Three Mycobacterium tuberculosis Rel Toxin-Antitoxin Modules Inhibit Mycobacterial Growth and Are Expressed in Infected Human Macrophages

Abstract

Mycobacterium tuberculosis protein pairs Rv1246c-Rv1247c, Rv2865-Rv2866, and Rv3357-Rv3358, here named RelBE, RelFG, and RelJK, respectively, were identified based on homology to the Escherichia coli RelBE toxin:antitoxin (TA) module. In this study, we have characterized each Rel protein pair and have established that they are functional TA modules. Overexpression of individual M. tuberculosis rel toxin genes relE, relG, and relK induced growth arrest in Mycobacterium smegmatis; a phenotype that was completely reversible by expression of their cognate antitoxin genes, relB, relF, and relJ, respectively. We also provide evidence that RelB and RelE interact directly, both in vitro and in vivo. Analysis of the genetic organization and regulation established that relBE, relFG, and relJK form bicistronic operons that are cotranscribed and autoregulated, in a manner unlike typical TA modules. RelB and RelF act as transcriptional activators, inducing expression of their respective promoters. However, RelBE, RelFG, and RelJK (together) repress expression to basal levels of activity, while RelJ represses promoter activity altogether. Finally, we have determined that all six rel genes are expressed in broth-grown M. tuberculosis, whereas relE, relF, and relK are expressed during infection of human macrophages. This is the first demonstration of M. tuberculosis expressing TA modules in broth culture and during infection of human macrophages.

MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs

“…Consistent with observations of other TA systems, we also observed relative abundance of mazF transcripts in comparison with mazE transcripts (Fig. 1c-e) 32,41,42 . Compared with early log phase bacteria, no differences were observed in the transcript levels of mazE3, mazF3, mazE6, mazF6, mazE9 and mazF9 in mid-log and late-log growth stages (Fig.…”

“…These differences in the transcript levels may be attributed to the differential stability of the transcripts or the expression of mazF is driven from multiple promoters. Similar post-transcriptional regulation of TA modules has also been observed in E. coli and during infection of macrophages and mice with Mtb 32, 41,42 .…”

“…TA systems are differentially induced under adverse environmental conditions such as amino acid starvation, oxidative stress and hypoxia, and in macrophages and host tissues 24,32,[40][41][42] . The present study was conducted to investigate the role of MazEF TA systems in the physiology, drug-tolerance and virulence of Mtb.…”

Mycobacterial toxin MazF-mt6 inhibits translation through cleavage of 23S rRNA at the ribosomal A site

“…Third, TA systems have intriguing connections to stress responses and persistence, both in E. coli (1,2,11,12) and in M. tuberculosis (9,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Various TA loci in M. tuberculosis are induced during heat shock (23), hypoxia (9,21), DNA damage (20), nutrient starvation (13), macrophage infection (9,14,18), and antibiotic treatment (19,22), whereas other TA loci are down-regulated during macrophage infection (16) or salicylate treatment (15). Finally, 10 TA loci are induced in M. tuberculosis persister cells (17), whereas three RelE toxins increase M. tuberculosis persister recovery when expressed and two RelE toxins decrease persister recovery in response to specific drugs when deleted (22).…”

Early diagnosis and effective treatment regimens are the keys to tackle antimicrobial resistance in tuberculosis (TB): A report from Euroscicon's international TB Summit 2016

“…57 Korch chose to focus on the RelBE system which inhibits translation through mRNA cleavage. 58,59 By generating strains that overexpress the toxins followed by antitoxin rescue they demonstrated a persistence phenotype wherein only 20% of total translation was observed. 60 This model will enable further investigation into the stages of persistence namely, entrance, maintenance and exit.…”