ANTI-BIOFILM ACTIVITY OF TERPENES AND THEIR MECHANISM AGAINST CRYPTOCOCCUS NEOFORMANS USING INTEGRATED OMICS APPROACH

Abstract

Cryptococcosis is a multifaceted and potentially fatal systemic fungal infection entailing a global burden of ~1 million clinical cases leading to 625,000 losses per year. Cryptococcus neoformans, encapsulated yeast is majorly responsible for causing this life-threatening infection, involving central nervous system (CNS), pulmonary and cutaneous sites. The need to address the problem of cryptococcosis has significantly increased in past years due to acquired immunodeficiency syndrome (AIDS) epidemic, intensive chemotherapy of cancer patients, and extensive use of immunosuppressive drugs. The most acute manifestation of cryptococcal infection is meningoencephalitis which is further challenged due to the colonization of C. neoformans inside the CNS to form biofilm-like cryptococcomas. Moreover, the ability of this fungus to adhere and form biofilm on indwelling medical devices highlights the significance of its biofilm as a critical pathogenic condition The biofilm formation and pathogenesis of C. neoformans is attributed to glucuronoxylomannan (GXM); a key component of its polysaccharide capsule. Biofilms are well-structured surface attached sessile cryptococcal cells embedded in a self-produced polysaccharide-rich extracellular polymeric matrix (EPM), responsible for its recalcitrant and invasive nature. The EPM provides mechanical stability, strong cell-cell communication and serves as a nutrient source for biofilms. This high resistance of biofilm to antifungal drugs compared to their planktonic counterparts is therefore of great clinical relevance. The currently available treatment strategy for cryptococcosis includes three classes of antifungal agents including polyenes, azoles, and pyrimidine analog. However, only a few drugs show limited-to-good efficacy against C. neoformans biofilms. Though the biofilm forms are susceptible to polyenes and its lipid formulations, the effective concentrations transcend the therapeutic range leading to severe nephrotoxicity and emergence of drug-resistance in clinical strains. Cryptococcal biofilms are highly tolerant to azole antifungals and do not inhibit biofilm formation as these drugs are unable to prevent GXM release, a crucial step involved in yeast adhesion and later biofilm formation. Thus, due to the incompetence of these standard antifungal drugs in effectively curing biofilm infections, the next stage of treatment is often restricted to device replacement that incurs a heavy cost, surgical procedures leading to pain, and further challenged by the development of resistance.In view of the current scenario, there is an imperative need to develop alternative natural drug therapies that are not only effective against C. neoformans biofilms but also safe and costeffective. In this regard, terpenes, the major active secondary metabolites present in essential oils of aromatic plants have been reported to possess well-established antimicrobial and anti-biofilm potential against different pathogens like pathogenic bacteria and fungi. An updated literature review on the antimicrobial potential and the mode of action of phytochemicals including essential oils (EOs) and their active components (EO-ACs) has been well described in the present study. Further, the utilization of omics approaches (transcriptomics, proteomics, and metabolomics) for a comprehensive understanding of the mechanism of action of the unknown drug, pharmacological response to drug action and identification of new candidates for drug development have also been discussed. The previous studies in this field have majorly focused on the planktonic form, leaving behind a lacuna in the activity of terpenes against recalcitrant Cryptococcus biofilms. Keeping the above facts in mind, the first objective of the study was to investigate the antifungal and antibiofilm activity of selected essential oil EO-ACs against C. neoformans and C. laurentii. A total of six EO-ACs based on their strong activity against different human pathogens were selected. The EO-ACs were classified into three different classes with terpenic phenols which included thymol and carvacrol; terpenic aldehydes and alcohol included citral and menthol respectively while phenylpropanoid included eugenol and cinnamaldehyde. The above EO-ACs was screened for their activity against three infectious forms; planktonic cells, biofilm formation and preformed biofilm of C. neoformans and C. laurentii as compared to standard drugs. The data showed that anti-biofilm activity of the tested EO-ACs were in the order: thymol>carvacrol>citral>eugenol=cinnamaldehyde>menthol respectively. The three most potent terpenes, thymol, carvacrol, and citral showed excellent anti-biofilm activity at a much lower concentration against C. laurentii in comparison to C. neoformans indicating the resistant nature of the latter. Effect of the potent terpenes on the biofilm morphology was visualized using SEM which revealed the absence of EPM and alteration in the surface morphology of biofilm cells. Additionally, CLSM analysis complemented with COMSTAT analysis showed a reduction in substratum coverage, mean thickness, biomass and viability of biofilm cells. Further, to realize the efficacy of the terpenes in terms of human safety, cytotoxicity assays and co-culture model were evaluated. Thymol and carvacrol as compared to citral were the most efficient in terms of human safety in keratinocyte- Cryptococcus sp. co-culture infection model suggesting that these two can be further exploited as cost-effective and non-toxic anti-cryptococcal drugs. Taking into account the potency of the above three terpenes it was important to illustrate the biofilm inhibition mechanisms of phenolic and aldehydic terpenes against C. neoformans at the phenotypic level. In this respect, an integrative biophysical and biochemical approach was adopted 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 the terpenes acted through ergosterol biosynthesis inhibition, the phenolic terpenes also selectively interacted via ergosterol binding. Further, alterations in fatty acid profile in response to terpenes resulted in the decrement of UFA/SFA ratio with a drastic increase in palmitic acid content. This variation in fatty acid composition 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 lead to increased lipid peroxidation and activation of the antioxidant defense system. Indeed, the oxidative stress caused a significant decline in the amount of EPM and capsule sugars (mannose, xylose and glucuronic acid) leading to a reduced capsule size and overall negative charge on the cell surface. This comprehensive data revealed the mechanistic insights of terpenes in the biofilm inhibition at the cellular level, which could be further beneficial for formulating novel anti-biofilm agents. Further, to gain deep insights into the molecular responses and to elucidate the mechanism of these terpenes action against C. neoformans biofilm cells an “integrated omics approach” comprising of metabolomics and proteomics and supplemented with RT-PCR of key target genes was employed. The phenolic terpenes acting at the level of membrane ergosterol caused an increase in cell wall thickness while all the terpenes penetrated through cellular and organelle membrane causing severe damage and high vacuolization. The phenolic terpenes (thymol/carvacrol) treated biofilm cells showed differential levels of 30/31 metabolites and 46/45 proteins, while aldehydic terpene (citral) caused alterations in the abundance of 28 and 51 metabolites and proteins respectively All the three terpenes followed a multifaceted action affecting different metabolic pathways. In common, downregulation of glycolysis was substantiated with concomitant upregulation of PPP to enhance reducing equivalents required for maintenance of cellular homeostasis. The deactivation of the TCA cycle and downregulation of F1ATPase and COX19 disrupted ETC resulting in decreased energy metabolism and increased ROS generation leading to mitochondrial dysfunction. To cope up with high energy demands, glyoxylate shunt was in-turn activated in thymol and citral treated biofilm cells. The upregulation of ADH and downregulation of GLO1 suggested ethanol and methylglyoxal toxicity in biofilm cells treated with phenolic and aldehydic terpenes respectively. All the terpenes downregulated ergosterol biosynthesis pathway with fatty acid biosynthesis pathway specifically targeted by phenolic terpenes while citral treatment upregulated glycerol biosynthesis. The phenolic terpenes also selectively caused calcium burst evident from upregulation of CNB1 protein/gene leading to disruption of calcium homeostasis. Further, the trehalose pathway, a key antifungal target was severely affected by terpenes causing downregulation of tps1 protein and TPS1 gene supplemented with the metabolomics data showing significant reduction in trehalose, UDPglucose and maltose content. Based on integrated omics approach we observe that the action of terpenes was multifaceted acting at the level of lipid metabolism, amino acid metabolism, trehalose pathway, and energy metabolism. The above course of events caused a metabolic switch from respiration to fermentation with the induction of oxidative stress resulting in downregulation of EPM biosynthesis-related proteins and HSP90 which led to reduction in capsule formation and thus biofilm inhibition. To conclude, the present study describes the strong antifungal and anti-biofilm potential of phenolic and aldehydic terpenes against C. neoformans biofilm and unravels their biofilm inhibition mechanism mediated through oxidative stress. Future studies can focus on the transcriptomic analysis of C. neoformans biofilm cells in response to terpenes treatment and its integration with proteomics and metabolomics data that could help in getting a much clear picture of the mechanism of these terpenes action against biofilm cells.