S1, XLSX file, 0.1 MB.
TABLE S2, XLSX file, 0.04 MB.
TABLE S3, XLSX file, 0.03 MB.
TABLE S4, XLSX file, 0.01 MB.
TABLE S5, XLSX file, 0.01 MB.
TABLE S6, XLSX file, 0.01 MB.
TABLE S7, XLSX file, 0.01 MB.
ACKNOWLEDGMENTS
We thank members of the Skaar laboratory for reviewing the manuscript.
Work in E.P.S.’s laboratory was supported by Public Health Service grants AI101171,
AI069233, R01 AI138581, AI150701, and AI073843 and the Defense Advanced Research
Projects Agency (DARPA). L.J.J. was supported by American Heart Association grant
15PRE25060007 and Public Health Service award T32 GM07347 from the National
Institute of General Medical Studies for the Vanderbilt Medical-Scientist Training Pro-
gram. L.J.J. was a P.E.O. Scholar. W.N.B. was supported by American Heart Association
grant 18POST34030426. Work in A.B.B.’s laboratory was supported by NIH ES010563,
and in addition K.J.H. was supported by T32 ES007028. Work in J.M.B.’s laboratory was
supported by grants 1R01AI139100-01 from the NIH, a CAREER award from the National
Science Foundation, and multistate research fund project NE1028 from the USDA. S. aureus
transposon mutants were provided by the Network on Antimicrobial Resistance in Staph-
ylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH: Nebraska Trans-
poson Mutant Library (NTML) Genetic Toolbox, NR-48850. Staphylococcus aureus 8325-4
was obtained through the Network of Antimicrobial Resistance in S. aureus (NARSA)
program supported under NIAID/NIG contract no. HHSN272200700055C.
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