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Real-time probing of membrane transport in living microbial cells using single nanoparticle optics and living cell
Xiao-Hong Nancy Xu1, William J Brownlow, Sophia V Kyriacou
1Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia 23529, USA. xhxu@odu.edu
Abstract:
Membrane transport plays a leading role in a wide spectrum of cellular and subcellular pathways, including multidrug resistance (MDR), cellular signaling, and cell-cell communication. Pseudomonas aeruginosa is renowned for its intriguing membrane transport mechanisms, such as the interplay of membrane permeability and extrusion machinery, leading to selective accumulation of specific intracellular substances and MDR. Despite extensive studies, the mechanisms of membrane transport in living microbial cells remain incompletely understood. In this study, we directly measure real-time change of membrane permeability and pore sizes of P. aeruginosa at the nanometer scale using the intrinsic color index (surface plasmon resonance spectra) of silver (Ag) nanoparticles as the nanometer size index probes. The results show that Ag nanoparticles with sizes ranging up to 80 nm are accumulated in living microbial cells, demonstrating that these Ag nanoparticles transport through the inner and outer membrane of the cells. In addition, a greater number of larger intracellular Ag nanoparticles are observed in the cells as chloramphenicol concentration increases, suggesting that chloramphenicol increases membrane permeability and porosity. Furthermore, studies of mutants (nalB-1 and DeltaABM) show that the accumulation rate of intracellular Ag nanoparticles depends on the expression level of the extrusion pump (MexAB-OprM), suggesting that the extrusion pump plays an important role in controlling the accumulation of Ag nanoparticles in living cells. Moreover, the accumulation kinetics measured by Ag nanoparticles are similar to those measured using a small fluorescent molecule (EtBr), eliminating the possibility of steric and size effects of Ag nanoparticle probes. Susceptibility measurements also suggest that a low concentration of Ag nanoparticles (1.3 pM) does not create significant toxicity for the cells, further validating that single Ag nanoparticles (1.3 pM) can be used as biocompatible nanoprobes for the study of membrane transport kinetics in living microbial cells.
Insights
Silver nanoparticles reveal how membrane transport and multidrug resistance occur in Pseudomonas aeruginosa. This study uses nanoparticles to measure real-time changes in bacterial membrane permeability and pore size.
Area of Science:
- Microbiology
- Nanotechnology
- Biophysics
Background:
- Membrane transport is crucial for cellular functions like multidrug resistance (MDR).
- Pseudomonas aeruginosa exhibits complex membrane transport, contributing to its MDR.
- Understanding membrane transport in living microbes remains a challenge.
Purpose of the Study:
- To directly measure real-time changes in membrane permeability and pore size in P. aeruginosa.
- To investigate the role of silver nanoparticles (Ag NPs) as probes for bacterial membrane transport.
- To elucidate the influence of chloramphenicol and efflux pumps on membrane transport.
Main Methods:
- Utilized silver nanoparticles (Ag NPs) with sizes up to 80 nm as nanoprobes.
- Measured changes in Ag NP accumulation and intracellular size distribution.
- Analyzed P. aeruginosa mutants (nalB-1, DeltaABM) with varying MexAB-OprM expression.
- Compared Ag NP kinetics with a fluorescent dye (EtBr).
Main Results:
- Ag NPs were observed to transport through the inner and outer membranes of P. aeruginosa.
- Increased chloramphenicol concentration led to greater accumulation of larger intracellular Ag NPs, indicating increased membrane permeability.
- Ag NP accumulation rate correlated with MexAB-OprM efflux pump expression levels.
- Ag NP accumulation kinetics mirrored those of EtBr, ruling out probe-specific size effects.
Conclusions:
- Silver nanoparticles serve as effective, biocompatible nanoprobes for studying membrane transport in living microbial cells.
- Chloramphenicol enhances membrane permeability and porosity in P. aeruginosa.
- The MexAB-OprM efflux pump significantly influences Ag NP accumulation and membrane transport control.
