Periodic Reporting for period 4 - BACTIN (Shaping the bacterial cell wall: the actin-like cytoskeleton, from single molecules to morphogenesis and antimicrobials)
Reporting period: 2023-08-01 to 2025-01-31
Little is known about the mechanisms determining cell shape, and the larger questions concerning morphogenesis are the same in prokaryotic and eukaryotic systems. How is structural information acquired and maintained? How is cell shape spatially and temporally regulated? In bacteria, the external cell wall is the major determinant of cell shape. As a hallmark of microbial life, the cell wall is the most conspicuous macromolecule expanding in concert with bacterial cell growth. Because it is unique to bacteria, it is also the most prominent target for antibiotics. Despite decades of study, the mechanism of cell wall morphogenesis remains poorly understood. In rod-shaped bacteria, actin-like MreB proteins shape the cylindrical cell wall by orienting the movement of cell wall-synthesizing enzymes . However, the ultrastructure of MreB assemblies and the mechanistic details underlying their morphogenetic function remain to be elucidated. Also, the broad picture emerging is that MreB proteins significantly differ from eukaryotic actin, but their biochemical characterisation is still in its infancy.
The aim of BACTIN is to combine ground-breaking light microscopy and spectroscopy techniques with cutting-edge genetic, biochemical and systems biology approaches to elucidate how MreB and cell wall biosynthetic enzymes collectively act to build a bacterial cell. The overall objectives are to determine new features of MreB assemblies in vivo and in vitro, and to develop a “toolbox” of approaches to determine the modes of action of antibiotics targeting cell wall synthesis.
Research on the bacterial cytoskeleton is important because many mechanisms may be conserved in higher organisms, and bacteria are still privileged experimental systems for the exploration of basic molecular processes. Conversely, research on the bacteria-specific cell wall is also highly important
because it provides potential strategies for the development of antimicrobials at a time when multiple antibiotics resistance has become a major health concern.
The aim of BACTIN is to combine ground-breaking light microscopy and spectroscopy techniques with cutting-edge genetic, biochemical and systems biology approaches to elucidate how MreB and cell wall biosynthetic enzymes collectively act to build a bacterial cell. The overall objectives are to determine new features of MreB assemblies in vivo and in vitro, and to develop a “toolbox” of approaches to determine the modes of action of antibiotics targeting cell wall synthesis.
Research on the bacterial cytoskeleton is important because many mechanisms may be conserved in higher organisms, and bacteria are still privileged experimental systems for the exploration of basic molecular processes. Conversely, research on the bacteria-specific cell wall is also highly important
because it provides potential strategies for the development of antimicrobials at a time when multiple antibiotics resistance has become a major health concern.
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.
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.
Until the end of the project, we expect to completely resolve the ultrastructure of MreB assemblies in vivo and to elucidate the dynamics of MreB and cell wall enzymes in the bacterial membrane with unprecedented temporal resolution. In vitro, we will further characterize the biochemical and polymerization properties of MreB proteins, and we expect to overcome the challenge of imaging single MreB filaments and their dynamics in synthetic membrane systems. We will also revisit our second and third levels of screen for MreB inhibitors and perform another round of analysis with the 17 candidate compounds identified in our primary screen. In case of success, the most interesting candidate drugs could also be the subject of a downstream “hits to leads” program to identify, through combinatorial chemistry, highly specific and efficient molecules targeting MreB that could be patented as new families of antibiotics. Finally, our study of the effect of antibiotics targeting the cell wall will provide new insight into the process of cell wall biogenesis as well as into the modes of action of current antibiotics targeting it, while developing a “toolbox” of integrated approaches to study the effects of antimicrobials.
Altogether, we will expand the toolbox of high-resolution fluorescence-based approaches available in the field to go beyond the state-of-the art and obtain information on the actors involved in bacterial cell morphogenesis at unprecedented levels.
Altogether, we will expand the toolbox of high-resolution fluorescence-based approaches available in the field to go beyond the state-of-the art and obtain information on the actors involved in bacterial cell morphogenesis at unprecedented levels.