Enterobactin- And salmochelin-β-lactam conjugates induce cell morphologies consistent with inhibition of penicillin-binding proteins in uropathogenicEscherichia coliCFT073

Article abstract of DOI:10.1039/d0sc04337k

The design and synthesis of narrow-spectrum antibiotics that target a specific bacterial strain, species, or group of species is a promising strategy for treating bacterial infections when the causative agent is known. In this work, we report the synthesi

Full text of DOI:10.1039/d0sc04337k

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Enterobactin- and salmochelin-b-lactam
conjugates induce cell morphologies consistent
with inhibition of penicillin-binding proteins in
uropathogenic Escherichia coli CFT073†
Cite this: Chem. Sci., 2021, 12, 4041
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
a
a
bcd
Artur Sargun,
Timothy C. Johnstone,
Hui Zhi,b Manuela Raffatellu
a
*
and Elizabeth M. Nolan
The design and synthesis of narrow-spectrum antibiotics that target a specific bacterial strain, species, or
group of species is a promising strategy for treating bacterial infections when the causative agent is
known. In this work, we report the synthesis and evaluation of four new siderophore-b-lactam
conjugates where the broad-spectrum b-lactam antibiotics cephalexin (Lex) and meropenem (Mem) are
covalently attached to either enterobactin (Ent) or diglucosylated Ent (DGE) via a stable polyethylene
glycol (PEG3) linker. These siderophore-b-lactam conjugates showed enhanced minimum inhibitory
concentrations against Escherichia coli compared to the parent antibiotics. Uptake studies with
uropathogenic E. coli CFT073 demonstrated that the DGE-b-lactams target the pathogen-associated
catecholate siderophore receptor IroN. A comparative analysis of siderophore-b-lactams harboring
ampicillin (Amp), Lex and Mem indicated that the DGE-Mem conjugate is advantageous because it
targets IroN and exhibits low minimum inhibitory concentrations, fast time-kill kinetics, and enhanced
stability to serine b-lactamases. Phase-contrast and fluorescence imaging of E. coli treated with the
siderophore-b-lactam conjugates revealed cellular morphologies consistent with the inhibition of
penicillin-binding proteins PBP3 (Ent/DGE-Amp/Lex) and PBP2 (Ent/DGE-Mem). Overall, this work
illuminates the uptake and cell-killing activity of Ent- and DGE-b-lactam conjugates against E. coli and
supports that native siderophore scaffolds provide the opportunity for narrowing the activity spectrum of
antibiotics in clinical use and targeting pathogenicity.
Received 7th August 2020
Accepted 31st December 2020
DOI: 10.1039/d0sc04337k
rsc.li/chemical-science
infections such as those caused by Clostridioides difficile.5,6
Coupled with the scarcity of new antibiotics in the drug pipe-
Introduction
line, society is faced with the reality that bacterial infections
that were once of minor concern can become lethal.7 To address
this emerging public health crisis, novel therapies that exhibit
narrow-spectrum activity must be developed and implemented
in combination with rapid diagnostics.8–11 Such pathogen-
selective antibiotics are predicted to reduce the occurrence of
antibiotic resistance in microbial populations and have smaller
impact on the host microbiome.
Infections caused by Gram-negative bacteria can be difficult to
treat due to the semipermeable outer-membrane (OM) that
serves as an efficient barrier against most antibiotics.1,2 The
overuse of broad-spectrum antibiotics in the treatment of such
bacterial infections facilitates the selection of resistant strains,
which causes antibiotics to lose effectiveness over time.3,4
Moreover, broad-spectrum antibiotics can damage the
commensal microbiota and trigger life-threatening secondary
To date, various strategies of narrowing the activity spectrum
of antibiotics in clinical use have been explored.9,12–14 In this
regard, essential nutrient transporters located in the OM of
Gram-negative bacteria provide opportunities for selective
recognition and intracellular delivery of antibacterial mole-
cules.15,16 Iron is an essential nutrient for the vast majority of
bacterial species; thus, acquiring adequate levels of iron is
important for survival and host colonization.17–19 To scavenge
iron from the host, many bacteria biosynthesize small-molecule
Fe3+ chelators called siderophores. These secondary metabo-
lites are biosynthesized in the cytoplasm, exported to the
aDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, MA
02139, USA. E-mail: lnolan@mit.edu; Tel: +1-617-452-2495
bDivision of Host-Microbe Systems and Therapeutics, Department of Pediatrics,
University of California San Diego, La Jolla, CA 92093, USA
cCenter for Microbiome Innovation, University of California San Diego, La Jolla, CA
92093, USA
dChiba University-UC San Diego Center for Mucosal Immunology, Allergy, and
Vaccines, La Jolla, CA 92093, USA
† Electronic supplementary information (ESI) available: Supplementary
experimental, Tables S1–S4, Schemes S1 and S2, Fig. S1–S13 and supplementary
references. See DOI: 10.1039/d0sc04337k
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extracellular space to coordinate Fe3+ and then returned to the (Fig. S1†). In addition to producing Ent, a number of Gram-
bacterial cell via specialized OM receptors.20–23 Several decades negative pathogens including uropathogenic E. coli and
of work showed that the siderophores and siderophore uptake Salmonella enterica also biosynthesize salmochelins, C-
machinery of Gram-negative bacteria can be leveraged to deliver glucosylated analogs of Ent. For instance, salmochelin S4,
toxic cargos into the bacterial periplasm and cytoplasm.15,24–33 hereaer named diglucosylated Ent (DGE), is a C5,C50-diglu-
Recent examples demonstrated that the activity of broad- cosylated analogue of Ent.42 DGE is deployed by pathogenic
spectrum antibiotics (e.g. b-lactams and uoroquinolones) can bacteria to acquire iron and evade the Ent-scavenging host-
be narrowed by targeting select species or a group of strains defense protein lipocalin-2.43–46 [Fe(DGE)]3ꢁ is transported
within
a
species through covalent attachment to into the periplasm by IroN.43,47 Whereas all E. coli produce Ent
a siderophore.34–36
and express FepA, the production of salmochelins and expres-
Enterobactin (Ent) is
a
tris-catecholate siderophore sion of IroN are mostly pathogen-associated and require the
produced by Gram-negative species including Escherichia coli, iroA gene cluster.42,47
Salmonella enterica and Klebsiella pneumoniae.37,38 The high
In prior work, we attached the aminopenicillins ampicillin
)
39,40 allows bacteria to (Amp) and amoxicillin (Amx) to the Ent scaffold via a poly-
Fe3+-binding affinity of Ent (Ka ꢀ 1049 Mꢁ1
scavenge Fe3+ from the host environment in the form of ethylene glycol (PEG3) linker installed at the C5 position of one
[Fe(Ent)]3ꢁ
.
In E. coli, [Fe(Ent)]3ꢁ is recognized by the OM catechol ring.34 These Ent-b-lactam conjugates afforded 100–
41
receptors FepA and IroN, and is transported into the periplasm 1000-fold increases in antimicrobial activity against E. coli
utilizing the energy provided by the TonB–ExbB–ExbD complex expressing FepA compared to the unmodied b-lactams. We
Fig. 1 Chemical structures of the Ent- and DGE-b-lactam conjugates with Amp (1, 2), Lex (3, 4), and Mem (5, 6). Glc, glucose. The curved line on
the b-glycosidic bond of glucose (b-Glc) represents the point of attachment to the catechol.
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then demonstrated that C-glucosylation of the two remaining can render broad-spectrum antibiotics selective towards path-
catechol rings, yielding DGE-b-lactam conjugates, afforded ogenic bacteria on the basis of siderophore-uptake
selective killing of E. coli strains that harbor the iroA gene machineries.
cluster.35 The antibacterial activity of the Ent/DGE-b-lactam
conjugates was attributed to the b-lactam cargos because b-
lactam hydrolysis of the Ent congeners abrogated this activity.34
Results and discussion
Although it is accepted that the b-lactam cargo of side-
Design and synthesis of Ent/DGE-b-lactam conjugates
rophore-b-lactam conjugates is responsible for bacterial cell
harboring Lex and Mem
death, studies addressing the mechanism of antimicrobial
activity of siderophore-b-lactam conjugates are limited.48–50
Generally, b-lactam antibiotics enter Gram-negative bacteria via
OM porins, bind to penicillin-binding proteins (PBPs) in the
periplasm and thereby inhibit cell wall (CW) biosynthesis.51,52 In
bacteria, there are numerous PBP enzymes, which are classied
according to their enzymatic function. In E. coli, class A PBPs
such as PBP1a and PBP1b catalyze both transglucosylation
(polymerization) of the peptidoglycan and transpeptidation
(cross-linking) of the glycan strands. Class B enzymes such as
PBP2 and PBP3 are monofunctional transpeptidases with roles
in elongation and cell division, respectively. Class C enzymes,
which include PBP4, PBP4b, PBP5, PBP6, PBP6b, and AmpH,
carry out various peptidase reactions that facilitate maturation,
remodeling, and metabolism of PG.53,54 Consequently, inhibi-
tion of essential PBPs by b-lactams induces a diverse array of
cellular morphologies, reecting the roles that the enzymes play
in coordinating cell division and elongation. Thus, studying the
cellular morphologies that occur as a result of exposing E. coli to
siderophore-b-lactam conjugates is likely to provide insight into
the observed antibacterial activity and cellular target(s).
In this work, we report the synthesis of four new side-
rophore-b-lactam conjugates harboring the antibiotics cepha-
lexin (Lex) and meropenem (Mem) (Fig. 1). These conjugates are
based on our original Ent/DGE-b-lactam design (e.g. Ent/DGE-
Amp) and extend the scope of antibiotic cargos that can be
effectively attached to Ent/DGE. These molecules show side-
rophore receptor-dependent antibacterial activity against E. coli.
Importantly, Ent-Mem also retains antibacterial activity against
a serine b-lactamase-producing strain. For the rst time, we
obtain microscopic evidence that Ent/DGE-b-lactam conjugates
exert antibacterial activity by inhibiting essential PBP enzymes.
This study further demonstrates that the Ent and DGE scaffolds
We prepared and evaluated the Ent/DGE conjugates 3–6
carrying two additional broad-spectrum b-lactam antibiotics,
Lex and Mem (Fig. 2A). Lex is a rst-generation cephalosporin
analog of Amp, whereas meropenem is a carbapenem with
enhanced resistance to extended-spectrum b-lactamases and
cephalosporinases.55–60 From a structural standpoint, Amp, Lex
and Mem differ by the size and nature of the ring fused with the
b-lactam core: thiazolidine (Amp), dihydrothiazine (Lex), and
thiazoline (Mem) (Fig. 1). Compared to Amp, the 4,6-fused ring
system of Lex provides enhanced stability to acid hydrolysis,55,61
whereas the 4,5-ring system of Mem, in which the endocyclic S
atom of the 5-membered ring is replaced by C, confers superior
resistance to serine b-lactamases.58–60 Additionally, these struc-
tural differences (i) determine the rate with which Amp, Lex,
and Mem diffuse through bacterial porins62 and (ii) induce
variations in b-lactam reactivity.61
The syntheses of conjugates 3–6 were carried out in one step
from either Ent-PEG3-N3 10 or DGE-PEG3-N3 11 and one of the
acylated b-lactams 7–9, using the methods developed for the
assembly of Ent/DGE-Amp/Amx (Schemes 1 and S1†).34,35 Each
b-lactam was acylated with hex-5-ynoyl chloride to provide the
alkyne functionality for the coupling reactions with azides 10
and 11 (Scheme S2†). The preparation of Lex-alkyne 8 was
similar to that developed for Amp-alkyne 7 due to the compa-
rable hydrophilicities of Amp and Lex (log PAmp ¼ 1.35, log PLex
¼ 0.65),63–65 which allows for an efficient extraction of 7 and 8
from acidied solution. Because Mem is more hydrophilic and
difficult to extract from an aqueous phase (log P ¼ ꢁ0.6),66 the
acylation reaction of Mem was optimized to avoid acid-catalyzed
hydrolysis of Mem-alkyne 9 during extraction from an acidic
solution. Thus, the water–acetone system employed for prepa-
ration of Amp/Lex-alkyne 7, 8 was replaced by MeOH. Removal
of MeOH under reduced pressure eliminated the need for
Scheme 1 Synthesis of Ent/DGE-Mem 5, 6. The curved line on the b-glycosidic bond of glucose (b-Glc) represents the point of attachment to
the catechol. Syntheses for the Amp and Lex conjugates are provided in Scheme S1.†
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Fig. 2 Antibacterial activity of Ent/DGE-b-lactam conjugates 1–6 against E. coli K-12 and CFT073. (A) and (B) Activity of Ent-Amp/Lex/Mem 1, 3,
and 5; (C) and (D) activity of DGE-Amp/Lex/Mem 2, 4, and 6; (E) and (F) activity of the parent antibiotics Amp, Lex and Mem. All assays were
performed in modified M9 medium (20 h, 30 ꢂC; mean ꢃ standard deviation, n ¼ 3). Data for E. coli UTI89 are presented in Fig. S2.† A summary of
all MIC values for conjugates 1–6 is provided in Table S4.†
extraction from acidied solvent, which avoided hydrolysis of medium. These assays were conducted in modied M9 medium
Mem-alkyne 9 during workup. that provides iron-limiting conditions (ꢀ0.3 mM Fe, Table S2†),
The resulting b-lactam alkynes 7–9 were coupled to Ent- or causing E. coli to express the Ent/DGE uptake machineries.
DGE-PEG3-N3 10 and 11 via copper-catalyzed azide–alkyne Under iron limitation, the three E. coli strains were more
cycloaddition (Schemes 1 and S1†). TBTA (tris[(1-benzyl-1H- susceptible to Ent-Amp/Lex/Mem (Fig. 2A, B and S2†) than to
1,2,3-triazol-4-yl)methyl]amine) was used in all cycloaddition the corresponding parent antibiotics (Fig. 2E and F). Ent-Amp 1
reactions to chelate copper and thereby prevent copper- showed minimum inhibitory concentration (MIC) values that
mediated decomposition of the b-lactam ring.34,35 The azide– were lower than Amp (10ꢁ5 M) by 1000-fold for both K-12 and
alkyne cycloaddition reactions yielded 40–70% Ent/DGE-b-lac- CFT073, and by 100-fold for UTI89 (Fig. S2†). These ndings are
tam conjugates on a milligram scale.
in overall agreement with our prior results obtained in MHB
medium supplemented with the iron chelator 2,20-bipyridine
(Bpy).34 Ent-Mem 5 afforded an MIC value of 10ꢁ8 M in K-12,
CFT073 and UTI89 (Fig. 2A, B and S2†), which represents
a 10-fold reduction when compared to Mem (10ꢁ7 M) (Fig. 2E
and F). In contrast to the Ent-Amp and -Mem conjugates, Ent-
Lex 3 exhibited a relatively modest enhancement in the AMA
compared to parent Lex (Fig. 2A, B and S2†). This fact may be
attributed to the lower potency of Lex against the strains
employed in this study.78 It is also possible that the cell la-
mentation readily induced by Lex (vide infra) may impact the
relationship between OD600 and cell viability.
The AMA of the DGE-b-lactams 2, 4 and 6 paralleled that of
the corresponding Ent-b-lactams against E. coli CFT073
(Fig. 2D) and UTI89 (Fig. S2†). For instance, in CFT073 and
UTI89, DGE-Mem 6 afforded an MIC value of 10ꢁ8 M, and the
MIC values of DGE-Amp 2 were 10ꢁ8 M in CFT073 and 10ꢁ7 M in
UTI89, whereas DGE-Lex 4 showed a modest MIC value (10ꢁ5 M)
consistent with that of Ent-Lex 3 (Fig. 2B, D and S2†). In
contrast, the DGE-b-lactam conjugates, especially DGE-Amp 2
and DGE-Mem 6, exhibited attenuated AMA against K-12
Conjugation to Ent/DGE enhances the antimicrobial activity
of parent b-lactams and affords selectivity towards pathogenic
E. coli independent of b-lactam structure
To evaluate the antimicrobial activity (AMA) of the Ent/DGE-b-
lactam conjugates against E. coli, we employed three E. coli
strains: K-12, CFT073, and UTI89. E. coli K-12 (ref. 67) is a non-
pathogenic laboratory strain that lacks the iroA gene cluster and
relies on FepA for Ent-mediated iron uptake. Uropathogenic E.
coli CFT073 (ref. 68–70) and UTI89 (ref. 71) harbor the iroA gene
cluster and thus biosynthesize DGE and express IroN.72–75
CFT073 also harbors the iha gene, which encodes an outer
membrane receptor, Iha, that has been shown to transport
[Fe(Ent)]3ꢁ
.
76,77
We performed AMA assays using a 10-fold dilution series to
compare the AMA of Ent/DGE-Amp/Lex/Mem 1–6 and the
parent antibiotics Amp/Lex/Mem (Fig. 2A–F and S2†). In these
assays, the bacteria were treated with the apo siderophore-b-
lactam conjugates, which can scavenge iron from the culture
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(Fig. 2C). This result agrees with our prior observations for DGE-
Amp/Amx35 and, taken with the results for CFT073, indicates
that expression of IroN is required for DGE-b-lactam conjugates
to exert AMA. We attribute the growth inhibition observed at
10ꢁ5 M DGE-Amp/Lex/Mem to iron limitation that results from
DGE-b-lactams sequestering Fe3+ in the growth medium.
Ent/DGE-b-lactam conjugates are transported across the OM
by Ent and salmochelin receptors
To ascertain the contributions of the Ent and salmochelin OM
transporters for the uptake of the Ent/DGE-b-lactam conjugates,
we examined the AMA of Ent/DGE-b-lactam conjugates 1–6
against E. coli CFT073. We selected E. coli CFT073 as a case
study because this strain exhibits high susceptibility to all six
conjugates and expresses FepA, IroN, and Iha.76,77 We took
a genetic approach using six OM and IM receptor mutants.
Namely, mutants in fepA, iroN, fepA iroN, and fepA iroN iha were
employed to probe conjugate recognition and transport across
the OM (Fig. 4 and S3†). A fepC mutant (ATPase) and a fepDG
double mutant (IM translocase) were used to investigate
whether transport across the IM affects the AMA of the Ent/
DGE-b-lactams (Fig. S4†).
AMA assays under iron limitation revealed that single dele-
tion of either fepA or iroN has negligible effect on the AMA of
Ent-Mem 5 (Fig. 4A). This result is in agreement with our
expectations and is explained by uptake of the Ent-based
conjugate through both FepA and IroN.87 In the case of DGE-
Mem 6, deletion of fepA had no effect on the MIC value,
whereas deletion of iroN attenuated the AMA by 1000-fold
(Fig. 4B). This result indicates that IroN is essential for trans-
port of the DGE-b-lactam conjugates. Overall, these results are
in agreement with prior siderophore competition studies that
indicated that both FepA and IroN provide transport of Ent-
Amp/Amx into the periplasm, whereas DGE-Amp/Amx can
only be transported through IroN.35
The AMA of both Ent- and DGE-Mem conjugates 5 and 6
(Fig. 4A and B) was attenuated against a fepA iroN double
mutant as well as a fepA iroN iha triple mutant. Moreover, the
similar susceptibility of both mutants to the conjugates
suggests that Iha is not involved in conjugate transport. The
origin of the residual AMA for Ent/DGE-Mem is unclear. One
possible explanation is that these mutant strains are more
susceptible to conjugate-mediated Fe3+ sequestration from the
growth medium compared to the parent strain because they
lack multiple siderophore receptors. Alternatively, E. coli
CFT073 might express an additional OM receptor that can
Ent-b-lactam conjugates retain b-lactamase stability of parent
b-lactams
Hydrolysis of b-lactams by b-lactamases is a major mechanism
of resistance to these antibiotics.51,79–81 Of the three b-lactams
considered in this work, Mem is the most resistant to b-lac-
tamases as it is only a substrate for carbapenemases (metallo-
b-lactamases).59,60,82–84 In contrast, Amp and Lex are readily
hydrolyzed by serine b-lactamases.85,86 To determine whether
conjugation to the Ent scaffold affects the susceptibility of the
b-lactam cargos to hydrolysis by a serine b-lactamase, we
evaluated the AMA of Ent-Amp/Lex/Mem 1, 3, 5 against E. coli
ATCC 35218, a strain that expresses a class A serine b-lacta-
mase (Fig. 3A and B). As expected, neither Amp nor Lex dis-
played AMA against E. coli ATCC 35218, whereas Mem retained
its activity (MIC ¼ 10ꢁ7 M) (Fig. 3B). In agreement with prior
work,34 Ent-Amp 1 exhibited negligible AMA against ATCC
35218 (Fig. 3A). Similarly to Ent-Amp 1, Ent-Lex 3 showed no
AMA against E. coli ATCC 35218 (MIC > 10ꢁ5 M) (Fig. 3A and B).
In contrast, Ent-Mem 5 retained potent AMA against E. coli
ATCC 35218, showing a 10-fold enhancement of the MIC value
relative to Mem (10ꢁ8 M and 10ꢁ7 M, respectively) (Fig. 3A and
B). Thus, conjugation of Mem to Ent has negligible effect on
the stability of the warhead to a class A serine b-lactamase. A
genome search indicated that E. coli ATCC 35218 does not
harbor iroN. In agreement with this analysis, none of the DGE-
b-lactams inhibited the growth of this strain over the concen-
tration range tested, presumably due to lack of uptake (Table
S4†). Taken together with the potent AMA of the Ent/DGE-Mem
conjugates 5, 6, we conclude that Mem would be the most
promising broad-spectrum b-lactam cargo for further in vitro
and in vivo studies.
Fig. 3 Antibacterial activity of (A) Ent-Amp 1, -Lex 3, and -Mem 5 and (B) Amp, Lex, and Mem against the class A serine b-lactamase producer E.
coli ATCC 35218. Assays were performed in modified M9 medium (20 h, 30 ꢂC; mean ꢃ standard deviation, n ¼ 3). A summary of all MIC values,
including those for the DGE-b-lactam conjugates, are provided in Table S4.†
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Fig. 4 Antibacterial activity of (A) Ent-Mem 5, (B) DGE-Mem 6 and (C) unmodified Mem against uropathogenic E. coli CFT073 wild-type and its
isogenic OM receptor mutants in fepA, iroN, fepA iroN, and fepA iroN iha. All assays were performed in modified M9 medium (20 h, 30 ꢂC; mean
ꢃ standard deviation, n ¼ 3). The AMA plots for the OM receptor mutants treated with Ent/DGE-Amp/Lex 1–4 are shown in Fig. S3,† whereas the
IM receptor mutants fepC and fepDG are shown in Fig. S4.†
transport the Ent/DGE conjugates across the membrane (e.g. of FepCDG inuences the antibacterial activity of the conju-
IreA),88 albeit less efficiently than FepA and IroN. Lastly, it is gates, we examined the susceptibility of a fepC mutant and
also possible that CFT073 secretes hydrolases that linearize Ent/ a fepDG mutant. Neither deletion of fepC nor fepDG affected the
DGE and subsequently imports the resulting iron-bound antibacterial activity of the Ent- and DGE-Mem 5, 6 conjugates
hydrolyzed conjugates into the periplasm via Cir or Fiu. These (Fig. S4†). These results indicate that, if any transport of the
possibilities warrant further investigation, which could uncover conjugates through FepCDG occurs, this process has negligible
yet-unknown pathways of Ent/DGE-b-lactam uptake that explain impact on AMA against E. coli CFT073. Therefore, we presume
the increased sensitivity of CFT073.
that the majority of the molecules are being trapped in the
As expected, unmodied Mem exhibited potent AMA (MIC of periplasm following covalent attachment to the PBPs. This
10ꢁ7 M) against the six CFT073 OM mutants (Fig. 4C). This conclusion agrees with our prior analysis of the E. coli K-12 fepC
activity is consistent with porin-mediated uptake of unmodied mutant, which showed comparable susceptibility to Ent-Amp/
b-lactams, which is unaffected by siderophore receptor Amx as the parent strain.34
expression.
Next, we investigated whether conjugate transport into the
cytoplasm affects the antibacterial activity of Ent/DGE-Mem 5 Siderophore conjugation accelerates the killing of E. coli to
and 6. Both the carrier and the cargo moieties of the Ent- and a greater extent for Amp than for Mem
DGE-b-lactam conjugates have binding partners in the peri-
plasm. The b-lactam warheads target PBPs and form covalent
acyl-enzyme species with an active site serine residue, whereas
the siderophore moiety can be captured by FepB for delivery to
In previous studies, we found that conjugation of Amp/Amx to
either Ent or DGE accelerated the rate of killing of E. coli CFT073
and UTI89 relative to the unmodied b-lactams.34,35 We there-
fore sought to determine whether siderophore modication
also enhances the time-kill kinetics of other b-lactams, and
the IM transporter FepCDG. To determine whether the presence
Fig. 5 Time-kill kinetics of (A) Ent/DGE-Amp 1, 2 and (B) Ent/DGE-Mem 5, 6 against E. coli CFT073 (ꢀ108 CFU mLꢁ1) treated with 50 mM Ent/
DGE-b-lactams and 100 mM unmodified Amp/Mem. All assays were performed in modified M9 medium (37 ꢂC; mean ꢃ standard deviation, n ¼
3).
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Fig. 6 Phase-contrast micrographs of mixed cultures of E. coli CFT073 (rods) with S. aureus ATCC 25923 (cocci) treated with MIC doses of Ent-
b-lactam conjugates. All cell cultures were incubated with the conjugates in 50% MHB in the presence of 100 mM Bpy for 20 h at 30 ꢂC prior to the
microscopy imaging. Scale bar: 10 mm. The micrographs of the mixed cultures treated with DGE-b-lactam conjugates are provided in Fig. S7.†
compared the time-kill kinetics of the conjugates with the attenuates the AMA of these b-lactams against S. aureus. The
lowest MIC values, Ent/DGE-Amp 1, 2 and Ent/DGE-Mem 5, 6, latter outcome is in agreement with our previous studies on
against E. coli CFT073.
HardyCHROM™ UTI plates that showed both E. coli selective
Of the two b-lactams, Mem is more hydrophilic than Amp killing and attenuated AMA against S. aureus ATCC 25923 for
(vide supra for log P values) and is transported by OM porins Ent-Amp/Amx.34
more readily.89–92 Indeed, the time-kill curves of Amp (Fig. 5A)
and Mem (Fig. 5B) show that Mem reduces OD600 of E. coli
CFT073 almost to baseline within 1 h, whereas Amp requires 5 h
to reach a similar OD600 value. Conjugation to the Ent/DGE
scaffolds changes the uptake pathway of the b-lactam warhead
different class B high-molecular-weight PBPs when adminis-
from passive diffusion through porins to TonB-powered active
transport, which results in acceleration of killing for Ent/DGE-
Amp 1, 2. Consequently, Ent/DGE-Amp/Mem 1, 2, 5, and 6
reduce the OD600 of CFT073 to almost baseline within 1–2 h
whereas Mem targets PBP2 that participates in cell elonga-
(Fig. 5A and B). Hence, these results suggest that the rate of
bacterial cell killing will be accelerated to a greater extent for
induce bacterial cell lamentation, whereas Mem induces the
more lipophilic b-lactams such as Amp.
Ent/DGE-Amp/Lex induce lamentous growth, whereas Ent/
DGE-Mem cause formation of spheroplasts, in E. coli
The three b-lactam antibiotics used as cargos in this study target
tered at sub-MIC doses, inducing different bacterial cell shapes
as a result of inhibition of the corresponding PBPs.53,54 Amp and
Lex primarily bind to PBP3 that participates in cell division,93–96
tion.93,97,98 Consequently, at sub-MIC quantities, Amp and Lex
formation of spheroplasts, which are CW-decient cells. At
quantities equal to or greater than the MIC, all three b-lactams
induce cell lysis due to concomitant inhibition of multiple
PBPs.54,99,100 To further examine the consequences of Ent- and
Ent/DGE-b-lactam conjugates selectively kill E. coli in co-
culture with S. aureus
To investigate species selectivity of the new Ent-b-lactams, we DGE-b-lactam treatment on E. coli and thereby inform mecha-
performed mixed-species assays to ascertain if the conjugates nism, we employed phase-contrast microscopy to visualize the
selectively kill E. coli CFT073 in co-culture with another bacte- cellular morphologies caused by these conjugates (Fig. 7).
rial species. We employed phase-contrast microscopy and
E. coli CFT073 and its fepA iroN and fepA iroN iha mutants
selected to co-culture E. coli CFT073 with the Gram-positive were incubated with the Ent-Amp/Lex/Mem conjugates 1, 3, 5 at
pathogen Staphylococcus aureus ATCC 25923. The cell shape of a high cell density (ꢀ108 CFU mLꢁ1) to facilitate microscopic
E. coli (rod) is distinct from the cell shape of S. aureus (coccus), visualization of the bacterial cells. To probe the effect of the
which facilitates microscopic differentiation of these species. conjugates at sub-MIC quantities, the bacteria were treated with
Since S. aureus did not grow in the modied M9 medium, we 50 mM of Ent-Amp/Mem 1 and 5 and 100 mM of Ent-Lex 3.
performed these assays in 50% MHB containing 100 mM Bpy, Signicant cell elongation was observed for E. coli CFT073
which provides iron limitation and promotes expression of the treated with Ent-Amp 1 and Ent-Lex 3 (Fig. 7B and C) compared
siderophore transport machinery. The co-cultures were treated to the untreated control (Fig. 7A). In contrast, E. coli CFT073
with MIC doses of the Ent/DGE-b-lactams or parent antibiotics cells treated with 50 mM Ent-Mem 5 transformed into round or
adjusted with respect to the cell density used in each imaging ovoid-shaped spheroplasts (Fig. 7D). These observations are
experiment. In the co-cultures treated with the unmodied consistent with the inhibition of the primary PBP targets of the
antibiotics (Fig. S6†), both E. coli and S. aureus were killed. In three b-lactams. Treatment of the double and triple mutants
contrast, the Ent/DGE-b-lactam conjugates selectively killed E. fepA iroN and fepA iroN iha with sub-MIC concentrations of the
coli as evidenced by the presence of cocci and absence of rod- Ent-b-lactam conjugates showed that some of the cells exhibit
shaped bacteria remaining in cultures following treatment slight elongation following exposure to Ent-Amp/Lex 1, 3
(Fig. 6 and S7†). Together with LIVE/DEAD viability assays (vide (Fig. 7F, G, J and K) and that some cells form spheroplasts
infra, Fig. S8†), these results indicate that Ent/DGE conjugation following exposure to Ent-Mem 5 (Fig. 7H and L). The obser-
narrows the AMA spectrum of Amp, Lex and Mem and vation of these morphologies is consistent with the weak AMA
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Fig. 7 Full-field view micrographs of E. coli CFT073 WT (A–D) and the indicated outer-membrane receptor mutants (E–L) treated with sub-MIC
amounts of Ent-b-lactams in modified M9 medium for 20 h at 30 ꢂC. Scale bar: 10 mm. The micrographs of E. coli and outer-membrane receptor
mutants treated with sub-MIC amounts of DGE-b-lactams are shown in Fig. S9.†
observed for these mutants in the AMA assays (Fig. 4), which as for the cells treated with Ent-b-lactams 1, 3, and 5 (Fig. S9†).
might suggest the existence of alternative pathways of Ent-b- Thus, the b-lactam warhead induces specic morphological
lactam uptake in E. coli CFT073.
changes to the bacterial cell shape independent of the carrier to
When the DGE-b-lactam conjugates 2, 4, and 6 were added to which it is attached. Curiously, the triple-mutant fepA iroN iha
bacteria at sub-MIC doses, similar morphologies were observed appears to be more sensitive to the action of DGE-b-lactams
(Fig. S9J–L†) than to the corresponding Ent-b-lactams (Fig. 7J–
L). Further investigation into this nding may uncover addi-
tional mechanisms of Ent/DGE uptake and better explain the
enhanced sensitivity of E. coli CFT073 to the Ent/DGE
conjugates.
Next, we treated E. coli CFT073 with an estimated MIC dose
of Ent-Amp or DGE-Amp (100 mM) determined by extrapolating
to ꢀ108 CFU mLꢁ1 of the MIC values obtained in the AMA
assays. This concentration of the conjugates resulted in cell
lysis, indicated by the cell debris observed in the micrographs
(Fig. 8B and C). Moreover, the presence of enlarged cells in the
bacterial cultures treated with Ent/DGE-Amp 1, 2 (Fig. 8B and C)
Fig. 8 Full-field view micrographs of E. coli CFT073 WT (A–C) treated
with MIC amounts of Ent/DGE-Amp 1, 2 in modified M9 medium for
is consistent with the concomitant inhibition of multiple
essential PBPs by Amp at doses that are equal or exceeding the
20 h at 30 ꢂC. Scale bar: 10 mm. The micrographs of E. coli CFT073
MIC.54 Similar cell shapes and debris were observed at MIC
treated with MIC amounts of Ent/DGE-Lex/Mem 3–6 are shown in
doses of Ent/DGE-Lex 3, 4 and Ent/DGE-Mem 5, 6 (Fig. S10†).
Fig. S10.†
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Fig. 9 Fluorescence micrographs acquired after incubating E. coli CFT073 and the fepA iroN and fepA iroN iha mutants with the LIVE/DEAD dyes
at 30 ꢂC for 15 min. The cell cultures were incubated with the Ent/DGE conjugates in modified M9 for 20 h at 30 ꢂC prior to staining. Scale bar: 10
mm. The micrographs acquired after incubation of DGE-Amp/Mem are shown in Fig. S11.†
Overall, phase-contrast microscopy imaging corroborated result of Ent/DGE-b-lactam treatment. This assay utilizes uo-
the AMA assays and illuminated the differences in cell rescent dyes to distinguish between cells with intact OM (stain
morphologies due to variable affinities of the b-lactam green with SYTO 9) and cells with compromised OM (stain red
warheads towards PBP2 and PBP3. It should be noted, however, with propidium iodide, PI).110
that sub-MIC doses of b-lactam antibiotics can also inhibit
As expected, elongation/lamentation was detected in all
nonessential PBPs (e.g. PBP4)101 in Gram-positive and Gram- strains treated with sub-MIC quantities of the PBP3 inhibitors
negative bacterial strains.54,101–105 Inhibition of nonessential Ent-Amp/Lex 1, 3 and DGE-Amp/Lex 2, 4 (Fig. 9B–D and S11B†),
PBPs leads to more subtle morphological effects than inhibition whereas the PBP2 inhibitors Ent/DGE-Mem 5, 6 led to sphero-
of PBP1, 2 and 3;106–109 thus, further studies are needed to plast formation (Fig. 9E and S11C†). Similarly to the phase-
elucidate whether the Ent/DGE-b-lactam conjugates also inhibit contrast microscopy and the AMA assays, lamentation
nonessential PBPs similar to the parent b-lactams.
(Fig. 9G–I, L–N and S11E, H†) and spheroplasts formation
(Fig. 9J, O and S11F, I†) were also observed, to different degrees,
in the fepA iroN and fepA iroN iha mutants.
Inhibition of PBP2 and PBP3 by the Ent/DGE-b-lactam
conjugates concludes with cell lysis
All untreated cells stained green with SYTO 9, indicating
viability (Fig. 9A, F and K). In contrast, addition of sub-MIC
quantities of the conjugates lead to a mixture of green and
Next, we employed the LIVE/DEAD viability assay to examine the
fate of the bacterial cell laments and spheroplasts formed as
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red cells (Fig. 9B–E, G–J, L–O and S11†). The latter indicated that
In contrast to Ent/DGE-Amp 1, 2, the treatment of E. coli
cell lysis occured for a subset of the cells under these condi- CFT073 with 100 mM Ent-Mem 3 (Fig. 10) is accompanied by
tions. Thus, aer undergoing morphological change as deter- explosive lysis that starts within the rst 60 min and is virtually
mined by inhibition of either PBP3 (Ent-Amp/Lex 1, 3 and Ent/ complete by 4 h. Thus, targeting of different primary PBPs by
DGE-Lex 2, 4) (Fig. 9B–D, G–I, L–N and S11B†) or PBP2 (Ent/ the Ent/DGE-b-lactam conjugates determines not only the
DGE-Mem 5, 6) (Fig. 9E, J, O and S11C†), the bacterial cells morphologies of the cells treated with sub-MIC doses, but also
lose OM integrity and can be considered dead based on the PI the character of lysis when MIC amounts are reached.
staining. Lastly, the partial red staining of the fepA iroN
(Fig. 9G–J) and fepA iroN iha (Fig. 9L–O) cells is another indi-
cation that the conjugates exert some AMA against these cells
Perspectives on siderophore-b-lactam conjugates
The growing problem of antibiotic resistance and the insuffi-
cient amount of new antibiotics in the drug pipeline pose
a serious threat to public health. Development of narrow-
spectrum antibiotics that kill pathogenic species and spare
commensal microbes is considered a promising solution in the
face of infections caused by multidrug-resistant Gram-negative
bacteria. Leveraging the essential nutrient uptake machineries
of such bacteria for a targeted delivery of antibiotics has been
reported for a number of different compounds.111–113 Recently,
FDA approved the rst antibiotic that is reported to hijack Fe3+
uptake machinery to deliver a cephalosporin cargo to the peri-
plasm of Gram-negative bacteria in the treatment of compli-
and are likely transported into the periplasm.
Overall, the LIVE/DEAD assay showed that the treatment of
E. coli CFT073 and its mutants with sub-MIC quantities of
conjugates leads to the death of both lamentous and CW-
decient cells as result of OM rupture. Therefore, the b-lactam
warhead is responsible for the bactericidal effect of the Ent/
DGE-b-lactam conjugates 1–6 as it inhibits the target PBPs
causing characteristic phenotypes and compromises OM
integrity conducive to cell lysis.
Ent-Mem induces explosive cell lysis in E. coli
To obtain a visual depiction of the course of cell killing by the cated urinary tract infections.114–116
Ent/DGE conjugates, we added MIC amounts of Ent/DGE-Amp
Our approach consists of using the native siderophore scaf-
1, 2 and Ent-Mem 5 to E. coli CFT073 and acquired time-lapse folds Ent and DGE as antibiotic carriers.34–36 The Ent/DGE-Lex/
images of the process at 37 ꢂC. The Ent/DGE-Lex 3, 4 conju- Mem conjugates synthesized and studied in this work expand
gates were not studied due to their relatively high MIC values. the arsenal of clinically-approved broad-spectrum antibiotics
The images of CFT073 cells treated with 100 mM Ent/DGE-Amp that can be endowed with selectivity towards pathogenic strains
1, 2 show that the cells ceased to grow and divide. Moreover, that express IroN. This selectivity is achieved by targeting the
these cells did not elongate or undergo explosive lysis over the enterobactin/salmochelin uptake machinery of E. coli and is not
course of 4 h. These observations indicate that non-explosive affected by the b-lactam structure. Moreover, conjugation to Ent/
rupture of the cell membranes occurs and results in cell lysis DGE further enhances the MIC of the parent b-lactams by 10–
without bursting (Fig. 10) similar to the red-stained cells in the 1000-fold. The time-kill kinetics of the conjugated b-lactams is
LIVE/DEAD assay (Fig. 9).
enhanced to a greater extent for the more lipophilic antibiotics
Fig. 10 Time-lapse phase-contrast micrographs of E. coli CFT073 incubated with 100 mM Ent/DGE-Amp 1, 2 and Ent-Mem 5 in modified M9
medium (37 ꢂC). Scale bar: 10 mm.
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such as Amp due to the change of the uptake route from porins siderophore receptors FepA and IroN. Furthermore, the side-
(Amp) to TonB-powered FepA or IroN (Ent-Amp and DGE-Amp). rophore-b-lactam conjugates presented in this work efficiently
These results indicate that the Ent/DGE scaffold could serve as kill pathogenic E. coli regardless of the structure and polarity of
a versatile tool for pharmaceutical development of antibacterial the b-lactam cargo. Targeting pathogenesis with such conju-
therapeutics that target IroN-expressing strains (e.g. DGE conju- gates could reduce the onset of secondary infections and slow
gates), enhancing the MIC values and decreasing the time the evolution of antibiotic resistance. In future work, studies
needed to exert its bactericidal effect.
using mouse models will aid the understanding of the stability
The three b-lactams, Amp, Lex, and Mem, conjugated to the and efficacy of such siderophore-b-lactam conjugates in vivo.
Ent/DGE scaffolds in this work, exert AMA by inhibiting class B Lastly, it will be important to determine whether this strategy is
high-molecular-weight PBP2 and PBP3 as indicated by the applicable to other bacterial pathogens such as Salmonella
phase-contrast microscopy and LIVE/DEAD staining assays. enterica that cause human disease and utilize both Ent and DGE
These results along with the AMA assays of the OM receptor for Fe3+ acquisition.
mutants demonstrated that the b-lactam cargo is indeed
responsible for the killing of bacteria while the uptake of the
Ent/DGE conjugates with different b-lactam cargos takes place
Experimental section
Synthetic reagents
via FepA and IroN. In prior work, we hypothesized that Iha may
contribute to the sensitivity of E. coli CFT073 to the Ent/DGE-b-
lactams. However, the AMA assays and microscopy study of the
fepA iroN and fepA iroN iha mutants indicated that Iha does not
play an important role in the transport of either Ent- or DGE-b-
lactams. Nevertheless, we have observed weak AMA and signs of
uptake of the conjugates by the fepA iroN and the fepA iroN iha
mutants, which suggests that CFT073 possesses other yet-
unknown routes of Ent/DGE uptake.
Anhydrous N,N-dimethylformamide (DMF) and dichloro-
methane (DCM) were purchased from VWR. Anhydrous
dimethyl sulfoxide (DMSO) was purchased from Millipore
Sigma and used as received. All other chemicals were purchased
from Millipore Sigma or VWR and used as received. The
syntheses of Ent-PEG3-N3 10,34,35,119 DGE-PEG3-N3 11,35 Amp-
alkyne 7,34,35 Ent-Amp 1,34,35 and DGE-Amp 2 (ref. 35) are re-
ported elsewhere.
Although uptake through the OM siderophore receptors allows
the b-lactam cargos of the Ent/DGE conjugates to exert antibacte-
rial activity, there may be additional mechanisms in place that
General methods and instrumentation
explain the enhancement of the MIC values of conjugates relative EMD TLC silica gel 60 F254 plates were used for analytical thin
to parent antibiotics. For instance, it has been shown that accu- layer chromatography. EMD PLC silica gel 60 F254 plates of
mulation of apo Ent in the periplasm of E. coli leads to deleterious 2 mm thickness were used for preparative TLC. Sigma-Aldrich
effects for the bacterial cells.117 However, under the assay condi- silica gel (70–230 mesh, 60 A) was used for ash column
˚
tions employed in this work, we did not observe any of the chromatography.
abnormal morphologies that were attributed to the accumulation
of Ent in the bacterial periplasm.117
1H NMR spectra were collected on a Bruker Avance III DPX 400
MHz spectrometer operated at ambient probe temperature (293 K).
Of the four b-lactam cargos we have thus far conjugated to Q-LC/MS and Q-ToF MS analyses were carried out on an Agilent
Ent/DGE – Amp, Amx,34,35 Lex, and Mem – Mem seems to be the 6125B mass spectrometer attached to an Agilent 1260 Innity LC
most promising for further evaluation. The exceptional stability and a high-resolution Agilent 6545 mass spectrometer, respec-
of the carbapenem ring to the action of extended spectrum b- tively. For all LC/MS analyses, solvent A was 0.1% formic acid/H2O
lactamases, its enhanced MIC upon conjugation to Ent/DGE, and solvent B was 0.1% formic acid/MeCN (LC/MS grade, Millipore
and induction of explosive cell lysis at nanomolar concentra- Sigma). The samples were analyzed using a solvent gradient of 5–
tions make Mem an outstanding warhead for siderophore 95% B over 6 min with a ow rate of 0.4 mL minꢁ1
conjugation. However, use of Mem as warhead could be limited
Analytical and semi-preparative HPLC were performed using
.
by the spread of carbapenemase-producing strains, which an Agilent 1200 series HPLC system outtted with a Clipeus
render Mem inactive.80,82 Likewise, the selectivity afforded by reversed-phase C18 column (5 mm pore size, 4.6 ꢄ 250 mm,
conjugation of antibiotics to Ent/DGE could be compromised by Higgins Analytical) at a ow rate of 1 mL minꢁ1 and an Agilent
the existence of commensals that can cross-feed using Ent/DGE Zorbax reversed-phase C18 column (5 mm pore size, 9.4 ꢄ 250
as xenosiderophores.118 Therefore, further studies are needed to mm) at a ow rate of 4 mL minꢁ1. The multi-wavelength
better understand the host microbiome and evaluate the detector was set to detect absorbance at 220, 280, and
potential of Ent/DGE antibiotic conjugates as pathogen- 316 nm. HPLC-grade acetonitrile (MeCN) and triuoroacetic
selective therapeutics.
acid (TFA) were purchased from Millipore Sigma. For HPLC
analysis, solvent A was 0.1% v/v TFA/H2O and solvent B was
0.1% v/v TFA/MeCN. The percentage of TFA in the HPLC
solvents used for antibiotic conjugate purication was 0.005%
Conclusion and outlook
In closing, this work further demonstrates that conjugation of to avoid b-lactam ring hydrolysis. The HPLC solvents were
b-lactam antibiotics to the Ent/DGE scaffolds is an effective prepared with HPLC grade MeCN and TFA, with Milli-Q water
strategy to convert broad-spectrum antibiotics into compounds (18.2 MU cm), and ltered through a 0.2 mm lter before use. To
that selectively target bacteria expressing the catecholate evaluate conjugate purity by analytical HPLC, the entire portion
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of each HPLC-puried compound was dissolved in a 1 : 1 solution in DMSO, 0.825 mmol) were combined and 100 mL of
mixture of MeCN/H2O and an aliquot was taken for HPLC DMSO was added. An aliquot of aqueous CuSO4 (50 mL of
analysis. The remaining solution was subsequently lyophilized. a 90 mM solution, 4.5 mmol) and TBTA (100 mL of a 50 mM
Optical absorption spectra were recorded on a Beckman solution in DMSO, 5 mmol) were combined to give a blue solu-
Coultier DU800 spectrophotometer (1 cm quartz cuvettes, tion, to which sodium ascorbate (NaAsc, 100 mL of a 180 mM
Starna). A BioTek Synergy HT plate reader was used to record solution in water, 18.0 mmol) was added. The color of the
absorbance at 600 nm (OD600) for antimicrobial activity assays solution immediately changed from blue to pale yellow
and time-kill kinetics.
(reduction of Cu2+ to Cu+) and the mixture was added to the
alkyne/azide solution. The reaction was gently mixed on
a bench-top rotator for 2.5 h at 25 ꢂC (150 rpm). Then, the
reaction mixture was diluted with 1 : 1 MeCN/water (3ꢄ the
volume of solution) and puried by semi-preparative HPLC (0–
100% B over 30 min, 4 mL minꢁ1; 0.005% TFA). Lyophilization
of the HPLC fractions afforded the corresponding conjugates
(1–6) as white or off-white powders. Analytical data and the
yields of the Ent/DGE conjugates are provided in Table S3.†
Synthesis of Lex-alkyne, 8
Hex-5-ynoyl chloride (0.352 g, 2.70 mmol) was added dropwise to
a 0 ꢂC solution of Lex sodium salt (0.500 g, 1.35 mmol) and
NaHCO3 (0.567 g, 6.75 mmol) in 6.75 mL of water under vigorous
stirring. The reaction mixture was warmed to room temperature
and le to react for 1 h under stirring. Then, 5 mL of water was
added to the solution and the resulting mixture was washed with
EtOAc (2 ꢄ 10 mL). The aqueous layer was acidied to pH 2 using
1 M HCl and the product extracted with EtOAc (3 ꢄ 10 mL). The
organic layer was dried over anhydrous Na2SO4 and the solvent was
removed under reduced pressure to yield a yellow solid. The crude
product was triturated with hexane, then puried by semi-
preparative HPLC (0–100% B over 30 min, 4 mL minꢁ1; 0.005%
TFA). Lyophilization of the HPLC fractions afforded 0.390 g (65%)
of Lex-alkyne, 8, as an off-white powder. Analytical HPLC Rt ¼
17.5 min (0–100% B over 30 min, 1 mL minꢁ1; 0.005% TFA).1H
NMR (DMSO-d6, 400 MHz): d, ppm 13.20 (br, 1H), 9.25 (d, J ¼
8.5 Hz, 1H), 8.56 (d, J ¼ 8.1 Hz, 1H), 7.44 (d, J ¼ 7.6 Hz, 2H), 7.25–
7.36 (m, 3H), 5.68 (d, J ¼ 8.2 Hz, 1H), 5.62 (dd, J ¼ 4.7, 8.2 Hz, 1H),
4.96 (d, J ¼ 4.7 Hz, 1H), 3.47 (d, J ¼ 18.9 Hz, 1H), 3.28 (d, J ¼
18.2 Hz, 1H), 2.78 (s, 1H), 2.31 (t, J ¼ 7.3 Hz, 2H), 2.13–2.22 (m, 2H),
1.99 (s, 3H), 1.67 (m, 2H). QToF-MS (m/z): mass calcd for
(C22H23N3O5S + Na+) ¼ 464.1256; found 464.1253.
Storage and handling of siderophores and siderophore-b-
lactam antibiotic conjugates
All precursors and Ent/DGE conjugates were stored as either
powders or DMSO stock solutions at ꢁ20 ꢂC. The stock solution
concentrations for the Ent/DGE conjugates ranged between 1–
2 mM. These values were determined by diluting the DMSO
stocks in MeOH and using the reported extinction coefficient
for enterobactin in MeOH (3316 ¼ 9500 Mꢁ1 cmꢁ1).39 To mini-
mize multiple freeze–thaw cycles, the resulting solutions were
divided into 50 mL aliquots and stored at ꢁ20 ꢂC. The b-lactam
antibiotic cargos as well as Ent/DGE are prone to hydrolysis, and
aliquots were routinely analyzed by HPLC to conrm the
integrity of the samples.
General microbiology materials and methods
Information pertaining to all bacterial strains used in this study
is listed in Table S1.† Freezer stocks of all E. coli strains were
prepared from single colonies in 25% glycerol/lysogeny broth
(LB) medium. Lysogeny broth (tryptone 10 g Lꢁ1, yeast extract
Synthesis of Mem-alkyne, 9
Hex-5-ynoyl chloride (0.101 g, 0.78 mmol) was added dropwise
to a 0 ꢂC solution of Mem (0.150 g, 0.39 mmol) and Et3N (1.522
mL, 1.105 g, 10.92 mmol) in 4 mL of MeOH under vigorous
stirring. The reaction mixture was warmed to room temperature
and le to react for 1 h under stirring. Then, the solvent was
removed under reduced pressure and the crude was puried by
5 g Lꢁ1, NaCl 10 g Lꢁ1), modied M9 (Na2HPO4 6.8 g Lꢁ1
,
KH2PO4 3 g Lꢁ1, NaCl 0.5 g Lꢁ1, NH4Cl 1 g Lꢁ1, 0.4% glucose,
0.2% casein amino acids, 2 mM MgSO4, 0.1 mM CaCl2, 16.5 mg
mLꢁ1 thiamine hydrochloride)36 and agar were obtained from
Becton Dickinson (BD). Mueller–Hinton broth (MHB; beef
extract powder 2 g Lꢁ1, casein hydrolysate 17.5 g Lꢁ1, and
soluble starch 1.5 g Lꢁ1) was purchased from Sigma-Aldrich.
The iron chelator 2,20-bipyridine (Bpy) was purchased from
Sigma-Aldrich. All growth media and Milli-Q water used for
bacterial cultures or for preparing stock solutions were steril-
ized in an autoclave. The modied M9 medium was lter-
sterilized through a 0.22 mm lter. A 200 mM Bpy stock solu-
tion was prepared in anhydrous DMSO and utilized in bacterial
growth inhibition assays. Working solutions of Ent and Ent/
DGE-antibiotic conjugates were prepared by 10-fold serial
dilutions in 10% DMSO/H2O. For all microbiology assays, the
nal cultures contained 1% v/v DMSO. Sterile polypropylene
culture tubes and sterile polystyrene 96-well plates used for
semi-preparative HPLC (0–100% B over 30 min, 4 mL minꢁ1
;
0.005% TFA). Lyophilization of the HPLC fractions containing
product afforded 0.093 g (50%) of Mem-alkyne, 9, as a yellow
powder. Analytical HPLC Rt ¼ 14.0 min (0–100% B over 30 min,
1 mL minꢁ1; 0.005% TFA). The 1H NMR (CD3OD, 400 MHz):
d, ppm 4.24 (t, J ¼ 9.1 Hz, 1H), 4.07–4.17 (m, 1H), 3.82–3.92 (m,
1H), 3.53–3.64 (m, 1H), 3.48 (t, J ¼ 10.2 Hz, 1H), 3.32 (dd, J ¼ 2.7,
7.1 Hz, 2H), 3.19 (s, 3H), 3.05 (s, 1H), 2.98 (s, 3H), 2.73–2.84 (m,
1H), 2.33–2.60 (m, 2H), 2.24–2.31 (m, 4H), 1.77–1.87 (m, 2H),
1.31 (d, J ¼ 6.7 Hz, 3H), 1.28 (d, J ¼ 7.2 Hz, 3H). QToF-MS (m/z):
mass calcd for (C23H31N3O6S + H+) ¼ 478.2012; found 478.2010.
General synthesis of Ent/DGE-Amp, Ent/DGE-Lex, Ent/DGE-
Mem (1–6)
b-lactam alkyne 7–9 (50 mL of a 50 mM solution in DMSO, 2.5 culturing were purchased from VWR and Corning Inc., respec-
mmol) and Ent/DGE-PEG3-N3 10, 11 (73 mL of an 11.3 mM tively. The optical density at 600 nm (OD600) was recorded on
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a Beckmann Coulter DU800 spectrophotometer or by using and 6 for E. coli CFT073 is equal to 1 ꢄ 10ꢁ8 M (Table S4†). For
a BioTek Synergy HT plate reader.
microscopy experiments, 5 ꢄ 108 CFU mLꢁ1 of mid-log phase
CFT073 cultures were incubated with the Ent/DGE-b-lactams 1,
2, 5, and 6. Hence, the MIC of Ent/DGE-Amp/Mem 1, 2, 5, and 6
per 5 ꢄ 108 CFU mLꢁ1 was estimated to be 100 mM. No cells
were detected aer incubating 5 ꢄ 108 CFU mLꢁ1 of E. coli
CFT073 with >100 mM of the Ent/DGE-Amp/Mem 1, 2, 5, and 6.
For Ent/DGE-Lex 3 and 4, MIC ¼ 1 ꢄ 10ꢁ5 M per 5 ꢄ 104 CFU
mLꢁ1. To observe lamentous growth, 100 mM of Ent/DGE-Lex
3, 4 was incubated per 5 ꢄ 106 CFU mLꢁ1 (sub-MIC).
General procedure for antimicrobial activity (AMA) assays
Overnight bacterial cultures were prepared in 15 mL poly-
propylene tubes by inoculating 5 mL of growth medium
(modied M9) with the appropriate freezer stock. The overnight
cultures were then incubated at 37 ꢂC for 16–18 h on a rotating
wheel set at 150 rpm. Each overnight culture was diluted in a v/v
ratio of 1 : 100 into 5 mL of fresh modied M9 medium and
incubated at 37 ꢂC and 150 rpm until OD600 reached ꢀ0.6 (mid-
log phase). Each culture was subsequently diluted into fresh
Phase-contrast microscopy
modied M9 to achieve a nal OD600 of 0.001. A 90 mL aliquot of Phase-contrast microscopy imaging was carried out at the W. M.
the diluted culture was combined with a 10 mL aliquot of a 10ꢄ Keck Biological Imaging Facility of the Whitehead Institute
solution of the antibiotic or antibiotic conjugate in a 96-well (Cambridge, Massachusetts). The incubation of cell cultures
plate. The covered plate was wrapped in a wet paper towel and with the Ent/DGE-b-lactams was performed on 96-well plates
Saran wrap, then incubated at 30 ꢂC with shaking at 150 rpm for (100 mL sample per well) using 5 ꢄ 108 CFU mLꢁ1 and 50 or 100
20 h in a tabletop incubator. Bacterial growth was determined mM of Ent/DGE conjugates. For the Ent/DGE-Lex conjugates, the
by measuring OD600 (end point analysis) on a BioTek Synergy initial cell density was 5 ꢄ 106 CFU mLꢁ1 to account for the
HT plate reader. Each well condition was prepared in duplicate higher MIC values of the Lex conjugates and use less compound.
(technical replicate) and at least three independent replicates Following a 20 h incubation (30 ꢂC, 150 rpm), a 5 mL sample from
(biological replicate) utilizing at least two synthetic batches of each culture was pipetted on an agarose (Bio-Rad, PCR grade)
each conjugate were conducted on different days. The resulting pad (1% w/w agarose/Milli-Q water) positioned on a microscope
mean OD600 values are reported, and the error bars are the slide. The sample was then covered with a coverslip and imaged
standard deviation of the mean (SDM) obtained from the bio- using a Zeiss Axioplan2 upright microscope equipped with
logical replicates.
a 100ꢄ oil-immersion objective lens. For each type of microscopy
experiment, each condition was repeated in at least three inde-
pendent replicates on different days. Representative micro-
graphs for each condition are shown in the gures.
Time-kill kinetics
Overnight cultures were prepared by inoculating 5 mL of
modied M9 medium with bacterial freezer stocks. The over-
night cultures were diluted 1 : 100ꢂinto 5 mL of fresh modied
Mixed-species microscopy assays
M9 medium and incubated at 37 C with shaking at 150 rpm All bacterial strains were cultured in 50% MHB in the presence
until OD600 reached ꢀ0.3. The culture was centrifuged of 100 mM of 2,20-bipyridine to an OD600 ꢀ 0.6 (mid-log phase)
(3000 rpm ꢄ 10 min) and the resulting pellet was re-suspended (S. aureus ATCC 25923 does not grow in the modied M9
in modied M9 and centrifuged (3000 rpm ꢄ 10 min). The medium). Bacterial suspensions were then adjusted to 1 ꢄ 107
resulting pellet was re-suspended again in fresh modied M9 CFU mLꢁ1, mixed in a 1 : 1 ratio (107 : 107 CFU mLꢁ1) and
and the OD600 adjusted to 0.3. A 90 mL aliquot of the resulting incubated with 50 mM Ent/DGE-Amp/Mem per 100 mL sample in
culture was combined with a 10 mL aliquot of a 10ꢄ solution of 96-well plates (20 h, 30 ꢂC, 150 rpm). For Ent/DGE-Lex, bacterial
parent antibiotic or Ent/DGE conjugate in a 96 well plate, which suspensions were adjusted to 1 ꢄ 106 CFU mLꢁ1 initial cell
was wrapped in Saran lm and incubated at 37 ꢂC with shaking density and incubated with 200 mM Ent/DGE-Lex. A 5 mL sample
at 150 rpm. The OD600 values were recorded at 1 h intervals. from each culture was pipetted on an agarose pad (1% w/w
Each well condition was prepared in duplicate (technical agarose/Milli-Q water) positioned on a microscope slide. The
replicate) and at least three independent replicates (biological specimen was then covered with a coverslip and imaged using
replicate) utilizing at least two synthetic batches of each a Zeiss Axioplan2 upright microscope equipped with a 100ꢄ oil-
conjugate were conducted on different days. The resulting immersion objective lens.
mean OD600 values are reported, and the error bars are the
standard deviation of the mean (SDM) obtained from the bio- Time-lapse microscopy
logical replicates.
The time-lapse studies were carried out using poly-D-lysine
coated MatTek glass-bottom Petri dishes of 35 mm, with
Calculation of Ent/DGE-b-lactam MIC for microscopy assays
a 14 mm microwell and a No. 1.5 cover glass. The untreated
The MIC values (Table S4†) of the Ent/DGE-Amp/Lex/Mem bacteria (5 ꢄ 108 CFU mLꢁ1) were mixed on 96-well plates (100
conjugates 1–6 were determined in modied M9 medium for mL sample per well) to give a nal concentration of 100 mM Ent/
mid-log phase (OD600 ꢀ 0.6) cell cultures diluted to OD600
¼
DGE conjugate. Once the conjugate was added to the culture, a 5
0.001 (vide supra). Plating of the diluted cultures onto LB agar mL sample from each suspension was pipetted out onto the
plates gave an average count of 5 ꢄ 104 CFU mLꢁ1 (n > 3). Thus, 14 mm microwell and covered with an agarose pad (1% w/w
at 5 ꢄ 104 CFU mLꢁ1, the MIC of Ent/DGE-Amp/Mem 1, 2, 5, agarose/Milli-Q water). The Petri dish with the sample was
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´
then immediately covered with the No. 1.5 coverslip and placed
on the microscope stage for imaging at 37 ꢂC. Image acquisition
was carried out at 5 min intervals over 4.5–5 h. The time-lapse
images were collected on a Nikon-TE 2000 U wide-eld inver-
ted microscope equipped with a 100ꢄ oil-immersion objective
lens.
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ꢂ
(30 C, 150 rpm). Subsequently, the cells were pelleted and re-
suspended in 0.85% NaCl (centrifugation at 10 000g for 10
min). These NaCl-suspended cells were further incubated in 96-
well plates wrapped in aluminum foil with the SYTO 9-propi-
dium iodide (PI) dye mixture (1 : 5, 12 mM SYTO 9 : 60 mM PI) for
15 min (30 ꢂC, 150 rpm). Following incubation, 5 mL of cell
cultures were pipetted on agarose pads (1% w/w agarose/Milli-Q
water) placed on microslides and covered with glass coverslip.
Fluorescence microscopy images were recorded on the Zeiss
Axioplan2 microscope. The Texas Red (lEx ¼ 532–587 nm; lEm
¼
608–683 nm) and GFP (lEx ¼ 457–487 nm; lEm ¼ 502–538 nm)
channels were utilized to obtain the images of the DEAD and
LIVE cells, respectively.
Image analysis
The microscopy images were processed using the FIJI soware.
For uorescence images, uorescence background subtraction
was performed using a rolling ball method with a radius of 150
pixels. 8-bit image types were analyzed and the brightness
intensity was set between 0 and 350.
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Conflicts of interest
E. M. N. and M. R. hold patents related to this work.
20 L. D. Palmer and E. P. Skaar, Annu. Rev. Genet., 2016, 50, 67–
91.
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Acknowledgements
This work was supported by the NIH (R01 5R01AI114625 and R21
AI126465 to E. M. N and M. R.). NMR and MS instrumentation is
housed by the MIT Department of Chemistry Instrumentation
Facility. The ICP-MS instrument at MIT is maintained by the MIT
Center for Environmental Health Sciences (NIH P30-ES002109).
We thank Dr Wendy Salmon for assistance with microscopy
experiments. Work in M. R. lab is also supported by Public Health
Service Grants AI126277, AI114625, AI145325, by the Chiba
University-UCSD Center for Mucosal Immunology, Allergy, and
Vaccines, and by the UCSD Department of Pediatrics. M. R. also
holds an Investigator in the Pathogenesis of Infectious Disease
Award from the Burroughs Wellcome Fund.
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