The Function And Regulating Mechanism Of The WhiB3 Regulator In Mycobacterium Tuberculosis

For responding all kinds of environmental stresses within the host cells during infection process, Mycobacterium tuberculosis must timely regulate expression of many important genes. It has been shown that whiB family genes play important roles in mycobacterial growth and environmental adaptation, but the mechanism remains unclear. In this study, we pay close attention to the transcriptional regulation and functional mechanism of the WhiB3 protein. The major results are as follows:(1) We constructed a whiB3 random-mutation library and found that several important functional sites located in the a region and the AT-hook domain in C-terminus of WhiB3 protein using bacterial one-hybrid assay, bacterial two-hybrid assay, EMSA and complementation experiments. (2) Mycobacterium smegmatis WhiB has been confirmed to possess DNA-binding activity; (3) Different MS△whiB recombinant strains have been constructed, whose growth curve under stresses of 1.5mM H2O2 were surveyed. We show that MSwhiB△C without C-terminus can not re-establish the resistance phenotype of wide type MS in the MS△whiB comeplementary strains. In contrast, three recombinant strains, including MtbwhiB3full, MSwhiBfull, MtbwhiB3△C presented complemental phenotype; (4) Using WhiB3 as a bait, lots of proteins have been found to interact with WhiB3 by screening pTRG-MtbORF library and these interactions were further verified by Pull-down and SPR experiments; (5) WhiB3 could interact with RNA a subunit in vivo, which interaction may has positive effect to DNA binding activity of WhiB3.In mycobacteria, whiB family genes are universally conserved. In this study, we have verified WhiB3’s DNA binding activity, and confirmed its functional amino acid redidues, and many proteins were found to interact with WhiB3. In particular, functional differences between two WhiB3 genes in pathogenic Mtb and nonpathogenic Ms has been for the first time figured out. These studies could help increase our understanding of the function of whib3 gene and its contribution to the virulence in Mtb.