Gallium(III)-Salophen as a Dual Inhibitor of Pseudomonas aeruginosa Heme Sensing and Iron Acquisition

Article abstract of DOI:10.1021/acsinfecdis.0c00138

Pseudomonas aeruginosa is an opportunistic bacterium that causes life-Threatening infections in immunocompromised patients. In infection, it uses heme as a primary iron source and senses the availability of exogenous heme through the heme assimilation sys

Full text of DOI:10.1021/acsinfecdis.0c00138

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Article
Gallium (III) Salophen as a Dual Inhibitor of Pseudomonas
aeruginosa Heme Sensing and Iron Acquisition
Garrick Centola, Daniel Deredge, Kellie Hom, Yong Ai, Alecia Dent, Fengtian Xue, and Angela Wilks
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.0c00138 • Publication Date (Web): 17 Jun 2020
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Gallium (III) Salophen as a Dual Inhibitor of Pseudomonas aeruginosa Heme Sensing and
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Iron Acquisition
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Garrick Centola, Daniel J. Deredge, Kellie Hom, Yong Ai, Alecia T. Dent, Fengtian Xue*,
Angela Wilks*
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Author Affiliations: Department of Pharmaceutical Sciences, University of Maryland School of
Pharmacy. Baltimore, Maryland 21201.
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Pseudomonas aeruginosa is an opportunistic bacterium that causes life-threatening infections in
immunocompromised patients. In infection, it uses heme as a primary iron source and senses the
availability of exogenous heme through the heme assimilation system (Has), an extra cytoplasmic
function (ECF) -factor system. A secreted hemophore HasAp scavenges heme and, on
interaction with the outer-membrane receptor HasR, activates a signaling cascade which in turn
creates a positive feedback loop critical for sensing and adaptation within the host. The ability to
sense and respond to heme as an iron source contributes to virulence. Consequently, the inhibition
of this system will lead to a disruption in iron homeostasis, decreasing virulence. We have
identified a salophen scaffold that successfully inhibits the activation of the Has signaling system
while simultaneously targeting iron uptake via xenosiderophore receptors. We propose this dual
mechanism wherein free Ga3+-salophen reduces growth through uptake and iron mimicry. A dual
mechanism targeting extracellular heme signaling and uptake together with Ga3+-induced toxicity
following active Ga3+salophen uptake provides a significant therapeutic advantage while reducing
the propensity to develop resistance.
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Keywords. Antimicrobials, heme sensing, heme uptake, metallotherapeutics, Pseudomonas
aeruginosa
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Multidrug-resistant Pseudomonas aeruginosa has been classified by the CDC as a “serious
threat” to public health owing to its ability to overcome many current treatment strategies.1 It
frequently infects immunocompromised patients and is a leading cause of nosocomial infections.2–
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It is particularly problematic for cystic fibrosis (CF) patients, who suffer from life-threatening
chronic infection where even direct delivery of the antibiotic tobramycin to the lung is becoming
ineffective.3,4 While new antibiotics are entering the pipeline, resistance often emerges within a
few years of introduction as many candidates in Phase I-III trials are typically improved versions
of existing inhibitors rather than new classes of antibiotics and further have minimal activity
against P. aeruginosa.5,6
Recently, the development of antivirulence agents that reduce the ability of bacteria to
colonize or spread has gained traction with the hope that such strategies will slow resistance
development by exerting less selective evolutionary pressure.7 The regulation of many virulence
factors is dependent on iron acquisition from the host, which also serves as a necessary nutrient
for survival.8–11 Host iron levels are kept tightly regulated, minimizing the availability of labile
iron in circulation for bacterial consumption.12 This innate immune response often referred to as
“nutritional immunity” aims to keep the availability of essential nutrients at concentrations below
what would permit colonization and growth.
Though P. aeruginosa and many other pathogens secrete iron-chelating siderophores, the
low abundance of labile iron shifts the preference to heme, which is more abundant in a host.13–15
Furthermore, chronic infections in CF patients show evolution towards heme as a preferred iron
source along with a decrease in production of pyoverdine, a high-affinity siderophore.16,17 We
have previously characterized the non-redundant heme uptake systems in P. aeruginosa, which
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involve a primary heme transporter Phu (Pseudomonas heme uptake) system and the heme sensing
Has (Heme assimilation system) system.18 While the Phu system is the predominant uptake
transporter, the Has system relies on the secretion of an extracellular hemophore, HasAp, to deliver
exogenous heme through the outer-membrane receptor HasR. The release of heme to HasR in turn
activates the ECF -factor HasI that binds to the has promoter recruiting RNA polymerase and
upregulating the has operon.19,20 These recent studies have further shown that the heme metabolite
biliverdin IXβ (BVIX β) positively regulates HasAp translation providing a positive feedback loop
that is highly tunable and rapidly responsive to fluctuating extracellular heme levels.
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The complex transcriptional and post-transcriptional regulation over the has system is
consistent with its significance in infection where it has been shown to be highly upregulated in
transcriptomic analysis of an acute mouse lung infection (~300-fold for hasAp, ~70-fold for
hasR).21 Furthermore, deletion of hasR significantly reduced bacterial load, highlighting the
importance of heme sensing and adaptation in infection and suggesting that HasAp is an attractive
therapeutic target. Targeting the Has system combines the advantages of disrupting heme
acquisition while inhibiting the ability to sense the extracellular environment. Indirectly, the
reduction in heme uptake caused by inhibition of the heme signaling cascade will lead to lower
levels of BVIXβ and further repress the heme signaling ability.19,20 Moreover, HasAp is an
extracellular target that circumvents typical roadblocks of penetrating gram-negative membranes
and multidrug efflux pumps that enhance antibiotic resistance.22,23
Though structural work has extensively characterized HasAp, the HasAp/HasR interaction
is still under investigation which complicates the development of molecules targeting the protein-
protein interaction.24–29 Alternatively, recent reports have demonstrated that synthetic iron
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complexes bind in the heme site of HasAp and are coordinated by the same residues, opening the
door to heme mimicry as a targeting strategy.30,31 Of note is the N,N’-disalicylal-1,2-
phenylenediamine (“salophen”) complex, which is synthetically modular and more soluble than
many of the larger macrocycles. The use of metallosalophen complexes has also been investigated
for potential anticancer applications, establishing their bioactivity as metallotherapeutics.32–34
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The design of antimicrobials that inhibit heme sensing and iron uptake have a significant
advantage over traditional strategies targeting solely iron uptake. Presently, strategies aimed at
targeting iron uptake have shown gallium to be an effective iron mimic due to its similar size to
iron and redox-inactivity.35–40 Currently, formulations of gallium including the FDA-approved
Ganite (Ga(NO3)3) are in clinical trials for efficacy as antibiotics, though reports of gallium
inducing virulence factor production have also emerged.41–43 Since the binding of the
metallosalophens to HasAp has been confirmed, we sought to further characterize the salophen-
HasAp complex and its effect on the Has system in P. aeruginosa and found that both Fe3+-
salophen (FeSal) and Ga3+-salophen (GaSal) complexes inhibited transcriptional activation of the
has operon. The use of GaSal also inhibited growth as a result of uptake as a xenosiderophore
through a currently unidentified receptor. Therefore, GaSal inhibits heme sensing while
simultaneously causing toxicity following active uptake through the siderophore receptors. This
dual mechanism of action has several advantages not the least of which is the reduced propensity
to develop resistance. We hypothesize inhibiting heme sensing and intracellular iron homeostasis
will ultimately lead to dysregulation of related metabolic and virulence pathways.
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Results
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Salophen Complexes Bind to HasAp
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Following reports that several synthetic iron complexes with heme-like features bind to
HasAp in the heme-binding site and that larger macrocycles such as phthalocyanine (Pc) could be
transported through HasR, we identified the o-phenylenediaminesalicylaldehyde (“salophen”)
scaffold for its synthetic accessibility and potential for derivatization.30,31 The salophen complexes
were reported to bind through the same coordination and in the heme-binding site, including
overlaps of the pyrrole rings of the porphyrin with the aromatic rings of the salophen.30 We
synthesized the FeSal and GaSal analogs, characterized their binding affinities, and found that
although salophen binds more weakly than heme, the metal substitution from Fe3+ to Ga3+ has less
of an impact on affinity than switching the coordinating scaffold from protoporphyrin IX to
salophen (Table 1).
Table 1. Binding Affinities of Ligands to HasAp as Determined by Fluorescence Quenching
Compound
KD (nM, ± SD)
heme
GaPPIX
FeSal
350 ± 50
280 ± 30
1050 ± 100
2050 ± 280
GaSal
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FeSal Inhibits Has-dependent Heme Signaling and Is a Substrate for Iron Uptake
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FeSal was previously reported as a heme uptake inhibitor.30 To test whether FeSal would
affect the activation of the Has signaling cascade and further study the mechanism, we employed
a β-galactosidase (β-Gal) transcriptional reporter assay previously constructed by our lab wherein
activation of the HasR promoter following ligand release to HasR would lead to a colorimetric
readout.20 P. aeruginosa PAO1 hasR-lacZ was supplemented with heme-HasAp or FeSal-HasAp
(1 μM) and aliquots were harvested and assayed for β-Gal activity during log-phase to observe
maximal transcriptional activation of the Has system. Further, as the signaling activation is
dependent on HasAp levels, the preformed ligand-HasAp complexes were used rather than wait
for natively expressed HasAp to accumulate in the extracellular media, leading to a more effective
observation of the transcriptional response. Cultures supplemented with FeSal-HasAp showed
decreased signaling activation (Figure 1A), but had no effect on growth, indicating that FeSal
could be used as an iron source (Figure 1B). Previous characterization of the Has signaling cascade
revealed that transcriptional activation requires heme release to HasR and that apo-HasAp, despite
its ability to interact with HasR, does not initiate the signaling cascade.20 The FeSal-HasAp
complex had improved overall growth relative to the unbound salophen ligand, most likely due to
increased bioavailability or solubility (Figure 1B). We next tested growth in the presence of either
FeSal or FeSal-HasAp on the PAO1 hasRphuR strain that lacks both heme receptors and
observed no effect on growth whereas supplementation with heme, or holo-HasAp is highly
dependent on the presence of HasR and PhuR (Figure 1B, Figure S2). The lack of transport
through the heme receptors, suggests that the FeSal scaffold is recognized as a xenosiderophore
and taken in through such receptors. It is not uncommon for bacteria to scavenge iron-siderophores
they do not secrete, as such xenosiderophore acquisition or piracy provides a viable survival and
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virulence trait.44 For instance, the recent FDA-approved iron-chelating cephalosporin, Cefiderocol
is taken up by the P. aeruginosa ferric iron transport systems though it is linked to an artificial
iron-chelating group and not any native or exogenous siderophore.45
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Figure 1. A) Transcriptional Activation of the hasR Promoter with Holo- and FeSal-HasAp. FeSal
shows decreased transcriptional activation relative to heme. Both cultures were supplemented
with pre-formed HasAp complexes and data represent the average of three independent
experiments*, p < 0.05; **, p < 0.005 by t-test between holo and FeSal-HasAp. B) Growth of
PAO1 Wild-Type and the hasRphuR Strain with FeSal and FeSal-HasAp. Cultures were grown
in a 96-well plate with 1 µM supplements as described in the experimental section.
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GaSal Inhibits Has Signaling and Growth
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Figure 2. A) Transcriptional Activation of the hasR Promoter with Holo- and GaSal-HasAp.
GaSal shows decreased transcriptional activation relative to heme. Both cultures were
supplemented with pre-formed HasAp complexes and represent the average of three independent
experiments. *, p < 0.05; **, p < 0.005 by t-test between holo and GaSal-HasAp. B) GaSal
antibacterial activity in PAO1WT and hasRphuR strain. Data represent the average of three
independent cultures with blanks (media only) subtracted. Cultures were grown in M9 minimal
media with 1 µM supplements. C) HasAp titration alleviates GaSal toxicity in PAO1 ΔhasAp.
Cultures were prepared in M9 minimal media with FeCl3 (400 nM) and GaSal (7 μM), with HasAp
supplemented at increasing concentrations. Growth was calculated relative to gallium-free
cultures in stationary phase growth and data represent the average of three independent exeriments
*, p < 0.05; **, p < 0.005 by t-test between 0 µM HasAp and specified concentration.
The salophen complex was then synthesized to include a gallium metal center to prevent
its utility as an iron source and increase inhibitory potential based on the utility of gallium salts
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and nanoparticles as antibacterial agents.46,47 We tested GaSal-HasAp and found similar inhibition
profile for has transcriptional activation relative to that of holo-HasAp complex (Figure 2A). In
contrast to FeSal, GaSal was inhibitory to growth when added on its own as well as when
complexed with HasAp (Figure 2B). To determine if GaSal toxicity is independent of the signaling
cascade and results from active uptake by a siderophore-dependent mechanism, we employed the
P. aeruginosa ΔhasAp strain that does not produce HasAp protein. As the maximum OD600 of
the P. aeruginosa strains in iron-deplete media (M9 alone) is low, we measured the effect of GaSal
inhibition in cultures supplemented with 400 nM FeCl3. This concentration was selected as it
yields a maximum OD600 midway between that observed for growth in iron-deplete or iron-
replete conditions (Figure S3). Under these conditions of partial iron-restriction, the concentration
of GaSal required to inhibit growth by 50% was 7 μM (Figure S3). We hypothesized that if gallium
toxicity is due to uptake of unbound GaSal, rather than Ga stripped by endogenous siderophores,
then titrating HasAp to GaSal supplemented P. aeruginosa ΔhasAp cultures would alleviate the
inhibition of growth. Employing the previously optimized conditions, P. aeruginosa ΔhasAp
cultures containing 400 nM FeCl3 and 7 μM GaSal were supplemented with increasing
concentrations of HasAp protein. As shown in Figure 2C, increasing HasAp concentrations
alleviated the toxicity of GaSal, confirming that GaSal is transported intact into the cell when not
bound to HasAp. The formation of the GaSal-HasAp complex in the extracellular medium was
also confirmed by Saturation Transfer Difference (STD)-NMR (Figure 3). We further confirmed
that native siderophores are not stripping the metals from the salophen using a mutant strain that
lacks the gene for pyoverdine biosynthesis (Figure S4). As pyoverdine has an incredibly high
affinity for ferric iron, the sustained growth in the siderophore-deficient strain and no prevention
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of toxicity with GaSal shows that pyoverdine, and by extension pyochelin, are not responsible for
metal uptake.48
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Figure 3. A) 1H NMR of GaSal in M9 Minimal Media. Proton peaks were assigned and labeled
as depicted for comparison in the STD spectrum. B) STD NMR using GaSal (1 mM) and HasAp
(10 μM) in PAO1 ΔhasAp supernatant.
In the STD experiment, a selective saturation pulse is delivered exclusively to HasAp and
the saturation distributes throughout the protein by intramolecular spin diffusion. The saturation
is transferred to any bound ligands, which are detected upon dissociation from the protein. In the
1
resultant difference spectrum, only ligand H peaks receiving saturation from the protein are
observed and their intensities are proportional to their proximity to the protein, which is a useful
observation of ligand binding epitopes.49 The top spectrum (Figure 3A) is a 1H-NMR spectrum of
GaSal in M9 minimal media to serve as a reference spectrum for the STD experiment. The bottom
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spectrum (Figure 3B) is GaSal in PAO1 ΔhasAp supernatant with added HasAp. This result shows
saturation transfer to GaSal and confirms formation of the GaSal-HasAp complex in the
extracellular media. Protons from the diamine ring (a and b) buried in the back of the heme binding
site are clearly represented. Protons e and f are also distinguishable and could be useful sites to
build outward from the salophen core to enhance binding affinity as they are pointed away from
the binding site in the crystal structure of FeSal-HasAp.30 The planarity and symmetry of GaSal
also enhances the STD signals and permits binding from either face of the molecule. While these
experiments show that GaSal binds to HasAp, they do not explicitly show the binding site. Based
on the binding of GaPPIX and GaPc in combination with the crystallized FeSal-HasAp structure,
GaSal is likely coordinated by the same ligand set in the heme binding site.30,31
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Has Cell-Surface Signaling is Scaffold-Dependent
Figure 4. A) Transcriptional Activation of the hasR Promoter with Holo- and GaPPIX-HasAp.
GaPPIX shows increased (2 h) or similar transcriptional activation to heme. Cultures were
supplemented with pre-formed HasAp complexes and data represent the average of three
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independent experiments. **, p < 0.005 by t-test between holo and GaPPIX-HasAp. B) Growth
of PAO1 Supplemented with either 1 µM Heme or GaPPIX. Supplementation with GaPPIX
inhibits growth in M9 Minimal Media. Cultures were grown in a 96-well plate as described in the
methods section.
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Gallium protoporphyrin IX (GaPPIX) is a common heme mimic with antibacterial
properties owing to the redox-inactive nature of Ga3+ versus Fe3+.37,38,50 However, unlike GaSal,
GaPPIX activates the Has signaling cascade despite causing toxicity to cultures (Figure 4). This
result confirms that, even though GaPPIX is toxic, the protoporphyrin IX scaffold permits
signaling and transport through HasR, which limits the potential of GaPPIX as a therapeutic
strategy as the signal activation will likely overcome initial toxicity and increase HasAp levels
while exerting selective pressure towards siderophore-based iron acquisition to circumvent
GaPPIX uptake. A gallium-based strategy that targets iron-uptake pathways while inhibiting heme
sensing is therefore less likely to develop resistance. The use of the salophen scaffold for signaling
inhibition not observed with the porphyrin scaffold is therefore a starting point for further
development but also for understanding the mechanism of signaling activation between HasAp
and HasR.
As the signaling inhibition is dependent on the scaffold and not the metal, we sought to test
whether GaSal-HasAp prevented interaction with HasR and thus signaling. As signaling activation
requires a protein-protein interaction, conformational changes, ligand transfer and energy-
dependent uptake, we recognize that any in vitro method is not a physiological characterization of
the HasAp/HasR interaction but rather a useful determination of the relative associations of the
HasAp complexes with HasR. We measured the binding affinities of holo-, apo-, and GaSal-
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HasAp to HasR by surface plasmon resonance (SPR) and found that the holo- and apo-HasAp bind
HasR with similar affinity (Table 2). In contrast the binding of GaSal-HasAp to HasR was weaker
although we could only estimate the KD value to be greater than 1 M as at higher concentrations
we observed non-specific binding most likely originating from heterogeneity introduced as a
consequence of aggregation of the HasR lipid nanodiscs. The KD values were determined by
steady-state association measurements at various analyte concentrations (Figure S6). This result
indicates that the HasAp/HasR interaction, while weaker for the GaSal-HasAp complex, is not
completely inhibited and suggests the nature of the scaffold is critical in facilitating the correct
protein-protein interaction for ligand release and transcriptional activation.
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Table 2. Binding Affinities of HasAp to HasR as Measured by SPR.
Ligand
KD (nM, ± SD)
Apo-HasAp
Holo-HasAp
GaSal-HasAp
810 ± 50
479 ± 20
>1000
Structural Studies of the Salophen-HasAp Complex Reveal Differences in Solution
Conformations that May Affect Signaling
The salophen-HasAp effectively inhibits the activation of the Has signaling cascade
without completely inhibiting the binding of HasAp to HasR, indicating that there must be
differences in ligand release that prevent the transfer and uptake. We next sought to investigate if
differences in the solution conformations of HasAp could lead to the inability to initiate ligand
transfer despite nearly superimposable crystal structures. HasAp undergoes a large conformational
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rearrangement of the H32 loop upon heme binding (Figure 5A, RMSD between apo and holo =
5.7 Å, PDB 3MOK and 3ELL).51 In the FeSal-HasAp crystal structure, this loop is also in the
closed position and reveals less significant differences in the overall fold between holo-HasAp and
the FeSal-HasAp complexes (Figure 5B, RMSD = 0.81 Å, PDB ID 3ELL and 3W8M).30 We
expect that if the GaSal-HasAp is more conformationally flexible relative to holo-HasAp and is
otherwise identical to FeSal-HasAp structurally, then these differences may be responsible for
impaired ligand release despite maintained HasR binding. We analyzed the solution structures of
apo-, holo-, and GaSal-HasAp by HDX-MS. Differences in deuterium uptake over time are
reflective of how readily backbone amide hydrogens of a protein exchange with the solvent
hydrogens or deuterons in a manner dependent on structure, solvent-exposure and flexibility. We
have previously used this technique to describe ligand induced structural and dynamic changes of
the heme binding protein PhuS and HemO.52,53 Here, heme binding resulted in significant
protection from deuteration throughout the heme binding site, consistent with loop closure and
decreased solvent exposure (Figure 5C). Similar protection of the heme site was observed upon
GaSal binding further suggesting that GaSal binds to HasAp in the heme site and is coordinated
with the same ligands (Figure 5D). As GaSal-HasAp is not crystallized, we have used the FeSal-
HasAp structure as a representative example for mapping structural changes and visualizing the
bound ligand.
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Figure 5. Conformational changes of HasAp upon ligand binding. A) Alignment of apo, holo and
FeSal-HasAp highlighting closure of the H32 loop from the apo (orange, PDB 3MOK) to the holo
(blue, PDB 3ELL) and FeSal-HasAp (red, PDB 3W8M) forms. B) Alignment of holo and FeSal-
HasAp highlighting the closed-loop conformation with minimal structural differences. Relative
Deuteration of HasAp bound to heme (C) and GaSal (D). Individual peptides are plotted from N
to C-terminus based on first residue number. For each peptide, differences in percent deuteration
at each time point are color coded according to the legend with the sum of all differences integrated
over time are represented in gray bars. 98% confidence intervals are represented as dashed (for
individual time points) and solid (for total sums) lines. Peptides exceeding both confidence
intervals were considered to display a statistically significant difference in deuterium uptake
between the apo and ligand bound form and were mapped on to the crystal structure of holo-HasAp
(panel C, inset; PDB 3ELL). Positive values indicate protection from deuteration upon ligand
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binding and negative values indicate increased deuteration of a region upon ligand binding.
Significant regions are highlighted and color coded on the HasAp structure (panel C, inset).
In contrast with the crystallographic comparison of FeSal-HasAp and holo-HasAp, the H32
and Y75 loops contained the greatest differences in deuterium uptake between the heme-bound
and GaSal-bound states (Figure 6A). On the H32 loop, the GaSal binding increased protection
from deuteration relative to the apo form, but not at strongly as heme binding (Figure 6B). GaSal
is a smaller scaffold that does not have as extensive interactions with the protein, particularly those
between the backbone of the H32 loop and heme (Figure 6C). Notably, in the heme bound crystal
structure, the His loop is observed to engage in several possible interactions with the porphyrin,
including hydrogen bonds between the amide hydrogens of P34 and G35 with the heme
propionates, or the V37 carbonyl with the heme methyl groups that are absent in the salophen-
bound complex. The lack of these interactions and increased flexibility of the heme site do not
prevent HasR binding, but, combined with the structural differences in the salophen ligand, likely
hinder the initiation of ligand release and uptake through HasR.
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Figure 6. Deuteration differences between ligand-bound forms. A) Percent Deuteration of GaSal-
HasAp minus holo-HasAp. Positive peaks represent greater deuteration in the salophen-bound
form, negative peaks indicate increased deuteration in the heme-bound form. Inset: representation
of protein regions that are different between all three HasAp states, using FeSal-HasAp as a
representation of GaSal-HasAp. B) Deuteration of a Peptide Comprising Residues 26-54. C)
Heme binding site with heme (blue) and FeSal (red) overlaid. Potential contacts between the H32
loop and heme are drawn in red.
To assess whether this observation carried a thermodynamic consequence, we measured
the melting temperatures of the three HasAp complexes by circular dichroism. All three HasAp
o
spectra show similar degrees of folding at 25 C, indicating that the overall structure is not
significantly impacted by ligand binding (Figure 7A). The melting temperature of the apo form
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(58 C) was significantly increased by heme binding where the protein only began to unfold at
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temperatures >90 oC (Figure 7B). HasAp bound to GaSal still showed increased thermal stability
(65 oC), but not nearly to the extent on heme binding. Taken together the HDX-MS and CD data
indicate significant differences between the holo and GaSal-HasAp complexes not reflected in the
FeSal-HasAp crystal structure that may be responsible for the lack of signaling. These differences
in solution carry physiological consequences beyond inhibiting signaling, wherein the decreased
stability of the GaSal-HasAp compared to holo-HasAp may increase susceptibility to proteolytic
cleavage in the host.
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Figure 7. Thermal denaturation of HasAp bound to heme or GaSal. A) Comparison of three
ligand states at 25 oC. B) Thermal denaturation profiles at 222 nm fit to a sigmoidal distribution.
CD spectra were recorded as described in materials and methods.
Discussion
As an extracellular protein, HasAp must be secreted in high amounts to counteract both
diffusion from the site of infection and host defense mechanisms such as proteolysis to effectively
scavenge heme. Its relative abundance and role in virulence coupled with the advantage of an
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extracellular target make HasAp an attractive therapeutic target that can lead to the dysregulation
of heme sensing and host adaptation. Though GaPPIX is a potent redox-inactive heme mimic that
enters the cell through the Has and Phu systems where it is causes toxicity, the levels of
transcriptional activation of the Has system are not decreased. This will lead to a downstream
increase in HasAp levels which will, over time, counteract the inhibitory effect and lead to
increased heme-scavenging abilities. Additionally, the lack of iron obtained through this pathway
will also lead to adaptation towards siderophore-based iron acquisition as the heme systems lead
to gallium toxicity, limiting the potential of GaPPIX as a long-term therapeutic option.
Structurally, GaPPIX is identical to heme, which also increases the potential for off-target effects
and inhibition of human heme binding proteins such as heme oxygenase, highlighting a need for
new scaffolds with greater selectivity towards HasAp.54
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Previous heme mimetic approaches reported that FeSal and FePhthalocyanine (FePc) were
inhibitors of heme uptake when cultures were not supplied with holo-HasAp as an iron source.30
However, in the presence of holo-HasAp, only FePc was inhibitory, suggesting that the larger
phthalocyanine macrocycle blocks HasR and inhibits heme uptake. More recent studies showed
that GaPc activity results from active transport through HasR and not inhibition of heme uptake,
as previously described.31 Based on these conflicting reports, we sought to revisit the mechanism
of inhibition by the salophen complexes as potential therapeutics due to their synthetic accessibility
and modularity. While the salophen scaffold binds to HasAp, unlike the PPIX scaffold it is less
likely that these molecules will be generic inhibitors of heme proteins, given the significance of
both the extensive hydrophobic and salt bridge interactions afforded by the propionate groups of
PPIX.55,56
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We found that, unlike GaPPIX, both FeSal and GaSal inhibited the activation of the cell
surface signaling cascade. We have previously demonstrated that heme-dependent activation of
the signaling cascade requires the release of heme from HasAp to HasR.20 The lack of signaling
suggests that FeSal and GaSal are either not released to HasR or, if they are released, do not induce
the requisite conformational change in the HasR signaling domain required to trigger the cascade
or to be transported. We also found that the metallosalophens retained activity in the ΔhasRΔphuR
mutant, indicating that the uptake is independent of the heme receptors. As metal chelates, the
salophen complexes are likely a substrate for xenosiderophore receptors expressed by P.
aeruginosa to utilize siderophores from other bacteria, a strategy that has also been used to develop
siderophore-drug conjugates.44,57,58 The development of Cefiderocol, an FDA-approved iron-
chelating cephalosporin that differs from typical siderophore-drug conjugates, was found to be
transported despite containing no natural siderophore and only included iron-chelating features
linked to a cephalosporin antibiotic.44,45 The current data would support a similar mechanism of
uptake that will be explored in future studies. As GaSal displayed antimicrobial activity consistent
with previous reports of gallium-based therapies, this strategy conveniently combines the use of
gallium salts and a porphyrin mimetic into a single compound that targets both iron and heme
pathways without leading to the upregulation of the heme sensing system.41,59
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We further characterized the mechanism through which GaSal is inhibitory to better
understand what features could be explored in later metallosalophen derivatives. While the
salophen complexes have weaker binding affinities than the PPIX counterparts, we found while
GaSal-HasAp has a weaker binding affinity than holo-HasAp to HasR, the interaction was not
completely inhibited. To further investigate the weaker binding and decreased transcriptional
activation by the metallosalophen complexes, we studied the HasAp complexes by HDX MS.
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These experiments showed increased conformational flexibility of the H32 loop with GaSal-
HasAp relative to holo-HasAp, which is consistent with the loss of stabilizing interactions with
the PPIX macrocycle.25 The lack of these interactions is also consistent with the decreased binding
affinity of the metallosalophens relative to their PPIX counterparts. The increased flexibility of
the H32 loop may therefore prevent HasR binding in a manner that triggers the conformational
change and concerted ligand release. The increased flexibility of the loop is also reflected in the
lower Tm values. Since the melting temperature of GaSal-HasAp is also much closer to apo-HasAp
than holo-HasAp, GaSal binding leads to a less-stable and more proteolytically susceptible HasAp
complex, further diminishing the ability of P. aeruginosa to acquire and sense heme.
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With the utility of GaSal, we envision that future derivatives will have increased HasAp
affinity. New designs could also extend molecules anchored to the heme site into the HasAp/HasR
interaction region with the goal of blocking binding to HasR entirely. Conveniently, approaches
that either block the HasR interaction entirely or prevent ligand release from HasAp are likely to
have merit. Based on our findings, the development of an inhibitor that is transported through
HasR should be avoided as this will lead to the activation of the signaling cascade. Additionally,
these inhibitors should maintain affinity to iron uptake pathways to not target the heme sensing
pathway exclusively, which would be overcome by adaptation to iron acquisition.
Conclusion
Various gallium formulations have been employed for antimicrobial applications. The use
of GaSal represents a more specific approach that targets the extracellular hemophore HasAp and
the heme-sensing ability of P. aeruginosa in addition to targeting iron uptake for antimicrobial
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activity. The biophysical consequences of salophen binding versus heme build on the structural
characterization of the Has system previously reported. This work provides a platform for the
development of new heme mimics that can target bacterial hemophore signaling as well as iron
uptake. These dual mechanisms have the propensity to slow the development of resistance as the
simultaneous evolution of mutations in separate pathways is likely to lead to reduced fitness and
survival of the bacteria. Furthermore, that these mechanisms are critical for infection but not
survival outside of the host further lowers selective pressure for the development of resistance.
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Methods
Bacterial Strains
Pseudomonas aeruginosa (PAO1 and mutants) strains were stored as glycerol stocks in LB at -80
°C and were freshly streaked on Pseudomonas isolation agar (BD Biosciences) before transferring
to liquid culture medium. PAO1 (wild type) was used as reported.60 PAO1 LacZ fusions and
hasAp deletion strains were constructed as described previously.20 The heme receptor deletion
strain (PAO1 ΔhasRΔphuR) was constructed as reported.18 The pyoverdine biosynthesis deletion
strain (PAO1 ΔpvdA) was constructed as reported.17
Expression and Purification of WT HasAp
HasAp was prepared from freshly transformed E. coli BL21(DE3) competent cells harboring the
pET11a plasmid with the full-length hasAp gene as previously described with an additional
purification step.20 Briefly, a single colony was cultured for 16 h in LB medium (50 mL)
containing 100 μg/mL ampicillin. The cells were harvested by centrifugation, resuspended in M9
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media, and divided evenly among 4 1-L cultures in M9 containing 100 μg/mL ampicillin and
grown to an A600 of ~1.0. The cells were once again pelleted and resuspended in 4 fresh 1-liter M9
cultures and induced with 1 mM final concentration of isopropyl-β-D-thiogalactopyranoside and
grown for 16 h at 30 °C. Cells were harvested by centrifugation and resuspended in 40 mL of lysis
buffer (20 mM Tris-HCl (pH 7.5), 20 mM NaCl, 1 mM EDTA) containing a protease inhibitor
cocktail tablet (cOmpleteTM EDTA-Free, Roche Applied Science), 1 mg/mL DNase, and 25
mg/mL lysozyme and passed through an LM-20 microfluidizer at 20,000 p.s.i. The suspension
was centrifuged at 25,000 rpm for 1 h to separate the cell debris. The supernatant was applied to
a Q-Sepharose column (2.6 × 10 cm) pre-equilibrated with 20 mM Tris-HCl (pH 7.5) and 20 mM
NaCl). The column was washed (3-5 column volumes) with buffer and the protein eluted over a
gradient from 20 to 600 mM NaCl in 20 mM Tris-HCl (pH 7.5). The purity of the eluted fractions
was determined by SDS-PAGE, and those containing HasAp were pooled. To separate the apo-
protein, the pooled fractions were concentrated to ~5 mL and exchanged into 50 mM sodium
phosphate buffer with 0.7 M ammonium sulfate (pH 7.0). The concentrate was loaded onto a Butyl
Sepharose Fast Flow (GE Healthcare, 2.6 × 10 cm) column equilibrated with the same buffer.
Weakly bound contaminants and holo-HasAp were eluted with 2-3 bed volumes of 50 mM sodium
phosphate containing 0.5 M ammonium sulfate. Apo-HasAp was then eluted with a linear gradient
of sodium phosphate (50 to 20 mM) containing ammonium sulfate (0.5 to 0 M). Fractions were
once again analyzed by SDS-PAGE and the apo-HasAp was pooled, concentrated (Spin-X UF 10k
MWCO, Corning) and exchanged into 20 mM sodium phosphate buffer.
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Expression and Purification of HasR
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HasR was prepared as reported with modifications to prepare the protein in lipid discs.1 Briefly,
a single colony of freshly transformed E. coli BL21 (DE3) cells harboring the pHasR22b plasmid
was selected to inoculate 50 mL of noninducing MDAG-135 media containing 100 μg/mL Amp
and grown overnight at 37 °C and 225 rpm. This culture was used to inoculate 4 1-L flasks of
autoinducing MDA-5052 media containing 100 μg/mL Amp and grown for 10 h at 25 °C. Cells
were harvested by centrifugation for 15 min at 7,000 rpm at 4 °C.
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Pellets were resuspended in 40 mL lysis buffer and passed through an LM-20
microfluidizer at 20,000 p.s.i. Debris was removed by centrifugation at 12,000 rpm for 15 min
and the supernatant was centrifuged for 1 h at 25,000 rpm to pellet the cellular membranes.
Pelleted membranes were resuspended in 30 mL lysis buffer with an added EDTA-free protease
inhibitor tablet overnight. The cytoplasmic membrane proteins were solubilized by addition of
2% (v/v) Triton X-100 (Sigma) and 0.5% (v/v) N-Lauroylsarcosine sodium salt (Sigma). The
membrane fractions were stirred at room temperature for 1 h and pelleted at 25,000 rpm for 1 h at
4 °C. The resulting supernatant containing only the cytoplasmic membrane proteins was
discarded. The pelleted OM fraction was then resuspended in 30 mL of lysis buffer containing an
EDTA-free protease inhibitor cocktail tablet and stirred at 4 °C overnight.
The RC DC protein assay (Bio-Rad) was used to determine total protein concentration.
The OM fragments were diluted to at least 10 mg/mL final concentration. The styrene-maleic acid
copolymer (Xiran SL30010 P20) was then added to the OM suspension to a final concentration of
2.5% (v/v) and inverted continuously at r.t. for 1 h. The suspension was frozen in liquid nitrogen
and thawed at 42 oC a total of 5 times followed by passage through a microfluidizer at 20,000 p.s.i.
This cycle was then repeated once more. The final suspension was centrifuged at 25,000 rpm for
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1 h at 4 oC and the supernatant containing the HasR in lipid nanodisc (HasR-smalp) was collected.
The supernatant was concentrated and the HasR concentration was determined using the extinction
coefficient of 126 mM-1 cm-1 after subtracting the blank containing the filtrate (to account for
absorption of residual lipids and polymer).
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Chemical Synthesis
All the reagents and solvents were purchased from commercial sources and used as received,
unless otherwise stated. The 1H and 13C NMR spectra were obtained in DMSOd6 on a 400 MHz
spectrometer with chemical shifts referenced to tetramethylsilane (TMS). Purity (>95%, HPLC)
of FeSal and GaSal were confirmed by HPLC as only singular peaks were detected.
Synthesis of 2,2'-((1E,1'E)-(1,2-phenylenebis(azaneylylidene))bis(methaneylylidene))diphenol
(Salophen). O-phenylenediamine (5.4 g, 50 mmol) was dissolved in dichloromethane (100 mL).
To the resulting solution was added salicylaldehyde (12.2 g, 100 mmol). The mixture was stirred
at room temperature for 8 h before removing solvent via rotary evaporation, to afford an orange
solid (10.5 g, 99%). 1H NMR (400 MHz, DMSOd6): δ 12.94 (s, 2 H), 8.941 (s, 2 H), 7.68-7.66 (d,
2 H), 7.48-7.40 (m, 6 H), 7.00-6.95 (t, 4 H).61
Synthesis of FeSal Chloride. To a solution of salophen (0.316 g, 1 mmol) in ethanol (3 mL) was
o
added ferric chloride (0.16 g, 1 mmol). The reaction mixture was heated at 65 C in a sealed
pressure tube overnight. A black precipitate was washed with cold ether and collected (70 mg,
17%). NMR peaks broadened beyond detection due to paramagnetic effects of iron. HRMS (ESI)
m/z: [M -Cl]+ Calc’d for C20H14N2O2Fe 370.0405; Found 370.0443.
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Synthesis of GaSalophen Nitrate. To a solution of salophen (0.316 g, 1 mmol) in ethanol (10 mL)
was added Gallium (III) nitrate hydrate (0.274 g, 1 mmol). The reaction mixture was refluxed for
2 h and cooled to room temperature. The final product was collected by filtration and washed with
1
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ethanol and diethyl ether to afford yellow crystals (0.2 g, 50%). H NMR (400 MHz, DMSOd6):
δ 9.37 (s, 2 H), 8.16-8.13 (m, 2 H), 7.68-7.66 (dd, 2 H), 7.66-7.51 (m, 4 H), 7.02-6.99 (d, 2 H),
6.90-6.86 (t, 2 H). 13C NMR (400 MHz, DMSOd6): δ 167.9, 162.9, 137, 136.6, 135.4, 129, 122.3,
118.3, 117.1, 117. HRMS (ESI) m/z: [M -NO3]+ Calc’d for C20H14N2O2Ga 383.0311; Found
383.0301.
Preparation of HasAp Complexes
Heme and GaPPIX were purchased from Frontier Scientific. Heme solutions were prepared as
described previously immediately before use and concentrations determined via pyridine
hemochrome assay.62 GaPPIX solutions were prepared identically, except concentrations were
determined using the extinction coefficient at 404 nm (90.6 mM cm-1). Apo-HasAp was prepared
o
at a desired concentration in 20 mM sodium phosphate buffer (pH 7.4, 25 C). To this solution
was added a 3-fold molar excess of ligand prepared in the same buffer solution. Excess unbound
ligand was removed using 7K MWCO centrifugal desalting columns (ZebaTM Spin, Thermo Fisher
Scientific) according to manufacturer’s recommendations.
Binding Affinity Determination by Fluorescence Quenching
Binding affinities were determined on an ISS K2 multifrequency fluorometer in L-format using 1
cm quartz cuvettes. To a 1 µM solution of apo-HasAp in 20 mM sodium phosphate buffer (pH
7.4, 25 oC) was titrated ligand of interest. Emission spectra were recorded (300-500 nm) following
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excitation at 295 nm. The decrease in maximum emission was plotted against the ligand
concentration (accounting for dilution) and fit to one-site binding using GraphPad Prism 8.
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P. aeruginosa strains were grown overnight in 50 mL LB Broth with shaking at 37 oC. Cells were
harvested by centrifugation and resuspended in M9 minimal medium. Strains were then inoculated
at A600 = 0.05 in 10 mL M9 minimal medium and grown for 3 h to deplete bacterial iron stores.20
Cultures were then plated in 96-well plates (Costar clear, flat-bottom with lid, Corning Inc) at A600
≈ 0.05 to a final volume of 200 μL in M9 medium. Supplements (2 µL) were added as 100× stock
solutions. Growth curves were recorded at 600 nm with a Biotek SynergyTM HT plate reader over
16 h of growth with shaking at 37 oC. To account for background absorbance, blank wells were
prepared with the same supplements but only M9 medium without culture. The blank wells were
subtracted from the average of three growth wells per condition.
Transcriptional Reporter Assay
The chromosomal fusions of the PhasR-lacZ and promoterless lacZ gene were previously
constructed and used to report on transcriptional activation of the Has signaling cascade.20 For all
β-gal assays, strains were grown overnight in 50 mL LB broth with shaking at 37 oC. Cells were
harvested by centrifugation and resuspended in M9 media. Strains were then inoculated at A600
=
0.05 in triplicate in 25 mL of M9 minimal medium and cultured for 3 h to deplete bacterial iron
stores.20 Growth cultures were then supplemented with 1 µM of either holo-HasAp, GaPPIX-
HasAp, FeSal-HasAp or GaSal-HasAp. Aliquots (1 mL) at 0 (just prior to supplementation), 2
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ACS Infectious Diseases
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and 5 h post supplementation were harvested and assayed for β-gal activity as described
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previously.63
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Circular Dichroism
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Circular dichroism experiments were performed on a Jasco J-810 spectropolarimeter using 1 mm
quartz cuvettes. All samples were recorded with 10 μM HasAp in 10 mM potassium phosphate
(pH 7.4) at 25 °C from 190 to 260 nm at a scan rate of 50 nm/min, with each spectrum representing
5 accumulations. Data were acquired at 0.2-mm resolution and 10 nm bandwidth. The mean
residue ellipticity (degrees cm2 dmol-1) was calculated using CDPRO software as recommended
by Jasco. Thermal denaturation studies were performed over a temperature range of 20–90 °C at
222 nm. Melting temperatures were determined by fitting denaturation data to a Boltzmann-
Sigmoidal distribution in GraphPad Prism 8.
Saturation Transfer Difference NMR
PAO1 ΔhasAp was cultured overnight from a single colony in LB Broth. The cells were harvested
by centrifugation and resuspended in M9 Minimal Media. 10 mL of M9 were inoculated at A600
~ 0.05 and grown for 3 h. Cell-free supernatant was obtained by centrifugation and subsequent
filter-sterilization by a 0.22 µM syringe filter. Supernatant was then used as the solvent for
subsequent STD experiments. Final sample volume contained 600 µL of supernatant with or
without 10 µM HasAp (as a negative control) and 5% D2O for solvent locking.
The STD experiments were performed at 25 oC on an Agilent DD2 500 MHz spectrometer.
The vendor supplied pulse sequences, dpfgse_satxfer.c was used, the on- and off-resonance fids
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