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Zinc depletion induces ribosome hibernation in mycobacteria.

ABSTRACT: Bacteria respond to zinc starvation by replacing ribosomal proteins that have the zinc-binding CXXC motif (C+) with their zinc-free (C-) paralogues. Consequences of this process beyond zinc homeostasis are unknown. Here, we show that the C- ribosome in Mycobacterium smegmatis is the exclusive target of a bacterial protein Y homolog, referred to as mycobacterial-specific protein Y (MPY), which binds to the decoding region of the 30S subunit, thereby inactivating the ribosome. MPY binding is dependent on another mycobacterial protein, MPY recruitment factor (MRF), which is induced on zinc depletion, and interacts with C- ribosomes. MPY binding confers structural stability to C- ribosomes, promoting survival of growth-arrested cells under zinc-limiting conditions. Binding of MPY also has direct influence on the dynamics of aminoglycoside-binding pockets of the C- ribosome to inhibit binding of these antibiotics. Together, our data suggest that zinc limitation leads to ribosome hibernation and aminoglycoside resistance in mycobacteria. Furthermore, our observation of the expression of the proteins of C- ribosomes in Mycobacterium tuberculosis in a mouse model of infection suggests that ribosome hibernation could be relevant in our understanding of persistence and drug tolerance of the pathogen encountered during chemotherapy of TB.

SUBMITTER: Li Y

PROVIDER: S-EPMC6094098 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Zinc depletion induces ribosome hibernation in mycobacteria.

Li Yunlong Y Sharma Manjuli R MR Koripella Ravi K RK Yang Yong Y Kaushal Prem S PS Lin Qishan Q Wade Joseph T JT Gray Todd A TA Derbyshire Keith M KM Agrawal Rajendra K RK Ojha Anil K AK

Proceedings of the National Academy of Sciences of the United States of America 20180723 32

Bacteria respond to zinc starvation by replacing ribosomal proteins that have the zinc-binding CXXC motif (C+) with their zinc-free (C-) paralogues. Consequences of this process beyond zinc homeostasis are unknown. Here, we show that the C- ribosome in <i>Mycobacterium smegmatis</i> is the exclusive target of a bacterial protein Y homolog, referred to as mycobacterial-specific protein Y (MPY), which binds to the decoding region of the 30S subunit, thereby inactivating the ribosome. MPY binding i ...[more]

PMID: 30038002

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Project description:Hibernation-promoting factor (HPF) is a ribosomal accessory protein that inactivates ribosomes during bacterial starvation. In Pseudomonas aeruginosa, HPF protects ribosome integrity while the cells are dormant. The sequence of HPF has diverged among bacteria but contains conserved charged amino acids in its two alpha helices that interact with the rRNA. Here, we characterized the function of HPF in P. aeruginosa by performing mutagenesis of the conserved residues and then assaying mutant HPF alleles for their ability to protect ribosome integrity of starved P. aeruginosa cells. The results show that HPF functionally tolerates point mutations in charged residues and in the conserved Y71 residue as well as a C-terminal truncation. Double and triple mutations of charged residues in helix 1 in combination with a Y71F substitution reduce HPF activity. Screening for single point mutations that caused impaired HPF activity identified additional substitutions in the two HPF alpha helices. However, alanine substitutions in equivalent positions restored HPF activity, indicating that HPF is tolerant to mutations that do not disrupt the protein structure. Surprisingly, heterologous HPFs from Gram-positive bacteria that have long C-terminal domains functionally complement the P. aeruginosa Δhpf mutant, suggesting that HPF may play a similar role in ribosome protection in other bacterial species. Collectively, the results show that HPF has diverged among bacteria and is tolerant to most single amino acid substitutions. The Y71 residue in combination with helix 1 is important for the functional role of HPF in ribosome protection during bacterial starvation and resuscitation of the bacteria from dormancy.IMPORTANCE In most environments, bacteria experience conditions where nutrients may be readily abundant or where nutrients are limited. Under nutrient limitation conditions, even non-spore-forming bacteria may enter a dormant state. Dormancy is accompanied by a variety of cellular physiological changes that are required for the cells to remain viable during dormancy and to resuscitate when nutrients become available. Among the physiological changes that occur in dormant bacteria is the inactivation and preservation of ribosomes by the dormancy protein, hibernation-promoting factor (HPF). In this study, we characterized the activity of HPF of Pseudomonas aeruginosa, an opportunistic pathogen that causes persistent infections, and analyzed the role of HPF in ribosome protection and bacterial survival during dormancy.

Project description:The translationally silent 100S ribosome is a poorly understood form of the dimeric 70S complex that is ubiquitously found in all bacterial phyla. The elimination of the hibernating 100S ribosome leads to translational derepression, ribosome instability, antibiotic sensitivity, and biofilm defects in some bacteria. In Firmicutes, such as the opportunistic pathogen Staphylococcus aureus, a 190-amino acid protein called hibernating-promoting factor (HPF) dimerizes and conjoins two 70S ribosomes through a direct interaction between the HPF homodimer, with each HPF monomer tethered on an individual 70S complex. While the formation of the 100S ribosome in gammaproteobacteria and cyanobacteria is exclusively induced during postexponential growth phase and darkness, respectively, the 100S ribosomes in Firmicutes are constitutively produced from the lag-logarithmic phase through the post-stationary phase. Very little is known about the regulatory pathways that control hpf expression and 100S ribosome abundance. Here, we show that a general stress response (GSR) sigma factor (SigB) and a GTP-sensing transcription factor (CodY) integrate nutrient and thermal signals to regulate hpf synthesis in S. aureus, resulting in an enhanced virulence of the pathogen in a mouse model of septicemic infection. CodY-dependent regulation of hpf is strain specific. An epistasis analysis further demonstrated that CodY functions upstream of the GSR pathway in a condition-dependent manner. The results reveal an important link between S. aureus stress physiology, ribosome metabolism, and infection biology.IMPORTANCE The dimerization of 70S ribosomes (100S complex) plays an important role in translational regulation and infectivity of the major human pathogen Staphylococcus aureus Although the dimerizing factor HPF has been characterized biochemically, the pathways that regulate 100S ribosome abundance remain elusive. We identified a metabolite- and nutrient-sensing transcription factor, CodY, that serves both as an activator and a repressor of hpf expression in nutrient- and temperature-dependent manners. Furthermore, CodY-mediated activation of hpf masks a secondary hpf transcript derived from a general stress response SigB promoter. CodY and SigB regulate a repertoire of virulence genes. The unexpected link between ribosome homeostasis and the two master virulence regulators provides new opportunities for alternative druggable sites.

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Zinc depletion induces ribosome hibernation in mycobacteria.

Publications

Zinc depletion induces ribosome hibernation in mycobacteria.

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