Distribution of fluoroquinolones in the two aqueous compartments of Escherichia coli

Pandeya, Ankit; Alegun, Olaniyi; Cai, Yuguang; Wei, Yinan

doi:10.1016/j.bbrep.2020.100849

Cited by 4 publications

(3 citation statements)

References 42 publications

(79 reference statements)

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“…In the recent 25 years, research focus shifted to the involvement of DP in the cell surface behavior in response to different environmental stimuli including charge, ionic strength, and pH [53][54][55][56][57][58][59][60]. In addition, the role of DP on compound accumulation in bacterial cells attracted research interest, in a bid to solve the fast-evolving problem of antibiotic resistance in bacteria [61][62][63][64].…”

Section: Membrane Potential (Mp) Regulates Antibiotic Transport Across the Om Of Gram-negative Bacteriamentioning

confidence: 99%

Donnan Potential across the Outer Membrane of Gram-Negative Bacteria and Its Effect on the Permeability of Antibiotics

et al. 2021

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The cell envelope structure of Gram-negative bacteria is unique, composed of two lipid bilayer membranes and an aqueous periplasmic space sandwiched in between. The outer membrane constitutes an extra barrier to limit the exchange of molecules between the cells and the exterior environment. Donnan potential is a membrane potential across the outer membrane, resulted from the selective permeability of the membrane, which plays a pivotal role in the permeability of many antibiotics. In this review, we discussed factors that affect the intensity of the Donnan potential, including the osmotic strength and pH of the external media, the osmoregulated periplasmic glucans trapped in the periplasmic space, and the displacement of cell surface charges. The focus of our discussion is the impact of Donnan potential on the cellular permeability of selected antibiotics including fluoroquinolones, tetracyclines, β-lactams, and trimethoprim.

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Section: Membrane Potential (Mp) Regulates Antibiotic Transport Across the Om Of Gram-negative Bacteriamentioning

confidence: 99%

Donnan Potential across the Outer Membrane of Gram-Negative Bacteria and Its Effect on the Permeability of Antibiotics

et al. 2021

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“…Periplasm and cytoplasm were separated according to the osmotic shock method we described previously [31]. Briefly, the pellet obtained after incubation with compounds and centrifugation was resuspended in 100 μL periplasm preparation buffer (200 mM Tris-Cl, 1 mM EDTA, 20% sucrose, 1 mg/mL lysozyme, pH 8.0).…”

Section: Fractionation Of Periplasm and Cytoplasmmentioning

confidence: 99%

Biotinylation as a tool to enhance the uptake of small molecules in Gram-negative bacteria

et al. 2021

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Antibiotic resistance is a major public health concern. The shrinking selection of effective antibiotics and lack of new development is making the situation worse. Gram-negative bacteria more specifically pose serious threat because of their double layered cell envelope and effective efflux systems, which is a challenge for drugs to penetrate. One promising approach to breach this barrier is the “Trojan horse strategy”. In this technique, an antibiotic molecule is conjugated with a nutrient molecule that helps the antibiotic to enter the cell through dedicated transporters for the nutrient. Here, we explored the approach using biotin conjugation with a florescent molecule Atto565 to determine if biotinylation enhances accumulation. Biotin is an essential vitamin for bacteria and is obtained through either synthesis or uptake from the environment. We found that biotinylation enhanced accumulation of Atto565 in E. coli. However, the enhancement did not seem to be due to uptake through biotin transporters since the presence of free biotin had no observable impact on accumulation. Accumulated compound was mostly in the periplasm, as determined by cell fractionation studies. This was further confirmed through the observation that expression of streptavidin in the periplasm specifically enhanced the accumulation of biotinylated Atto565. This enhancement was not observed when streptavidin was expressed in the cytoplasm indicating no significant distribution of the compound inside the cytoplasm. Using gene knockout strains, plasmid complementation and mutagenesis studies we demonstrated that biotinylation made the compound a better passenger through OmpC, an outer membrane porin. Density functional theory (DFT)-based evaluation of the three-dimensional geometries showed that biotinylation did not directly stabilize the conformation of the compound to make it favorable for the entry through a pore. Further studies including molecular dynamics simulations are necessary to determine the possible mechanisms of enhanced accumulation of the biotinylated Atto565.

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“…Yet, MIC values and permeability levels can deviate for a number of reasons, including altered engagement with the target; moreover, there are established examples where there is an inconsistency between MIC values and accumulation . The general lack of a strong correlation between MIC values and cellular accumulation has been described in the past. − …”

Section: Introductionmentioning

confidence: 99%

Measurement of Small Molecule Accumulation into Diderm Bacteria

et al. 2022

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Some of the most dangerous bacterial pathogens (Gram-negative and mycobacterial) deploy a formidable secondary membrane barrier to reduce the influx of exogenous molecules. For Gram-negative bacteria, this second exterior membrane is known as the outer membrane (OM), while for the Gram-indeterminate Mycobacteria, it is known as the “myco” membrane. Although different in composition, both the OM and mycomembrane are key structures that restrict the passive permeation of small molecules into bacterial cells. Although it is well-appreciated that such structures are principal determinants of small molecule permeation, it has proven to be challenging to assess this feature in a robust and quantitative way or in complex, infection-relevant settings. Herein, we describe the development of the bacterial chloro–alkane penetration assay (BaCAPA), which employs the use of a genetically encoded protein called HaloTag, to measure the uptake and accumulation of molecules into model Gram-negative and mycobacterial species, Escherichia coli and Mycobacterium smegmatis, respectively, and into the human pathogen Mycobacterium tuberculosis. The HaloTag protein can be directed to either the cytoplasm or the periplasm of bacteria. This offers the possibility of compartmental analysis of permeation across individual cell membranes. Significantly, we also showed that BaCAPA can be used to analyze the permeation of molecules into host cell-internalized E. coli and M. tuberculosis, a critical capability for analyzing intracellular pathogens. Together, our results show that BaCAPA affords facile measurement of permeability across four barriers: the host plasma and phagosomal membranes and the diderm bacterial cell envelope.

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Distribution of fluoroquinolones in the two aqueous compartments of Escherichia coli

Cited by 4 publications

References 42 publications

Donnan Potential across the Outer Membrane of Gram-Negative Bacteria and Its Effect on the Permeability of Antibiotics

Donnan Potential across the Outer Membrane of Gram-Negative Bacteria and Its Effect on the Permeability of Antibiotics

Biotinylation as a tool to enhance the uptake of small molecules in Gram-negative bacteria

Measurement of Small Molecule Accumulation into Diderm Bacteria

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