# Therapeutic Potential of Gramicidin S in the Treatment of Root Canal Infections
## 2 Results
### GS Penetration into the Bacterial Cells and Binding Affinity to Nucleotides  
Microbiological assays helped to elucidate the complex action of GS at the cellular level. To understand the anti-biofilm activity at the molecular level, we first studied the membrane-penetrating ability of GS, in order to find out whether it has any access to intracellular targets. For comparative fluorescent microscopy, we used the dye 5(6)-carboxy-fluorescein-\(N\)-hydroxysuccinimide ester (CFSE) as a control, which cannot penetrate the cell membrane. To detect GS by fluorescence microscopy, the photo-switchable GS analog GS-sw(FP) was used, which possesses an intrinsic green fluorescence upon irradiation with visible light [20]. The fluorescence image of _E. faecalis_ TRE2 stained with CFSE showed strong fluorescence of only the cell envelope (Figure 3A). On the other hand, bacteria that were incubated with GS peptide for 30 min (Figure 3B) showed an even fluorescence internally. The same result was obtained by staining the other _E. faecalis_ strains (data not shown).  
The access of cationic GS to the cytoplasm suggests that it may engage in electrostatic interactions with intracellular anionic targets. A key target could be the alarm ppGpp, which regulates biofilm formation [21]. Indeed, the addition of ppGpp to GS at different molar ratios led to their co-precipitation as a result of mutual binding. At ppGpp:GS ratios of 2:1, 1:1 and 1:2 (Figure 3C), floating white flakes could be seen, suggesting the presence of large aggregates. A further excess of GS led to the formation of a stable milky opalescent suspension, which contained smaller nano-rods (Figure 3D). \({}^{31}\)P-NMR analysis of the intrinsic ppGpp phosphate signals confirmed the binding and aggregation, as sharp signals were observed only in the presence of low concentrations of GS (Figure 3E). The broadening and disappearance of the \({}^{31}\)P-NMR signals with excess peptide indicated the precipitation of the alarmome/GS complex. Electron microscopy of the milky suspension at a molar ratio of ppGpp:GS 1:8 (Figure 3D) revealed the assembly of GS with ppGpp into nano-rods with 50 nm width and 200 nm length. The ability of GS to bind to ppGpp and thus to deplete its free cytoplasmic pool may explain the strong inhibition of biofilm formation by GS described above (see MBIC\({}_{90}\) values in Table 1).  
Notably, ppGpp is not the only possible intracellular target of GS. In the literature, complex formation of GS with nucleic acids and adenosine phosphates has already been described [22; 23]. Because the triphosphates GTP and ATP are utilized in the biosynthesis of ppGpp, their binding to GS may further enhance the anti-biofilm effect of the peptide. Using the same \({}^{31}\)P-NMR approach as described above, we also compared the ability of several other phosphorus-containing nucleotides to bind GS. Judged by the molar ratio at which the \({}^{31}\)P-NMR signal disappeared (see Figures in Supplementary Materials), the studied nucleotides exhibited GS binding affinities in the following order: ADP < ppGpp < ATP = GTP < GDP. In particular, this means that the ppGpp precursors represent even stronger complex partners for GS, and their binding to GS should additionally contribute to the overall inhibitory and bactericidal effects in bacterial biofilms.