Ribosome hibernation: a new molecular framework for targeting nonreplicating persisters of mycobacteria

Abstract

Treatment of tuberculosis requires a multi-drug regimen administered for at least 6 months. The long-term chemotherapy is attributed in part to a minor subpopulation of nonreplicating Mycobacterium tuberculosis cells that exhibit phenotypic tolerance to antibiotics. The origins of these cells in infected hosts remain unclear. Here we discuss some recent evidence supporting the hypothesis that hibernation of ribosomes in M. tuberculosis, induced by zinc starvation, could be one of the primary mechanisms driving the development of nonreplicating persisters in hosts. We further analyse inconsistencies in previously reported studies to clarify the molecular principles underlying mycobacterial ribosome hibernation.

Keywords: Mycobacterium tuberculosis; cryo-EM structure analysis; drug tolerance; nonreplicating persisters; ribosome hibernation; zinc starvation.

Fig. 1.

Comparing various forms of ribosome hibernation factors. (a) Schematic representation of domain organization in short and long variants of Hpf (named Hpf^short and Hpf^long, respectively), YfiA and PSRP1. It should be noted that despite an extended C-terminal region in PSRP1 (also referred to as protein Y), it forms a hibernating 70S ribosome. This is also observed for the ribosome hibernation factor in mycobacteria (MSMEG_1878), which is therefore named mycobacterial protein Y (Mpy). (b) Alignment of the protein sequences of representative ribosome hibernation factors from each of the categories shown in (a). An additional N-terminal extension of variable length in PSRP1 and Mpy is notable.

Fig. 2.

Binding of Mpy requires Mrf-associated ribosomes. (a) Immunoblot analysis of the presence of Mpy in ribosomes purified from a mixture containing recombinant Mpy (rMpy) and crude ribosomes from indicated strains (in parenthesis) grown under either low-zinc (1 µM TPEN) or high-zinc (1 mM ZnSO4) Sauton’s medium for 4 days. The type of ribosomes obtained from each strain under each growth condition is indicated above the lane as C+ or C-. The strain pYL53 constitutively expressed C- ribosomes, even under high-zinc conditions. Crude ribosomes equivalent to OD 0.1 were mixed with 2.4 picomoles of rMpy in a 50 µl reaction and the mixture was incubated at 37 ^∘C for 1 h. Then the ribosomes were separated from the unbound rMPY by ultracentrifugation of the mixture on a 32 % sucrose cushion at 100 000 r.p.m. for 3 h in Beckman TLA 100.3 rotor. The pellets containing the ribosomes were analysed for the presence of rMpy. S13 was probed as the loading control. (b) Quantitative difference between the level of rMpy in the indicated ribosome samples, relative to low-zinc C- ribosomes (normalized as 1) obtained from immunoblots including the one shown in (a). Data represent the average of two independent binding experiments from biologically independent preparations of crude ribosomes and rMPy. * and ** represent P-value (t-test) <0.05 and 0.01, respectively.

Fig. 3.

Comparison of the density corresponding to Mpy within the 3.11 Å resolution cryo-EM map of the 70S-Mpy complex (EMD-23076) with atomic models of Mpy and MSMEG_3935. (a) Overall fitting of the atomic model (red) of the N-terminal domain of Mpy into the corresponding cryo-EM density (semitransparent green) shown from two opposite sides of the molecule. (b) Magnified views of some of the modelled segments of Mpy (red) (E74 – N82 and C102 - R127) based on the secondary structural elements and side-chain information inferred from the cryo-EM map (EMD-23076). (c) Magnified views of the analogous segments from the MSMEG_3935 model (blue) docked into the same cryo-EM map (semitransparent green). Red circles highlight the regions of incompatibility between the atomic model of MSMEG_3935 and the cryo-EM density corresponding to Mpy in our 70S-Mpy complex.

Fig. 4.

Comparison of atomic models of Mpy and MSMEG_3935 with the corresponding cryo-EM density of Mishra and coworkers (EMD 6921: PDBID 5ZEP) [102]. Atomic models of (a) Mpy (red) or (b) MSMEG_3935 (blue) could be docked equally well into the cryo-EM density (semitransparent orange).

Fig. 5.

A working hypothesis for changing zinc availability to Mtb during progressive TB. Following initial infection, intracellular growth of Mtb is accompanied by enrichment of zinc in the phagosomes [117–119], leading to the expression of C+ribosomes in the pathogen. Infiltrating neutrophils during granuloma formation employ various anti-bacterial mechanisms, including the secretion of zinc-chelating calprotectin (CP) as a component of nutritional immunity [121]. Subsequent lysis of infected macrophages by Mtb-derived factors and Mtb-specific cytotoxic T-cells would cause release of bacilli in extracellular space, where free zinc concentration is expected to be reduced by CP. As a result, Mtb cells will sense zinc starvation and induce C- ribosome expression to conserve intracellular zinc. Induced expression of the C- ribosome is perhaps followed by its hibernation upon further zinc deprivation during exuberant extracellular growth of bacilli.