Subcellular Quantification of Uptake in Gram-Negative Bacteria

Abstract

Infections by Gram-negative pathogens represent a major health care issue of growing concern due to a striking lack of novel antibacterial agents over the course of the last decades. The main scientific problem behind the rational optimization of novel antibiotics is our limited understanding of small molecule translocation into, and their export from the target compartments of Gram-negative species. To address this issue, a versatile, label-free assay to determine the intracellular localization and concentration of a given compound has been developed for Escherichia coli and its effluximpaired TolC mutant. The assay applies a fractionation procedure to antibiotic-treated bacterial cells to obtain periplasm, cytoplasm and membrane fractions of high purity, as demonstrated by Western Blots of compartment-specific marker proteins. This is followed by an LC-MS/MS-based quantification of antibiotic content in each compartment. Antibiotic amounts could be converted to antibiotic concentrations by assuming that an E. coli cell is a cylinder flanked by two half spheres and calculating the volumes of bacterial compartments. The quantification of antibiotics from different classes, namely ciprofloxacin, tetracycline, trimethoprim and erythromycin demonstrated pronounced differences in uptake quantities and distribution patterns across the compartments. For example, in the case of ciprofloxacin a higher amount of compound was located in the cytoplasm than in the periplasm (592 pg ±50 pg vs 277 ±13 pg per 3.9x10 9 cells), but owing to the smaller volume of the periplasmic compartment, its concentration in the cytoplasm was much lower (37 ±3 pg/µl vs. 221 ±10 pg/µl for the periplasm). For erythromycin and tetracycline, differences in MICs between WT and TolC mutant strains were not reflected by equal differences in uptake, illustrating that additional experimental data are needed to predict antibiotic efficacy. We believe that our assay, providing the antibiotic concentration at the compartment in which the drug target is expressed, constitutes an essential piece of information for a more rational optimization of novel antibiotics against Gram-negative infections.

Fluorescent macrolide probes – synthesis and use in evaluation of bacterial resistance

“…5 Where intracellular distribution studies have been conducted, they have largely relied on radioactive analogues, which are costly, difficult to access, and require specialist equipment. [10][11][12][13][14][15][16][17][18][19] Neither of these techniques readily allow for analysis at single-cell resolution, though some single cell sequencing reports are beginning to appear. 20,21 Despite the success of these approaches, there remains a significant appeal in simple tools that are readily accessible and do not require a large time or resource investment, or which can be utilised in combination with more modern technology, such as highresolution microscopy and single cell microfluidics.…”

Dynamic interaction of fluoroquinolones with magnesium ions monitored using bacterial outer membrane nanopores

“…[1][2][3] One of the current bottlenecks in the development of molecules active against Gram-negative bacteria is their low permeability across the bacterial cell wall, composed of an outer membrane (OM) and inner membrane (IM). 4,5 Methods to investigate the accumulation of antimicrobial agents inside a cell can be in vivo viability assay, 6 liposome swelling assay, 7 mass spectrometry and uorometry related approaches, [8][9][10] and electrophysiology. 5,11 Nanopore based single molecule tools could already investigate a single nucleotide segment, 12,13 and nucleotide base lesion analogues, 14,15 peptides [16][17][18][19] etc.…”

Distribution of fluoroquinolones in the two aqueous compartments of Escherichia coli

“…The reported volume of the periplasmic space of Gram-negative bacteria ranges from 7% to 40% [ 20 , [25] , [26] , [27] , [28] , [29] , [30] , [31] ]. A seminal study by Stock et al reported that the periplasmic space in E. coli and S. typhimurium constitute of approximately 20–40% of the total cell volume [ 25 ].…”

“…When we first started this project, no publications were available to our knowledge that studied the accumulation of antibiotics in subcellular fractions of Gram-negative bacteria. A very recent report described fractionation and quantification of the antibiotic accumulation using mass spectroscopy [ 20 ]. The accumulation of four antibiotics from different classes were investigated.…”