Abstract:
The WHO has identified several Candida species including C. albicans as critical priority fungal pathogens due to greater infection prevalence and formation of recalcitrant biofilms. Resistance to antifungal drugs and increased rates of infection highlight an urgent need for novel antifungal agents. Antimicrobial peptides (AMPs) are a potential alternative to antifungal drugs due to their novel modes of action and broad-spectrum activity. However, further therapeutic development of many AMPs is halted by inactivation in physiological concentrations of salts, serum, and plasma. Tryptophan end-tagging was identified as a structural modification that increases the activity of AMPs in these physiological environments. In this study, Os-C was tagged with tryptophan residues to form Os-C(W5) and the effect of tryptophan end-tagging on the structural characteristics, anticandidal activity and mode of action of Os-C was investigated.
Mechanistic insight into the structural characteristics of Os-C(W5) compared with Os-C is provided by circular dichroism (CD) spectroscopy and molecular dynamics (MD) simulations. Steady state analysis using CD spectroscopy shows that tryptophan end-tagging alters the secondary structure in Tris buffer and sodium dodecyl sulfate. In silico, MD simulations of peptides were performed with a C. albicans model membrane consisting of the lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol (POPI), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and ergosterol. Like the CD data, MD simulation data reveals changes in the secondary structure of Os-C after end-tagging. Furthermore, MD simulations show that tryptophan end-tagging reduces interactions with and insertion into a model C. albicans membrane and promotes peptide aggregation at its surface.
Antiplanktonic assays indicate that tryptophan end-tagging enhances the activity of Os-C which decreases the growth and viability of C. albicans. More in-depth mode of action studies reveal that Os-C(W5) does not cause membrane permeabilisation. Instead, the antifungal activity correlates with the induction of reactive oxygen species and changes in cell morphology.
Further antibiofilm studies show that Os-C(W5) prevents biofilm formation and eradicates preformed biofilms. Reduced cell adhesion and viability contribute to reduced biofilm extracellular matrix formation. Although reduced, Os-C(W5) retains some antibiofilm activity in RPMI-1640 supplemented with 50% foetal bovine serum and in a synthetic wound medium.
In conclusion, this study demonstrates that tryptophan end-tagging is a simple modification that transforms a salt-sensitive AMP (Os-C) into a peptide (Os-C(W5)) with antifungal activity in physiologically relevant environments.
