| Mycobacterium tuberculosis(M.tuberculosis)is an intracellular pathogen and the causative agent of Tuberculosis(TB).Around one third of the world population has been infected by the M.tuberculosis.However,most of the people carry the bacteria harmlessly in their body without any signs and symptoms called latent TB(LTB).Currently,LTB shows extensive reactivation and spread of the disease across the globe.The clear mechanism of which is currently unknown.Moreover,M.tuberculosis is getting resistance drastically to the currently available antibiotics,which offers a wide stand to M.tuberculosis to infect and spread in the human population.Furthermore,there is no good diagnostic tool to early detect the disease.Meanwhile,the currently available vaccine for TB is only effective in children.To get more understanding of mechanisms of drugresistance,virulence and reactivation and to develop more effective anti-tuberculosis drugs and vaccine,we need to know about the functional genomics study of M.tuberculosis.However,the functional genomics study of M.tuberculosis has been hampered by the complicated and time-consuming genetic manipulation techniques used for it.Therefore,in our current work,we have been focused on two aspects.(1)We reprogrammed M.tuberculosis endogenous type III-A CRISPR-Cas10 system for simple and efficient gene editing,RNA interference and genome wide CRISPR interference(CRISPRi)screening via simple delivery of a plasmid harboring a miniCRISPR array,thereby avoiding the introduction of exogenous proteins and minimizing proteotoxicity.Previously we demonstrated that M.tuberculosis genes were efficiently and specifically knocked-in/out by this system,which was confirmed by whole-genome sequencing.This system was further employed for single and simultaneous multiple-gene RNA interference.Now,we used this system for genome-wide CRISPR interference(CRISPRi)screening to identify growth-regulating genes during in-vitro growth and growth inside macrophages.Our in-vitro growth screening results were largely overlapped with the reported growth-essential genes.In addition,we found that upon knocked down a set of genes lead to the increase in growth of M.tuberculosis during in-vitro growth.We called these genes as M.tuberculosis growth repressing genes.These genes were included toxins gene,stress related genes and dormancy related genes.These genes could be a potential host directed anti –M.tuberculosis drug targets and vaccine candidates.Moreover,we found certain families of genes that are suppressing or enhancing the growth of M.tuberculosis in different environmental conditions.We also employed this tool to screen for the genetic requirements of M.tuberculosis during growth inside macrophages.Which demonstrated that asset of genes is dispensable for the for the in-vitro growth of M.tuberculosis but highly essential for the growth inside macrophages.These genes could be the vaccine candidate genes.(2)We have screened a small library of human endocrine hormones against M.tuberculosis to check the response of M.tuberculosis toward these hormones.The human endocrine hormones especially the catecholamine hormones have been studied as a source of communication among human micro biota and their host.Therefore,we hypothesized that M.tuberculosis would also sense certain types of human endocrine hormones and respond to it accordingly.In our results,we found that Mt.b is growing faster in in-vitro culture in serum based medium and inside macrophages upon treatment with catecholamine hormones.The transcription profile of epinephrine treated M.tuberculosis showed that many genes involved in virulence and metabolism were highly induced.More interestingly we found that the two component system genes(mpr B(Rv 0982)and mtr B(Rv 3245c)were also significantly up regulated.Upon Insilco analysis,we found that Rv 0982 gene could be the possible hormone sensing gene in M.tuberculosis.However the over expression of this gene and Knocked down in M.tuberculosis didn't support this hypothesis.We are further applying forward genetic tools mainly RNA seq and our currently introduced CRISPR case system to know the correct target and mechanism.Finally we will confirm our study in a proper animal's model to know the effect of these hormones on reactivation of latent M.tuberculosis.Together,we introduced a simple and efficient genome-wide CRISPRi screening method for M.tuberculosis and presented a new mechanism for the M.tuberculosis host interaction. |
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