Biology subjects

Wilmaerts, D.

Publications and source records attributed to Wilmaerts, D..

3 recordsLinked to original sources

bioRxiv2021

Transcription-coupled DNA repair underlies variation in persister awakening and the emergence of resistance

Persisters constitute a population of temporarily antibiotic-tolerant variants in an isogenic bacterial population and are considered an important cause of relapsing infections. It is currently unclear how cellular damage inflicted by antibiotic action is reversed upon persister state exit and how this relates to antibiotic resistance development. We demonstrate that persisters, upon fluoroquinolone treatment, accumulate oxidative damage which is repaired through nucleotide excision repair. Detection of the damage occurs via transcription-coupled repair using UvrD-mediated backtracking or Mfd-mediated displacement of the RNA polymerase. This competition results in heterogeneity in persister awakening lags. Most persisters repair the oxidative DNA damage, displaying a mutation rate equal to the untreated population. However, the promutagenic factor Mfd increases the mutation rate in a persister subpopulation. Our data provide in-depth insight in the molecular mechanisms underlying persister survival and pinpoints Mfd as an important molecular factor linking persistence to resistance development.

microbiology↗

bioRxiv2021

Alternative dimerization is required for activity and inhibition of the HEPN ribonuclease RnlA

The rnlAB toxin-antitoxin operon from Escherichia coli functions as an anti-phage defense system. RnlA was recently identified as a member of the HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) superfamily of ribonucleases. The activity of the toxin RnlA requires tight regulation by the antitoxin RnlB, the mechanism of which remains unknown. Here we show that RnlA exists in an equilibrium between two different homodimer states: an inactive resting state and an active canonical HEPN dimer. Mutants interfering with the transition between states show that canonical HEPN dimerization via the highly conserved RX4-6H motif is required for activity. The antitoxin RnlB binds the canonical HEPN dimer conformation, inhibiting RnlA by blocking access to its active site. Single-alanine substitutions mutants of the highly conserved R255, E258, R318 and H323 show that these residues are involved in catalysis and substrate binding and locate the catalytic site near the dimer interface of the canonical HEPN dimer rather than in a groove located between the HEPN domain and the preceding TBP-like domain. Overall, these findings elucidate the structural basis of the activity and inhibition of RnlA and highlight the crucial role of conformational heterogeneity in protein function.

biophysics↗

bioRxiv2021

The dynamic transition of persistence towards the VBNC state during stationary phase is driven by protein aggregation

Decades of research into bacterial persistence has been unable to fully characterize this antibiotic-tolerant phenotype, thereby hampering the development of therapies effective against chronic infections. Although some active persister mechanisms have been identified, the prevailing view is that cells become persistent because they enter a dormant state. We therefore characterized starvation-induced dormancy in Escherichia coli. Our findings indicate that dormancy develops gradually; persistence strongly increases during stationary phase and decreases again as persisters enter the viable but nonculturable (VBNC) state. Importantly, we show that dormancy development is tightly associated with progressive protein aggregation, which occurs concomitantly with ATP depletion during starvation. Persisters contain protein aggregates in an early developmental stage while VBNC cells carry more mature aggregates. Finally, we show that at least one persister protein, ObgE, works by triggering aggregation and thereby changing the dynamics of persistence and dormancy development. These findings provide evidence for a genetically-controlled, gradual development of persisters and VBNC cells through protein aggregation.

microbiology↗