Figure 3.

PIPD1 reduces surface display of 6-azido-TMMs in spheroplasts, indicating inhibition of TMM flipping across the IM. A, schematic representation illustrating the utility of 6-azido-trehalose to probe TMM topology in the inner membrane (IM) of spheroplasts. Spheroplasts were incubated with 6-azido-trehalose to allow synthesis of 6-azido-TMM, which were subsequently labeled with alkyne-containing biotin (sDIBO-biotin) via bioorthogonal chemistry. Surface-exposed biotin-TMM were recognized by Alexa Fluor 488-conjugated streptavidin and visualized by fluorescence microscopy. B, representative bright-field and fluorescence microscopy images are shown following sDIBO-biotin/Alexa Fluor 488-streptavidin labeling of spheroplasts synthesizing TMM (negative control) or 6-azido-TMM in the presence of DMSO (no drug treatment), 4× MIC BM212, and 4× MIC PIPD1. Scale bars, 2 μm. The fluorescence microscopy images shown are after subtraction of median fluorescence of the negative control. C, fluorescence intensity per unit area for randomly picked individual spheroplasts (n = 100) in each condition is plotted, with the medians and interquartile ranges indicated. Mann–Whitney test. ***, p < 0.001 compared with the “no drug treatment” control.