The Toxin-Antitoxin Systems of Mycobacterium tuberculosis and their Role in Persistence
A key impediment to the control of tuberculosis is the ability of the causative agent, Mycobacterium tuberculosis (MTB), to adopt a persistent, non-replicating state that renders the bacteria resistant to both host and antibiotic killing. The expression of toxin-antitoxin (TA) systems in other bacteria regulate the generation of persister subpopulations and act as stress response elements that curtail bacterial replication under adverse conditions. We have comprehensively identified putative TA systems in M. tuberculosis and identified 30 that are functional when expressed in M. smegmatis. Why M. tuberculosis has so many TA systems and what role they play in the unique biology of the pathogen is unknown.
Comparative genomic analysis revealed that the vast majority of these systems are conserved in the M. tuberculosis complex (MTBC), but largely absent from other mycobacteria, including close relatives of M. tuberculosis. Expression profiling demonstrated that four systems are specifically activated during stresses likely encountered in vivo, including hypoxia and phagocytosis by macrophages. Given that hypoxia is an in vitro condition that induces the persistent state in M.tuberculosis, we sought to further characterize the two TA systems induced during hypoxia, Rv2009-2010 and Rv1955-1956.
Intriguingly, both of these systems are part of a horizontally-acquired genomic island that contains other hypoxia-regulated genes. A knock-out mutant of Rv2009-2010 is able to out-compete wild type M. tuberculosis during competitive growth assays under hypoxic conditions. Additionally, this mutant appears to have an altered transcription profile during hypoxia, as compared to wild type bacteria. Further examination of this region of the genome revealed that an adjacent gene, Rv2008c, is misregulated in cells lacking the Rv2009-2010 operon and may contribute to some of the phenotypic changes observed in the mutant.
In contrast to canonical two-gene TA systems, the Rv1955-Rv1956 locus encodes four genes. Using multiple protein interaction techniques, we have shown that one of these accessory proteins, Rv1957, interacts directly with that antitoxin and the TA complex. We show that Rv1955-1957 is transcribed by at least two promoters that are differentially regulated by stress. Additionally, we provide evidence that Rv1957 is required for full repression of the TA system operon. The complex architecture and regulation suggest that Rv1955-1957 via a novel mechanism to promote persistence in a major human pathogen.