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+ζ, Ag-CALNNE NPs−4ζ), 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 (ΔBmrA), 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 ΔBmrA 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 ΔBmrA 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 ΔBmrA, 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 ΔBmrA increases nearly proportionally to the NP concentration. The accumulation of negatively charged NPs in ΔBmrA, 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.

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

“…26,32,56 The biocompatibility of the functionalized Ag NPs (AgMUNH 2 NPs) could be attributed to the surface protecting layer of MUNH 2 and the low concentration of AgMUNH 2 NPs, as we reported previously. 33,40,57 At a low concentration, Ag NPs and functionalized Ag NPs do not inhibit bacterial growth and can be used to study the efflux function of single live bacterial cells. [29][30][31][32]58,59 Further, our previous studies also show that the positively charged peptide functionalized Ag NPs are more biocompatible with the zebrash embryonic development than negatively charged peptide functionalized Ag NPs.…”

“…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).…”

Advanced Nanoscale Approaches to Single-(Bio)entity Sensing and Imaging

“…No labelling with fluorophores is needed compared to fluorescence microscopy, which is perhaps the most employed technique for cellular imaging. Processes as diverse as the transport of single silver nanoparticles in zebrafish embryos [284], formation of lipid bilayers during exo- or endocytosis events [285], imaging and characterisation of single receptor molecules [286], transport kinetics in membrane [287] have been studied by LSPR monitoring of plasmonic nanoparticles. Intracellular p53 protein dynamics in HeLa cells was studied using a nanoscale surface plasmon-polariton fibre-tip-probe [288].…”

Single gold nanoparticle plasmonic spectroscopy for study of chemical-dependent efflux function of single ABC transporters of single live Bacillus subtilis cells

“…Localized surface plasmon resonance (LSPR) spectra of single NPs highly depend upon their sizes, shapes, dielectric constants, and surrounding environments, 22–30 which enables us to use size-dependent LSPR spectra of single NPs to quantitatively determine sizes of single NPs in solution, in single live cells and embryos in situ in real time. 26, 30–43…”

“…subtilis cells (Gram-positive bacterium). 31, 42, 43 Currently, 78 ABC transporters have been identified in B. subtilis , 44–46 making B. subtilis a widely used model organism to study efflux function of ABC membrane transporters. We have also used size-dependent LSPR spectra of single Ag NPs to study transport dynamics of single MexAB-OprM membrane transporters in single live Pseudomonas aeruginosa cells (Gram negative bacterium).…”