Functional studies of five toxin-antitoxin modules in Mycobacterium tuberculosis H37Rv

Abstract

Toxin-antitoxin (TA) systems, which consist of an intracellular toxin and its antidote (antitoxin), are encoded by ubiquitous genetic modules in prokaryotes. Commonly, the activity of a toxin is inhibited by its antitoxin under normal growth conditions. However, antitoxins are degraded in response to environmental stress, and toxins liberated from antitoxins consequently induce cell death or growth arrest. In free-living prokaryotes, TA systems are often present in large numbers and are considered to be associated with the adaptation of pathogenic bacteria or extremophiles to various unfavorable environments by shifting cells to a slow growth rate. Genomic analysis of the human pathogen Mycobacterium tuberculosis H37Rv (Mtb) revealed the presence of a large number of TA systems. Accordingly, we investigated five uncharacterized TA systems (Rv2019-Rv2018, Rv3697c-Rv3697A, Rv3180c-Rv3181c, Rv0299-Rv0298, and Rv3749c-Rv3750c) of Mtb. Among these, the expression of the Rv2019 toxin inhibited the growth of Escherichia coli, and M. smegmatis and this growth defect was recovered by the expression of the Rv2018 antitoxin. Interestingly, Rv3180c was toxic only in M. smegmatis, whose toxicity was neutralized by Rv3181c antitoxin. In vivo and in vitro assays revealed the ribosomal RNA (rRNA) cleavage activity of the Rv2019 toxin. Moreover, mRNAs appeared to be substrates of Rv2019. Therefore, we concluded that the ribonuclease activity of the Rv2019 toxin triggers the growth defect in E. coli and that the Rv2018 antitoxin inhibits the ribonuclease activity of the Rv2019 toxin.