Elongation factor P: Function and effects on bacterial fitness

Doerfel, Lili K.; Rodnina, Marina V.

doi:10.1002/bip.22341

Cited by 23 publications

(28 citation statements)

References 78 publications

(173 reference statements)

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“…The same appears to be true for M. tuberculosis. This system may have evolved for rapid remodeling of the cellular proteome in response to changing environmental conditions, such as carbon source (43,44). The highly conserved organization of genes for proline-specific aminopeptidases and EF-P in bacteria thus supports a potential role for pepQ in regulating maturation and/or turnover of specific proteins in a manner that is sensitive to the metabolic status of M. tuberculosis.…”

Section: Discussionmentioning

confidence: 98%

Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis

Almeida

Ioerger

Tyagi

et al. 2016

Antimicrob Agents Chemother

181

142

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fThe novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuberculosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multidrug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, >4-fold upward shifts in bedaquiline MIC during treatment have been attributed to non-target-based mutations in Rv0678 that putatively increase bedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presumably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofazimine cross-resistance in Mycobacterium tuberculosis: loss-of-function mutations in pepQ (Rv2535c), which corresponds to a putative Xaa-Pro aminopeptidase. pepQ mutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with or without clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parental H37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant and parent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR (qPCR) revealed no significant differences in expression of Rv0678, mmpS5, or mmpL5 between mutant and parent strains. Complementation of a pepQ mutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is sufficient for reduced susceptibility both in vitro and in mice. Although the mechanism by which mutations in pepQ confer bedaquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant further evaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene. Multidrug-resistant tuberculosis (MDR-TB) is a major threat to global control of tuberculosis (TB). When multidrug resistance is not diagnosed, patients respond poorly to standardized first-line regimens and additional resistance may develop. When MDR-TB is diagnosed, current second-line regimens require prolonged treatment durations and are less effective, more toxic, and far more expensive than first-line therapy (1).The diarylquinoline drug bedaquiline (B) received accelerated approval from the U.S. Food and Drug Administration as part of combination therapy for MDR-TB when other alternatives are not available (2). It is now being studied as a component of novel short-course regimens for MDR as well as drug-susceptible TB (ClinicalTrials.gov identifiers NCT02333799, NCT02193776, NCT02589782, NCT02409290, and NCT02454205 [https://clinicaltrials.gov/]). For new drugs such as bedaquiline, it is essential to define and catalog the mechanisms conferring bacterial resistance in order to design appropriate diagnostic tests (including rapid molecular tests), to better manage the treatment of patients who fail ...

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Section: Discussionmentioning

confidence: 98%

Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis

Almeida

Ioerger

Tyagi

et al. 2016

Antimicrob Agents Chemother

181

142

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“…Translational efficiency was determined by attributing the nTE score to each codon 55 . Residues that were found to stall the ribosome, based on previous investigation 20,56,57 , were lysine, arginine, glutamate, aspartate, proline and glycine. Because of variation in specific motifs, and uncertainty in whether these motifs are additive, we simply compared the total number of these residues in the indicated 10 residue spans.…”

Section: Extended Datamentioning

confidence: 91%

Cotranslational signal-independent SRP preloading during membrane targeting

Chartron

Hunt

Frydman

2016

Nature

160

228

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Ribosome-associated factors must faithfully decode the limited information available in nascent polypeptides to direct them to their correct cellular fate1. It is unclear how the low complexity information exposed by the nascent chain suffices for accurate recognition by the many factors competing for the limited surface near the ribosomal exit site2,3. Questions remain even for the well-studied cotranslational targeting cycle to the endoplasmic reticulum (ER), involving recognition of linear hydrophobic Signal Sequences (SS) or Transmembrane Domains (TMD) by the Signal Recognition Particle (SRP)4,5. Intriguingly, SRP is in low abundance relative to the large number of ribosome nascent chain complexes (RNCs), yet it accurately selects those destined to the ER6. Despite their overlapping specificities, SRP and the cotranslational Hsp70 SSB display exquisite mutually exclusive selectivity in vivo for their cognate RNCs7,8. To understand cotranslational nascent chain recognition in vivo, we interrogated the cotranslational membrane targeting cycle using ribosome profiling (herein Ribo-seq)9 coupled with biochemical fractionation of ribosome populations. Unexpectedly, SRP preferentially binds secretory RNCs before targeting signals are translated. We show non-coding mRNA elements can promote this signal-independent SRP pre-recruitment. Our study defines the complex kinetic interplay between elongation and determinants in the polypeptide and mRNA modulating SRP-substrate selection and membrane targeting.

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“…During the elongation phase of protein synthesis in prokaryotes, ribosomes are assisted by at least two cofactors that bind and unbind during each translation cycle (1). EF-Tu mediates the proper binding of the correct aminoacyl-tRNA (aa-tRNA) to the ribosomal A site.…”

Section: Introductionmentioning

confidence: 99%

Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live Escherichia coli

Mohapatra

Choi

et al. 2017

mBio

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In vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Ribosome profiling shows that stalling at Pro-Pro-X triplets is especially severe but is largely alleviated in Escherichia coli by the action of elongation factor EF-P. EF-P and its eukaryotic/archaeal homolog IF5A enhance the peptidyl transfer step of elongation. Here, a superresolution fluorescence localization and tracking study of EF-P–mEos2 in live E. coli provides the first in vivo information about the spatial distribution and on-off binding kinetics of EF-P. Fast imaging at 2 ms/frame helps to distinguish ribosome-bound (slowly diffusing) EF-P from free (rapidly diffusing) EF-P. Wild-type EF-P exhibits a three-peaked axial spatial distribution similar to that of ribosomes, indicating substantial binding. The mutant EF-PK34A exhibits a homogeneous distribution, indicating little or no binding. Some 30% of EF-P copies are bound to ribosomes at a given time. Two-state modeling and copy number estimates indicate that EF-P binds to 70S ribosomes during 25 to 100% of translation cycles. The timescale of the typical diffusive search by free EF-P for a ribosome-binding site is τfree ≈ 16 ms. The typical residence time of an EF-P on the ribosome is very short, τbound ≈ 7 ms. Evidently, EF-P binds to ribosomes during many or most elongation cycles, much more often than the frequency of Pro-Pro motifs. Emptying of the E site during part of the cycle is consistent with recent in vitro experiments indicating dissociation of the deacylated tRNA upon translocation.

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Elongation factor P: Function and effects on bacterial fitness

Cited by 23 publications

References 78 publications

Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis

Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis

Cotranslational signal-independent SRP preloading during membrane targeting

Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live Escherichia coli

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