Genome-wide transcriptome analyses of E. coli treated with ribosome-targeting antibiotics
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ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE80251 | GEO | 2016/09/13
SECONDARY ACCESSION(S): PRJNA318398
REPOSITORIES: GEO
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Project description:Bacterial growth environment strongly influences the efficacy of antibiotic treatment, with slow growth often being associated with decreased susceptibility. Yet in many cases, the connection between antibiotic susceptibility and pathogen physiology remains unclear. We show that for ribosome-targeting antibiotics acting on Escherichia coli, a complex interplay exists between physiology and antibiotic action; for some antibiotics within this class, faster growth indeed increases susceptibility, but for other antibiotics, the opposite is true. Remarkably, these observations can be explained by a simple mathematical model that combines drug transport and binding with physiological constraints. Our model reveals that growth-dependent susceptibility is controlled by a single parameter characterizing the ‘reversibility’ of ribosome-targeting antibiotic transport and binding. This parameter provides a spectrum classification of antibiotic growth-dependent efficacy that appears to correspond at its extremes to existing binary classification schemes. In these limits, the model predicts universal, parameter-free limiting forms for growth inhibition curves. The model also leads to nontrivial predictions for the drug susceptibility of a translation mutant strain of E. coli, which we verify experimentally. Drug action and bacterial metabolism are mechanistically complex; nevertheless, this study illustrates how coarse-grained models can be used to integrate pathogen physiology into drug design and treatment strategies.
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Project description:The ribosome is one of the major targets in the cell for clinically used antibiotics. However, the increase in multidrug resistant bacteria is rapidly reducing the effectiveness of our current arsenal of ribosome-targeting antibiotics, highlighting the need for the discovery of compounds with new scaffolds that bind to novel sites on the ribosome. One possible avenue for the development of new antimicrobial agents is by characterization and optimization of ribosome-targeting peptide antibiotics. Biochemical and structural data on ribosome-targeting peptide antibiotics illustrates the large diversity of scaffolds, binding interactions with the ribosome as well as mechanism of action to inhibit translation. The availability of high-resolution structures of ribosomes in complex with peptide antibiotics opens the way to structure-based design of these compounds as novel antimicrobial agents.
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Project description:Two-component regulatory systems (TCSSs) are key regulatory elements responsible for the adaptation of bacteria to environmental stresses. A classical TCSS is typically comprised of a sensory histidine kinase and a corresponding response regulator. Here, we used homologous recombination to construct a Mycobacterium smegmatis mutant defective in the synthesis of cytosolic histidine kinase PdtaS (Msmeg_1918). The resulting ΔpdtaS mutant strain was tested in the Phenotype Microarray screening system, which allowed us to identify aminoglycoside antibiotic sensitivity, tetracyclines antibiotic resistance as well as membrane transport and respiration, as the main processes affected by removal of pdtaS. The antibiotic sensitivity profiles were confirmed by survival assessment and complementation studies. To gain insight into the molecular mechanisms responsible for the observed phenotype, we compared ribosomal RNA and protein profiles of the mutant and wild-type strains. We carried out Northern blotting and qRT-PCR to compare rRNA levels and analyzed ribosome sedimentation patterns of the wild-type and mutant strains on sucrose gradients. Isolated ribosomes were further used to estimate relative abundance of individual proteins in the ribosomal subunits using label free mass spectrometry analysis. Additionally, the ΔpdtaS mutant revealed lower activity of the respiratory chain as measured by the rate of TTC (triphenyltetrazolium chloride) reduction, while at the same time showing only insignificant changes in the uptake of aminoglycosides. We postulate that deficiency of PdtaS affects the oxidative respiration rates and ribosomal composition causing relevant changes to intrinsic resistance or susceptibility to antibiotics targeting ribosomes, which are commonly used to treat mycobacterial infections.
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Project description:Cationic intrinsically disordered antimicrobial peptides (CIDAMPs) belong to a novel class of epithelial peptide antibiotics with microbicidal activity against various pathogens, including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Here we show that treatment of distinct bacteria with different hornerin (HRNR)-derived CIDAMPs cause formation of unique cytoplasmic protein aggregates, suggesting a common intracellular mode of action. We further found that, unlike most amphipathic antimicrobial peptides, HRNR traverses bacterial membranes energy-dependently and accumulates within the cytoplasm. Strikingly, certain structurally different, HRNR-based CIDAMPs were found to bind to an identical panel of distinct bacterial ribosomal proteins, thereby manifesting features of several known classes of antibiotics. This may cause the formation of aberrant proteins and toxic protein aggregates in HRNR-treated pathogens which eventually may induce its death. Our study reveals evidence that structurally distinct CIDAMPs of an abundant body surface protein simultaneously target multiple sites of the bacterial protein synthesis machinery.
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Project description:Understanding how antibiotics inhibit bacteria can help to reduce antibiotic use and hence avoid antimicrobial resistance-yet few theoretical models exist for bacterial growth inhibition by a clinically relevant antibiotic treatment regimen. In particular, in the clinic, antibiotic treatment is time-dependent. Here, we use a theoretical model, previously applied to steady-state bacterial growth, to predict the dynamical response of a bacterial cell to a time-dependent dose of ribosome-targeting antibiotic. Our results depend strongly on whether the antibiotic shows reversible transport and/or low-affinity ribosome binding ('low-affinity antibiotic') or, in contrast, irreversible transport and/or high affinity ribosome binding ('high-affinity antibiotic'). For low-affinity antibiotics, our model predicts that growth inhibition depends on the duration of the antibiotic pulse, and can show a transient period of very fast growth following removal of the antibiotic. For high-affinity antibiotics, growth inhibition depends on peak dosage rather than dose duration, and the model predicts a pronounced post-antibiotic effect, due to hysteresis, in which growth can be suppressed for long times after the antibiotic dose has ended. These predictions are experimentally testable and may be of clinical significance.
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Project description:The rise of multidrug-resistant bacterial infections is a cause of global concern. There is an urgent need to both revitalize antibacterial agents that are ineffective due to resistance while concurrently developing new antibiotics with novel targets and mechanisms of action. Pathogen associated resistance-conferring ribosomal RNA (rRNA) methyltransferases are a growing threat that, as a group, collectively render a total of seven clinically-relevant ribosome-targeting antibiotic classes ineffective. Increasing frequency of identification and their growing prevalence relative to other resistance mechanisms suggests that these resistance determinants are rapidly spreading among human pathogens and could contribute significantly to the increased likelihood of a post-antibiotic era. Herein, with a view toward stimulating future studies to counter the effects of these rRNA methyltransferases, we summarize their prevalence, the fitness cost(s) to bacteria of their acquisition and expression, and current efforts toward targeting clinically relevant enzymes of this class.
| S-EPMC10131624 | biostudies-literature
Project description:BackgroundThe ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3' -CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity.ResultsThe present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3'-CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability.ConclusionBoth protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.
| S-EPMC4085065 | biostudies-literature
Project description:The present work examines the evolutionary trajectories of replicate Pseudomonas aeruginosa cultures in presence of the ribosome-targeting antibiotics tobramycin and tigecycline. It is known that large number of mutations across different genes - and therefore a large number of potential pathways - may be involved in resistance to any single antibiotic. Thus, evolution toward resistance might, to a large degree, rely on stochasticity, which might preclude the use of predictive strategies for fighting antibiotic resistance. However, the present results show that P. aeruginosa populations evolving in parallel in the presence of antibiotics (either tobramycin or tigecycline) follow a set of trajectories that present common elements. In addition, the pattern of resistance mutations involved include common elements for these two ribosome-targeting antimicrobials. This indicates that mutational evolution toward resistance (and perhaps other properties) is to a certain degree deterministic and, consequently, predictable. These findings are of interest, not just for P. aeruginosa, but in understanding the general rules involved in the evolution of antibiotic resistance also. In addition, the results indicate that bacteria can evolve toward higher levels of resistance to antibiotics against which they are considered to be intrinsically resistant, as tigecycline in the case of P. aeruginosa and that this may confer cross-resistance to other antibiotics of therapeutic value. Our results are particularly relevant in the case of patients under empiric treatment with tigecycline, which frequently suffer P. aeruginosa superinfections.
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Project description:RNA sequencing was performed on E. coli K12 MG1655 on three media (M9, CA-MHB, R10LB) treated with four antibiotics (Ciprofloxacin, Trimethoprim-sulfamethoxazole, Ceftriaxone, Meropenem) at their media-specific MIC90s
2021-08-05 | GSE159494 | GEO
Project description:Escherichia coli possesses three stalled-ribosome rescue factors, tmRNA·SmpB (primary factor), ArfA (alternative factor to tmRNA·SmpB), and ArfB. Here, we examined the susceptibility of rescue factor-deficient strains from E. coli SE15 to various ribosome-targeting antibiotics. Aminoglycosides specifically decreased the growth of the ΔssrA (tmRNA gene) strain, in which the levels of reactive oxygen species were elevated. The decrease in growth of ΔssrA could not be complemented by plasmid-borne expression of arfA, arfB, or ssrAAA to DD mutant gene possessing a proteolysis-resistant tag sequence. These results highlight the significance of tmRNA·SmpB-mediated proteolysis during growth under aminoglycoside stress. In contrast, tetracyclines or amphenicols decreased the growth of the ΔarfA strain despite the presence of tmRNA·SmpB. Quantitative RT-PCR revealed that tetracyclines and amphenicols, but not aminoglycosides, considerably induced mRNA expression of arfA. These findings indicate that tmRNA·SmpB, and ArfA exert differing functions during stalled-ribosome rescue depending on the type of ribosome-targeting antibiotic.
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