Unveiling the Modulating Role of Extracellular pH in Permeation and Accumulation of Small Molecules in Subcellular Compartments of Gram-negative Escherichia coli using Nonlinear Spectroscopy

Abstract

Quantitative evaluation of small molecule permeation and accumulation in Gram-negative bacteria is important for drug development against these bacteria. While these measurements are commonly performed at physiological pH, Escherichia coli and many other Enterobacteriaceae infect human gastrointestinal and urinary tracts, where they encounter different pH conditions. To understand how external pH affects permeation and accumulation of small molecules in E. coli cells, we apply second harmonic generation (SHG) spectroscopy using SHG-active antimicrobial compound malachite green as the probe molecule. Using SHG, we quantify periplasmic and cytoplasmic accumulations separately in live E. coli cells, which was never done before. Compartment-wise measurements reveal accumulation of the probe molecule in cytoplasm at physiological and alkaline pH, while entrapment in periplasm at weakly acidic pH and retention in external solution at highly acidic pH. Behind such disparity in localizations, up to 2 orders of magnitude reduction in permeability across the inner membrane at weakly acidic pH and outer membrane at highly acidic pH are found to play key roles. Our results unequivocally demonstrate the control of external pH over entry and compartment-wise distribution of small molecules in E. coli cells, which is a vital information and should be taken into account in antibiotic screening against E. coli and other Enterobacteriaceae members. In addition, our results demonstrate the ability of malachite green as an excellent SHG-indicator of changes of individual cell membrane and periplasm properties of live E. coli cells in response to external pH change from acidic to alkaline. This finding, too, has great importance, as there is barely any other molecular probe that can provide similar information.

Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria

“…SHG is a nonlinear optical technique that is sensitive to molecular species at interfaces and has previously been applied to the study of small molecules interacting with both model (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and living cell membranes (21)(22)(23)(24)(25)(26)(27)(28)(29). In SHG, incoming light at a frequency u with sufficient intensity induces a second-order polarization in the sample.…”

Determination of Bacterial Surface Charge Density Via Saturation of Adsorbed Ions

“…This characteristic mechanism has previously been employed to monitor molecular adsorption and transport across phospholipid membranes in biomimetic model liposomes (12)(13)(14)(15)(16)(17)(18)(19) and living cells. (20)(21)(22)(23)(24)(25)(26)(27)(28) When SHG-active molecules adsorb onto the exterior surface of the membrane, they align with one another due to the similarity of the interaction driving the adsorption process. This oriented ensemble of SHG-active molecules gives rise to a coherent SHS response, the intensity of which scales as the square of the molecular surface density.…”

Molecule‐Membrane Interactions in Biological Cells Studied with Second Harmonic Light Scattering

“…Indeed, other groups have recently begun adopting this approacht oc haracterize molecular adsorption and membrane transport in living cells, primarily bacteria. [29,30] Prior to this, the patch clamp and av ariety of fluorescence-based techniques were the standard go-to methods for studying molecular transport across membranes. While the patch clamp technique is routinelyu sed to measurem embrane transport of ions, [31] it is not well suited for dealing with neutral molecules or cells with multiple membranes,s uch as bacteria.…”

“…This study demonstrated an alternative label‐free method for detecting adsorption and transport of small molecules at the membranes of biological cells. Indeed, other groups have recently begun adopting this approach to characterize molecular adsorption and membrane transport in living cells, primarily bacteria . Prior to this, the patch clamp and a variety of fluorescence‐based techniques were the standard go‐to methods for studying molecular transport across membranes.…”