Unveiling Mtb's Secret Weapon: How a New Toxin Hijacks Protein Synthesis to Drive TB Persistence

Tuberculosis (TB), caused by the formidable bacterium Mycobacterium tuberculosis (Mtb), remains a devastating global health crisis, responsible for more deaths annually than any other single infectious agent. This ancient pathogen has evolved an astounding ability to resist both antibiotics and the host immune system, largely thanks to complex survival strategies. One key to its resilience lies in its unusually high number of type II toxin-antitoxin (TA) systems, which act as critical stress sensors, enabling Mtb to endure harsh conditions and establish persistent infections.

            Recent research in the Woychik Lab at Rutgers University, Robert Wood Johnson Medical School has shed light on an 11th member of the MazEF family of Mtb toxins, dubbed MazF-mt11, revealing its precise target and a novel mechanism by which it can shut down protein production in the bacterium. This discovery offers insights into how Mtb might achieve its notorious persistent state, which characterizes latent TB infections in over a quarter of the world's population.

         MazF toxins are a class of bacterial proteins that function as single-strand, sequence-specific endoribonucleases that can recognize and cut specific RNA sequences. Mtb’s genome is packed with these systems, boasting around 70 type II TA systems, with 11 belonging to the MazEF family. In a toxin-antitoxin (TA) system, a toxin (like MazF) is normally kept in check by a cognate antitoxin. However, under stress conditions, the antitoxin's activity can be disrupted, unleashing the toxin to act on its targets within the bacterial cell. Previous studies had identified 10 MazEF members, but the exact cellular target for most remained unknown.

         The new study set out to characterize MazF-mt11, identified from the Rv3098A gene. The researchers employed a multi-pronged approach to uncover its function:

• Using primer extension on a viral RNA substrate (MS2 enterobacteriophage RNA), they first identified a single cleavage site: between a C and an A (C↓A).

• Defining the Consensus Sequence: To get a broader picture, they utilized MORE (Mapping by Overexpression of an RNase in E. coli) RNA-seq. This genome-scale tool allowed them to pinpoint the full RNA cleavage consensus sequence recognized by MazF-mt11 as C↓ACCU, confirming that cleavage occurs specifically between the C and A.

• While MORE RNA-seq in E. coli provides the consensus sequence, identifying the actual physiological target in Mtb requires expression in a mycobacterial host. When MazF-mt11 was expressed in Mycobacterium smegmatis (a close relative of Mtb with identical 16S rRNA sequences in the relevant region), 5'-OH RNA-seq revealed 16S rRNA within the 30S ribosomal subunit as the only MazF-mt11 RNA target with the C↓ACCU consensus sequence. This specificity highlights the precision of this toxin.

         The location of MazF-mt11's single cleavage site in 16S rRNA maps just before the anti-Shine–Dalgarno (aSD) sequence at the 3' end of the 16S rRNA.

            The aSD sequence (specifically CCUCCU in mycobacteria) initiates protein synthesis. It normally binds to a complementary Shine-Dalgarno (SD) sequence found in most messenger RNAs (mRNAs), effectively directing the ribosome to the correct start codon for translation.

            By selectively cutting the 16S rRNA and removing the terminal 11 nucleotides, including this critical aSD sequence, MazF-mt11 leads to a nearly complete inhibition of protein synthesis. Experimental evidence showed new protein synthesis levels plummeted to just 19% after 6 hours and 5% after 24 hours of toxin induction in M. smegmatis. This reduction in protein production directly correlates with the strong growth arrest phenotype observed in mycobacterial cells expressing MazF-mt11.

         This discovery strongly suggests that MazF-mt11 plays an important role in the establishment and/or maintenance of the nonreplicating persistent state in Mtb. This is a dormant, non-replicating phase that allows the bacterium to evade the host immune system and survive antibiotic treatment, leading to latent TB infections.

            While many antibiotics target and perturb the ribosome, none are currently known to specifically target the 16S rRNA helix 45 and its terminal nucleotides that include the aSD sequence. This unique mode of action by MazF-mt11 provides a novel mechanism for inhibiting protein synthesis in Mtb.

            While the exact physiological triggers for MazF-mt11 activation in Mtb during infection are still under investigation, this research opens new avenues for understanding Mtb’s stress response and persistence. Unraveling the ways these bacterial toxins shut down essential cellular processes could pave the way for developing new strategies to combat drug-resistant TB and tackle the persistent nature of this challenging pathogen.

Sherrier TW, Barth VC, Schifano JM, Greendyk JR, Woychik NA. Targeted removal of the 16S rRNA anti-Shine-Dalgarno sequence by a Mycobacterium tuberculosis MazF toxin. J Biol Chem. 2025 Jul;301(7):110323. doi: 10.1016/j.jbc.2025.110323. Epub 2025 May 30. PMID: 40451429; PMCID: PMC12274833.

https://www.jbc.org/article/S0021-9258(25)02173-8/fulltext