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  • Clinician Researcher
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  • PhD Student
  • Physician
  • Post-doc
  • Prize
  • Project Manager
  • Research Associate
  • Research Engineer
  • Retired scientist
  • Technician
  • Undergraduate Student
  • Veterinary
  • Visiting Scientist
  • Deputy Director of Center
  • Deputy Director of Department
  • Deputy Director of National Reference Center
  • Deputy Head of Facility
  • Director of Center
  • Director of Department
  • Director of Institute
  • Director of National Reference Center
  • Group Leader
  • Head of Facility
  • Head of Operations
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  • Honorary President of the Departement
  • Labex Coordinator
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About

In the lab, we are focusing on bacterial mechanisms of survival in hostile environments containing sub-lethal antibiotic concentrations.

The emergence of antibiotic resistance is increasingly associated with low antibiotic concentrations in the aquatic environment, such as lakes and rivers neighboring hospitals and pharmaceutical industries in certain countries, and in aquaculture farms, where antibiotics are regularly used. These environments are host to various bacteria (among which Vibrios). Low antibiotic concentrations can also be found in different compartments of the body upon antibiotic treatment: for example, in the lungs where they diffuse at different rates, or in the urine at the end of antibiotic treatment.

Research on antibiotic resistance usually focuses on resistance mechanism to high antibiotic doses which kill bacterial cells. Because we are studying the effects of low antibiotic doses, we can identify new targets involved in antibiotic resistance development.One new factor in antibiotic tolerance that we have recently identified involves bacterial RNA modifications.

RNA molecules constitute the template and machineries to produce proteins, therefore RNA modifications impact the production of bacterial proteins. Proteins are the building blocks of the cells and allow bacteria to survive.

Presence or absence of some RNA modifications confer, not resistance, but increased or decreased tolerance to antibiotics, which may lead to treatment failure, and development of antibiotic resistance. Our project addresses antimicrobial resistance, under the prism of the less well studied “observable resistance”, where populations of bacteria start tolerating antibiotics and become resistant, without the presence of known genetic resistance determinants in their chromosomes.

The diversity of RNA modifications, their specific influence on bacterial traits and behaviors, and the possibility that they are tuned by the conditions where bacteria grow, make them now a promising field of study.

Based on this, one can also envisage applying identified associations between RNA modifications and antibiotic resistance phenotypes, for the analysis of available bacterial collections with established genome sequences and resistance profiles, for which observed antibiotic resistance does not always correlate with known resistance factors.

Former Members

2000
2000
Name
Position
2019
2021
Anamaria Babosan
Post-doctoral researcher

Projects

Phenotypic adaptation to antibiotics: t/rRNA modifications

Free-living bacteria commonly cope with and adapt to changing environments. We have shown that various families of antibiotics are capable, at concentrations as low as 1% of the MIC, to activate stress responses in pathogenic Gram-negative bacteria from different genera, and that such responses can lead to bacterial adaptation and emergence of resistance to antibiotics. One such response is the SOS DNA damage response.

Our observation that antibiotics targeting translation (aminoglycosides, chloramphenicol, tetracycline) can induce SOS in various bacteria was intriguing.  We thus started to study the mechanisms of these inductions. We demonstrated that exposure to sub­-MIC of aminoglycosides was leading to oxidative DNA damage, sufficiently to trigger SOS in V. cholerae. Furthermore, we showed that the RpoS dependent general stress response plays a role in the protection against the stress caused by the presence of antibiotics.

We found that protection against this oxidative stress is more efficient in E. coli than in V. cholerae, due to a more efficient RpoS response. Importantly, we found that the unresponsiveness of E. coli to non-genotoxic antibiotics regarding SOS induction, was very specific to this species. These studies have thus revealed that V. cholerae could be a good model for proteobacteria for understanding the effects of low doses of antibiotics commonly found in the environment, and the mechanisms by which these molecules trigger antibiotic resistance.

To understand the mechanisms of the SOS induction by aminoglycosides in V. cholerae, we adopted genetic and high throughput approaches. These led to the identification of genes involved in transcription restart at R-loops, in the response to sub­MIC antibiotics.

The results also highlight the need for efficient translation transcription coupling upon exposure to sub-MIC aminoglycosides. Thus, sub­lethal aminoglycoside stress is sufficient to interfere with the DNA repair and replication machineries, and the RNA metabolism, through several processes: first through increased reactive oxygen species formation, which can directly damage DNA, but also through impaired translation (and transcription) which can lead to R-loop formation and DNA breaks, and general proteotoxic stress.

Sub-MIC aminoglycosides may not affect all ribosomes equally, leading to heterogeneity of responses within a clonal population. Single cell approaches would be complementary and suitable in future research to compare behaviors and responses at both sub-population/whole population levels.

The study of the response of bacteria to sub-MIC antibiotics can reveal new tolerance/resistance mechanisms to lethal doses. Studying the effects of non-lethal aminoglycoside concentrations, we uncovered a new role for RaiA in mediating ribosome protection under its 70S monomeric (active) form, which seems to enhance the appearance of persisters to aminoglycosides, thus at high antibiotic doses.

We have recently adopted parallel large-scale approaches to evaluate the effects of low doses of antibiotics from different families on gene expression (transcriptomic study through RNA-Seq) and the factors involved in the response to this stress (transposon insertion sequencing, (TN-Seq), allowing the search for genes that become essential or important for survival in the presence of low doses of antibiotics. Comparisons also allow the identification of common and specific responses to antibiotics with different modes of actions and targeting different processes.

We study the effects of sub-MIC antibiotic doses from 2% to 50% of the minimal inhibitory concentration (MIC) required to completely prevent bacterial growth. We also use data obtained from the study of sub-MICs to evaluate the involvement of identified pathways in tolerance to higher/lethal doses.

Until now, we have studied mostly the effects of sub-MICs of the aminoglycoside family of antibiotics, and occasionally fluoroquinolones for studies involving genotoxic stress. Aminoglycosides target the ribosome, leading to mistranslation and eventually cell death, whereas fluoroquinolones cause DNA damage and chromosome breakage. Both classes are bactericidal.

The preliminary data obtained by the above-mentioned approaches and projects started earlier allows us now to specifically focus on the role of epigenetics and RNA modifications in the response to antibiotic stress.

The goal of our project is to study the role of RNA modifications and variations in translation fidelity and efficiency in mediating the response to sub-MIC antibiotic stress. We to explore the link between sub-lethal antibiotic stress, t/rRNA modifications and modulation of translation fidelity/codon decoding, and contribution to antibiotic resistance.

We are especially interested in the role of queuosine modification in tRNA (phenotypically uncharacterized in prokaryotes) in the response to sub-lethal antibiotic stress, as well as dihydrouridine and pseudouridine.

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