## 2 Results  
### Design of Peptides  
HRZN 13-17 was designed by database filtering technology (DFT) with the additional positional analysis (PA) filter to design a set of new peptides. The Giant Repository of AMP Activities (GRAMPA) database [43] consists of 6760 unique sequences and 51,345 MIC values against various organisms. We collected 643 peptides that reported activity against _A. baumantii_ with reported MIC values. Dataset 1 (374 sequences) was obtained by removing duplicate sequences from that list. DFT was performed on Dataset 1 by MIC selection, and then size selection and positional analysis [40; 48], which resulted in HRZN-13 and 14. Further processing using positional analysis based on different iterations yielded three additional peptides. HRZN-15, 16, and 17 were generated by processing on peptide lengths of 13, 13 \(\pm\) 3, and 13 \(\pm\) 4 amino acids, respectively [40]. All peptides were synthesized by ChinaPeptides with high purity (\(\geq\)98%) and checked by reverse-phase high-performance liquid chromatography. Secondary structures of the newly designed HRZN peptides (Table 1) were predicted by AlphaFold2, and all peptides were predicted to adopt \(\alpha\)-helical structures [49]. The \(\alpha\)-helical structure is linked to the ability of peptides to lyse membranes and contributes to antibacterial activity, as in magainin 2 [50], and was one of our design parameters. Analysis of physicochemical characteristics revealed that HRZN peptides have an overall hydrophobicity ratio of 54-62% of the amino acids and an overall positive charge of +3\(\rightarrow\)6. This is consistent with our previous results, which found that gram-negative active AMPs have hydrophobicity of around 50% and an optimal net charge between +2 and +4 [40]. HRZN-13 and -14 sequences differ in one residue at position 11 (K in HRZN-13 and R in HRZN-14); hence, they both have +5 charge and 54% total hydrophobicity. HRZN-15 and HRZN-16 have the highest hydrophobic ratio of 62% and the highest hydrophobic moment, but they differ in the net charge, with HRZN-15 having +3 and +5 for HRZN-16. HRZN-17 has the highest net charge of +6 and similar hydrophobicity to HRZN-13 and -14, but the highest hydrophobic moment. The Heliquest program ([[https://heliquestipmc.cnrs.fr](https://heliquestipmc.cnrs.fr)]([https://heliquestipmc.cnrs.fr](https://heliquestipmc.cnrs.fr)), accessed on 29 September 2021) was used to generate the helical wheel projections, illustrating the amphipability and the hydrophobic face of each peptide and calculating the hydrophobic moment. These newly designed peptides all represent new and novel sequences, none of which had been previously identified in any of the databases we searched. For example, the HRZN-15 sequence was searched in APD, DBAASP, and YADAMP, and there were no identical sequences. Peptide alignment of HRZN-15 was performed in APD and resulted in a 69.23% similarity of HRZN-15 to Temporin L and Temporin-1 Ob. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Peptide**} & \multirow{2}{*}{**Sequence**} & \multirow{2}{*}{**Charge**} & \multicolumn{2}{c}{**\%**} & \multirow{2}{*}{**Predicted Structure**} \\  & & & & **Hydrophobicity** & **AlphaFold2** \\ \hline HRZN-13 & FLWRISKFLGKKL-NH\({}_{2}\) & \multirow{2}{*}{+5} & \multicolumn{2}{c}{54\%} \\  & & NH\({}_{2}\) & & (0.537) & \\ \hline HRZN-14 & FLWRISKFLGRKL-NH\({}_{2}\) & \multirow{2}{*}{+5} & \multicolumn{2}{c}{54\%} \\  & & NH\({}_{2}\) & & (0.539) & \\ \hline HRZN-15 & FLPWISKFLGKIL-NH\({}_{2}\) & \multirow{2}{*}{+3} & \multicolumn{2}{c}{62\%} \\  & & NH\({}_{2}\) & & (0.659) & \\ \hline HRZN-16 & FLKKIWKLLGKLL-NH\({}_{2}\) & \multirow{2}{*}{+5} & \multicolumn{2}{c}{62\%} \\  & & NH\({}_{2}\) & & (0.877) & \\ \hline HRZN-17 & KLWKLLKKLGRLL-NH\({}_{2}\) & \multirow{2}{*}{+6} & \multicolumn{2}{c}{54\%} \\  & & NH\({}_{2}\) & & (0.804) & \\ \hline IDR-1018 & VRLIVAVRIWRR-NH\({}_{2}\) & \multirow{2}{*}{+5} & \multicolumn{2}{c}{67\%} \\  & & NH\({}_{2}\) & & (0.271) & \\ \hline \hline \end{tabular}
\end{table}