Session: Session 3

Control of bacterial permeability to antibiotics by a stress-induced envelope factor

Numerous multidrug resistant bacteria are Gram-negative. Their semi-permeable envelope consists of a multilayer including an inner membrane and an outer membrane (OM). The OM prevents entry of many antibiotics allowing nutrient diffusion. Its function requires integral OM proteins (OMPs) to be correctly assembled into the OM. The essential BAM complex (ß-Barrel Assembly Machinery) inserts OMPs into the OM and is thus crucial to preserve the OM permeability barrier. Using E. coli as a model organism, we have recently identified a 20kDa lipoprotein, DolP, that interacts with BAM and promotes proper folding and function of its central subunit BamA. DolP forms large clusters at midcell that are relocated under envelope stress or when BAM is upregulated.  Similar to BAM, DolP is crucial for OM integrity however its molecular mechanism is unknown.

Here I present an analysis of DolP alone or BAM-DolP complexes using multiple biochemical and structural mass-spectrometry (MS) approaches.  Upon purification of DolP and BAM as separate components and reconstitution of their interaction in detergent micelles, I isolated complexes by size exclusion chromatography to perform Hydrogen Deuterium exchange (HDX)-MS. This method allows the mapping of interactions sites and potential allosteric changes by comparing the solvent accessibility of proteins before and after formation of complexes and by identifying regions that are differentially exposed to deuterium exchange. Preliminary data reveal the HDX heat map of DolP, BAM and BAM-DolP complexes. Additional native MS and mass photometry assays will expand this structural characterization. To test the physiological role of these interactions, I will disrupt them by mutagenesis to study their contribution to BamA folding, BAM activity and maintenance of the E. coli OM permeability barrier.

In conclusion, combining powerful MS tools and in vivo physiological assays my project will help understand how bacteria resist to antibiotics.