The Cross Interactions Between The Mycobacterium Tuberculosis MazEF Proteins

Mycobacterium tuberculosis has multiple MazEF toxin-antitoxin systems. It has been confirmed that these MazEF systems may play important roles during the pathogen’s persistent infection. However, it has been unknown whether these different MazEF systems can coordinate to regulate the survival of M. tuberculosis in the host cell environment. It is important for studying the interaction networks among MazEF proteins and other essential proteins in the pathogen.In the current study, we studied the interaction between seven pairs of M. tuberculosis MazEF proteins, and their regulation on the function of topoisomerase Rv3646c (TopA) and also on the ssRNA cleavage functions of toxin (MazF) proteins. The results are as follows:(1) Using a bacterial two-hybrid experiment, we have constructed a protein interaction network involved in MazEFs and TopA protein, and confirmed some of thses interactions by Pull-Down/Western blot technique, for example, the physical interactions between Rv1495 and TopA,other MazEF. (2) We found that MazE, MazF, and their complex protein could inhibit TopA activity, but the complex between MazE and MazF negatively regulated the inhibition through a physical interaction. (3) Several MazF proteins were demonstrated to contain ssRNA endonuclease activities, and the activities were similarily inhibited by the MazE and MazF complex. (4) Using a random mutation technique in combination with bacterial two-hybrid assays, we have successfully identified the critical interaction residues sites between Rv1495 and Rv0660c, and between Rv1495 and TopA. Intyerestingly, these Rv1495 mutants also lost their inhibitory activities on the topoisomerase, TopA.We constructed a MazEF protein interaction network of M. tuberculosis and confirmed their cross-regulation relationship. These results imply that different MazEFs in M. tuberculosis may coordinate to play a role in persistent infection pathogenesis.