Design of Protease-Responsive Antifungal Liposomal Formulation Decorated with a Lipid-Modified Chitin-Binding Domain.

Candida albicans is a prevalent fungal pathogen that displays antibiotic resistance. The polyene antifungal amphotericin B (AmB) has been the gold standard because of its broad antifungal spectra, and its liposomal formulation, AmBisome, has been used widely and clinically in treating fungal infections. Herein, we explored enhancing the antifungal activity of AmBisome by integrating a small chitin-binding domain (LysM) of chitinase A derived from Pteris ryukyuensis. LysM conjugated with a lipid (LysM-lipid) was initially prepared through microbial transglutaminase (MTG)-mediated peptide tag-specific conjugation of LysM with a lipid-peptide substrate. The AmBisome formulation modified with LysM-lipid conjugates had a size distribution that was comparable to the native liposomes but an increased zeta potential, indicating that LysM-lipid conjugates were anchored to AmBisome. LysM-lipid-modified AmBisome exhibited long-term stability at 4 °C while retaining the capacity to bind chitin. Nevertheless, the antifungal efficacy of LysM-lipid-modified AmBisome against C. albicans was modest. We then redesigned a new LysM-lipid conjugate by introducing a peptide linker containing a thrombin digestion (TD) site at the C-terminus of LysM (LysM-TD linker-lipid), thereby facilitating the liberation of the LysM domain from AmBisome upon the addition of thrombin. This new AmBisome formulation anchored with LysM-TD linker-lipid exhibited superior performance in suppressing C. albicans growth in the presence of thrombin compared with the LysM-lipid formulation. These results provide a platform to design stimuli-responsive AmBisome formulations that respond to external environments and thus advance the treatment of pathogenic fungi infections.

Liposomal Amphotericin B Formulation Displaying Lipid-Modified Chitin-Binding Domains with Enhanced Antifungal Activity.

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.

Preparation of amphotericin B-loaded hybrid liposomes and the integration of chitin-binding proteins for enhanced antifungal activity.

Amphotericin B (AMB) is a gold standard antifungal drug because of its broad-spectrum activity toward pathogenic yeasts and molds. Because of its low solubility in water and toxicity toward humans, several lipid-based formulations that either increase the aqueous solubility or decrease the side effects have been employed in practical use. In our previous research, we found that the combination of AMB with an artificial palmitoylated chitin-binding domain from Pteris ryukyuensis chitinase (LysM-Pal) resulted in synergistic antifungal action against Trichoderma viride. Herein, we prepared hybrid liposomal formulations by combining a commercially available AMB formulation and liposomes with different surface charges to explore key factors in the antifungal activity. The characterization of AMB-loaded liposomal formulations (AMB-LFs), including particle size distribution and zeta potential, showed that anionic and neutral AMB-LFs could stably encapsulate AMB. The combination of either anionic or neutral AMB-LFs with unmodified LysM decreased the minimum inhibitory concentration of AMB. The combination of neutral AMB-LF with LysM-Pal resulted in a further decrease in the MIC, up to 15-fold compared with that of the neutral AMB-LF alone. Our results demonstrate the potential utility of lipid-based liposomal formulations of AMB combined with lipid-modified proteinaceous binders to tackle fungal infections.

Enhancement of the Antifungal Activity of Chitinase by Palmitoylation and the Synergy of Palmitoylated Chitinase with Amphotericin B.

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.

Orthogonal Enzymatic Conjugation Reactions Create Chitin Binding Domain Grafted Chitinase Polymers with Enhanced Antifungal Activity.

Enzymatic reaction offers site-specific conjugation of protein units to form protein conjugates or protein polymers with intrinsic functions. Herein, we report horseradish peroxidase (HRP)- and microbial transglutaminase (MTG)-catalyzed orthogonal conjugation reactions to create antifungal protein polymers composed of Pteris ryukyuensis chitinase-A (ChiA) and its two domains, catalytic domain, CatD, and chitin-binding domain, LysM2. We engineered the ChiA and CatD by introducing a peptide tag containing tyrosine (Y-tag) at N-termini and a peptide tag containing lysine and tyrosine (KY-tag) at C-termini to construct Y-ChiA-KY and Y-CatD-KY. Also, LysM2 with Y-tag and KY-tag (Y-LysM2-KY) or with a glutamine-containing peptide tag (Q-tag) (LysM2-Q) were constructed. The proteins with Y-tag and KY-tag were efficiently polymerized by HRP reaction through the formation of dityrosine bonds at the tyrosine residues in the peptide tags. The Y-CatD-KY polymer was further treated by MTG to orthogonally graft LysM2-Q to the KY-tag via isopeptide formation between the side chains of the glutamine and lysine residues in the peptide tags to form LysM2-grafted CatD polymer. The LysM2-grafted CatD polymer exhibited significantly higher antifungal activity than the homopolymer of Y-ChiA-KY and the random copolymer of Y-CatD-KY and Y-LysM2-KY, demonstrating that the structural differences of artificial chitinase polymers have a significant impact on the antifungal activity. This strategy of polymerization and grafting reaction of protein can contribute to the further research and development of functional protein polymers for specific applications in various fields in biotechnology.