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

Abstract

ATP-binding cassette (ABC) membrane transporters exist in all living organisms and play key roles in a wide range of cellular and physiological functions. The ABC transporters can selectively extrude a wide variety of structurally and functionally unrelated substrates, leading to multidrug resistance. Despite extensive study, their efflux molecular mechanisms remain elusive. In this study, we synthesized and characterized purified silver nanoparticles (Ag NPs) (97 ± 13 nm in diameter), and used them as photostable optical imaging probes to study efflux kinetics of ABC membrane transporters (BmrA) of single live cells (B. subtillis). The NPs with concentrations up to 3.7 pM were stable (non-aggregated) in a PBS buffer and biocompatible with the cells. We found a high dependence of accumulation of the intracellular NPs in single live cells (WT, Ct-BmrA-EGFP, ΔbmrA) upon the cellular expression level of BmrA and NP concentration (0.93, 1.85 and 3.7 pM), showing the highest accumulation of intracellular NPs in ΔbmrA (deletion of BmrA) and the lowest ones in Ct-BmrA-EGFP (over-expression of BmrA). Interestingly, the accumulation of intracellular NPs in ΔbmrA increases nearly proportionally with the NP concentration, while those in WT and Ct-BrmA-EGFP do not. This suggests that the NPs enter the cells via passive diffusion driven by concentration gradients and are extruded out of cells by BmrA transporters, similar to conventional pump substrates (antibiotics). This study shows that such large substrates (84-100 nm NPs) can enter into the live cells and be extruded out of the cells by BmrA, and the NPs can serve as nm-sized optical imaging probes to study the size-dependent efflux kinetics of membrane transporters in 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

“…Furthermore, we used the LSPR spectra and scattering intensity of single NPs to characterize the size and number of single NPs, and monitor the peptides functionalized on the surface of the NPs in situ in real time as they are incubated with the cells and transported across the cellular membrane over time using the similar approaches as we reported previously. 29–32, 43–44 …”

“…The results show that 94% of WT and 95% of ΔBmrA cells incubated with the positively charged NPs over 2 h are alive, and 98% of either WT or ΔBmrA cells incubated with the negatively charged NPs over 2 h are alive (Figure 8C), which are similar to what we observed previously using citrated Ag NPs. 29, 43 Notably, the cells with the NPs are all alive. This result shows that both positively and negatively charged Ag-peptide NPs (1.4 nm) are biocompatible to the cells, and the NPs are well suited to serve as optical imaging probes for real-time study of charged-dependent efflux function of BmrA membrane transporter of single live cells The results further demonstrate that accumulation of the NPs in single live cells is not attributed to the disintegration of cellular membrane due to the cell death, because the cells with the NPs are alive.…”

“…25–26, 29–38 Furthermore, we have demonstrated that we can use size-dependent LSPR (localized surface plasmon resonance) spectra and size-dependent scattering intensity of single NPs to quantitatively determine the sizes of single NPs in solution, in single live cells and single living embryos in real time, and we can use single Ag NPs as photostable nanometer sized optical probes to study the size-dependent efflux kinetics of multidrug membrane transporters in single live cells in real time, and to image single molecules of interest on/in single live cells for a desired period of time with both nm spatial and ms temporal resolutions. 25, 29–36, 39–43 …”

“…30–32, 44 We have also used size-dependent LSPR spectra of the noble metal NPs to study the dependence of efflux kinetics of single BmrA membrane transporters in B. subtilis (a Gram-positive bacterium) upon the substrate size and the cellular expression of BmrA. 29, 43 We have constructed two new strains of B. subtilis , BmrA fused with EGFP via its C- or N- terminus (Ct-BmrA-EGFP or Nt-BmrA-EGFP), and found that both strains retain the efflux function of BmrA. 18, 29 …”

Size-dependent inhibitory effects of antibiotic nanocarriers on filamentation ofE. coli

“…We have demonstrated that we can image and characterize the sizes of single Ag NPs at nanometer (nm) resolution in single live cells in situ in real time using size-dependent localized surface plasmon resonance (LSPR) spectra of single Ag NPs by dark-eld optical microscopy and spectroscopy (DFOMS). [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] We have used the size-dependent LSPR spectra of single Ag NPs to track the transport of single NPs in and out of single live cells and to determine the pore sizes of membrane transporters. 29,30 In our previous studies, we found dose and size dependent inhibitory effects of antibiotic nanocarriers (AgMUNH-Ox NPs) against Pseudomonas aeruginosa (Gram-negative bacterium) and Bacillus subtilis (Gram-positive bacterium).…”