Phenotypic plasticity in Cryptococcus: clinical outcome & alternative antifungal therapy

Abstract

Cryptococcosis, caused by pathogenic yeasts of the Cryptococcus neoformans/ gattii complex, is a fungal disease associated with high mortality rates due in part to an incomplete knowledge of host-pathogen interactions, and a lack of effective antifungals. The aims of this thesis were therefore firstly to investigate the ability of Cryptococcus for phenotypic variation and determine how this affects pathogenicity and clinical outcome, and secondly, to evaluate and develop the antimicrobial compound lactoferrin (LF) as an antifungal for use in future treatments against Cryptococcus. In the first part of this study, virulence-associated phenotypes were found to differ in a species-specific manner in the C. gattii complex. In C. neoformans, an association between the capacity to make different cell types and clinical outcome was found, indicating that strains capable of greater variation may be more able to sustain infection and resist the host response. Together, these results illustrate the importance of cellular plasticity to host-pathogen interactions. In the second part of this study, LF was found to be active across yeast species primarily due to iron chelation, however synergy with antifungal drug amphotericin B (AMB) was independent of iron. In combination, LF+AMB disrupted biofilms, reduced Cryptococcus cell and capsule size and successfully treated infection in an in vivo wax moth model. Enzymatic digestion of LF produced a hydrolysate with substantially improved synergy and a novel 30-residue peptide dubbed lactofungin (LFG) was identified and synthesised. Antifungal susceptibility testing revealed LFG to be a successful synergent with AMB, outperforming whole LF and requiring lower AMB dosages. Overall, this thesis has provided insight into the relationship between phenotypic variation and virulence with potential significance to clinical outcome and has identified a novel drug lead with the potential to be developed for use in future antifungal therapies.