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The bioactivity of the geobacillin II analogues produced in this study was tested using an agar diffusion growth inhibition assay (Figure 4.15). The purified modified peptides were incubated with GluC in order to remove the leader peptide (see Figures 4.16 and 4.17 and Table 4.3 for mass spectra). GluC cleaved at the −5 position leaving a four amino acid overhang at the N-terminus of core peptide. This overhang does not inhibit the antibacterial activity of geobacillin II as described above (39). The cleavage products were tested for biological activity. All analogues resulted in comparable activity as wild type geobacillin II treated in the same manner, except the A-ring analogues with amino acid compositions Dhb-Ala-Ile-Val-Cys (zone 13) and Dhb-Ala-Arg-Val-Cys (zone 14) resulting in methyllanthionine bridges, and the B-ring analogues (zones 4 and 5) constructed in this study (Figure 4.15). The modified core peptide of the methyllanthionine A-ring analogues (Dhb-Ala-Arg-Val-Cys and Dhb-Ala-Ile-Val-Cys) eluted as two separate peaks while the core peptide for lanthionine A-ring analogues (Dhb-Ala- Ile-Val-Cys and Dha-Gly-Ile-Val-Cys) eluted as a single peak on RP-HPLC (Figure 4.18). The other modified geobacillin II analogues produced in this study also eluted as single peak (Figure 4.19). In order to further investigate the identity of the two peaks, the geobacillin II analogue GeoAII-S1T/T2A/I3R was produced in larger quantities. The two peaks obtained in RP-HPLC after GluC cleavage of the corresponding modified peptide were collected separately and lyophilized. The lyophilized peptides were ran on a RP-HPLC column to determine their purity (Figure 4.20). The isolated peptides were hydrolyzed and derivatized for GC/MS analysis. Peak 1 contained a mixture of DL and LL stereoisomers of MeLan whereas only a very small signal for MeLan was observed in the analysis of peak 2 (Figure 4.21). The lack of cyclization of ring A

was also confirmed by alkylation of free cysteines in these peptides with iodoacetamide (IAA) using a previously published protocol (3). MALDI-MS spectra showed no IAA adducts were present in the reaction containing the product of the peptide corresponding to peak 1 whereas one IAA adduct was present in the reaction containing the product of the peptide corresponding to peak 2 (Figure 4.22). Similar results upon incubation with IAA were obtained for the geobacillin II analogue GeoAII-S1T/T2A that also consisted of two peaks by HPLC. The observed incomplete cyclization upon mutation of these residues in the A-ring suggests that the wild type sequence of this ring is important for efficient modification by GeoM.

For both analogues, the cyclized material (peak 1) inhibited the growth of B. subtilis whereas the uncyclized material (peak 2) resulted in no inhibition (Figure 4.23). When the cyclized and uncyclized material were applied together, no zone of inhibition was observed (zone 4 and 5, Figure 4.23). This observation suggests that the uncyclized material serves as antagonist of the fully cyclized peptides. In turn, this finding suggests formation of an oligomeric active complex involving multiple geobacillin II molecules may be required for antibacterial activity. For instance, the lantibiotic nisin has been shown to form a multimeric complex in the cell membrane upon lipid II binding (40). Formation of an inactive complex between fully cyclized and partially cyclized compounds, and potentially a target molecule may explain the lack of activity of geobacillin II S1T/T2A/I3R and geobacillin II S1T/T2A. Since ring B analogues had identical stereochemistry as wild-type geobacillin II and eluted as single peaks, the reduction in biological activity observed in zones 4 and 5 in Figure 4.15 may arise from mutation of the Dha residue to a Dhb residue. This mutation may inhibit binding to the target, which may specifically require a Lan at this position. Alternatively, a Lan B-ring may be

important for interactions between geobacillin II molecules. Deeper investigation of these results requires additional mechanism-of-action studies.

Figure 4.15. Agar diffusion growth inhibition assay of geobacillin II analogues produced in this study. Zone 1:

nisin (10 µL of 75 µM), Zone 2: nisin (10 µL of 50 µM). Zones 3-14 represent inhibition by geobacillin II and the analogues produced in this study (20 µL of 150 µM solution from GluC cleavage of each modified precursor peptide). Zone 3: geobacillin II, Zone 4: geobacillin II with ring B amino acid sequence Dhb-Leu-Arg-Ile-Cys, Zone 5: geobacillin II with ring B amino acid sequence Dhb-Thr-Arg-Ile-Cys, Zone 6: geobacillin II with ring C amino acid sequence Dhb-Leu-Arg-Phe-Cys, Zone 7: geobacillin II with ring C amino acid sequence Dhb-Thr-Arg-Phe- Cys, Zone 8: geobacillin II with ring D amino acid sequence Dhb-Phe-Lys-Val-Arg-Cys, Zone 9: geobacillin II with ring D amino acid sequence Dhb-Thr-Lys-Val-Arg-Cys, Zone 10: geobacillin II with ring A amino acid sequence Dha-Ala-Ile-Val-Cys, Zone 11: geobacillin II with ring A amino acid sequence Dha-Gly-Ile-Val-Cys, Zone 12: geobacillin II with ring A amino acid sequence Dhb-Dhb-Ile-Val-Cys, Zone 13: geobacillin II with ring A amino acid sequence Dhb-Ala-Ile-Val-Cys, and Zone 14: geobacillin II with ring A amino acid sequence Dhb-Ala- Arg-Val-Cys.

Figure 4.16. MALDI-MS spectra of modified His6-GeoAII and A-ring analogues generated by coexpression of the corresponding precursor peptide with untagged GeoM in E. coli. The amino acid sequences of the A-rings were: (A) Dha-Dhb-Ile-Val-Cys (wild type), (B) Dhb-Dhb-Ile-Val-Cys, (C) Dhb-Ala-Ile-Val-Cys, (D) Dhb-Ala-Arg-Val-Cys, (E) Dha-Ala-Ile-Val-Cys, and (F) Dha-Gly-Ile-Val-Cys.

Figure 4.17. MALDI-MS spectra of modified His6-GeoAII analogues of rings B, C, and D generated by coexpression of the corresponding precursor peptide with untagged GeoM in E. coli. The amino acid sequence of the B-ring analogues were: (A) DhbLeuArgIleCys, and (B) Dhb-Thr-Arg-Ile-Cys; analogues of the C-ring were: (C) Dhb-Leu-Arg-Phe-Cys, and (D) Dhb-Thr-Arg-Phe-Cys; and analogues of the D-ring were: (E) Dhb-Phe-Lys-Val- Arg-Cys, and (F) Dhb-Thr-Lys-Val-Arg-Cys.

Figure 4.18. Analytical HPLC traces of modified His6-GeoAII and A-ring analogues after GluC cleavage. The

amino acid sequences of the A-ring were: wild type Dha-Dhb-Ile-Val-Cys (black), Dhb-Dhb-Ile-Val-Cys (red), Dha- Ala-Ile-Val-Cys (blue), Dha-Gly-Ile-Val-Cys (pink), Dhb-Ala-Ile-Val-Cys (green), and Dhb-Ala-Arg-Val-Cys (navy).

Figure 4.19. Analytical HPLC traces of modified His6-GeoAII and analogues of the B, C, and D rings after GluC cleavage. The amino acid sequences of the B-ring were: Dhb-Leu-Arg-Ile-Cys (green), and Dhb-Thr-Arg-Ile-Cys (navy). The amino acid sequences of the C-ring sequences were: Dhb-Leu-Arg-Phe-Cys (blue), and Dhb-Thr-Arg- Phe-Cys (pink). The amino acid sequences of the D-ring were: Dhb-Phe-Lys-Val-Arg-Cys (black), and Dhb-Thr- Lys-Val-Arg-Cys (red).

Figure 4.20. Analytical HPLC trace of modified wild type His6-GeoAII-S1T/T2A/I3R (blue) after GluC cleavage. Peak 1 and Peak 2 were collected separately and lyophilized. The lyophilized powder was dissolved in water and analyzed by analytical HPLC. The black and red traces correspond to purified peak 1 and peak 2.

Figure 4.21. Stereochemical configuration of hydrolyzed and derivatized Lan/MeLan residues of peptides present in peak 1 and peak 2 (Figure 4.20). (A) Hydrolyzed and derivatized MeLan residue in sample generated

from peak 1 (black trace) and peak 2 (red trace). (B) Hydrolyzed and derivatized Lan residue in samples generated from peak 1 (black trace) and peak 2 (red trace).

Figure 4.22. MALDI-TOF mass spectra of peptides present in peak 1 and peak 2 after treatment with 5 mM TCEP

and 50 mM iodoacetamide. The peak corresponding to M-4 H2O + 9 is generated by one IAA adduct of dethiomethyl geobacillin II. Dethiomethyl geobacillin II is generated by loss of part of the side chain of an oxidized methionine (UNIMOD accession number 526) or by the reaction of the methionine side chain with IAA, followed by elimination resulting in a mass shift of −48 Da.

Figure 4.23. Bioassay with the indicator strain B. subtilis ATCC6633. Zone 1: 20 µL of GluC treated geobacillin

II-G−8K/T2A (150 µM). Zone 2: 10 µL of geobacillin II-S1T/T2A/I3R corresponding to peak 2 (150 µM) + 10 µL sterile water. Zone 3: 10 µL of geobacillin II-S1T/T2A/I3R corresponding to peak 1 (150 µM) + 10 µL sterile water. Zone 4: 10 µL each of geobacillin II-S1T/T2A/I3R corresponding to peak 1 and 2 (150 µM each). Zone 5: 10 µL each of geobacillin II-S1T/T2A corresponding to peak 1 and 2 (150 µM each). Zone 6: 10 µL of geobacillin II- S1T/T2A corresponding to peak 1 (150 µM) + 10 µL sterile water. Zone 7: 10 µL of geobacillin II-S1T/T2A corresponding to peak 2 (150 µM) + 10 µL sterile water.