Loss of the lipopolysaccharide (LPS) inner core increases the electrocompetence of Escherichia coli

Soh, Sandrine M.; Jang, Hyochan; Mitchell, Robert J.

doi:10.1007/s00253-020-10779-6

Cited by 9 publications

(9 citation statements)

References 36 publications

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“…Salmonella enterica serovar Typhimurium and E. coli waaD deletion mutants also showed a similar (2 to 5-fold) increase in NPN uptake in comparison to the respective parent strains. 55,56 Using colorimetric quantification of the hydrolysis of nitrocefin, a βlactam probe, by a periplasmic β-lactamase constitutively expressed from pBR322 as another proxy for outer membrane perturbation, we further confirmed the increased outer membrane permeability of the waaD::Tn mutant in comparison to its parent strain (Figure 3C), in agreement with previous reports. 57 Given that outer membrane permeabilization of the deep rough mutant (Figure 3B−C) is sufficient to enable macrolides to cross that barrier (Figure 1A, Figure 2C−D, 41 ), further membrane perturbation may not lead to enhancement of the macrolide activity.…”

Section: Acs Infectious Diseasessupporting

confidence: 90%

Polyamine-Mediated Sensitization of Klebsiella pneumoniae to Macrolides through a Dual Mode of Action

Adams,

Moulding,

El-Halfawy

2024

ACS Infect. Dis.

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Chemicals bacteria encounter at the infection site could shape their stress and antibiotic responses; such effects are typically undetected under standard lab conditions. Polyamines are small molecules typically overproduced by the host during infection and have been shown to alter bacterial stress responses. We sought to determine the effect of polyamines on the antibiotic response of Klebsiella pneumoniae, a Gram-negative priority pathogen. Interestingly, putrescine and other natural polyamines sensitized K. pneumoniae to azithromycin, a macrolide protein translation inhibitor typically used for Gram-positive bacteria. This synergy was further potentiated in the physiological buffer, bicarbonate. Chemical genomic screens suggested a dual mechanism, whereby putrescine acts at the membrane and ribosome levels. Putrescine permeabilized the outer membrane of K. pneumoniae (NPN and β-lactamase assays) and the inner membrane (Escherichia coli β-galactosidase assays). Chemically and genetically perturbing membranes led to a loss of putrescine−azithromycin synergy. Putrescine also inhibited protein synthesis in an E. coliderived cell-free protein expression assay simultaneously monitoring transcription and translation. Profiling the putrescine− azithromycin synergy against a combinatorial array of antibiotics targeting various ribosomal sites suggested that putrescine acts as tetracyclines targeting the 30S ribosomal acceptor site. Next, exploiting the natural polyamine−azithromycin synergy, we screened a polyamine analogue library for azithromycin adjuvants, discovering four azithromycin synergists with activity starting from the low micromolar range and mechanisms similar to putrescine. This work sheds light on the bacterial antibiotic responses under conditions more reflective of those at the infection site and provides a new strategy to extend the macrolide spectrum to drug-resistant K. pneumoniae.

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Section: Acs Infectious Diseasessupporting

confidence: 90%

Polyamine-Mediated Sensitization of Klebsiella pneumoniae to Macrolides through a Dual Mode of Action

Adams,

Moulding,

El-Halfawy

2024

ACS Infect. Dis.

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“…Figure S24 shows a strong correlation between the bioluminescence from the E. coli strain and its surviving population, implying the bioluminescence can be used as a proxy to evaluate viability. Subsequently, the efficacy of PEGtide[25.5.0] was evaluated against a number of E. coli strains that produce LPS of varying lengths (Figure d). , When compared against the wild-type cells, those strains producing the shortest LPS, i . e ., the Δ rfaC , Δ rfaD , Δ rfaE , Δ rfaF , and Δ rfaG mutants, were all more susceptible to PEGtide[25.5.0].…”

Section: Results and Discussionmentioning

confidence: 99%

“…Subsequently, the efficacy of PEGtide[25.5.0] was evaluated against a number of E. coli strains that produce LPS of varying lengths (Figure 2d). 38,39 When compared against the wild-type cells, those strains producing the shortest LPS, i.e., the ΔrfaC, ΔrfaD, ΔrfaE, ΔrfaF, and ΔrfaG mutants, were all more susceptible to PEGtide[25.5.0]. These results show that PEGtide[25.5.0] remained as effective against these mutants as the wild-type strain.…”

Section: Nmr and Gpc (Figures S8−s12)mentioning

confidence: 99%

Antimicrobial PEGtides: A Modular Poly(ethylene glycol)-Based Peptidomimetic Approach to Combat Bacteria

et al. 2021

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Despite their high potency, the widespread implementation of natural antimicrobial peptides is still challenging due to their low scalability and high hemolytic activities. Herein, we address these issues by employing a modular approach to mimic the key amino acid residues present in antimicrobial peptides, such as lysine, leucine, and serine, but on the highly biocompatible poly(ethylene glycol) (PEG) backbone. A series of these PEG-based peptides (PEGtides) were developed using functional epoxide monomers, corresponding to each key amino acid, with several possessing highly potent bactericidal activities and controlled selectivities, with respect to their hemolytic behavior. The critical role of the composition and the structure of the PEGtides in their selectivities was further supported by coarse-grained molecular dynamic simulations. This modular approach is anticipated to provide the design principles necessary for the future development of antimicrobial polymers.

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“…The permeability of the outer membrane was analyzed by using fluorescent probe 1- N -phenylnaphthylamine (NPN) as previously described ( Soh et al, 2020 ). Briefly, bacterial cells were centrifuged and washed twice with 5 mM HEPES buffer (pH 7.2) containing 1 mM sodium azide.…”

Section: Methodsmentioning

confidence: 99%

Incorporation of Plasmid DNA Into Bacterial Membrane Vesicles by Peptidoglycan Defects in Escherichia coli

et al. 2021

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Membrane vesicles (MVs) are released by various prokaryotes and play a role in the delivery of various cell-cell interaction factors. Recent studies have determined that these vesicles are capable of functioning as mediators of horizontal gene transfer. Outer membrane vesicles (OMVs) are a type of MV that is released by Gram-negative bacteria and primarily composed of outer membrane and periplasm components; however, it remains largely unknown why DNA is contained within OMVs. Our study aimed to understand the mechanism by which DNA that is localized in the cytoplasm is incorporated into OMVs in Gram-negative bacteria. We compared DNA associated with OMVs using Escherichia coli BW25113 cells harboring the non-conjugative, non-mobilized, and high-copy plasmid pUC19 and its hypervesiculating mutants that included ΔnlpI, ΔrseA, and ΔtolA. Plasmid copy per vesicle was increased in OMVs derived from ΔnlpI, in which peptidoglycan (PG) breakdown and synthesis are altered. When supplemented with 1% glycine to inhibit PG synthesis, both OMV formation and plasmid copy per vesicle were increased in the wild type. The bacterial membrane condition test indicated that membrane permeability was increased in the presence of glycine at the late exponential phase, in which cell lysis did not occur. Additionally, quick-freeze deep-etch and replica electron microscopy observations revealed that outer-inner membrane vesicles (O-IMVs) are formed in the presence of glycine. Thus, two proposed routes for DNA incorporation into OMVs under PG-damaged conditions are suggested. These routes include DNA leakage due to increased membrane permeation and O-IMV formation. Additionally, our findings contribute to a greater understanding of the vesicle-mediated horizontal gene transfer that occurs in nature and the utilization of MVs for DNA cargo.

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Loss of the lipopolysaccharide (LPS) inner core increases the electrocompetence of Escherichia coli

Cited by 9 publications

References 36 publications

Polyamine-Mediated Sensitization of Klebsiella pneumoniae to Macrolides through a Dual Mode of Action

Polyamine-Mediated Sensitization of Klebsiella pneumoniae to Macrolides through a Dual Mode of Action

Antimicrobial PEGtides: A Modular Poly(ethylene glycol)-Based Peptidomimetic Approach to Combat Bacteria

Incorporation of Plasmid DNA Into Bacterial Membrane Vesicles by Peptidoglycan Defects in Escherichia coli

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