LAUSR

The lack of homologues of the mechanosensitive (MS) channel of large conductance (MscL) in the human genome makes it a novel antimicrobial target. Specifically, MscL activators have been proposed as potential antibacterial species as they perturb cellular membrane potential and/or provide an alternative route of entry for antimicrobials into the cytosol. An important question regarding the mechanism of drug-induced activation in MscL is: Does the activated channel also allow other large species (i.e., secondary-molecules such as antibiotics) to pass through? To investigate this question, we have developed a label-free optical technique, second-harmonic light scattering (SHS), for real-time analysis of channel activity in a living organism. Using the principle of competitive transport [1], we apply time-resolved SHS to quantitatively characterize the transport rates of the second-harmonic active cation, malachite green (MG) across the cytoplasmic membrane (CM) of living E. coli in the presence and absence of an MscL activator. MG is known to slowly diffuse across the hydrophobic core of the CM [2]. Subsequently, if MG transport across an activated MscL is permitted, the measured transport rate should increase substantially. As proof-of-concept, we used two molecules recently suggested to activate MscL, streptomycin (Strp) and dihydrostreptomycin (dStrp) [3]. Our results reveal that application of either activator results in a significant increase in the MG transport rate, indicating that both Strp and dStrp are inducing a more efficient route for MG transport across the CM. These results support the notion that Strp and dStrp are indeed MS channel activators. This study also shows that the SHS technique is a useful experimental paradigm for in vivo study of channel activators, which is of particular interest in antimicrobial drug discovery.References[1]. Sharifian Gh. et al. “Label-Free Optical Method for Quantifying Molecular Transport Across Cellular Membranes In Vitro”, J. Phys. Chem. Lett. (2016), 7:3406.[2]. Wilhelm et al. “Gram's Stain Does Not Cross the Bacterial Cytoplasmic Membrane”, ACS Chem. Biol. (2015), 10:1711.[3]. Wray et al. “Dihydrostreptomycin Directly Binds to, Modulates, and Passes through the MscL Channel Pore”, PLoS Biol. (2016), 14:e1002473.