Characterization of the Interaction and Cross-Regulation of Three Mycobacterium tuberculosis RelBE Modules

Abstract

RelBE represents a typical bacterial toxin-antitoxin (TA) system. Mycobacterium tuberculosis H37Rv, the pathogen responsible for human tuberculosis, contains three RelBE-like modules, RelBE, RelFG, and RelJK, which are at least partly expressed in human macrophages during infection. RelBE modules appear to be autoregulated in an atypical manner compared to other TA systems; however, the molecular mechanisms and potential interactions between different RelBE modules remain to be elucidated. In the present study, we characterized the interaction and cross-regulation of these Rel toxin-antitoxin modules from this unique pathogen. The physical interactions between the three pairs of RelBE proteins were confirmed and the DNA-binding domain recognized by three RelBE-like pairs and domain structure characteristics were described. The three RelE-like proteins physically interacted with the same RelB-like protein, and could conditionally regulate its binding with promoter DNA. The RelBE-like modules exerted complex cross-regulation effects on mycobacterial growth. The relB antitoxin gene could replace relF in cross-neutralizing the relG toxin gene. Conversely, relF enhanced the toxicity of the relE toxin gene, while relB increased the toxicity of relK. This is the first report of interactions between different pairs of RelBE modules of M. tuberculosis.

Regulatory crosstalk between type I and type II toxin-antitoxin systems in the human pathogen Enterococcus faecalis

“…12,45,55,56 Recent studies have demonstrated that TA-II systems can affect positively or negatively the expression of other TA-II modules. 39,57,58 However in these studies, only changes in steady-state RNA levels have been shown, and RNA synthesis (transcription) and RNA stability effects cannot be distinguished. In our experiments, direct RNA stability measurements rule out stability effects of MazE and MazEF on RatA, since the RatA half-life is independent of MazE and MazEF levels.…”

Toxin-Antitoxin Systems of Mycobacterium smegmatis Are Essential for Cell Survival

“…In a separate study, 30 out of 38 putative TA systems from M. tuberculosis were functional when expressed in E. coli (61). Of the nine MazF homologs identified in M. tuberculosis, seven have been studied and shown to cause growth arrest when expressed in E. coli (49,62,63 (64,65). Further worked showed that expression of the RelE2 toxin in M. tuberculosis induced the formation of drug-specific persisters in vitro; however, deletion of relE2 did not affect the level of persisters of M. tuberculosis in rifampin-treated mice ruling out a role for relE2 in the generation of bacilli that survive multidrug therapy (66).…”

Radiation-sensitive Gene A (RadA) Targets DisA, DNA Integrity Scanning Protein A, to Negatively Affect Cyclic Di-AMP Synthesis Activity in Mycobacterium smegmatis

“…Surface Plasmon Resonance Analysis-Interaction between MsDisA and MsRadA in vitro was analyzed on a BIAcore 3000 instrument (GE Healthcare) as described previously (18,19). Briefly, the purified GST-tagged MsRadA fusion protein, to be used as the ligand, was diluted in HBS buffer (10 mM Hepes (pH 7.4), 150 mM NaCl, 50 M EDTA, 0.005% BIAcore surfactant P20).…”

“…Bacterial Two-hybrid Assay-Bacterial two-hybrid analysis was carried out according to the procedures described in a previous report (19). pBT and pTRG vectors containing the disA and radA genes were generated.…”