S3.4b Lactoferrin, a natural source of peptides that potentiate the antifungal activity of amphotericin B

Abstract

S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PMObjectivesIt is notoriously difficult to prevent and treat fungal infections, however, the natural world has come up with remedies that are non-toxic, effective, and evade resistance. Here we investigate lactoferrin, an iron-binding glycoprotein found in milk, tears, and sweat, for its capacity to inhibit fungi and to synergize with commonly used antifungal drugs, with the aim of determining its mode of action.MethodsLactoferrin (LF) was obtained from a commercial supplier and two dairy companies. LF was tested on suite of pathogenic yeast and mold species for inhibition using CLSI microdilution methods. Synergy was determined with antifungal drugs amphotericin B (AMB), nystatin (NYS), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), and 5-fluorocytosine (5FC). The effect of LF on fungal cells was analyzed using scanning electron microscopy (SEM). The active peptide/s within LF were then predicted from pepsin and in silico digestion, synthesized, and tested for synergy with amphotericin B (AMB). Tethered synthetic membranes were produced and were loaded with ergosterol or cholesterol to test the nature and specificity of membrane binding by LF and the synthetic peptide.ResultsLF demonstrated antifungal activity against yeast species Cryptococcus, Candida, and Saccharomyces and was much less effective against molds. Good synergy was achieved with AMB but not azole or echinocandin drugs. While the iron-chelating capacity of LF was important for the antifungal activity it was not involved in synergy. SEM revealed cell damage suggesting an interaction between AMB, LF and the fungal membrane or cell wall. A 30-residue peptide from the C lobe of LF was synthesized and tested for activity and synergy. This peptide, dubbed lactofungin (LFG) was inactive alone but was potently synergistic with AMB, indicating a direct role in augmenting AMB activity. Synthetic membranes loaded with ergosterol but not cholesterol were disrupted by AMB + LFG, demonstrating that activity was fungal-specific and was mediated through ergosterol binding.ConclusionLF is a complex molecule that causes fungal inhibition via iron binding and when cleaved by pepsin can produce active peptides. As AMB is a highly toxic treatment, the use of LFG as a synergent could help increase activity while lowering the effective dose, thereby reducing undesirable side effects. The action of AMB + LFG appears dependent on ergosterol, suggesting inhibition will be highly fungal-specific.