Co-cultivation induces elicitation of unique Fungus-derived Natural products that leads to thiol stress mediated killing of Mycobacterium tuberculosis
Tuberculosis, caused by Mycobacterium tuberculosis has remained a leading cause of death worldwide even after decades of it being declared as global health emergency by the WHO. Newer drugs with novel modes of action are urgently needed to combat the threats imposed by the constantly emerging drug resistant strains. Natural products (NPs) derived anti-mycobacterials appear lucrative because of their complex structural features and unique cellular targets they bind to. Herein, we employed co-cultivation approach to identify cryptic biosynthetic gene clusters (BGCs) from fungal genomes eliciting the expression of genes that are silent or poorly transcribed in axenic cultures. Fungi were isolated from sphagnum peat bog samples collected from different regions of North-eastern USA because aspects of this ecological niche reflect the critical microenvironment of the human tuberculosis granuloma and are a natural habitat for slow growing mycobacterial species that compete for limited nutrients with other microbes. Bioactivity-guided assay led us to identify three unique fungal isolates that selectively produce growth inhibitory metabolites during co-cultivation with Mtb. Fungal mRNA sequencing from co-cultured isolates facilitated the identification of elicited Type I Polyketide Synthase BGCs that were silent/cryptic in monoculture conditions. Bioinformatic analyses followed by chemical validation identified these molecules to be patulin, citrinin and nidulalin A. Interestingly, these induced fungal metabolites led to a highly responsive redox-stress homeostasis within Mtb. Our study thus, illustrates a co-cultivation mediated elicitation of unique fungal NPs resulting in a thiol-burst oxidative stress mediated killing of Mtb. We believe that the identification of vulnerable drug targets may yield insights into further understanding of this essential thiol stress mediated killing of the mycobacteria Overall design: In an effort to identify the intrinsic ability of microorganisms to produce prolific, highly specific and active small molecules against Mtb, we focused on utilizing the co-cultivation approach to identify cryptic BGCs from fungal genomes eliciting the expression of genes that are silent or poorly transcribed in axenic cultures. Fungi were isolated from sphagnum peat bog samples collected from different regions of North-eastern USA because aspects of this ecological niche reflect the critical microenvironment of the human tuberculosis granuloma like acidic pH, hypoxic and nutrient poor conditions. In addition, sphagnum peat bogs are a natural habitat for slow growing mycobacterial species that compete for limited nutrients with other microbes. A co-culture screen was utilized to identify metabolites specific for killing Mtb. Differential gene expression followed by bioinformatics analyses led us to identify unique biosynthetic gene clusters in these fungi expressed upon co-cultivation. Chemical approaches further facilitated the identification of Type I polyketide synthase derived nidulalin A and patulin responsible for specific anti-Mtb activity. Global transcriptomic profile of Mtb exposed to either of the induced fungal products at different concentrations and time was performed to understand the mechanism of action of killing. Total RNA from Mtb  was isolated from Control  (untreated) and Treatment (with Patulin and Nidulalin A containing fungal filtrates) groups in triplicates and utilized for mRNA sequencing. Differential gene expression analyses between either conditions would facilitate our understanding of the Mtb pathways influenced by these fungus derived anti-mycobacterials.