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Bioinformatic and mutational studies of related toxin-antitoxin pairs in Mycobacterium tuberculosis predict and identify key functional residues.

ABSTRACT: Mycobacterium tuberculosis possesses an unusually large representation of type II toxin-antitoxin (TA) systems, whose functions and targets are mostly unknown. To better understand the basis of their unique expansion and to probe putative functional similarities among these systems, here we computationally and experimentally investigated their sequence relationships. Bioinformatic and phylogenetic investigations revealed that 51 sequences of the VapBC toxin family group into paralogous sub-clusters. On the basis of conserved sequence fingerprints within paralogues, we predicted functional residues and residues at the putative TA interface that are useful to evaluate TA interactions. Substitution of these likely functional residues abolished the toxin's growth-inhibitory activity. Furthermore, conducting similarity searches in 101 mycobacterial and ∼4500 other prokaryotic genomes, we assessed the relative conservation of the M. tuberculosis TA systems and found that most TA orthologues are well-conserved among the members of the M. tuberculosis complex, which cause tuberculosis in animal hosts. We found that soil-inhabiting, free-living Actinobacteria also harbor as many as 12 TA pairs. Finally, we identified five novel putative TA modules in M. tuberculosis. For one of them, we demonstrate that overexpression of the putative toxin, Rv2514c, induces bacteriostasis and that co-expression of the cognate antitoxin Rv2515c restores bacterial growth. Taken together, our findings reveal that toxin sequences are more closely related than antitoxin sequences in M. tuberculosis Furthermore, the identification of additional TA systems reported here expands the known repertoire of TA systems in M. tuberculosis.

SUBMITTER: Tandon H

PROVIDER: S-EPMC6556569 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Toxin-antitoxin (TA) systems are ubiquitous genetic elements in prokaryotes, but their biological importance is poorly understood. Mycobacterium smegmatis contains eight putative TA systems. Previously, seven TAs have been studied, with five of them being verified as functional. Here, we show that Ms0251-0252 is a novel TA system in that expression of the toxin Ms0251 leads to growth inhibition that can be rescued by the antitoxin Ms0252. To investigate the functional roles of TA systems in M. smegmatis, we deleted the eight putative TA loci and assayed the mutants for resistance to various stresses. Deletion of all eight TA loci resulted in decreased survival under starvation conditions and altered fitness when exposed to environmental stresses. Furthermore, we showed that deletion of the eight TA loci decreased resistance to phage infection in Sauton medium compared with the results using 7H10 medium, suggesting that TA systems might have different contributions depending on the nutrient environment. Furthermore, we found that MazEF specifically played a dominant role in resistance to phage infection. Finally, transcriptome analysis revealed that MazEF overexpression led to differential expression of multiple genes, including those related to iron acquisition. Altogether, we demonstrate that TA systems coordinately function to allow M. smegmatis to adapt to changing environmental conditions. IMPORTANCE Toxin-antitoxin (TA) systems are mechanisms for rapid adaptation of bacteria to environmental changes. Mycobacterium smegmatis, a model bacterium for studying Mycobacterium tuberculosis, encodes eight putative TA systems. Here, we constructed an M. smegmatis mutant with deletions of all eight TA-encoding genes and evaluated the resistance of these mutants to environmental stresses. Our results showed that different TA systems have overlapping and, in some cases, opposing functions in adaptation to various stresses. We suggest that complementary TA modules may function together to regulate the bacterial stress response, enabling adaptation to changing environments. Together, this study provides key insights into the roles of TA systems in resistance to various environmental stresses, drug tolerance, and defense against phage infection.

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Bioinformatic and mutational studies of related toxin-antitoxin pairs in Mycobacterium tuberculosis predict and identify key functional residues.

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