pubs.acs.

org/journal/aidcbc Article

Overexpression of L,D‑Transpeptidase A Induces Dispensability of

Rod Complex in Escherichia coli
Rinki Gupta, Timsy Bhando, and Ranjana Pathania*
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ABSTRACT: Antimicrobial resistance (AMR) is a significant

Downloaded via INDIAN INST OF TECH ROORKEE on December 9, 2024 at 13:54:07 (UTC).

global threat, and the presence of resistance-determinant genes is

one of the major driving forces behind it. The bacterial rod
complex is an essential set of proteins that is crucial for cell survival
due to its role in cell wall biogenesis and shape maintenance.
Therefore, these proteins offer excellent potential as drug targets;
however, compensatory mutations in nontarget genes render this
complex nonessential. The MreB protein of this complex is an actin
homologue that rotates along the longitudinal axis of the cell to
provide rod shape to the bacteria. In this study, using chemical−
chemical interaction profiling and FtsZ suppression assay, we
identified the MreB targeting activity of IITR07865, a previously
discovered small molecule in our lab. Escherichia coli suppressors
against IITR07865 revealed mutations in two cell division-associated genes, min C and pal, that have not been previously implicated
in rod complex essentiality. IITR07865 resistant mutants were found to inactivate and render the rod complex nonessential, making
the rod complex inhibitors ineffective. Further, through transcriptome analysis, we reveal the primary cause of resistance in
suppressor strains to be the overexpression of an L, D-transpeptidase A enzyme, which is involved in peptidoglycan and Braun’s
lipoprotein cross-linking. Our results demonstrate a novel mechanism of resistance development in rod-shaped Gram-negative
bacterial pathogen E. coli involved in UTIs where mecillinam, a clinically used antibiotic that targets rod complex, is a drug of choice.
KEYWORDS: MreB inhibitor, antibacterial small molecule, L, D-transpeptidases, antimicrobial resistance, rod complex

The escalating mortality rate stemming from increasing coli.4,5 A22 [S-(3,4-dichlorobenzyl) isothiourea HCl] is
antimicrobial resistance (AMR) is a pressing clinical issue. another rod complex inhibitor that targets MreB, thereby
The rise in the frequency of multidrug resistant bacterial preventing lateral PG synthesis.6 Targeting E. coli cells with
pathogens has led to the failure of the therapeutic efficacy of mecillinam and A22 changes the shape of bacteria from rod to
existing antibiotics. Additionally, the innovation gap in spherical and eventually causes cell death.7 Additionally,
discovering novel antibacterials has been a setback in dealing various reports have shown that E. coli becomes resistant to
with growing antibiotic resistance issues. Therefore, it is rod complex inhibitors under certain conditions, thereby
imperative to discover novel antibacterials and identify the making the rod complex conditionally essential. This
critical physiological pathways/proteins that may act as phenomenon is seen when the cell division protein, FtsZ, is
resistance determinants. The bacterial rod complex is essential overexpressed or when target or certain nontarget genes are
for cell survival due to its involvement in cell wall formation mutated.8−13
and shape maintenance.1 Hence, the proteins of this complex Our study aimed to investigate the antibacterial potential
present promising drug targets. The complex is comprised of and mechanism of action of a novel small molecule IITR07865
six proteins (RodA, PBP2, MreB, MreC, MreD, and RodZ), (N-(3,4-dichlorophenyl)triimidodicarbonic diamide).
and all of these proteins, except MreB, are present in the inner IITR07865 demonstrated anti-Rod system activity. Analysis
membrane. MreB protein, which is a eukaryotic actin of E. coli IITR07865-resistant mutants uncovered point
homologue, is present on the cytoplasmic side of the inner
membrane.2 MreB monomers polymerize in an ATP-depend-
ent manner and move along the longitudinal axis of bacterial Received: July 24, 2024
cells.3 This movement helps in lateral peptidoglycan (PG) Revised: October 4, 2024
synthesis and provides characteristic morphology to rod- Accepted: October 10, 2024
shaped bacteria such as Escherichia coli. Mecillinam, a β-lactam Published: October 16, 2024
antibiotic, specifically targets PBP2 and is used as a first-line
treatment option for urinary tract infections caused by E.

© 2024 American Chemical Society https://doi.org/10.1021/acsinfecdis.4c00597

3928 ACS Infect. Dis. 2024, 10, 3928−3938
ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article

Figure 1. IITR07865 synergizes with peptidoglycan biogenesis inhibitors and targets rod complex in E. coli. (A) Chemical structures of A22 (MreB
inhibitor), compound 12, and IITR07865. (B) Bar graph representing combination ratio profiling of IITR07865 with cell wall biogenesis inhibitors
(Meropenem, Doripenem, A22, and Mecillinam) revealing their synergistic interactions. The dotted line represents the synergy cutoff value
(combination ratio = 0.25). Data are represented as Mean ± SD (N = 4). (C) Scanning electron micrographs representing altered E. coli ATCC
25922 cell morphology after treatment with IITR07865 (16 μg/mL), and A22 (8 μg/mL). The scale bar represents 1 μm. (D) Bar graph
representing FtsZ suppression assay with an increase in the MICs of IITR07865 and A22 against E. coli when FtsZ protein is overexpressed
(BW25113_pTB63), indicating that IITR07865, like A22 targets rod complex. Data are represented as Mean ± SD (N = 4). (E) Heat map
representing mecillinam toxicity suppression in the presence of IITR07865 (64 μg/mL) and A22 (64 μg/mL) in E. coli BW25113_pTB63 cells
when FtsZ is overexpressed depicting rod complex inhibitory activity of IITR07865. Mec (−), no mecillinam; Mec (+), 4 μg/mL mecillinam. The
color scale represents OD at 600 nm.

mutations in two cell division-related genes, not associated The small molecule exhibited higher MICs against various
with the rod complex and previously not implicated in rod strains of Acinetobacter baumannii (Gram-negative coccoba-
complex essentiality. Further exploration via transcriptomics cilli) and Staphylococcus aureus (Gram-positive), including
revealed overexpression of an L,D-transpeptidase gene in both clinical pathogens, indicating its specificity for Gram-negative
mutants as the primary cause of resistance to rod complex rods (Tables S1 and S2). Further, IITR07865 displayed
inhibitors. bactericidal potential against E. coli. The molecule reduced
bacterial burden by more than 3 Log10 CFU/mL at 128 μg/
■ RESULTS
Bactericidal Properties and Morphological Impact of
mL within 12 h of treatment (Figure S1A). The bactericidal
potential of antibiotics is crucial for effectively treating bacterial
IITR07865. IITR07865 is an antibacterial small molecule that infections. Bactericidal antibiotics are known to exert
exhibited growth inhibitory activity against a broad spectrum secondary lethal effects that enhance their effectiveness and
of Gram-negative rod-shaped bacteria, with a minimum bacterial eradication. They cause oxidative stress, generating
inhibitory concentration (MIC) of 4−16 μg/mL for E. coli reactive oxygen species (ROS) that help in damaging
strains (Table S1). This molecule was initially identified in our biomolecules like DNA, proteins, and lipids.15 Additionally,
laboratory through a forward chemical genetic screen for the these antibiotics alter membrane potential and integrity,
discovery of antibacterial small molecules targeting E. coli leading to ionic imbalance and loss of cellular homeostasis.16
ATCC 2592214 IITR07865 demonstrated significant anti- By targeting multicellular components, bactericidal antibiotics
bacterial potential, with MICs ranging from 4 to 256 μg/mL achieve effective pathogen eradication. Therefore, we explored
against a panel of clinical Gram-negative bacterial pathogens. the bactericidal mechanism of IITR07865, and these
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investigations revealed that IITR07865 induces oxidative stress control antibiotic.21 Bocillin FL is a fluorescent penicillin probe
in E. coli cells (Figure S1B). To ascertain that this ROS that binds to PBPs and can be visualized through a scanner
production is a secondary mechanism of its bactericidal using SDS-PAGE. The presence of a competitive inhibitor
activity, a ROS quenching assay was conducted in the presence reduces Bocillin FL binding to PBPs, thereby diminishing its
of vitamin C (ROS quencher). Our results showed that fluorescence and hence identifying PBP inhibitory molecules.
pretreatment of E. coli cells with vitamin C does not mitigate We found that IITR07865 does not bind to PBPs as there was
the lethal effect of IITR07865, whereas methyl viologen- no change in the intensity of the Bocillin-bound PBP bands
treated cells (control) were rescued (Figure S1C). This (Figure S2B); however, the PBP2 band was obscured on the
indicates that IITR07865 produces ROS as a consequence of gel due to a nonspecific fluorescent band of the same size
its bactericidal activity on E. coli. Further investigations (visible in no Bocillin control). To address this, we
revealed that IITR07865 disrupts membrane potential and overexpressed PBP2 in E. coli MHD79 strain, which allows
induces membrane damage, as evident from assays performed overexpression of the otherwise lethal PBP2 (Figure S2C).22
using molecular probes-DiBAC4(3) and Sytox OrangeTM, The Bocillin FL-bound PBP2 profile of IITR07865 demon-
respectively.17,18 DiBAC4(3) is a fluorescent probe that is strated that IITR07865 does not bind to PBP2 as well. The
used to measure the membrane potential of cells. It is a bis- lack of PBP inhibitory potential and microscopic indications
oxonal dye that enters depolarized cells, binds to intracellular motivated us to investigate the rod complex inhibitory
components, and emits fluorescence. The fluorescence potential of IITR07865. Therefore, we first performed an
intensity correlates with the degree of membrane depolariza- FtsZ suppression assay. E. coli cells overexpressing FtsZ
tion and provides a quantitative measurement of changes in the (FtsZUP) are tolerant to rod complex inhibitors. Therefore,
membrane potential. Our results showed a concentration- we assessed the MIC of IITR07865 and A22 in E. coli FtsZUP
dependent disruption of membrane potential after treatment cells. The E. coli FtsZUP cells exhibited tolerance to both
with IITR07865 (Figure S1D). Similarly, membrane integrity IITR07865 and A22, with a MIC value of 128 μg/mL for both
was measured by Sytox OrangeTM dye, which is a DNA- molecules (Figure 1D). Although FtsZUP renders the rod
binding molecular probe. The results demonstrated an complex nonessential; however, mecillinam which specifically
enhanced fluorescence of the dye upon treatment of cells targets PBP2, maintains its lethality by generating a futile cycle
with IITR07865, indicating a compromised state of the of PG precursor production and degradation.23 Therefore,
membrane (Figure S1E). inhibitors of other rod complex proteins should rescue bacteria
Chemical−chemical interaction profiling has been previously from the lethal effects of mecillinam by inactivating the rod
employed to elucidate the antibacterial mechanisms of complex in FtsZUP cells.24 Hence, we performed a mecillinam
unknown compounds based on their synergistic interactions suppression assay to validate the rod complex-targeting ability
with known bioactives.19 Further, IITR07865 is structurally of IITR07865 by relieving bacteria from mecillinam toxicity.
similar to A22, which targets the MreB protein of the rod To assess this, we preincubated E. coli FtsZUP cells with
complex (Figure 1A). subinhibitory concentrations of IITR07865 or A22 before
Therefore, we hypothesized that both molecules might have assessing the cellular tolerance to mecillinam. IITR07865 and
similar molecular targets, based on the structural similarity A22 were able to inhibit the lethal effects of mecillinam in
between them, and may also depict synergistic behavior. FtsZUP cells (Figure 1E). These results indicated that
Accordingly, we conducted a two-point dose matrix assay to IITR07865, like A22, targets a rod complex protein other
analyze the interactions of IITR07865 with other PG than PBP2.
biogenesis inhibitors. IITR07865 exhibited synergy with PG- Given these indications and the structural similarity between
targeting antibiotics such as meropenem, doripenem, mecilli- IITR07865 and A22, we investigated whether IITR07865
nam, and A22 against E. coli (Figure 1B). The structural interacts with MreB. For this, we purified E. coli MreB protein
resemblance of IITR07865 to A22, combined with its and studied its interaction with IITR07865 using dynamic light
antibacterial efficacy against E. coli and synergistic interactions scattering (DLS). DLS is used to determine the size
with PG biosynthesis inhibitors, prompted us to investigate its distribution of small particles in suspension or polymers in
ability to target PG. PG inhibitors, including rod complex and solution.25 MreB forms filaments after polymerization in the
penicillin-binding protein (PBP) inhibitors, typically cause presence of ATP. Using DLS, we showed that IITR07865
morphological changes in bacteria, altering their shape from interacts with MreB and reduces its polymerization similarly to
rod-like to round or oval.20 Microscopic analysis using A22 (Figure S2D). In the presence of IITR07865, we observed
scanning electron microscopy and fluorescence probe FM4- a decrease in the peak size as well as a shift of the peak to a
64 revealed that IITR07865 induces rounding of E. coli cells lower size region, indicating reduced polymer size of MreB
within 2 h of treatment, similar to the effects observed with compared to the untreated control. A similar trend was
A22 and meropenem treatments (Figures 1C and S2A, observed with MreB incubated with A22, where the peak
respectively). These findings implicated the role of shifted to a smaller size.
IITR07865 in the inhibition of PG biogenesis. Mutations in minC and pal Impart Resistance to MreB
IITR07865 Binds to MreB Protein of the Rod Inhibitors. In our attempt to validate the molecular target of
Complex. Meropenem is a β-lactam antibiotic that targets IITR07865 and assess the resistance generation capability of
PBPs, whereas A22 binds to the MreB protein of the rod bacteria, we exposed E. coli cells to subinhibitory concen-
complex. As shown previously from microscopy, inhibitors of trations of IITR07865 for 12 h repeatedly over a period of 30
both PBPs and rod complex can change the rod shape of E. coli days. It was observed that exposure to IITR07865 led to the
to a round morphology. Therefore, to determine whether generation of stable E. coli mutants resistant to IITR07865
IITR07865 binds to any of the PBPs, we conducted an SDS- after 9 days, which persisted until day 30 (Figure S2E). The
PAGE based PBP binding competition assay with Bocillin FL mutants obtained at the end of 30 days exhibited resistance
in the presence or absence of IITR07865 and meropenem as against IITR07865 when passaged in the absence of
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Figure 2. IITR07865 resistant minCG236A mutant of E. coli ATCC 25922 depicts growth and morphological defects. (A) Nucleotide mutations (and
corresponding amino acid mutations) identified through whole genome sequencing in three IITR07865 resistant mutants generated in E. coli
ATCC 25922. (B) Bar graph representing a decrease in susceptibility of E. coli ATCC 25922 resistant mutants (M1, M2, and M3) against
IITR07865 and A22 when compared to wild-type (WT) cells. Data are represented as Mean ± SD (N = 4). (C) Growth curve of E. coli ATCC
25922 WT, M1, and M2 strains representing a phenotypic growth defect in minCG236A mutant (M2) cells. Data are represented as Mean ± SD (N =
3). (D) Membrane potential assay (using DiBAC4 probe) in E. coli ATCC 25922 WT, M1, M2, and M3 cells depicting depolarized membrane of
mutant strains (M1, M2, and M3). Data are represented as Mean ± SD (N = 3). P values were determined by one-way ANOVA followed by
Dunnett’s multiple comparison test. ***, p < 0.001. (E) Scanning electron micrographs of E. coli ATCC 25922 WT, M1, M2, and M3 strains
representing morphological defects due to unequal cell division in minCG236A mutants (M2 and M3). The scale bar represents 2 μm. The arrows
indicate the elongated and mini-cell phenotypes of M2 and M3-resistant mutants.

IITR07865 stress revealing the development of stable resistant Table 1. Antibiotic Susceptibilities of E. coli ATCC 25922
mutants. Three colonies (denoted as M1, M2, and M3) from Wild-type (WT) and IITR07865 Resistant Mutants (M1 and
the resistant clones were selected at random, and their whole M2) Against Inhibitors of Rod Complex and Penicillin-
genomes were sequenced and compared with wild-type E. coli Binding Proteins (PBPs)a
ATCC 25922 (NCBI: CP009072.1). The results revealed
small molecule/ E. coli ATCC 25922 M1 (pal M2 (minC
mutations in two distinct genes: pal (peptidoglycan-associated antibiotics (WT) mutant) mutant)
lipoprotein) in M1 and minC (septum site-determining gene) IITR07865 8/16 256 256
in M2 and M3 (Figure 2A). A22 4 128 128
The mutations identified were missense variants at 347 and Mecillinam 8 >32 >32
236 nucleotide positions of pal and minC genes, respectively. Ampicillin 4 4 4
Since M2 and M3 exhibited the same mutations, therefore, Meropenem 0.03 0.03 0.03
most experiments were conducted only with M1 and M2. The Aztreonam 0.125 0.125 0.125
mutants demonstrated resistance to both IITR07865 and A22, Piperacillin 2 2 2
tolerating 16-32 and 32 times the concentration of IITR07865 Doripenem 0.03 0.03 0.03
and A22, respectively as compared to wild-type cells (Figure Cefotaxime 0.25 0.25 0.25
2B). These mutants were also assessed for their resistance a
minCG236A depicts physiological and morphological defects.
profile against other β -lactam antibiotics. The results showed
that the mutants were also resistant to mecillinam, a PBP2
targeting rod complex inhibitor (Table 1). Further, these piperacillin, doripenem, and cefotaxime (Table 1). This
resistant clones did not display resistance toward other β- indicated that the mutants exhibit resistant phenotype
lactam antibiotics such as ampicillin, meropenem, aztreonam, specifically to rod complex inhibitors.
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Both pal and minC genes encode for proteins associated with Table 2. MIC Analysis of E. coli Genetic Deletion Mutant
cell division. Pal belongs to the Tol−Pal complex, which helps Strains of Tol−Pal Complex Proteins (TolB, Pal, TolQ,
in OM invagination during cell division, whereas MinC helps TolA, TolR), MinC, RodZ, LpoB and Penicillin-Binding
in the localization of FtsZ ring and prevents nonpolar cell Proteins (PBPs) Against IITR07865 and A22
division.26,27 Given the crucial roles of these genes in cell
Strain (E. coli BW25113) IITR07865 (MIC, μg/mL) A22 (MIC, μg/mL)
division, we investigated potential physiological defects in
these mutants. Growth curve analysis revealed that E. coli Wild type 4 1
minCG236A (M2 mutant) exhibited a significant growth defect, ΔtolB 64 64
whereas the E. coli palA347G (M1 mutant) did not show any Δpal 128 128
growth impairment (Figure 2C). These findings were ΔtolQ 64 64
corroborated by reconstructing palA347G and minCG236A ΔtolA 64 128
mutations in E. coli BW25113 Δpal and E. coli BW25113 ΔtolR 64 128
ΔminC strains, respectively. The reconstructed mutants ΔrodZ 128 128
demonstrated similar phenotypes, including resistance to rod ΔminC 128 128
complex inhibitors and a growth defect in E. coli BW25113 ΔlpoB 4 1
minCG236A mutant (Figure S3A,B). Further, we performed a ΔmrcB (PBP1b) 4 1
ΔmrcA (PBP1a) 128 128
membrane potential assay to evaluate changes in membrane
ΔpbpC (PBP1c) 128 128
potential dynamics since these genes are also associated with
ΔdacB (PBP4) 128 128
the membrane. We found that all three mutants exhibited
ΔdacA (PBP5) 4 1
depolarized membrane (Figure 2D), indicating that mutations
ΔdacC (PBP6) 128 128
in the Pal and MinC proteins alter membrane potential in E.
ΔpbpG (PBP7) 128 128
coli. Considering these observations and the involvement of
these mutants in cell division, we performed SEM analysis to
assess morphological changes in the cells. Consistent with our With these observations and several genes identified
findings, E. coli minCG236A (M2 and M3) displayed altered previously in relation to resistance toward rod complex
morphology, whereas the E. coli palA347G (M1) did not show inhibitors, we further assessed whether other proteins of the
any morphological change compared to wild-type cells (Figure Tol−Pal complex and PBPs are also involved in this
2E). The E. coli minCG236A depicted irregular cell lengths phenomenon. We examined mutants of Tol−Pal complex
ranging from ∼0.5−8.6 μm (n = ∼ 100) due to the occurrence (ΔtolB, ΔtolQ, ΔtolA, ΔtolR), rod complex (ΔrodZ), PBPs
of nonpolar cell divisions. Further, we inspected whether these (ΔmrcA, ΔmrcB, ΔpbpC, ΔdacB, ΔdacA, ΔdacC, ΔpbpG), and
resistant mutants would exhibit altered round morphology ΔlpoB (PBP1b activator). These mutants were tested for their
upon treatment with IITR07865 and A22 at higher susceptibility to IITR07865 and A22. MIC results revealed that
concentrations (respective 2X MICs). SEM analysis showed deletion of most of these genes conferred resistance to rod
a round-shaped morphology in wild-type E. coli cells after complex inhibitors (Table 2). However, three gene deletion
treatment, consistent with our previous results; however, this mutants�lpoB, mrcB, and dacA�were susceptible to
alteration was not observed in the case of mutants (Figure IITR07865 and A22, exhibiting the same MIC values as
S3C). Instead, the mutants displayed damage to the lateral cell wild-type E. coli. It has been shown in the literature that the
wall following treatment with IITR07865 and A22, without upregulation of PBP2 and LpoB proteins confers resistance to
changes in their cell shape. As previously mentioned, the rod mecillinam in E. coli by rendering the rod complex
complex helps in lateral cell wall PG synthesis where MreB nonessential.30 Thus, the observed phenotype aligns with
moves along the lateral axis of the cell. Consequently, at higher these findings, suggesting that the presence of PBP2 and LpoB
concentrations, it appears that the lethal effects of MreB is necessary for the development of resistance to rod complex
inhibitors manifest as damage to the cell wall rather than inhibitors. Additionally, our observation that PBP5 (encoded
changes in cell morphology. by dacA), a carboxypeptidase, also plays a role in resistance
Mutated Tol−Pal Complex Proteins and PBPs Confer development, is notable since the ΔdacA mutant was found to
Rod Complex Nonessentiality. Mutations in a multitude of be susceptible to both A22 and IITR07865.
genes have been reported in the literature to render the rod Upregulation of LdtA Confers Resistance to Rod Complex
complex nonessential.13,28,29 These genes encode proteins Inhibitors. To gain a deeper understanding of the resistance
involved in diverse cellular pathways, including amino acid mechanism in mutants to rod complex inhibitors, we
metabolism, central metabolic regulation, cell division, and cell conducted transcriptome analysis. The results identified a
wall synthesis. Such reports have utilized both deletion total of 68 differentially expressed genes common to both
mutants and disruption mutants in the studies. The specific mutants (Table S3). Among these, only one cell wall-related
mutations in palA347G and minCG236A genes identified in our protein, i.e., L,D-transpeptidase A (LdtA, encoded by erf K), was
study have not been implicated in the functionality of rod found to be upregulated (Figure 3A). The overexpression of
complex previously. Consequently, we sought to determine LdtA in both mutants M1 (palA347G) and M2 (minCG236A) was
whether the observed resistance phenotype of mutants was confirmed through qPCR (Figure 3B,C). Ldt A is a periplasmic
solely due to these specific mutations or if deletion mutants of protein that is involved in the cross-linking of PG with OM
these genes would exhibit similar behavior. To investigate this, Braun’s lipoprotein (Lpp), as illustrated in Figure 3D.31
we obtained Δpal and ΔminC mutants from the Keio Further, we assessed the mutants for differential expression
collection library of E. coli and evaluated MICs of of genes such as mrcB, lpoB, and f tsZ since deletion mutants of
IITR07865 and A22 against these strains. Our results indicated these genes demonstrated susceptibility in MIC profiling, and
that Δpal and ΔminC mutants were also resistant to both upregulation of these genes has been previously associated with
molecules compared to the wild-type strain (Table 2). resistance to mecillinam. Additionally, we evaluated the relative
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Figure 3. LD-transpeptidase A encoded by erf K is upregulated in IITR07865-resistant mutants. (A) Transcriptome analysis of the E. coli ATCC
25922 WT, M1, and M2 resistant mutants revealed an upregulation of the erf K gene in mutants M1 (palA347G) and M2 (minCG236A) as compared to
WT cells. FC represents fold change. Real-time PCR analysis depicting overexpression of LdtA (erf K), PBP1b (mrcB), LpoB (lpoB), PBP5 (dacA),
and FtsZ (f tsZ) in M1 (B) and M2 (C) mutants as compared to WT cells. Data are represented as Mean ± SD (N = 3). P values were determined
by two-way ANOVA followed by Sidak’s multiple comparison test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, nonsignificant. (D) Graphical
representation of LD cross-links mediated by LD-transpeptidases (LDTs) as compared to DD cross-links mediated by penicillin-binding proteins
(PBPs) in E. coli cell wall.

gene expression of another gene, dacA, in mutants due to the complex inhibitors in E. coli, as elucidated by the chemical
observed altered MIC susceptibility of E. coli BW25113 ΔdacA probe IITR07865.
to A22 and IITR07865. Our findings indicated that all of these
genes, except f tsZ, were significantly upregulated in both
mutants; however, the differential expression of these genes
■ DISCUSSION
The present study explores a novel resistance mechanism
was not highlighted in transcriptomics. Therefore, to validate developed in E. coli against rod complex inhibitors, unveiling
the exclusive role of LdtA in conferring resistance to A22 and significant insights into genetic and regulatory underpinnings
IITR07865, we preliminarily evaluated the antibacterial of resistance development. The bacterial rod complex,
potential of these small molecules against IITR07865 resistant comprising of essential proteins such as MreB, is pivotal in
mutants (M1 and M2) in the presence of subinhibitory maintaining cell shape and integrity. Inhibition of MreB
concentrations of copper (CuSO4 and CuCl2). Ldts are known disrupts cell shape, transforming rod-shaped bacteria into
to possess serine in their active site and are inhibited by copper spherical forms and eventually leads to cell death. Our study
(Figure 4A).32 We found that inhibition of LdtA by copper introduced IITR07865, a chemical probe that targets MreB
rendered the mutants susceptible to A22 and IITR07865 and induces morphological changes in E. coli. A22 and similar
molecules are known to have an affinity toward MreB.
(Figure 4B−E).
Isothiourea and aryl halide moieties in A22-like molecules
Therefore, to confirm the primary role of LdtA, we have been identified as important entities for anti-Rod system
constructed the palA347G and minCG236A mutations in a deletion activity. Compound 12 is an A22-like molecule that was
mutant of LdtA in E. coli. When we assessed the susceptibility discovered in a previous screen for the discovery of antirod
of these mutants to A22 and IITR07865, we observed that system molecules; however, compound 12 did not exhibit
both mutants were as susceptible to small molecules as wild- antirod system activity, which was implicated due to the
type cells (Table 3). Collectively, these results emphasized the absence of the isothiourea group (Figure 1A).24 In this study,
role of LdtA overexpression in conferring resistance to rod we found that IITR07865, which is more similar to compound
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Figure 4. Inhibition of LdtA by copper reverts the resistant phenotype of mutants (M1 and M2) to the susceptible phenotype. (A) Illustration
depicting LdtA mediated cross-linking between m-DAP residue of tetrapeptide to the lysine residue of Braun’s lipoprotein Lpp. Reversal of M1 and
M2 susceptibility to IITR07865 and A22 in the presence of subinhibitory concentrations of CuSO4 (B,C) and CuCl2 (D,E). Data are represented
as Mean ± SD (N = 2).

Table 3. MIC Values of E. coli Mutant Strains Against compounds. Previously, a comprehensive study on mecillinam
IITR07865 and A22 When LdtA is Deleted in the Presence resistome conducted by Lai et al., reported mutations in ∼143
of palA347G and minCG236A Mutations loci that rendered the rod complex nonessential.28 In our
MIC (μg/mL)
study, we discovered two distinct mutations in pal and minC
genes, highlighting a peculiar resistance mechanism against rod
Strain IITR07865 A22 complex inhibitors. We found that the equivalent deletion
E. coli BW25113 8 2 mutants of these genes also conferred the resistant phenotype.
E. coli BW25113 Δpal::pal (347A > G) 64 128 These mutations inactivated the rod complex and rendered it
E. coli BW25113 ΔldtA pal::pal (347A > G) 4 2 nonessential, thus conferring resistance not only to IITR07865
E. coli BW25113 ΔminC::minC (236 G > A) 256 128 but also to other rod complex inhibitors such as A22 and
E. coli BW25113 ΔldtA minC::minC (236 G > A) 4 1
mecillinam. Interestingly, these mutants did not exhibit
resistance to β-lactam antibiotics that target PBPs, indicating
12 as opposed to A22, depicted antirod system activity despite a specific resistance pathway against rod complex inhibitors.
lacking the isothiourea group. The lack of MreB inhibitory Additionally, we found that individual deletion mutants of all
potential of compound 12 might have occurred due to the proteins of the Tol−Pal complex also resulted in a similar
requirement of a higher concentration than the concentration phenotype. These mutations, along with mutations in PBP5,
used for screening to elicit MreB inhibitory potential. Further, have not been implicated in rod complex inhibitor resistance
the MreB inhibitory activity of IITR07865 could also be phenomenon earlier. Moreover, minCG236A mutant demon-
attributed to slight deviations in the structure of IITR07865 strated altered morphology and growth kinetics. A previous
from compound 12, like the presence of an extra chlorine atom study reported multiple mutations in the N- and C-terminal
on the benzene ring and reduced nitrogen groups. These domains of MinC, which affected the bacterial morphology
observations indicate that isothiourea moiety may not be through loss of interaction between its counterpart FtsZ and
needed for antirod system activity; however, it may be required MinE proteins.33 The observed mutation in minC loci in our
to enhance the antirod system activity as observed in A22-like study also depicts the loss of interaction of MinC with its
3934 https://doi.org/10.1021/acsinfecdis.4c00597
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ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article

interacting partners, resulting in nonpolar divisions and ROS Quenching Assay. E. coli culture of O.D. ∼0.5 was
formation of mini cells, which can be further explored. The diluted 2000 times in MH broth. The culture was divided into
transcriptome analysis of the IITR07865 resistant mutants two tubes. In one tube, 5 mM of vitamin C was added to the
revealed the overexpression of LdtA in the resistant mutants, cells, and one was left only with the cells. Both tubes were then
suggesting its pivotal role in resistance development. Six types incubated at 37 °C, 180 rpm for 15 min. The cell suspension
of Ldts (Ldt A-F) are known in E. coli.34 LdtA, B, and C are was then added to 96 well plates in the presence or absence of
involved in the cross-linking of PG with Lpp in the OM, IITR07865. Methyl viologen treatment was used as the
whereas LdtD and LdtE mediate 3-3 cross-links in PG. LdtF positive control. The plate was incubated for 16 h at 37 °C
works antagonistically to LdtA−C and cleaves cross-links under static conditions in a Biotek synergy instrument, and
between Lpp and PG. LdtD-mediated 3-3 cross-links have OD600 was measured at an interval of 2 h.
been implicated in resistance to β-lactam antibiotics by Membrane Potential Assay. E. coli cells of O.D. ∼0.3
circumventing the conventional 4-3 cross-links.35 However, resuspended in 1× PBS were incubated for 15 min at room
there are no reports on the involvement of Ldts in rod complex temperature with 5 μM DiBAC4. In a 96-well black plate,
essentiality. Also, the carboxypeptidase activity of PBP5 is IITR07865 was added at the required concentrations, and the
required by Ldts for the generation of donor peptides by cell suspension was added to a final volume of 200 μL. The
hydrolysis of the bond between m-DAP and D-Ala of plate was incubated for 30 min, and fluorescence was measured
pentapeptide.35 This is likely the reason that we have seen at 490/518 nm in a Biotek synergy instrument. For IITR07865
an upregulation of PBP5 in mutants. The upregulation of LdtA mutants, cells were incubated with DiBAC4 were used, and no
in IITR07865-resistant mutants plausibly compensates for the treatment was given.
nonessential rod complex and maintains cell wall stability. The Membrane Damage Assay. E. coli cells of O.D. ∼0.3
findings of this study have profound implications for resuspended in 1× PBS were incubated with 1 μM Sytox
developing new therapeutic strategies. Understanding the Orange dye for 15 min at room temperature. In a 96-well black
resistance mechanisms against rod complex inhibitors paves plate, IITR07865 was added at the required concentrations,
the way for designing combination therapies that target and the cell suspension was added to a final volume of 200 μL.
resistance-determining genes and pathways. For instance, The plate was then incubated for 60 min, and fluorescence was
combining IITR07865/mecillinam with inhibitors of LdtA measured at 547/571 nm in a Biotek synergy instrument.
could restore the efficacy of rod complex inhibitors and reduce Polymyxin B treated sample was used as the positive control.
the emergence of resistant strains. This study highlights the Two-Point Dose Matrix Assay. The assay was conducted
critical role of rod complex in bacterial survival and the as has been described previously with slight modifications
emergence of novel resistance mechanisms against rod (19). Compounds were added at their respective 0.5× MIC,
complex inhibitors. alone or in combination, in 96-well plate wells at the final
volume of 100 μL in MHB. 100 μL of 1000 times diluted 0.5
■ METHODS
Bacterial Strains and Plasmids. All the details on the
McFarland E. coli culture in MHB was added to the wells and
incubated at 37 °C for 12 h. The combination ratio was
calculated as the growth percentage of bacteria treated with a
bacterial strains and plasmids used in the study are provided in combination of antibiotics and small molecules divided by the
Table S4. All the strains were grown in Mueller Hinton (MH) growth percentage of bacteria when treated with an alone
media. For cloning purposes, Luria−Bertani (LB) broth was antibiotic.
used for bacterial cultures. Fluorescence Microscopy. E. coli cells of O.D. ∼0.3 were
MIC Determination. Briefly, an overnight culture of E. coli taken and treated with IITR07865 for 2 h at 37 °C, 180 rpm.
was 1% subcultured and incubated at 37 °C, 180 rpm until it Cells were washed with 1X PBS after treatment and
reached McFarland 0.5. 100 μL culture was added to each well resuspended in 1× PBS, followed by staining with 1 μg/mL
containing 2-fold serially diluted drug concentrations in MH FM-4-64 for 15 min in the dark at room temperature. The cells
broth. The plate was incubated for 12−16 h, and OD600 was were subsequently washed and resuspended in 1× PBS. The
measured. The lowest concentration of the drug, which cell suspension was mounted onto a 1% agarose pad and
inhibited the growth of bacteria, was considered the MIC of observed under a Zeiss Axioscope A1 fluorescence microscope
the compound. Similarly, for the growth curve assay, the plate equipped with an AxioCam MRC digital camera using EC
was incubated in a Biotek Synergy plate reader, and OD600 was Plan-Neofluar 100× objective.
measured at specified time intervals. Scanning Electron Microscopy. E. coli cells of O.D. ∼0.3
Time Kill Kinetics Assay. E. coli culture of 0.5 McFarland were treated with IITR07865 for 2 h at 37 °C, 180 rpm. After
was treated with different concentrations of IITR007865. incubation, cells were washed with 1× PBS, followed by
Aliquots were taken at varied time intervals for 24 h and fixation in 2.5% glutaraldehyde and 4% formaldehyde at 4 °C
diluted in 1× PBS. The dilutions were spotted on MH agar for 2 h. The cells were subsequently washed and resuspended
plates and incubated overnight at 37 °C. in 1× PBS. Cell suspension was dried on clean-cut glass slides
ROS Determination Assay. E. coli cells of O.D. ∼0.3 and sequentially dehydrated with 30%, 50%, 70%, 90%, and
resuspended in 1× PBS were incubated with 100 μM DCFDA 100% ethanol. Samples were gold-coated and visualized under
probe for 30 min at 37 °C, 180 rpm in dark. In a 96-well black a scanning electron microscope.
plate, IITR07865 was added at required concentrations, and PBP Binding and Competition Assay. Membrane
cell suspension was added to a final volume of 200 μL. The protein isolation and Bocillin competition assay were
plate was then incubated for 60 min, and fluorescence was conducted as described previously with slight modifications.36
measured at 485/530 nm in a Biotek synergy instrument. Briefly, E. coli DH5α (OD600 ∼1) stationary phase cells were
Methyl viologen treated sample was used as the positive harvested by centrifugation. The cells were lysed using cell
control. disruptor (Constant System Ltd.) at a pressure of 25 psi, and
3935 https://doi.org/10.1021/acsinfecdis.4c00597
ACS Infect. Dis. 2024, 10, 3928−3938
ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article

cell debris was removed by centrifugation at 12,000g for 30 continued for 30 days. The cells from the resistant culture were
min. The supernatant was centrifuged at 150,000g for 60 min plated onto an MH agar plate, and genomic DNA was isolated
at 4 °C for isolation of membranes. The pellet was from three different colonies. Whole genome sequencing was
resuspended in 1× PBS (1 mL). For the PBP competition performed by Biokart India Pvt. Ltd. (India) using Illumina
assay, membrane preparations (10 μL each) were incubated Hiseq 4000, and E. coli ATCC 25922 (NCBI: CP009072.1)
with IITR07865 and control antibiotic at increasing concen- was used as the reference genome.
trations and incubated for 25 min, 30 °C. Later, Bocillin (15 Construction of Gene Mutation. E. coli ATCC 25922
μg/mL) was added and incubated for 25 min at 30 °C. After minC and pal mutated genes were PCR amplified from the
incubation, samples were electrophoresed using an SDS- IITR07865 resistant mutants and cloned in pUC18 vector with
polyacrylamide gel (12%) system. For PBP visualization, gels upstream 300bp (US300) and downstream 300 bp (DS300).
were scanned by the Typhoon FLA 9500 instrument using a An apramycin cassette (AprR) was introduced downstream to
488 nm excitation wavelength (emission, 520 to 530 nm) the gene and upstream to DS300. All the fragments were PCR
corresponding to the peak absorption of Bocillin FL. The same amplified (Table S2) separately and gel purified. The
gel was also stained with coomassie brilliant blue stain to fragments in the sequence US300-Gene-AprR-DS300 were
confirm equal loading of membrane protein in all lanes. cloned using TaKaRa In-fusion cloning mix as per
FtsZUP Suppression Assay. Plasmid pTB63 containing manufacturer’s instructions and transformed in pKD46
f tsZ under native promoter and tetracycline resistance marker carrying E. coli BW25113 Δpal or E. coli BW25113 ΔminC
was transformed into E. coli BW25113 competent cells using chemical competent cells for pal and minC mutants,
electroporation. The MIC of IITR07865, A22, and mecillinam respectively. The transformed mix was incubated at 30 °C,
was tested in E. coli BW25113 and BW25113 pTB63 cells, as 180 rpm for 1 h, followed by spreading onto an LB plate
mentioned, after 16 h of incubation at 30 °C. supplemented with 50 μg/mL apramycin. The plate was
Mecillinam Toxicity Suppression Assay. E. coli incubated overnight at 30 °C, and clones were confirmed by
BW25113 pTB63 cells (100 μL of 0.5 McFarland culture) PCR. Subsequently, the apramycin cassette was removed using
were preincubated with IITR07865 or A22 for 30 min at their the pCP20 plasmid, and the cells were cured of the plasmid at
respective 0.5× MIC at room temperature followed by 42 °C.
addition of 4 μg/mL of mecillinam to a total volume of 200 Gene Expression Analysis. Overnight cultures of E. coli
μL. The cells were incubated at 30 °C for 16 h, and OD600 was ATCC 25922 (WT and mutants) were subcultured to OD600 =
measured. 1. Subsequently, RNA was isolated using TRIzol reagent and
MreB Purification. MreB was purified as described
DNase treated to remove genomic DNA contamination. The
previously with some modifications.37 The gene encoding
DNase-treated RNA samples were then sent to miBiome
MreB was amplified from E. coli ATCC 25922 genomic DNA
Therapeutics LLP (India) for transcriptome analysis. The
using primers (Table S5). The gene was cloned in pET28 and
transcriptome profile of mutants was compared with wild-type
transformed into E. coli BL21 DE3. An overnight culture of E.
E. coli. For real-time PCR, an equal amount of DNase-treated
coli BL21 DE3 carrying pET28 cloned with MreB was
subcultured and incubated until it reached an OD of 0.6− RNA was reverse transcribed to cDNA using a TaKaRa cDNA
0.7. The culture was induced with 0.1 mM IPTG for 4 h, 180 synthesis kit as per the manufacturer’s instructions. The cDNA
rpm, 37 °C. After induction, cells were washed and was used to perform real-time PCR using the SYBR Green mix.
resuspended in buffer A without urea (50 mM Tris HCl, The change in gene expression was analyzed using ΔΔCt
500 mM NaCl, and 20 mM imidazole, pH 8.0), followed by method with 16S rRNA as the housekeeping control.
homogenization at 25 KPsi. The cell lysate was centrifuged at
12,000g, 30 min at 4 °C. The pellet was resuspended in 10 mL
Buffer A (50 mM Tris HCl, 500 mM NaCl, 8 M urea, and 20
■ ASSOCIATED CONTENT
Data Availability Statement
mM imidazole, pH 8.0). The suspension was loaded on a Ni-
The whole genome sequence data and transcriptome data of
NTA column equilibrated with buffer A, and protein was
the mutants are submitted on NCBI under the BioProject
eluted at increasing concentrations of imidazole (0−100%)
accession number PRJNA1121254. All other data included in
with buffer B (50 mM Tris HCl, 500 mM NaCl, 8 M urea and
the study are available upon request to the corresponding
500 mM imidazole, pH 8.0). The purified protein was dialyzed
with dialysis buffer (50 mM Tris HCl, 0.1 mM CaCl2and 0.5 author.
mM dithiothreitol, pH 8.0) at 4 °C and stored at −80 °C with *
sı Supporting Information

10% v/v glycerol for further experimentation. The Supporting Information is available free of charge at
Dynamic Light Scattering. The assay was conducted as https://pubs.acs.org/doi/10.1021/acsinfecdis.4c00597.
described previously with slight modifications.38 The purified 5
μM MreB was incubated with 200 μM ATP, 1 mM MgCl2, 100 [Figure S1, Antibacterial characteristics of IITR07865;
mM KCl, 100 μM CaCl2, and 50 mM Tris in a 1 mL reaction Figure S2, MreB targeting studies of IITR07865; Figure
in the presence or absence of 300 μM IITR07865 or A22. The S3, Phenotypic and morphological characterization of
sample was incubated at 20 °C for 30 min, and the particle size IITR07865 resistant mutants; Table S1, MIC values of
was measured using Malvern Panalytical DLS instrument. IITR07865 against various Gram-negative and Gram-
Resistant Mutant Beneration and Sequencing. E. coli positive bacterial pathogens; Table S2, MIC values of 5
ATCC 25922 resistant mutant of IITR07865 was generated by against clinical isolates; Table S3, list of differentially
serial passaging of cells in the presence of IITR07865 as expressed genes in IITR07865 resistant mutants; Table
described previously with slight modifications.39 The cells that S4, List of bacterial strains used in the study and their
survived the highest concentration of IITR07865 were source; Table S5, List of primers used in the study]
passaged and incubated at 37 °C for 12 h. This process was (PDF)

3936 https://doi.org/10.1021/acsinfecdis.4c00597
ACS Infect. Dis. 2024, 10, 3928−3938
ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article

■ AUTHOR INFORMATION
Corresponding Author
resistant Escherichia coli in a murine urinary tract infection model.
Int. J. Antimicrob. Agents 2020, 55 (2), 105851.
(6) Gitai, Z.; Dye, N. A.; Reisenauer, A.; Wachi, M.; Shapiro, L.
Ranjana Pathania − Department of Biosciences and MreB actin-mediated segregation of a specific region of a bacterial
Bioengineering, Indian Institute of Technology Roorkee, chromosome. Cell 2005, 120 (3), 329−341.
Roorkee, Uttarakhand 247 667, India; orcid.org/0000- (7) Cho, H.; Uehara, T.; Bernhardt, T. G. Beta-lactam antibiotics
0002-7965-8176; Email: [email protected] induce a lethal malfunctioning of the bacterial cell wall synthesis
machinery. Cell 2014, 159 (6), 1300−1311.
Authors (8) Vinella, D.; D’Ari, R.; Jaffé, A.; Bouloc, P. Penicillin binding
Rinki Gupta − Department of Biosciences and Bioengineering, protein 2 is dispensable in Escherichia coli when ppGpp synthesis is
Indian Institute of Technology Roorkee, Roorkee, induced. EMBO J. 1992, 11 (4), 1493−1501.
Uttarakhand 247 667, India (9) Vinella, D.; Joseleau-Petit, D.; Thévenet, D.; Bouloc, P.; D’Ari,
R. Penicillin-binding protein 2 inactivation in Escherichia coli results in
Timsy Bhando − Department of Biosciences and
cell division inhibition, which is relieved by FtsZ overexpression. J.
Bioengineering, Indian Institute of Technology Roorkee, Bacteriol. 1993, 175 (20), 6704−6710.
Roorkee, Uttarakhand 247 667, India; Present (10) Vinella, D.; Gagny, B.; Joseleau-Petit, D.; D’Ari, R.; Cashel, M.
Address: T.B.: Michael G. DeGroote Institute of Mecillinam resistance in Escherichia coli is conferred by loss of a
Infectious Disease Research, McMaster University, second activity of the AroK protein. J. Bacteriol. 1996, 178 (13),
Hamilton, Ontario L8S 4L8, Canada 3818−3828.
Complete contact information is available at: (11) Oppezzo, O. J.; Antón, D. N. Involvement of cysB and cysE
genes in the sensitivity of Salmonella typhimurium to mecillinam. J.
https://pubs.acs.org/10.1021/acsinfecdis.4c00597 Bacteriol. 1995, 177 (15), 4524−4527.
(12) Kruse, T.; Bork-Jensen, J.; Gerdes, K. The morphogenetic
Author Contributions MreBCD proteins of Escherichia coli form an essential membrane-
Conceptualization: R.P., R.G., and T.B.; Methodology: R.G.; bound complex. Mol. Microbiol. 2005, 55 (1), 78−89.
Resources: R.P.; Formal analysis: R.G; Funding acquisition: (13) Thulin, E.; Sundqvist, M.; Andersson, D. I. Amdinocillin
R.P.; Supervision: R.P.; Writing−original draft: R.G.; Writing− (Mecillinam) resistance mutations in clinical isolates and laboratory-
review and editing: R.G., T.B. and R.P. selected mutants of Escherichia coli. Antimicrob. Agents Chemother.
2015, 59 (3), 1718−1727.
Funding (14) Bhando, T.; Bhattacharyya, T.; Gaurav, A.; Akhter, J.; Saini, M.;
This work was funded by DST-SERB (Grant no. EMR/2016/ Gupta, V. K.; Srivastava, S. K.; Sen, H.; Navani, N. K.; Gupta, V.;
007116), Government of India. R.G. was supported by a Biswas, D.; Chaudhry, R.; Pathania, R. Antibacterial properties and in
fellowship from the Department of Biotechnology (DBT), vivo efficacy of a novel nitrofuran, IITR06144, against MDR
Government of India (DBT/2018/IIT-R/1144). T.B. was pathogens. J. Antimicrob. Chemother. 2019, 75 (2), 418−428.
supported by a fellowship from the Ministry of Human (15) Dwyer, D. J.; Belenky, P. A.; Yang, J. H.; MacDonald, I. C.;
Resource Development (MHRD), Government of India. Martell, J. D.; Takahashi, N.; Chan, C. T.; Lobritz, M. A.; Braff, D.;
Schwarz, E. G.; Ye, J. D.; Pati, M.; Vercruysse, M.; Ralifo, P. S.;
Notes Allison, K. R.; Khalil, A. S.; Ting, A. Y.; Walker, G. C.; Collins, J. J.
The authors declare no competing financial interest. Antibiotics induce redox-related physiological alterations as part of
their lethality. Proc. Natl. Acad. Sci. U.S.A. 2014, 111 (20), E2100−
■ ACKNOWLEDGMENTS
The authors would like to acknowledge Prof. Thomas
E2109.
(16) Kohanski, M. A.; Dwyer, D. J.; Hayete, B.; Lawrence, C. A.;
Collins, J. J. A common mechanism of cellular death induced by
Bernhardt, Harvard Medical School for kindly providing bactericidal antibiotics. Cell 2007, 130 (5), 797−810.
plasmid pTB63 and Prof. Waldemar Vollmer, Newcastle (17) Gaurav, A.; Gupta, V.; Shrivastava, S. K.; Pathania, R.
University Biosciences Institute for kindly providing the strain Mechanistic insights into synergy between nalidixic acid and
E. coli MHD79. The authors would also like to acknowledge tetracycline against clinical isolates of Acinetobacter baumannii and
Institute Instrumentation Centre (IIC) for Scanning Electron Escherichia coli. Commun. Biol. 2021, 4 (1), 542.
Microscopy facility and Central Instrumentation Facility (18) Dubey, V.; Gupta, R.; Pathania, R. Targeting Superoxide
available at the Centre for Nanotechnology, Indian Institute dismutase confers enhanced Reactive Oxygen Species mediated
of Technology Roorkee. eradication of Polymyxin B induced Acinetobacter baumannii
persisters. Antimicrob. Agents Chemother. 2021, 65 (5), 10−1128.

■ REFERENCES
(1) Typas, A.; Banzhaf, M.; Gross, C. A.; Vollmer, W. From the
(19) Farha, M. A.; Brown, E. D. Chemical probes of Escherichia coli
uncovered through chemical-chemical interaction profiling with
compounds of known biological activity. Chem. Biol. 2010, 17 (8),
regulation of peptidoglycan synthesis to bacterial growth and 852−862.
morphology. Nat. Rev. Microbiol. 2012, 10 (2), 123−136. (20) Cross, T.; Ransegnola, B.; Shin, J. H.; Weaver, A.; Fauntleroy,
(2) Shi, H.; Bratton, B. P.; Gitai, Z.; Huang, K. C. How to Build a K.; VanNieuwenhze, M. S.; Westblade, L. F.; Dörr, T. Spheroplast-
Bacterial Cell: MreB as the Foreman of E. coli Construction. Cell Mediated Carbapenem Tolerance in Gram-Negative Pathogens.
2018, 172 (6), 1294−1305. Antimicrob. Agents Chemother. 2019, 63 (9), 10−1128.
(3) Colavin, A.; Hsin, J.; Huang, K. C. Effects of polymerization and (21) Gupta, R.; Singh, M.; Pathania, R. Chemical genetic approaches
nucleotide identity on the conformational dynamics of the bacterial for the discovery of bacterial cell wall inhibitors. RSC Med. Chem.
actin homolog MreB. Proc. Natl. Acad. Sci. U.S.A. 2014, 111 (9), 2023, 14 (11), 2125−2154.
3585−3590. (22) Legaree, B. A.; Adams, C. B.; Clarke, A. J. Overproduction of
(4) den Blaauwen, T.; de Pedro, M. A.; Nguyen-Distèche, M.; Ayala, penicillin-binding protein 2 and its inactive variants causes
J. A. Morphogenesis of rod-shaped sacculi. FEMS Microbiol. Rev. morphological changes and lysis in Escherichia coli. J. Bacteriol.
2008, 32 (2), 321−344. 2007, 189 (14), 4975−4983.
(5) Zykov, I. N.; Frimodt-Møller, N.; Småbrekke, L.; Sundsfjord, A.; (23) Lobritz, M. A.; Andrews, I. W.; Braff, D.; Porter, C. B. M.;
Samuelsen, Ø. Efficacy of mecillinam against clinical multidrug- Gutierrez, A.; Furuta, Y.; Cortes, L. B. G.; Ferrante, T.; Bening, S. C.;

3937 https://doi.org/10.1021/acsinfecdis.4c00597
ACS Infect. Dis. 2024, 10, 3928−3938
ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article

Wong, F.; Gruber, C.; Bakerlee, C. W.; Lambert, G.; Walker, G. C.;
Dwyer, D. J.; Collins, J. J. Increased energy demand from anabolic-
catabolic processes drives β-lactam antibiotic lethality. Cell Chem. Biol.
2022, 29 (2), 276−286.e4.
(24) Buss, J. A.; Baidin, V.; Welsh, M. A.; Flores-Kim, J.; Cho, H.;
Wood, B. M.; Uehara, T.; Walker, S.; Kahne, D.; Bernhardt, T. G.
Pathway-Directed Screen for Inhibitors of the Bacterial Cell
Elongation Machinery. Antimicrob. Agents Chemother. 2019, 63 (1),
10−1128.
(25) Stetefeld, J.; McKenna, S. A.; Patel, T. R. Dynamic light
scattering: a practical guide and applications in biomedical sciences.
Biophys. Rev. 2016, 8 (4), 409−427.
(26) Yakhnina, A. A.; Bernhardt, T. G. The Tol-Pal system is
required for peptidoglycan-cleaving enzymes to complete bacterial cell
division. Proc. Natl. Acad. Sci. U.S.A. 2020, 117 (12), 6777−6783.
(27) Hu, Z.; Mukherjee, A.; Pichoff, S.; Lutkenhaus, J. The MinC
component of the division site selection system in Escherichia coli
interacts with FtsZ to prevent polymerization. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96 (26), 14819−14824.
(28) Lai, G. C.; Cho, H.; Bernhardt, T. G. The mecillinam resistome
reveals a role for peptidoglycan endopeptidases in stimulating cell wall
synthesis in Escherichia coli. PLoS Genet. 2017, 13 (7), No. e1006934.
(29) Barton, B.; Grinnell, A.; Morgenstein, R. M. Disruption of the
MreB Elongasome Is Overcome by Mutations in the Tricarboxylic
Acid Cycle. Front. Microbiol. 2021, 12, 664281.
(30) Thulin, E.; Andersson, D. I. Upregulation of PBP1B and LpoB
in cysB Mutants Confers Mecillinam (Amdinocillin) Resistance in
Escherichia coli. Antimicrob. Agents Chemother. 2019, 63 (10), 10−
1128.
(31) Sanders, A. N.; Pavelka, M. S. Phenotypic analysis of
Escherichia coli mutants lacking L,D-transpeptidases. Microbiology
2013, 159 (Pt_9), 1842−1852.
(32) Peters, K.; Pazos, M.; Edoo, Z.; Hugonnet, J. E.; Martorana, A.
M.; Polissi, A.; VanNieuwenhze, M. S.; Arthur, M.; Vollmer, W.
Copper inhibits peptidoglycan LD-transpeptidases suppressing β-
lactam resistance due to bypass of penicillin-binding proteins. Proc.
Natl. Acad. Sci. U.S.A. 2018, 115 (42), 10786−10791.
(33) LaBreck, C. J.; Conti, J.; Viola, M. G.; Camberg, J. L. MinC N-
and C-Domain Interactions Modulate FtsZ Assembly, Division Site
Selection, and MinD-Dependent Oscillation in Escherichia coli. J.
Bacteriol. 2019, 201 (4), 10−1128.
(34) Aliashkevich, A.; Cava, F. LD-transpeptidases: the great
unknown among the peptidoglycan cross-linkers. Febs j 2022, 289
(16), 4718−4730.
(35) Hugonnet, J. E.; Mengin-Lecreulx, D.; Monton, A.; den
Blaauwen, T.; Carbonnelle, E.; Veckerlé, C.; Brun, Y. V.; van
Nieuwenhze, M.; Bouchier, C.; Tu, K.; Rice, L. B.; Arthur, M. Factors
essential for L,D-transpeptidase-mediated peptidoglycan cross-linking
and β-lactam resistance in Escherichia coli. Elife 2016, 5, No. e19469.
(36) Zhao, G.; Meier, T. I.; Kahl, S. D.; Gee, K. R.; Blaszczak, L. C.;
Bocillin, F. L. BOCILLIN FL, a Sensitive and Commercially Available
Reagent for Detection of Penicillin-Binding Proteins. Antimicrob.
Agents Chemother. 1999, 43 (5), 1124−1128.
(37) Barkó, S.; Szatmári, D.; Bódis, E.; Türmer, K.; Ujfalusi, Z.;
Popp, D.; Robinson, R. C.; Nyitrai, M. Large-scale purification and in
vitro characterization of the assembly of MreB from Leptospira
interrogans. Biochim. Biophys. Acta 2016, 1860 (9), 1942−1952.
(38) Nurse, P.; Marians, K. J. Purification and characterization of
Escherichia coli MreB protein. J. Biol. Chem. 2013, 288 (5), 3469−
3475.
(39) Saini, M.; Gaurav, A.; Kothari, A.; Omar, B. J.; Gupta, V.;
Bhattacharjee, A.; Pathania, R. Small Molecule IITR00693 (2-
Aminoperimidine) Synergizes Polymyxin B Activity against Staph-
ylococcus aureus and Pseudomonas aeruginosa. ACS Infect Dis 2023, 9
(3), 692−705.

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