Mycobacterium Tuberculosis (MTB) Adaptive Gene Profiling And Identification Of Putative Key Regulator Of Drug Resistance Genes

Tuberculosis(TB)has become the leading infectious disease of 21 st century caused by Mycobacterium tuberculosis(Mtb).Resistance to anti-tuberculosis drugs is a formidable obstacle to effective TB prevention and control globally.One third population of the world is latently infected showing extensive reactivation and spread of the disease.New forms of multidrug-/extensive drug-/and total drug resistance have emerged drastically giving Mtb a wide platform to affect an extended range of human as well as animal population.Low quality drug,inadequate dosage and sub-optimal control of infection are the major contributor of developing antibiotic resistance and spread of multi resistant pathogens.Moreover,Mtb has diverse adaptability under variety of stress conditions making it a difficult task for drug development and hampering better control of its pathogenesis.These adaptations implicate induction of various regulatory networks through transcriptional expression of specific genes.Many areas regarding bacterial responsive adaptive gene network,the physiology involved in this process of adaptation and putative regulator gene which regulate and control this antibiotic resistance phenomenon remain elusive.In present study,we investigated Mtb's response and adaptation with reference to gene expression during sub-lethal kanamycin exposure.Mtb were cultured under sub-lethal drug and control conditions,where half were sub-cultured every 3-days to observe serial adaptation under same conditions and the remaining were subjected to RNA-seq.To identify differentially regulated genes,we assigned 5 folds change expression as threshold for each gene in treated sample as compared to control expression value.We identified 98 up-regulated and 198 down-regulated responsive genes compared to control through differential analysis,of which Ra1750 and Ra3160 were the most responsive genes.Adaptation analysis was performed through classifying genes expression in count table by K-Means clustering algorithm and find interested genes pattern.One group was selected for further study,where Ra1750,Ra3160,Ra3161,Ra3893 and Ra2492 showed up-regulation at early stage and gradually showed low expression levels at the later stages of drug exposure.The adaptive expression of Ra1750,Ra3160 and Ra3161 were further confirmed by real time q PCR.These results suggested that these genes contributed in Mtb's physiological adaptation during sub-lethal kanamycin exposure.To investigate key regulators of drug resistance genes,The protein-DNA interactions(PDIs)and protein-protein interaction(PPI)networks in Mtb were integrated by regulating TB data with drug resistance associated genes due to single nucleotide polymorphism(SNPs)and drug resistance mutation(DRM)database by cytoscape application and identified 30 candidate genes for putative regulator of drug resistance genes.Identification of that regulator may help us to explore their interconnectivity and establishing novel therapeutic approaches in drug development.These genes were amplified,cloned in a vector by homologous recombination and transformed in Mtb through electroporation.The candidate genes for drug resistance obtained through this network belonged to different functional categories which included regulatory functions,intermediary metabolism,information pathways,conserved hypothetical proteins,cell wall and cell processes and implicated in virulence.These genes were individually amplified,cloned in each vector and transformed through electroporation in Mtb genome.Each Mtb strain carried an additional overexpressed candidate gene except one as a control with Egfp.To measure the impacts of these genes on the growth of Mtb,Optical density(OD)of these different overexpressed Mtb strains was measured.Antibiotic screening of these Mtb strains were also performed with first line anti tuberculosis drugs isoniazid and rifampicin to identify their role in regulating drug resistance impact as compared to control.An interesting finding was the change in MIC of some overexpressed Mtb strains from 0.2?g/ml of isoniazid to 200?g/ml recorded whereas no change in MIC was observed with rifampicin screening.Our results further indicated that drug resistance in Mtb is complicated and under the control of a condensed interconnected gene network.Mtb strains with differential overexpressed genes resulting varied resistance response depending upon the overexpressed gene and the drug used during this process of antibiotic screening.Additionally,in vitro stress experimental models performed on Mtb up regulated genes were considered important targets for vaccine and drug development over down regulated genes under same stimulus.Here the unique feature of our study is the insight consideration of both up and down regulated genes to unveil their inter-connected potential pathways of these genes network involved in the process of bacterial response upon drug exposure and its connection with bacterial adaptation during sub-lethal kanamycin exposure.Mtb antibiotic adaptation and drug resistance patho-physiology remained the major challenges in drug development to control its pathogenesis.Our findings may aid to edify these potential targets for drug development against drug resistance tuberculosis.