Invasive fungal infections (IFIs) are challenging to treat, with mortality rates between 10% and 50%, even in developing countries with good health care. Amphotericin B (AmB) is one of the most effective anti-fungal drugs. AmB works by binding to ergosterol, the main sterol found in fungal cells, sequestering ergosterol and permeabilising the membrane. The strong structural similarity between cholesterol and ergosterol results in off-target binding, which causes high toxicity and is responsible for severe, dose-limiting side effects, particularly in immunocompromised patients. There is a need to find AmB derivatives or adjuvants to lower the toxicity of AmB while retaining its broad-spectrum antifungal activity.
Lactofungin (LFG) is a 30-residue peptide obtained from enzyme digests of the milk protein lactoferrin. LFG shows strong synergy with AmB, reducing the therapeutic dose 4-8-fold in a range of clinically relevant fungal pathogens1. LFG displays no cytotoxicity in mammalian or antifungal activity at concentrations as high as 16 times the synergistic dose, making the peptide a candidate for developing an adjuvant therapy to treat IFIs. However, the mechanism of synergy is unknown.
Using biophysical techniques and in sterol-containing phospholipid bilayers that mimic mammalian and fungal cell membranes, we show that the AmB-LFG synergy is lipid-dependent and ergosterol-specific. Specifically, LFG increases the membrane-disrupting activity of AmB 3-fold. LFG shows no membrane-disrupting activity on membranes with ergosterol or cholesterol, consistent with its lack of cytotoxic or anti-fungal activity. Data from RH421 fluorescence spectroscopy ITC suggest that LFG shows only weak binding to sterol-containing membranes and does not alter membrane properties. tBLM/EIS data also suggests that the synergy is mediated by direct interaction of the LFG and AmB, either in solution or on the membrane surface.