Gene regulation by CnpB in Mycobacterium tuberculosis. Mycobacterium tuberculosis

We have previously reported that Mycobacterium tuberculosis Rv2837c (cnpB) encodes a phosphodiesterase that specifically cleaves cyclic di-AMP (c-di-AMP) into AMP. Deletion of cnpB results in significant virulence attenuation in a mouse pulmonary infection model, which is very likely due to the significantly elevated c-di-AMP levels as overexpression of Mtb diadenylate cyclase, disA, also leads to a similar outcome. An earlier study also demonstrated that CnpB functions similarly to E. coli oligoribonuclease (Orn) that hydrolyzes 2-5-mer nanoRNAs (short oligonucleotides of five residues or shorter in length) except that CnpB prefers 2-mer nanoRNA as a substrate. Additionally, a recent report showed that CnpB also degrades cyclic di-GMP (c-di-GMP), although we demonstrated that CnpB prefers c-di-AMP to c-di-GMP according to an in vitro enzymatic kinetics analysis In this study, we initially attempted to determine c-di-AMP-mediated gene regulation in Mtb by comparing the expression profiles between WT and ∆cnpB using RNA-Seq. We found that the CRISPR-Cas system of M. tuberculosis was highly upregulated by deletion of cnpB. Overall design: This experiment is designed to study gene regulation by cnpB in Mycobacterium tuberculosis. We prepared RNA samples of M. tuberculosis WT and ∆cnpB with 3 sets of cultures. RNA samples were treated with DNase to remove contaminated DNA. rRNA was removed using Ribo-Zero magnetic kit (Epicentre). cDNA library was then generated using ScriptSeq Complete Kit for Bacteria (Epicentre). Samples 1, 3, 5, 7, 9, 11 were amplified with index PCR primers 1, 3, 5, 7, 9, 11, respectively, from Script Seq Index PCR primers (Epicentre). RNA-Seq was performed at the Next Generation Sequencing and Expression Analysis Core of University of Buffalo. >>> Grant << Funder: American Heart Association (AHA) Grant number: 12SDG12080067 Grantee: Guangchun Bai Title: Interaction between TB bacilli and host via cyclic nucleotides