PhD thesis defense in biological sciences by Marine Ote

Maintaining the barrier: cell envelope homeostasis balances stress resistance and fitness in Caulobacter vibrioides

Candidate

Marine Ote

Promoter

Prof. Jean-Yves Matroule, UNamur, Department of biology, Research Unit in Biology of Microorganisms (URBM)

Jury

• Prof. Francesco Renzi (UNamur), Président
• Prof. Jean-Yves Matroule (UNamur), Secrétaire
• Dr. Kathrin Fröhlich (Friedrich Schiller University, Jena, Germany)
• Prof. Patrick Viollier (University of Geneva, Switzerland)
• Prof. Liselot Dewachter (de Duve Institute, UCLouvain)

Summary

Antibiotic resistance is frequently driven by mutations that modify antibiotic targets, decrease membrane permeability, or enhance drug efflux. However, the physiological costs of overexpressing resistance mechanisms remain poorly understood. In this thesis, we investigate the fitness costs associated with constitutive overexpression of the RND efflux pump AcrAB2NodT in Caulobacter vibrioides. A ΔtipR mutant, which constitutively upregulates the RND efflux pump AcrAB2NodT, exhibits heightened sensitivity to copper, revealing a trade-off linked to excessive pump expression. Genetic deletion of acrAB2nodT or disruption of its transport activity restored cell envelope integrity and copper resistance, demonstrating that the observed vulnerability results from excessive pump activity rather than merely elevated protein abundance. Beyond copper, the ΔtipR mutant exhibited increased susceptibility to multiple transition metals, including zinc, nickel, and cadmium, highlighting a broader impact of efflux dysregulation on metal stress adaptation. Mechanistically, uncontrolled AcrAB2NodT activity imposed a significant energetic burden by dissipating the proton motive force and reducing cellular ATP levels, thereby compromising energy-dependent stress responses required for metal tolerance. These findings demonstrate that optimal efflux activity requires a delicate balance between resistance benefits and physiological costs. Importantly, this work reveals an additional regulatory layer controlling AcrAB2NodT expression. Although this pump was exclusively regulated at the transcriptional level by the TipR regulator, we identified a synonymous mutation in acrA that modulates pump levels through a post-transcriptional mechanism, enabling fine-tuning of efflux activity. This discovery highlights the complexity of bacterial adaptation and demonstrates that resistance determinants can be optimized through mechanisms beyond classical regulatory pathways. Finally, we show that exploiting the physiological vulnerabilities associated with efflux overactivation can enhance antibiotic activity. Combination treatment with copper and cefuroxime increased antibiotic potency by targeting membrane-related weaknesses in C. vibrioides. Overall, this thesis reveals the multifaceted role of efflux pumps beyond antibiotic resistance, demonstrating their central role in bacterial stress adaptation, metabolic homeostasis, and fitness optimization.