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

Abstract

Multidrug membrane transporters (efflux pumps) can selectively extrude a variety of structurally and functionally diverse substrates (e.g., chemotoxics, antibiotics), leading to multidrug resistance (MDR) and ineffective treatment of a wide variety of diseases. In this study, we have designed and constructed fusion gene (egfp-mexB) of N-terminal mexB with C-terminal egfp, inserted it into a plasmid vector (pMMB67EH), and successfully expressed it in ΔMexB (MexB deletion) strain of Pseudomonas aeruginosa to create a new strain that expresses MexA-(EGFP-MexB)-OprM. We characterized the fusion gene using gel electrophoresis and DNA sequencing, and determined their expression in live cells by measuring the fluorescence of EGFP in single live cells using fluorescence microscopy. Efflux function of the new strain was studied by measuring its accumulation kinetics of ethidium bromide (EtBr, a pump substrate) using fluorescence spectroscopy, which was compared with the cells (WT, ΔMexM, ΔABM, and nalB1) with various expression levels of MexAB-OprM. The new strain shows 6-fold lower accumulation rates of EtBr (15 μM) than ΔABM, 4-fold lower than ΔMexB, but only 1.1-fold higher than WT. As EtBr concentration increases to 40 μM, the new strain has nearly the same accumulation rate of EtBr as ΔMexB, but 1.4-fold higher than WT. We observed the nearly same level of inhibitory effect of CCCP (carbonyl cyanide-m-chlorophenylhydrazone) on the efflux of EtBr by the new strain and WT. Antibiotic susceptibility study shows that the minimum inhibitory concentrations (MICs) of aztreonam (AZT) and chloramphenicol (CP) for the new strain are 6-fold or 3-fold lower than WT, respectively, and 2-fold higher than those of ΔMexB. Taken together, the results suggest that the fusion protein partially retains the efflux function of MexAB-OprM. Modeled structure of the fusion protein shows that the position and orientation of the N-terminal fused EGFP domain may either partially block the translocation pore or restrict the movement of the individual pump domains, which leads to partially restrict efflux activity.

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

“…Thus, the PBS buffer (but not cell culture medium) was used to suspend the bacterial cells for the study of accumulation kinetics of pump substrates over time, as those reported previously. 14, 15, 24, 25, 33, 35, 37 Note that the cells would grow in the culture medium, and the number of the cells would change over time, making the study of the accumulation rates of single NPs in the cells over time impossible. Our studies demonstrate that the bacterial cells remain alive and retain their efflux functions in the PBS buffer, as shown in Figs.…”

“…9-15 These conventional methods enable one to study ensemble accumulation kinetics of bulk cells. We have also demonstrated that thin-layer total-internal reflection fluorescence microscopy and spectroscopy can be used to measure efflux kinetics of single membrane transporters of single live cells in real-time.…”

Single Nanoparticle Plasmonic Spectroscopy for Study of Charge-Dependent Efflux Function of Multidrug ABC Transporters of Single Live Bacillus subtilis Cells

“…14–15, 18–19, 29, 61 The previous studies have showed that the bacterial cells in the PBS buffer retain their efflux function over hours. 14–15, 18–19, 29, 61 Notably, the bacterial cells suspended in the PBS buffer retain their cellular growth stage and cellular function over hours, and their number remains the same over time.…”

“…14–15, 18–19, 29, 61 The previous studies have showed that the bacterial cells in the PBS buffer retain their efflux function over hours. 14–15, 18–19, 29, 61 Notably, the bacterial cells suspended in the PBS buffer retain their cellular growth stage and cellular function over hours, and their number remains the same over time. 14–15, 18–19, 29, 61 Therefore, in order to use the same growth stage and number of the cells over the duration of each experiment (2 h), it is essential to study the accumulation rates of the NPs in the cells that are suspended in the PBS buffer, but not in the cell culture medium which would lead to the cell growth and creation of the cells with various growth stages and functions.…”

Raman Spectroscopic Analysis of Signaling Molecules–Dopamine Receptors Interactions in Living Cells

“… 61 − 66 Some literature has also reported the changes in the biological property of the sample after fluorescence labeling. 67 , 68 Similarly, the radio ligand binding assay 69 produces toxic radioactive wastes which are extremely harmful to in vivo studies. Although the above-mentioned techniques are useful to screen the binding specificity of signaling molecules such as drugs and neurotransmitters, they suffer from some serious limitations.…”