Three Mycobacterium tuberculosis rel toxin-antitoxin modules inhibit mycobacterial growth and are expressed in infected human macrophages

Mycobacterium tuberculosis protein pairs Rvl246c-Rvl247c, Rv2865-Rv2866, and Rv3357-Rv3358, here named ReIBE, ReIFG, and RelJK, respectively, were identified based on homology to the Escherichia coli ReIBE 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 rel], respectively. We also provide evidence that ReIB 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 ReIJ 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.