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Structural analyses of the MazEF4 toxin-antitoxin pair in Mycobacterium tuberculosis provide evidence for a unique extracellular death factor.

ABSTRACT: The bacterial toxin-antitoxin MazEF system in the tuberculosis (TB)-causing bacterium Mycobacterium tuberculosis is activated under unfavorable conditions, including starvation, antibiotic exposure, and oxidative stress. This system contains the ribonucleolytic enzyme MazF and has emerged as a promising drug target for TB treatments targeting the latent stage of M. tuberculosis infection and reportedly mediates a cell death process via a peptide called extracellular death factor (EDF). Although it is well established that the increase in EDF-mediated toxicity of MazF drives a cell-killing phenomenon, the molecular details are poorly understood. Moreover, the divergence in sequences among reported EDFs suggests that each bacterial species has a unique EDF. To address these open questions, we report here the structures of MazF4 and MazEF4 complexes from M. tuberculosis, representing the first MazEF structures from this organism. We found that MazF4 possesses a negatively charged MazE4-binding pocket in contrast to the positively charged MazE-binding pockets in homologous MazEF complex structures from other bacteria. Moreover, using NMR spectroscopy and biochemical assays, we unraveled the molecular interactions of MazF4 with its RNA substrate and with a new EDF homolog originating from M. tuberculosis The EDF homolog discovered here possesses a positively charged residue at the C terminus, making this EDF distinct from previously reported EDFs. Overall, our results suggest that M. tuberculosis evolved a unique MazF and EDF and that the distinctive EDF sequence could serve as a starting point for designing new anti-tuberculosis drugs. We therefore conclude that this study might contribute to the development of a new line of anti-tuberculosis agents.

SUBMITTER: Ahn DH

PROVIDER: S-EPMC5704469 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Structural analyses of the MazEF4 toxin-antitoxin pair in Mycobacterium tuberculosis provide evidence for a unique extracellular death factor.

Ahn Do-Hwan DH Lee Ki-Young KY Lee Sang Jae SJ Park Sung Jean SJ Yoon Hye-Jin HJ Kim Soon-Jong SJ Lee Bong-Jin BJ

The Journal of biological chemistry 20171002 46

The bacterial toxin-antitoxin MazEF system in the tuberculosis (TB)-causing bacterium Mycobacterium tuberculosis is activated under unfavorable conditions, including starvation, antibiotic exposure, and oxidative stress. This system contains the ribonucleolytic enzyme MazF and has emerged as a promising drug target for TB treatments targeting the latent stage of M. tuberculosis infection and reportedly mediates a cell death process via a peptide called extracellular death factor (E ...[more]

PMID: 28972145

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Project description:The Mycobacterium tuberculosis genome harbors nine toxin-antitoxin (TA) systems that are members of the mazEF family, unlike other prokaryotes, which have only one or two. Although the overall tertiary folds of MazF toxins are predicted to be similar, it is unclear how they recognize structurally different RNAs and antitoxins with divergent sequence specificity. Here, we have expressed and purified the individual components and complex of the MazEF6 TA system from M. tuberculosis. Size exclusion chromatography-multiangle light scattering (SEC-MALS) was performed to determine the oligomerization status of the toxin, antitoxin, and the complex in different stoichiometric ratios. The relative stabilities of the proteins were determined by nano-differential scanning fluorimetry (nano-DSF). Microscale thermophoresis (MST) and yeast surface display (YSD) were performed to measure the relative affinities between the cognate toxin-antitoxin partners. The interaction between MazEF6 complexes and cognate promoter DNA was also studied using MST. Analysis of paired-end RNA sequencing data revealed that the overexpression of MazF6 resulted in differential expression of 323 transcripts in M. tuberculosis. Network analysis was performed to identify the nodes from the top-response network. The analysis of mRNA protection ratios resulted in identification of putative MazF6 cleavage site in its native host, M. tuberculosis. IMPORTANCE M. tuberculosis harbors a large number of type II toxin-antitoxin (TA) systems, the exact roles for most of which are unclear. Prior studies have reported that overexpression of several of these type II toxins inhibits bacterial growth and contributes to the formation of drug-tolerant populations in vitro. To obtain insights into M. tuberculosis MazEF6 type II TA system function, we determined stability, oligomeric states, and binding affinities of cognate partners with each other and with their promoter operator DNA. Using RNA-seq data obtained from M. tuberculosis overexpression strains, we have identified putative MazF6 cleavage sites and targets in its native, cellular context.

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Structural analyses of the MazEF4 toxin-antitoxin pair in Mycobacterium tuberculosis provide evidence for a unique extracellular death factor.

Publications

Structural analyses of the MazEF4 toxin-antitoxin pair in <i>Mycobacterium tuberculosis</i> provide evidence for a unique extracellular death factor.

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