Identification and characterization of pleiotropic high-persistence mutations in the beta subunit of the bacterial RNA polymerase
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ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE165446 | GEO | 2021/01/26
REPOSITORIES: GEO
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Project description:Mutations conferring resistance to bactericidal antibiotics reduce the average susceptibility of mutant populations. It is unknown, however, how those mutations affect the survival of individual bacteria. Since surviving bacteria can be a reservoir for recurring infections, it is important to know how survival rates may be affected by resistance mutations and by the choice of antibiotics. Here, we present evidence that (i) Escherichia coli mutants with 100 to 1,000 times increased frequency of survival in ciprofloxacin, an archetypal fluoroquinolone antibiotic, can be readily obtained in a stepwise selection; (ii) the high survival frequency is conferred by mutations in the switch region of the beta subunit of the RNA polymerase; (iii) the switch-region mutations are (p)ppGpp mimics, partially analogous to rpoB stringent mutations; (iv) the stringent and switch region rpoB mutations frequently occur in clinical isolates of E. coli, Acinetobacter baumannii, Mycobacterium tuberculosis, and Staphylococcus aureus, and at least one of them, RpoB S488L, which is a common rifampicin resistance mutations, dramatically increases the survival of a clinical methicillin-resistant S. aureus (MRSA) strain in ampicillin; and (v) the RpoB-associated high-survival phenotype can be reversed by subinhibitory concentrations of chloramphenicol.
| S-EPMC8522781 | biostudies-literature
Project description:Bacterial persistence, found in dormant and starved cells, is a health threat due to transient antibiotic tolerance. Harnessing a novel method for persister generation, we determined the proteome, metabolite levels and the physiology of E.coli persisters in and during entry into dormancy and starvation. In contrast to starved persisters, dormant persisters present in nutrient-rich conditions produced energy and grew, while both types had extremely low metabolite pools. The proteome of dormant cells governed by starvation response reached a unique state characterized by diminished anabolism, stress response and preservation of central metabolism protein levels. While starved cells approaches the same proteome, the limited carbon and energy source did not allow them to reach it, which caused their higher sensitivity to certain antibiotics. We present a conceptual model in which depleted metabolite pools resulting from initial persistence triggers provide a primitive, feed-forward starvation signal that sustains the growing persistent phenotype.
2016-09-27 | PXD001968 | Pride
Project description:Biofilms are heterogeneous bacterial communities featured by high persister prevalence, responsible for antibiotic tolerance. However, the mechanisms underlying persister formation within biofilms remained ambiguous. Here, by developing and utilizing a ribosomal RNA depleted bacterial single-cell RNA-seq method, RiboD-mSPLiT, we resolved biofilm heterogeneity and discovered pdeI as a marker gene for persister subgroup within biofilms. Remarkably, our findings elucidated that PdeI upregulates cellular levels of c-di-GMP, which acts as an antitoxin to modulate the toxicity of toxin protein HipH. HipH localizes on nucleoid and functions as a potent DNase, inducing cells into a viable but non-culturable state. Conversely, c-di-GMP interacts with HipH, reducing its genotoxic effects and enabling cells to enter a persister state, resulting in drug tolerance. Importantly, by targeting this toxin-antitoxin system, we repressed drug tolerance in Uropathogenic Escherichia coli infections, offering promising therapeutic strategies against chronic and relapsing infections.
2024-02-20 | GSE239504 | GEO
Project description:Biofilms are heterogeneous bacterial communities featured by high persister prevalence, responsible for antibiotic tolerance. However, the mechanisms underlying persister formation within biofilms remained ambiguous. Here, by developing and utilizing a ribosomal RNA depleted bacterial single-cell RNA-seq method, RiboD-mSPLiT, we resolved biofilm heterogeneity and discovered pdeI as a marker gene for persister subgroup within biofilms. Remarkably, our findings elucidated that PdeI upregulates cellular levels of c-di-GMP, which acts as an antitoxin to modulate the toxicity of toxin protein HipH. HipH localizes on nucleoid and functions as a potent DNase, inducing cells into a viable but non-culturable state. Conversely, c-di-GMP interacts with HipH, reducing its genotoxic effects and enabling cells to enter a persister state, resulting in drug tolerance. Importantly, by targeting this toxin-antitoxin system, we repressed drug tolerance in Uropathogenic Escherichia coli infections, offering promising therapeutic strategies against chronic and relapsing infections.
2024-02-20 | GSE239503 | GEO
Project description:Biofilms are heterogeneous bacterial communities featured by high persister prevalence, responsible for antibiotic tolerance. However, the mechanisms underlying persister formation within biofilms remained ambiguous. Here, by developing and utilizing a ribosomal RNA depleted bacterial single-cell RNA-seq method, RiboD-mSPLiT, we resolved biofilm heterogeneity and discovered pdeI as a marker gene for persister subgroup within biofilms. Remarkably, our findings elucidated that PdeI upregulates cellular levels of c-di-GMP, which acts as an antitoxin to modulate the toxicity of toxin protein HipH. HipH localizes on nucleoid and functions as a potent DNase, inducing cells into a viable but non-culturable state. Conversely, c-di-GMP interacts with HipH, reducing its genotoxic effects and enabling cells to enter a persister state, resulting in drug tolerance. Importantly, by targeting this toxin-antitoxin system, we repressed drug tolerance in Uropathogenic Escherichia coli infections, offering promising therapeutic strategies against chronic and relapsing infections.
2024-02-20 | GSE239502 | GEO
Project description:Persister cells are genetically identical variants in a bacterial population that have phenotypically modified their physiology to survive environmental stress. . To better define general persistence mechanisms that can be targeted to develop anti-persistence therapeutics, we performed transcriptomics analyses of Burkholderia thailandensis enriched for persisters using three methods: flow sorting by low proton motive force, meropenem treatment, and culture aging. Although the three persister populations generally expressed divergent gene expression profiles that underscores the multi-mechanistic nature of persistence, there were several common gene pathways expressed in two or more persister populations, including polyketide and non-ribosomal peptide synthesis, Clp proteases, mobile elements, enzymes involved in the lipid metabolism, and ATP-binding cassette (ABC) transporter systems. In particular, identification of genes that encode for polyketide synthases (PKS) and fatty acid catabolism indicate that generation of secondary metabolites, natural products, and complex lipids are needed for the maintenance of the persistence state. Persisters are markedly reduced in transposon mutants of the PKS gene BTH_I2366. Furthermore, treatment of multiple bacterial pathogens with the fatty acid synthesis inhibitor, CP-640186, potentiated antibiotic efficacy against the persisters. All together, our results suggest that bacterial persisters may exhibit an outward dormant physiology, but maintain active metabolic processes that are required to maintain persistence.
2019-03-06 | GSE127838 | GEO
Project description:Persister cells are a sub-population of all bacterial cultures which exhibit a non-inheritable, multi-drug tolerance when subjected to lethal antibiotic challenge. These persisters arise as a result of metabolic dormancy, and can resume growth subsequent to antibiotic challenge, leading to recalcitrance of bacterial infections. Overproduction of DosP, an oxygen sensing protein with phosphodiesterase activity, increases bacterial persistence. Here we performed a microarray to determine the expression profile induced by DosP as a means to elucidate mechanisms of persister cell formation. dosP was overexpressed in Escherichia coli K-12 BW25113 and compared to the empty vector.
2014-09-15 | E-GEOD-47427 | biostudies-arrayexpress
Project description:Persisters are a subpopulation of metabolically-dormant cells in biofilms that are resistant to antibiotics; hence, understanding persister cell formation is important for controlling bacterial infections. Previously we discerned that MqsR and MqsA of Escherichia coli are a toxin/antitoxin pair that influence persister cell production via their regulation of Hha, CspD, and HokA. Here, to gain more insights into the origin of persisters, we used protein engineering to increase the toxicity of toxin MqsR by reasoning it would be easier to understand the effect of this toxin if it were more toxic. We found that two mutations (K3N and N31Y) increase the toxicity four fold and increase persistence 73 fold compared to native MqsR by making the protein less labile. A whole transcriptome study revealed that the MqsR variant represses acid resistance genes (gadABCEWX and hdeABD), multidrug resistance genes (mdtEF), and osmotic resistance genes (osmEY). Corroborating these microarray results, deletion of rpoS as well as the genes that the master stress response regulator RpoS controls, gadB, gadX, mdtF, and osmY, increased persister formation dramatically to the extent that nearly the whole population became persistent. Therefore, the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS-based regulation of acid resistance, multidrug resistance, and osmotic resistance systems.
2012-04-03 | GSE31054 | GEO
Project description:Three types of phenotypic expression of ß-lactam resistance has been reported in MRSA: heterogeneous-, homogeneous-, and Eagle-type resistance. Heterogeneous to homogeneous (hetero-to-homo) conversion of ß-lactam resistance is postulated to be caused by a chromosomal mutation (chr*) together with mecA-gene expression. The Eagle-type resistance is a special pattern of chr* expression in the pre-MRSA strain N315 under the strong mecI-gene mediated repression of mecA gene transcription. Here, for the identification of chr*, experiments were conducted using an in-vitro derived homogeneously imipenem-resistant MRSA strain N315∆IPH5 (∆IPH5). The strain was selected with imipenem 8 mg/L from the heterogeneously imipenem-resistant MRSA strain N315∆IP (∆IP). The whole genome sequencing of ∆IPH5 revealed the presence of a unique mutation in the rpoB gene, rpoB(N967I), causing the amino-acid (AA) substitution of Asp by Ile at the 967th AA position of the RNA polymerase ß subunit. The effect of the mutation on the phenotypic change was confirmed by constructing and studying the phenotype of the strains H5rpoB(I967N), a ∆IPH5-derived strain cured of the rpoB mutation, and N315rpoB(N967I), a N315-derived strain introduced with the mutated rpoB gene. H5rpoB(I967N) regained the hetero-MRSA phenotype, and the mutant strain N315rpoB(N967I) showed an Eagle-type phenotype similar to that of N315h4. Furthermore, subsequent whole genome sequencing revealed that N315h4 also had a missense mutation in the rpoB gene rpoB(R644H). The rpoB mutations caused decreased autolysis, prolonged doubling-time, and tolerance to bactericidal concentrations of methicillin. We concluded that the certain rpoB mutations are chr* responsible for the hetero-to-homo phenotypic conversion of MRSA.
2013-08-27 | GSE42218 | GEO
Project description:Identification and characterization of pleiotropic high-persistence mutations in the beta subunit of the bacterial RNA polymerase
| PRJNA694627 | ENA