Fungal infections affect more than one billion people worldwide and cause more than one million deaths per year. Amphotericin B (AmB), a polyene antifungal drug, has been used as the gold standard for many years because of its broad antifungal spectrum, high activity, and low tendency of drug resistance. However, the side effects of AmB, such as nephrotoxicity and hepatotoxicity, have hampered its widespread use, leading to the development of a liposome-type AmB formulation, AmBisome. Herein, we report a simple but highly effective strategy to enhance the antifungal activity of AmBisome with a lipid-modified protein.The chitin-binding domain (LysM) of the antifungal chitinase, Pteris ryukyuensis chitinase A (PrChiA), a small 5.3 kDa protein that binds to fungal cell wall chitin, was engineered to have a glutamine-containing peptide tag at the C-terminus for the microbial transglutaminase (MTG)-catalyzed crosslinking reaction (LysM-Q). LysM-Q was site-specifically modified with a lysine-containing lipid peptide substrate of MTG with a palmitoyl moiety (Pal-K). The resulting palmitoylated LysM (LysM-Pal) exhibited negligible cytotoxicity to mammalian cells and can be easily anchored to yield LysM-presenting AmBisome (LysM-AmBisome). LysM-AmBisome exhibited a dramatic enhancement of antifungal activity toward Trichoderma viride and Cryptococcus neoformans, demonstrating the marked impact of displaying a cell-wall binder protein on the targeting ability of antifungal liposomal formulations. Our simple strategy with enzymatic protein lipidation provides a potent approach to upgrade other types of lipid-based drug formulations.

Combinations of antifungal drugs can have synergistic antifungal activity, achieving high therapeutic efficacy while minimizing the side effects. Amphotericin B (AMB) has been used as a standard antifungal drug for fungal infections; however, because of its high toxicity, new strategies to minimize the required dose are desirable. Chitinases have recently received attention as alternative safe antifungal agents. Herein, we report the combination of palmitoylated chitinase domains with AMB to enhance the antifungal activity. The chitin-binding domain (LysM) from Pteris ryukyuensis chitinase was site-specifically palmitoylated by conjugation reaction catalyzed by microbial transglutaminase. The palmitoylated LysM (LysM-Pal) exhibited strong antifungal activity against Trichoderma viride, inhibiting the growth completely at a concentration of 2 μM. This antifungal effect of LysM-Pal was mainly due to the effect of anchoring of palmitic acid motif to the plasma membrane of fungi. A combination of AMB with LysM-Pal resulted in synergistic enhancement of the antifungal activity. Intriguingly, LysM-Pal exhibited higher level of antifungal activity enhancement than palmitoylated catalytic domain (CatD) and fusion of LysM and CatD. Addition of 0.5 μM LysM-Pal to AMB reduced the minimal inhibition concentration of AMB to 0.31 μM (2.5 μM without LysM-Pal). The possible mechanism of the synergistic effect of AMB and LysM-Pal is destabilization of the plasma membrane by anchoring of palmitic acid and ergosterol extraction by AMB and destabilization of the chitin layer by LysM binding. The combination of LysM-Pal with AMB can drastically reduce the dose of AMB and may be a useful strategy to treat fungal infections.

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