Subcellular Quantification of Uptake in Gram-Negative Bacteria

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 efflux-impaired
ΔTolC 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 ± 50 pg vs 277 ±
13 pg per 3.9 × 109 cells), but owing to the smaller
volume of the periplasmic compartment, its concentration in the cytoplasm
was much lower (37 ± 3 vs 221 ± 10 pg/μL for the periplasm).
For erythromycin and tetracycline, differences in MICs between WT
and ΔTolC 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.