Characterization of PIN-domain Toxin-antitoxins VapBC from Mycobacterium tuberculosis

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2015.Bacterial toxin-antitoxin systems play important roles in plasmid inheritance, cellular adaptation to stress, the formation of persister cells and cell death. Type II toxin-antitoxin pairs, composed of protein toxins and antitoxins, exist in nearly all bacteria and are classified into different groups based on the structure of the toxins. The role of TA systems in dormancy and formation of drug resistant persisters make them medically important subjects for analysis. Dormancy appears to play an important role in the survival of M. tuberculosis (Mtb) during latent infections and this bacterium carries a large number of TA operons in its genome. The VapBC TA operons comprise the most numerous TA pairs in Mtb, yet their contribution to Mtb viability and pathogenesis remains relatively uncharacterized. We characterized a number of the putative VapC toxins in E. coli and M. smegmatis and found, consistent with previous observations, that many lack apparent toxic activity. Mtb VapC4 (Rv0595c) inhibits growth in both organisms and is antagonized by co-expression of its antitoxin VapB4. The activity of VapC4 requires acidic amino acids predicted from sequence homologies to be required for endoribonuclease activity. Expression of the toxin inhibits protein, but not RNA synthesis in M. smegmatis. However, unlike its enteric homologues, VapC4 does not appear cleave tRNAfMet in vivo. Generally, type II antitoxins bind with high specificity to their cognate toxins via a toxin-binding domain and endow the complex with DNA binding specificity via a DNA binding domain. Despite the ubiquity of VapBC systems and their critical role in the regulation of gene expression, few functional studies have addressed the details of VapB-VapC interactions. This thesis reports experiments designed to identify molecular determinants of the specificity of the Mycobacterium tuberculosis VapB4 antitoxin for its cognate VapC4 toxin. The results identify the minimal domain of VapB4 required for this interaction, as well as amino acid side chains required for binding to VapC4. These findings have important implications for the evolution of VapBC toxin-antitoxin systems and their potential as targets of small molecule protein-protein interaction inhibitors