Cell-wall synthesis and ribosome maturation are co-regulated by an RNA switch in Mycobacterium tuberculosis

Abstract

The success of Mycobacterium tuberculosis as a pathogen relies on the ability to switch between active growth and non-replicating persistence, associated with latent TB infection. Resuscitation promoting factors (Rpfs) are essential for the transition of M. tuberculosis to dormancy and for emergence from the non-replicating persistent state. But these enzymes are double-edged swords, as their ability to degrade the cell wall, is potentially lethal to the bacterium itself. Hence, Rpf expression is tightly regulated. We have identified a novel regulatory element in the 5’ untranslated region (UTR) of rpfB. We demonstrate that this element is a transcriptionally regulated RNA switch/riboswitch candidate, which is restricted to pathogenic mycobacteria, suggesting a role in virulence. Moreover, we have used translation start site mapping to re-annotate the RpfB start codon and identified and validated a ribosome binding site that is likely to be targeted by an RpfB antisense RNA. Finally, we show that rpfB is co-transcribed with downstream genes, ksgA and ispE. ksgA encodes a universally conserved methyl transferase involved in ribosome maturation and ispE encodes an essential ATP-dependent kinase involved in cell wall synthesis. This arrangement implies co-regulation of resuscitation, cell wall synthesis and ribosome maturation via the RNA switch. We propose that deregulation of this switch, associated with cell wall synthesis and ribosome function, presents a new target for anti-tuberculosis drug development.ImportanceThis work describes the identification and characterisation of a novel regulatory RNA element/attenuator that controls cell wall synthesis and ribosome function in Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB). By switching between two different conformations, this RNA switch can either enable or inhibit transcription of a tri-cistronic mRNA that encodes a cell-wall remodelling enzyme crucial for activation of latent TB, an RNA methytransferase that is important for ribosome function and a protein kinase essential for early steps in cell wall synthesis. This RNA switch is only present in a subset of pathogenic mycobacteria, and by regulating the expression of three genes associated with classical antimicrobial targets we believe that it offers a novel important target for future anti-tuberculosis drugs.

Regulatory RNA in Mycobacterium tuberculosis, back to basics

“…Similar to rpfA, the 176-nucleotide 5' leader of the rpfB mRNA harbours an RNA switch (or riboswitch candidate), and like rpfA, identification of the rpfB element precedes identification of its ligand. Unlike ydaO however, the rpfB switch has a recognisable intrinsic terminator structure, and also unlike ydaO, the rpfB switch appears to be restricted to a small subset of pathogenic mycobacteria (Schwenk et al, 2018). By extensive genetic and biochemical analysis, this switch has been shown to control rpfB transcription via an intrinsic terminator located immediately upstream of the TTG start codon, which was experimentally re-annotated in the same study.…”

“…By extensive genetic and biochemical analysis, this switch has been shown to control rpfB transcription via an intrinsic terminator located immediately upstream of the TTG start codon, which was experimentally re-annotated in the same study. The rpfB switch regulates a tri-cistronic operon, which also encodes the methyltransferase KsgA, crucial for ribosome biogenesis and IspE, essential for early steps in M. tuberculosis cell wall synthesis (Connolly et al, 2008, Schwenk et al, 2018.…”