CHARACTERIZATION OF A FRAME-SHIFT MUTATION IN CLINICAL ISOLATES OF MYCOBACTERIUM TUBERCULOSIS RV1738, A KEY GENE ENABLING DORMANCY AND PERSISTENT LATENCY

We used next generation sequencing to characterize clinical isolates of Mycobacterium tuberculosis (Mtb). Two clonal isolates from the same patient showed a frame-shift mutation in Mtb Rv1738. During hypoxic conditions Rv1738 is highly up-regulated by the DosR operon, Rv1738 becoming the most abundant protein in the cell. Rv1738 is a conserved small protein of 94 amino acids. Recently, the structure of this protein was determined and it revealed a surprising similarity to a family of stress proteins known as hibernation-promoting factors (HPFs) like E. coli YfiA. YfiA reversibly inhibit translation and protein synthesis by binding to a deep groove in the 30S ribosomal subunit. This suggests that the functional role of the up-regulated Rv1738 protein in non-replicating persistence of Mtb is to contribute to the shutdown of ribosomal protein synthesis.

The Rv1738 polypeptide is folded into a three-stranded antiparallel β-sheet packed against a long C-terminal amphipathic α-helix (residues 55–86). The protein forms an intimately associated homodimer in which the extended β-sheet wraps around the two antiparallel C-terminal α-helices, which pack against the β-sheet and make intimate side chain–side chain interactions with each other. The frame shift in codon 73 of Rv1738 with the introduction of a wrong amino acid and creation of a 100 amino acid long mutant protein, causes a severe disruption in the alpha helix and possibly may affect dimerization and binding to the ribosome. A significant question that needs answering is about the ability of these mutants to hibernate. A structural homology search revealed an additional 12 GenBank entries with identical Rv1738 frame shift insertions, making this a rare but potentially successful evolutionary strategy of unknown significance.