Figure 3.

Characterization of CDG-Tre labeling. (a) Confocal images (top row = bright field and bottom row = green fluorescence, Ex-490 nm/Em-520 nm) of freshly cultured E. coli (TOP10), S. aureus, C. diphtheriae and M. smegmatis incubated with 50 μM of CDG-Tre in PBS at 37 °C for 2 h. Scale bars indicate 5 μm. (b) Flow cytometry analysis of bacteria and fungi shown in (a). The unlabeled bacterial autofluorescence in PBS (F₀) was utilized to normalize and calculate the relative fluorescence intensity. (c) Inhibitory study with increasing concentrations of free trehalose at 1, 10, 100, 1000-fold over CDG-Tre (100 μM) with M. smegmatis. (d) Inhibitory study with clavulanate (2 mg/ml), avibactam (2 mg/ml), ebselen (200 μg/ml) and spermidine (2.5, 5, 7.5 mg/ml) in the presence of 50 μM CDG-Tre with M. smegmatis. M. smegmatis treated with CDG-Tre exhibited a 60-fold increase of mean fluorescence intensity over PBS, which was arbitrarily set as 1 to normalize the test samples and show percentage inhibition. (e) M. smegmatis was treated with rifampicin (RIF, 0.2 μg/mL) or isoniazid (INH, 10 μg/mL or 100 μg/mL) for 3 h before incubation with CDG-Tre (100 μM) for 1 h at 37 °C. Mean fluorescent intensity of CDG-Tre was arbitrarily set as 1 to normalize other samples with drugs treatment and show percentage inhibition. All error bars represent standard deviation of three independent measurements. ****=P<0.0001