The outer membrane (OM) of Gram-negative bacteria plays a key role in virulence as it protects cells from physical and chemical stressors including several antibiotics. Preserving such permeability barrier is a particularly challenging task as the OM is constantly exposed to external stressor(1). Furthermore, during cell division, the OM undergoes profound remodeling leading to cell separation, which must occur without compromising the permeability barrier. We have recently characterized the divisome-associated virulence factor DolP of Enterobacteriaceae (formerly YraP) an OM lipoprotein critical to preserve the cell permeability barrier to detergents and antibiotics. We have shown that DolP is crucial for fitness in cells that undergo OM stress. We have also demonstrated that DolP interacts with BamA, a catalyzer of protein assembly in the OM that is upregulated under stress conditions. DolP promotes proper folding of BamA, thus supporting protein assembly in the OM(2). The underlying mechanism by which DolP functions remains unclear.

This project aims at elucidating the molecular mechanisms by which DolP functions using a combination of genetics, cell biology, proteomics, and structural mass spectrometry methods.

The PhD student, based at the Institute of Pharmacology and Structural Biology in the team ProteoToul (headed by Odile Schiltz) and at the neighboring Center of Integrative Biology, will be co-supervised by Julien Marcoux (IPBS, Toulouse) and Raffaele Ieva (CBI/LMGM, Toulouse). The long-lasting expertise in Mass Spectrometry of the ProteoToul group team is devoted to the understanding of biological processes and include methodological developments in large-scale quantitative proteomics and structural MS strategies(3-6). The group hosts the Proteomics facility of Toulouse, one of the three nodes of the French Proteomics Infrastructure (ProFI) and is equipped with state-of-the-art MS instrumentation associated to dedicated bioinformatics tools. The project will consist in using a combination of classical bottom-up proteomics with Structural MS methods, including native MS, hydrogen-deuterium exchange coupled to MS (HDX-MS) and top-down proteomics, to unravel the molecular mechanism of action of DolP. The student will integrate the mass-spectrometry results with phenotypic analysis of E. coli mutant cells, performing cell fitness experiments and in vivo functional assays to test and validate mechanistic models of DolP function directly in bacterial cells.

References

1. D. Ranava, A. Caumont-Sarcos, C. Albenne, R. Ieva, Bacterial machineries for the assembly of membrane-embedded beta-barrel proteins. FEMS microbiology letters 365, (2018).
2. D. Ranava et al., Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope stress. eLife 10, (2021).
3. V. Chaptal et al., Drug-bound and -free outward-facing structures of a multidrug ABC exporter point to a swing mechanism. 2021.2003.2012.435132 (2021).
4. J. Lesne et al., Conformational maps of human 20S proteasomes reveal PA28- and immuno-dependent inter-ring crosstalks. Nature communications 11, 6140 (2020).
5. J. Marcoux, C. V. Robinson, Twenty years of gas phase structural biology. Structure (London, England : 1993) 21, 1541-1550 (2013).
6. J. Marcoux et al., Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump. Proceedings of the National Academy of Sciences of the United States of America 110, 9704-9709 (2013).