The recalcitrant
biofilm formed by fungus Cryptococcus neoformans is a life-threatening pathogenic condition responsible for further
intensifying cryptococcosis. Considering the enhanced biofilm resistance
and toxicity of synthetic antifungal drugs, the search for efficient,
nontoxic, and cost-effective natural therapeutics has received a major
boost. Phenolic (thymol and carvacrol) and aldehydic (citral) terpenes
are natural and safe alternatives capable of efficient microbial biofilm
inhibition. However, the biofilm inhibition mechanism of these terpenes
still remains unclear. In this study, we adopted an integrative biophysical
and biochemical approach to elucidate the hierarchy of their action
against C. neoformans biofilm cells.
The microscopic analysis revealed disruption of the biofilm cell surface
with elevation in surface roughness and reduction in cell height.
Although all terpenes acted through ergosterol biosynthesis inhibition,
the phenolic terpenes also selectively interacted via ergosterol binding.
Further, the alterations in the fatty acid profile in response to
terpenes attenuated the cell membrane fluidity with enhanced permeability,
resulting in pore formation and efflux of the K+/intracellular
content. Additionally, mitochondrial depolarization caused higher
levels of reactive oxygen species, which led to increased lipid peroxidation
and activation of the antioxidant defense system. Indeed, the oxidative
stress caused a significant decline in the amount of extracellular
polymeric matrix and capsule sugars (mannose, xylose, and glucuronic
acid), leading to a reduced capsule size and an overall negative charge
on the cell surface. This comprehensive data revealed the mechanistic
insights into the mode of action of terpenes on biofilm inhibition,
which could be exploited for formulating novel anti-biofilm agents.