Cryptococcosis
is a multifaceted and potentially fatal systemic fungal infection, globally responsible for  ? 1 million cases/year mostly occurring in
immunodeficient patients. This infection is further augmented due to the
ability of Cryptococcus neoformans to
form biofilm. Considering the enhanced biofilm resistance and toxicity concerns
of synthetic antifungal drugs, search for efficient and non-toxic natural therapeutics
have received a major boost in recent times. Hence, in the present study the anti-biofilm
potential of thymol, a monoterpene phenol of thyme oil and its mechanism of
action (MOA) against C. neoformans
were evaluated.

 

To test
the potency of thymol the minimum inhibitory concentration (MIC) and the
biofilm inhibitory/ eradicating concentration (BIC/BEC) were determined. The morphological
and physiological changes in response to thymol were analyzed using biophysical
techniques. NMR based metabolomic and Mass spectrometry based proteomic studies
were performed to assess the MOA of thymol. Real time qPCR was conducted to substantiate
the multiomics results. Finally, the toxic nature of thymol against human
cell-lines was evaluated using co-culture assays.

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Thymol
showed an antifungal and anti-biofilm activity of MIC 16 µg/mL and BIC/BEC 32/128
µg/mL respectively lower than fluconazole. Microscopic analysis revealed absence
of extracellular
polymeric matrix, reduction in capsule
size, and disruption of
surface/ultrastructure of biofilm cells. The metabolomic analysis evidenced for a decrease in TCA cycle
intermediates (fumarate, malate, citrate) and amino acid metabolism (branched
chain, aromatic) while an increase in the energy metabolites (ATP, NAD+)
suggesting thymol interference with the key metabolic pathways. These results
were in line with the proteomics data as it showed upregulation of stress proteins
while downregulation of carbohydrate metabolism and cell wall integrity related
proteins. RT-PCR studies established that stress responsive gene calcineurin, CNA1 is overexpressed and cell wall
integrity gene ergosterol, ERG11 is under
expressed, thus substantiating the above omics results. Furthermore, we
observed that thymol is minimally toxic as it showed selective killing of C. neoformans without affecting
keratinocytes in co-cultures.

 

This study provided mechanistic
insights depicting how thymol can modulate the membrane permeability and
regulatory pathways to invade C.
neoformans; and demonstrated its therapeutic potential to be formulated as
next-generation antifungal agent.

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