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

ABSTRACT: Multidrug membrane transporters can selectively extrude a wide variety of structurally and functionally unrelated substrates, and they are responsible for ineffective treatment of a wide range of diseases (e.g., infection and cancer). Their underlying molecular mechanisms remain elusive. In this study, we functionalized Ag NPs (11 nm in diameter) with two biocompatible peptides (CALNNK, CALNNE) to prepare positively and negatively charged Ag-peptide NPs (Ag-CALNNK NPs+?, Ag-CALNNE NPs-4?), respectively. We used them as photostable plasmonic imaging probes to study charge-dependent efflux kinetics of BmrA (ABC) membrane transporter of single live Bacillus (B.) subtilis cells. Two strains of the cells, normal expression of BmrA (WT) or devoid of BmrA (?BmrA), were used to study the charge-dependent efflux kinetics of single NPs upon the expression of BmrA. The NPs (1.4 nM) were stable (non-aggregated) in a PBS buffer and biocompatible to the cells. We found the high dependent accumulation of the intracellular NPs in both WT and ?BmrA upon the charge and concentration of NPs. Notably, the accumulation rates of the positively charged NPs in single live WT cells are nearly identical to those in ?BmrA cells, showing independence upon the expression of BmrA. In contrast, the accumulation rates of the negatively charged NPs in WT are much lower than in ?BmrA, showing high dependence upon the expression of BmrA and suggesting that BmrA extrude the negatively charged NPs, but not positively charged NPs, out of the WT. The accumulation of positively charged NPs in both WT and ?BmrA increases nearly proportionally to the NP concentration. The accumulation of negatively charged NPs in ?BmrA, but not in WT, also increases nearly proportionally to the NP concentration. These results suggest that both negatively and positively charged NPs enter the cells via passive diffusion driven by concentration gradients across the cellular membrane, and BmrA can only extrude the negatively charged NPs out of the WT. This study shows that single NP plasmon spectroscopy can serve as a powerful tool to identify single plasmonic NPs and to probe the charge-dependent efflux kinetics and function of single membrane transporters in single live cells in real time.

SUBMITTER: Browning LM 

PROVIDER: S-EPMC5899213 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature