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,Arg₁₀-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 Arg₁₀-teixobactin analogue. Lys(Rhod)₉,Arg₁₀-teixobactin is prepared by a regioselective labeling strategy that labels Lys₉ 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,Arg₁₀-teixobactin results in septal and lateral staining, which is consistent with an antibiotic targeting cell wall precursors. Concurrent treatment of Lys(Rhod)₉,Arg₁₀-teixobactin and BODIPY FL vancomycin results in septal colocalization, providing further evidence that Lys(Rhod)₉,Arg₁₀-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,Arg₁₀-teixobactin can thus serve as a functional probe to study Gram-positive bacteria and their interactions with teixobactin.

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 B. subtilis enables super-resolution visualization of the interaction of teixobactin with bacterial cell walls and permits the observation of aggregated clusters of the antibiotic on the bacteria. Förster resonance energy transfer (FRET) microscopy further elucidates the supramolecular assembly by showing that fluorescent teixobactin molecules co-localize within a few nanometers on B. subtilis. Fluorescence microscopy over time with a fluorescent teixobactin analogue and propidium iodide in B. subtilis reveals a correlation between cell death and binding of the antibiotic to cellular targets, followed by lysis of cells. Collectively, these studies provide new insights into the binding of teixobactin to Gram-positive bacteria, its supramolecular mechanism of action, and the lysis of bacteria that follows.