Cryptococcosis based proteomic studies were performed to assess

Cryptococcosisis a multifaceted and potentially fatal systemic fungal infection, globally responsible for  ? 1 million cases/year mostly occurring inimmunodeficient patients. This infection is further augmented due to theability of Cryptococcus neoformans toform biofilm. Considering the enhanced biofilm resistance and toxicity concernsof synthetic antifungal drugs, search for efficient and non-toxic natural therapeuticshave received a major boost in recent times. Hence, in the present study the anti-biofilmpotential of thymol, a monoterpene phenol of thyme oil and its mechanism ofaction (MOA) against C.

neoformanswere evaluated. To testthe potency of thymol the minimum inhibitory concentration (MIC) and thebiofilm inhibitory/ eradicating concentration (BIC/BEC) were determined. The morphologicaland physiological changes in response to thymol were analyzed using biophysicaltechniques. NMR based metabolomic and Mass spectrometry based proteomic studieswere performed to assess the MOA of thymol. Real time qPCR was conducted to substantiatethe multiomics results. Finally, the toxic nature of thymol against humancell-lines was evaluated using co-culture assays.

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 Thymolshowed an antifungal and anti-biofilm activity of MIC 16 µg/mL and BIC/BEC 32/128µg/mL respectively lower than fluconazole. Microscopic analysis revealed absenceof extracellularpolymeric matrix, reduction in capsulesize, and disruption ofsurface/ultrastructure of biofilm cells. The metabolomic analysis evidenced for a decrease in TCA cycleintermediates (fumarate, malate, citrate) and amino acid metabolism (branchedchain, aromatic) while an increase in the energy metabolites (ATP, NAD+)suggesting thymol interference with the key metabolic pathways. These resultswere in line with the proteomics data as it showed upregulation of stress proteinswhile downregulation of carbohydrate metabolism and cell wall integrity relatedproteins. RT-PCR studies established that stress responsive gene calcineurin, CNA1 is overexpressed and cell wallintegrity gene ergosterol, ERG11 is underexpressed, thus substantiating the above omics results. Furthermore, weobserved that thymol is minimally toxic as it showed selective killing of C.

neoformans without affectingkeratinocytes in co-cultures. This study provided mechanisticinsights depicting how thymol can modulate the membrane permeability andregulatory pathways to invade C.neoformans; and demonstrated its therapeutic potential to be formulated asnext-generation antifungal agent.

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