Progression from remodeling to hibernation of ribosomes in zinc-starved mycobacteria

Abstract

Zinc starvation in mycobacteria leads to remodeling of ribosomes, in which multiple ribosomal (r-) proteins containing the zinc-binding CXXC motif are replaced by their motif-free paralogues, collectively called C− r-proteins. We previously reported that the 70S C− ribosome is exclusively targeted for hibernation by mycobacterial-specific protein Y (Mpy), which binds to the decoding center and stabilizes the ribosome in an inactive and drug-resistant state. In this study, we delineate the conditions for ribosome remodeling and hibernation and provide further insight into how zinc depletion induces Mpy recruitment to C− ribosomes. Specifically, we show that ribosome hibernation in a batch culture is induced at an approximately two-fold lower cellular zinc concentration than remodeling. We further identify a growth phase in which the C− ribosome remains active, while its hibernation is inhibited by the caseinolytic protease (Clp) system in a zinc-dependent manner. The Clp protease system destabilizes a zinc-bound form of Mpy recruitment factor (Mrf), which is stabilized upon further depletion of zinc, presumably in a zinc-free form. Stabilized Mrf binds to the 30S subunit and recruits Mpy to the ribosome. Replenishment of zinc to cells harboring hibernating ribosomes restores Mrf instability and dissociates Mpy from the ribosome. Finally, we demonstrate zinc-responsive binding of Mpy to ribosomes in Mycobacterium tuberculosis (Mtb) and show Mpy-dependent antibiotic tolerance of Mtb in mouse lungs. Together, we propose that ribosome hibernation is a specific and conserved response to zinc depletion in both environmental and pathogenic mycobacteria.

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

“…The ambiguity was further underscored by the evidence of active C- ribosomes [100, 101], and the structure of hibernating C+ ribosomes purified from zinc-rich cultures [102]. A more recent study from our laboratory clarified this ambiguity by demonstrating that Mpy dissociates from the ribosome upon reintroduction of zinc to a suspension of mycobacterial cells in zinc-depleted phosphate-buffered saline [44]. Moreover, our findings also uncoupled ribosome remodelling from hibernation by showing that Mpy binding is induced at a lower zinc concentration than that inducing assembly of C- ribosomes [44].…”

“…A more recent study from our laboratory clarified this ambiguity by demonstrating that Mpy dissociates from the ribosome upon reintroduction of zinc to a suspension of mycobacterial cells in zinc-depleted phosphate-buffered saline [44]. Moreover, our findings also uncoupled ribosome remodelling from hibernation by showing that Mpy binding is induced at a lower zinc concentration than that inducing assembly of C- ribosomes [44]. The zinc-responsive Mpy binding to the ribosome requires Mpy-recruitment factor (Mrf), which likely binds to the 30S subunit of the C- ribosome upstream from Mpy binding and promotes Mpy recruitment to the ribosome [44].…”

“…Moreover, our findings also uncoupled ribosome remodelling from hibernation by showing that Mpy binding is induced at a lower zinc concentration than that inducing assembly of C- ribosomes [44]. The zinc-responsive Mpy binding to the ribosome requires Mpy-recruitment factor (Mrf), which likely binds to the 30S subunit of the C- ribosome upstream from Mpy binding and promotes Mpy recruitment to the ribosome [44]. Mrf protein is stabilized at a zinc level lower than that required for ribosome remodelling [43, 44], and its expression is regulated at both transcriptional and post-translational levels.…”

“…The Clp protease system in mycobacteria is a barrel-shaped hetero-tetradecameric peptidase complex of ClpP1P2 that partners with an ATPase (ClpA/ClpC or ClpX) and an adaptor protein (SspB or ClpS) to perform the essential function of degrading proteins with specific sequence determinants, called degrons [103–113]. Zinc binding to Mrf appears to be necessary for its interaction with ClpS, which delivers the protein to ClpCP1P2 complex for degradation [44, 114]. Derepression of Mrf transcription – an event likely preceded by loss of zinc stoichiometry in Zur – at a zinc concentration that is sufficient for its degradation by Clp suggests that Mrf may have greater zinc affinity than Zur, and that this difference may be crucial in separating the ribosome remodelling step from the hibernation process.…”

“…Because ribosome hibernation is induced at lower zinc concentration than that required for inducing C- r-proteins [44], it is highly likely that differences in local zinc concentrations in host tissues can produce a range of physiological states in Mtb bacilli depending on the ratio of active to Mpy-bound C- ribosomes in each cell. Arguably, Mtb cells harbouring peak-level of hibernating C- ribosomes are expected to have the slowest metabolism, while those with the least level would be most active.…”