Co-cultivation induces elicitation of unique Fungus-derived Natural products that leads to thiol stress mediated killing of Mycobacterium tuberculosis [F51]

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 and were screened using co-cultivation based approach for their specific anti-Mtb activity induced upon co-cultivation with Mtb as assessed by performing growth inhibition of reprter strain of Mtb using cell free media from mono and co-cultures. Unique fungi obtained in our study F2, F50, F51, C7 and F31 were taken forward for RNA seq to identify the differentially expressed gene clusters responsible for producing anti-Mtb metabolites in cocultivation conditions. Fungal spores were grown in PDB media for 24h following which they were either treated with phosphate buffer or Mtb. The mono- and co-cultures were incubated for different day points at 32'C. Mycelia was then harvested, ground by mortar pestle in Liquid N2 and the ground mycelia was used for Total RNA extraction. mRNA was then extracted from Total RNA following rRNA depletion method