Broad-Spectrum Anti-biofilm Peptide That Targets a Cellular Stress Response (original) (raw)
Figure 7
Peptide 1018 bound to ppGpp in vitro and led to degradation of (p)ppGpp in vivo.
(A) Binding of peptide 1018 to various nucleotides based on co-precipitation. Peptide 1018 (0.25 mM) was separately mixed with increasing amounts of ppGpp, GTP, ATP, GDP and ADP in buffer (50 mM Tris, pH 7.4) and the extent of co-precipitation was assessed by measuring the increase in absorbance at 620 nm. The amount of co-precipitation induced by 1018 appeared to correlate with an increased negative charge on the nucleotides. A separate sample containing NaH2PO4 revealed that phosphate ions did not induce precipitation of 1018 in the concentration range tested. (B) Anti-biofilm peptide 1018 preferentially bound to ppGpp compared to GTP as revealed by 31P-NMR spectroscopy. In the absence of 1018 (top panel), a mixture of 0.5 mM ppGpp and 0.5 mM GTP revealed unique signals corresponding to the phosphorous atoms in ppGpp and GTP (indicated by arrows). Upon the addition of 1 mM 1018 (bottom panel), the peak intensity from the ppGpp signals was almost completely abolished, while the signals from GTP were reduced but to a lesser degree. (C) Samples containing an equimolar mixture of ppGpp and GTP at intermediate concentrations of 1018 were used to further evaluate the preferential binding of 1018 to ppGpp. Examination of specific spectral regions unique to 31P signals from either ppGpp (∼−4.2 ppm) or GTP (∼−20 ppm) showed that the ppGpp peak intensity decreased more readily than those from GTP (peptide concentrations, in mM, are indicated above each trace). The preferential precipitation of ppGpp by 1018 suggests that the peptide had a higher affinity for ppGpp over GTP under these conditions (See also Fig. S5B). (D) The ppGpp levels in nucleotide extracts from P. aeruginosa PAO1 cultures induced with SHX and treated with 1018 were also measured using 31P-NMR spectroscopy. In these spectra, the ppGpp phosphorous signals were shifted because of the presence of 6.5 M formic acid used to extract the nucleotides from the PAO1 cells. The chemical shifts of GTP and ppGpp in 6.5 M formic acid were determined separately using samples of pure nucleotide (See Fig. S6). Only the region from 3 ppm to −0.5 ppm is shown as this contained a unique ppGpp phosphorous peak at 0.6 ppm (for comparison, the spectra of 0.5 mM ppGpp in 6.5 M formic acid is shown as the top trace). In the 31P spectrum of nucleotide extracts from PAO1 induced with SHX, the ppGpp peak at 0.6 ppm appeared as a shoulder on the large phosphate peak at 1.5 ppm (middle grey trace). This shoulder was absent in samples taken from PAO1 cells grown without SHX (lowest grey trace). When PAO1 induced with SHX was treated with 20 µg/ml 1018, the ppGpp peak was essentially lost (black trace) demonstrating that the addition of 1018 to bacteria leads to the degradation of ppGpp in vivo.