## 3 Results and Discussion  
### Designation and Antifungal Effects of HnMc Variants  
In a previous study, we reported the in vitro and in vivo antibacterial, anti-inflammatory, and antifungal activities and the mechanism of action of HnMc, a hybrid peptide connecting the N-terminal and C-terminal parts of HPA3 NT3 and melittin, respectively [22; 23]. We designed three model peptides, HnMc-W (F1W substitution), HnMc-WP1 (S9P substitution), and HnMc-WP2 (addition of PXXP motif), which changed the peptide structure in HnMc and demonstrated the non-toxicity and potent in vivo antibacterial effects of HnMc-WP2 in a drug-resistant _Pseudomonas aeruginosa_-infected mouse model [20]. In bacteria, the antibacterial mechanism appears to involve destruction of the cell membrane. However, because fungi are eukaryotic cells, this study was conducted with the expectation that the antifungal mechanisms may differ depending on their structure.  
First, their antifungal effects were compared using the MIC values from microdilution assays on pathogenic fungi consisting of three drug-susceptible yeasts, four drug-resistant yeasts, and five molds. As shown in Table 1, the MIC values of HnMc-W and HnMc-WP1 did not differ from that of HnMc; however, the antifungal activity of HnMc-WP2 was enhanced against most fungi. All four peptides exhibited potent antifungal activity against drug-resistant _C. albicans_ strains. This suggests that AMP may be a good alternative for overcoming drug resistance, although clinical studies on AMP are needed. Figure 1 shows their antifungal effects against _C. gloosporioides_ at the MIC and 12 MIC. Hollow hyphae, shrunken conidia, and swollen conidia were observed microscopically, indicating there was more than one mechanism acting on the fungus.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Fungal Cells**} & \multicolumn{6}{c}{**MIC (\(\mu\)M)**} \\ \cline{2-7}  & **HnMc** & **HnMc-W** & **HnMc-WP1** & **HnMc-WP2** & **Melittin** & **Fluconazole** \\ \hline \multicolumn{7}{c}{**Yeast**} \\ \hline _C. albicans_ & 16 & 16 & 16 & 8 & 8 & 0.8 \\ \hline _C. albicans_ & 16 & 16 & 16 & 8 & 4 & \textgreater{}128.0 \\ \hline _C. albicans_ & 16 & 16 & 16 & 8 & 4 & \textgreater{}128.0 \\ \hline _C. albicans_ & 16 & 16 & 16 & 8 & 4 & \textgreater{}128.0 \\ \hline _C. albicans_ & 16 & 16 & 16 & 8 & 4 & \textgreater{}128.0 \\ \hline _C. albicans_ & 16 & 16 & 16 & 8 & 4 & \textgreater{}128.0 \\ \hline _C. krusei_ & 8 & 8 & 8 & 4 & 4 & 32.0 \\ \hline _C. parapsilosis_ & 16 & 16 & 16 & 8 & 8 & 4.0 \\ \hline \multicolumn{7}{c}{**Mold**} \\ \hline _C. gloosporioides_ & 1 & 1 & 1 & 1 & 8 & - \\ \hline _F. gaminearum_ & 2 & 2 & 2 & 1 & 4 & - \\ \hline _F. oxysporum_ & 2 & 2 & 2 & 2 & 2 & - \\ \hline _F. solani_ & 2 & 2 & 2 & 2 & 2 & - \\ \hline _T. harzinum_ & 2 & 2 & 2 & 1 & 4 & - \\ \hline \hline \end{tabular}
\end{table}
Table 1: Antifungal activity of HnMc variants against yeast and mold species in low-ionic-strength buffer.  
To apply AMPs clinically, it is important that their antibacterial effect is maintained under physiochemical conditions. Except for HnMc-W, the antifungal activity of the three peptides was enhanced under high-salt conditions and was maintained under divalent ions (Figure 2). Although there are issues with peptide degradation by proteolytic enzymes and side reactions in clinical applications, physicochemical conditions had little influence on antifungal activity.  
Figure 1: Alterations in the microscopic morphologies of _C. gloosporioides_ with peptides. Red dotted squares, red dotted circles, and blue dotted circles indicate the hollow hyphae, shrunken conidia, and swollen conidia, respectively.  
Figure 2: Salt tolerance effects of HnMc variants on antifungal activity. MIC\({}_{90}\) is the minimum inhibitory concentration that inhibits fungal cell growth by 90%.