In order to gain new insight into the spatial organization of MreB proteins in bacterial cells, we are investigating the ultrastructure of MreB assemblies in Bacillus subtilis using super-resolution microscopy techniques. Two of these techniques (SIM-TIRF and STED) have allowed us to determine the length of MreB nanoassemblies in vivo and that their width is below 50-60 nm. We are currently using a super-resolution technique that goes further in resolution (PALM) to measure the width of MreB assemblies.
In order to gain new insight into the dynamics of MreB and associated proteins in the bacterial surface, we have transferred a new technology based on fluorescence correlation spectroscopy (FCS), which was developed mainly for eukaryotic research, to the field of microbiology. We have successfully implemented this technique, which allows to follow molecular dynamics with both spatial information and high statistics. We have now started to measure the dynamics of labelled proteins and lipids in the membrane of live bacterial cells.
To bridge the gap between cellular function and genetic information, it is also imperative to characterize the biochemical properties of the actin-like MreB proteins. We have succeed in purifying functional full-length MreB from B. subtilis and another Gram-positive bacterium, and shown that they polymerize into filaments as seen by electron microscopy. We also succeeded in obtaining the crystal structure of MreB in several nucleotide-bound states, and we have characterized MreB polymerization properties. In parallel, we are implementing methods for observation of MreB filaments in vitro using fluorescence microscopy. To this end, we have overcome the challenge of labelling purified MreB with fluorescent probes that do not affect MreB polymerization in vitro. Two different strategies are currently under development for single filament imaging.
We have also performed a screen for MreB inhibitors using two libraries of chemicals. There is a large set of chemicals available to characterize eukaryotic actin dynamics, including compounds that promote polymerisation, depolymerisation, stabilization or network dynamics, etc. However, to date no equivalent molecules have been identified for MreB from Gram-positive bacteria. Identification of such inhibitors would have a major impact both for the study of MreBs and for the identification of potential new antimicrobial drugs. We developed a reporter system for MreB loss-of-function and screened 2 560 compounds from two chemical libraries. 17 candidates specifically activated the reporter system while inhibiting cell growth. Interestingly, 2 of them were not previously known to have antibacterial effect, opening the way to their requalification. However, any of the 17 hits arising from this primary screen affected MreB polymerization or dynamics in the second and third levels of the screen involving fluorescence and electron microscopy, respectively. These assays should nevertheless optimized and thus further work is needed to conclude about the impact of the 17 candidate compounds on MreB.
Finally, we are taking advantage of the accessibility of antibiotics that specifically target different steps of cell wall biosynthesis to study their mode of action using an integrated approach that includes transcriptomics, chemical analysis of cell walls, and single-cell and single particle
imaging. We have performed an unprecedented, exhaustive transcriptomics analysis of cell wall perturbations as well as a systematic analysis of changes in cell wall composition upon antibiotic treatment. We have also monitored changes in cell morphology and growth, as well as the effect of the antibiotics on the MreB-associated cell wall synthesizing machineries. Overall, a huge amount of publishable data has been generated in this study, which will result in several publications and feed research in the lab for a good number of years.
