Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2023

Unravelling the mechanism of lactofungin, a peptide that shows potent synergy with the antifungal drug amphotericin (93655)

Yumeng Hao 1 , Anneka C Pereira Schmidt 1 , Lissy M Hartmann 1 , Kenya Fernandes 2 , Samir Kulkarni 3 , Richard J Payne 3 , Dee Carter 2 , Evelyne Deplazes 1
  1. University of Queensland, Brisbane, QLD, Australia
  2. School of Life and Environmental Sciences, Univerity of Sydney, Sydney, NSW, Australia
  3. School of Chemistry, University of Sydney, Sydney, NSW, Australia

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, particularly for treating multi-drug resistant IFIs but the drug’s high toxicity to mammalian cells is dose-limiting and causes severe side effects, particularly in immunocompromised patients.

AmB works by binding to ergosterol, the main sterol found in fungal cells, subsequently sequestering ergosterol and permeabilising the membrane. The strong structural similarity between cholesterol and ergosterol results in off-target binding, causing the drug’s toxicity to mammalian cells. There is thus an ongoing effort 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 up to 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 synergy of LFG and AmB is lipid-dependent and ergosterol-specific. Specifically, tethered lipid bilayer membranes (tBLM) electrical impedance spectroscopy (EIS) experiments show that 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.