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FRET microscopy experiments demonstrate supramolecular assembly of teixobactin molecules on Bacillus subtilis, providing further evidence that teixobactin is a supramolecular antibiotic.

Teixobactin has been the source of intensive study and interest as a promising antibiotic, because of its excellent activity against drug-resistant Gram-positive pathogens and its novel but not yet fully understood mechanism of action that precludes drug resistance. Recent studies have demonstrated that the mode of action of teixobactin is more complicated than initially thought, with supramolecular assembly of the antibiotic appearing to play a critical role in the binding process. Further studies of the interactions of teixobactin with bacteria and its molecular targets offer the promise of providing deeper insights into its novel mechanism of action and guiding the design of additional drug candidates and analogues. The current study reports the preparation and study of teixobactin analogues bearing a variety of fluorophores. Structured illumination microscopy of the fluorescent teixobactin analogues with

This report describes the first synthesis and application of a fluorescent teixobactin analogue that exhibits antibiotic activity and binds to the cell walls of Gram-positive bacteria. The teixobactin analogue, Lys(Rhod)(9),Arg(10)-teixobactin, has a fluorescent tag at position 9 and an arginine in place of the natural allo-enduracididine residue at position 10. The fluorescent teixobactin analogue retains partial antibiotic activity, with minimum inhibitory concentrations of 4–8 μg/mL across a panel of Gram-positive bacteria, as compared to 1–4 μg/mL for the unlabeled Arg(10)-teixobactin analogue. Lys(Rhod)(9),Arg(10)-teixobactin is prepared by a regioselective labeling strategy that labels Lys(9) with an amine-reactive rhodamine fluorophore during solid-phase peptide synthesis, with the resulting conjugate tolerating subsequent solid-phase peptide synthesis reactions. Treatment of Gram-positive bacteria with Lys(Rhod)(9),Arg(10)-teixobactin results in septal and lateral staining, which is consistent with an antibiotic targeting cell wall precursors. Concurrent treatment of Lys(Rhod)(9),Arg(10)-teixobactin and BODIPY FL vancomycin results in septal co-localization, providing further evidence that Lys(Rhod)(9),Arg(10)-teixobactin binds to cell wall precursors. Controls with either Gram-negative bacteria, or an inactive fluorescent homologue with Gram-positive bacteria, showed little or no staining in fluorescence micrographic studies. Lys(Rhod)(9),Arg(10)-teixobactin can thus serve as a functional probe to study Gram-positive bacteria and their interactions with teixobactin.