Study of the Multidrug Membrane Transporter of Single Living Pseudomonas aeruginosa Cells Using Size-Dependent Plasmonic Nanoparticle Optical Probes

Abstract

Multidrug membrane transporters (efflux pumps) in both prokaryotes and eukaryotes are responsible for impossible treatments of a wide variety of diseases, including infections and cancer, underscoring the importance of better understanding their structures and functions for design of effective therapies. In this study, we designed and synthesized two silver nanoparticle (Ag NP) solution with average diameters of 13.1 ± 2.5 nm (8.1–38.6 nm) and 91.0 ± 9.3 nm (56–120 nm), and used the size-dependent plasmonic spectra of single NPs to probe the size-dependent transport kinetics of MexAB-OprM (multidrug transporter) in Pseudomonas (P.) aeruginosa in real-time at nanometer resolution. We found that the accumulation of intracellular NPs in wild-type (WT) cells was higher than in nalB1 (over-expression of MexAB-OprM), but less than ΔABM (deletion of MexAB-OprM). In the presence of proton ionophores (CCCP, inhibitor of proton-motive-force), we found that intracellular NPs in nalB1 were nearly doubled. These results suggest that the MexAB-OprM is responsible for the extrusion of NPs out of cells and NPs (orders of magnitude larger than conventional antibiotics) are the substrates of the transporter, which indicates that the substrates may trigger the assembly of the efflux pump optimized for the extrusion of the encountered substrates. We found that the smaller NPs stayed longer inside the cells than larger NPs, suggesting the size-dependent efflux kinetics of the cells. This study shows that multi-sized NPs can be used to mimic various sizes of antibiotics for probing the size-dependent efflux kinetics of multidrug membrane transporters in single living cells.

Single nanoparticle plasmonic spectroscopy for study of the efflux function of multidrug ABC membrane transporters of single live cells

“…The purified Ag NPs were stable in DI water for months, as we reported previously. 20, 24, 33 The high-resolution transmission electron microscopy (HRTEM) images and histogram of size distribution of single NPs show the polygon NPs with average sizes of (97 ± 13) nm, ranging from 72 to 147 nm with aspect ratios of 0.97-3.9 (Figure 1A and B). The sizes of the NPs were determined by averaging the length and width of individual NPs.…”

“…We determined their sizes as 84, 91 and 100 nm in diameter, respectively, using their size-dependent LSPR spectra and the calibration curves of λ max of LSPR spectra of single NPs versus their sizes, as we reported previously. 20, 24 …”

Design and study of the efflux function of the EGFP fused MexAB-OprM membrane transporter in Pseudomonas aeruginosa using fluorescence spectroscopy

“…37 Therefore, EtBr has been used to monitor and characterize the efflux kinetics of membrane transporters in live cells in real time. 33, 35-36, 38, 40 …”

“…29-30, 33-36 Fluorescent dyes (EtBr) emit weak fluorescence in aqueous solution (outside cells) and become strongly fluorescent in non-polar and hydrophobic environments, especially as they enter the cells and intercalate with DNA. 37 Thus, time-dependent (time-course) fluorescence intensity of EtBr is particularly suitable for real-time monitoring of efflux kinetics of multidrug membrane transporters in live cells.…”

“…37 Thus, time-dependent (time-course) fluorescence intensity of EtBr is particularly suitable for real-time monitoring of efflux kinetics of multidrug membrane transporters in live cells. 29-30, 33-36, 38 Minimum inhibitory concentrations (MICs) of antibiotics that are specific substrates of the transporters and show the specific susceptibility toward given strains of bacterial cells have also been used to study efflux function of multidrug membrane transporters. 39 …”