CE15 - Immunologie, Infectiologie et Inflammation

Looking for proteins regulating Peptidoglycan synthases – LookingForPegase

Looking for proteins regulating Peptidoglycan synthases

The characterization of the mode of action of the PBP regulators studied in this project will contribute to a better understanding of the inner-workings of bacterial cell morphogenesis. In applied terms, it will open new avenues towards the rational design of new antibacterial drugs and new strategies to combat bacterial infections, notably antibiotic resistant-strains.

toward the understanding of the bacterial cell wall assembly

The major objective of this project is to investigate the cellular function of new PBP regulators in the assembly of the bacterial cell wall using two different gram-positive bacterial models. In addition, we will also test if these regulators can affect bacterial resistance to ß-lactam antibiotics.

Combination of bacterial genetics, computational approaches and protein biochemistry, to cutting-edge imaging methods, including electron microscopy and single-molecule localization microscopy.

After 18 months, our efforts have essentially focused on the proteins CozEa, CozEb and TseB. In S. pneumoniae, our results show that the interplay between PBP1a and the cell size regulators CozEa and CozEb is required for the maintenance of cell size and shape. Analysis of muropeptides of cozE mutants is currently in progress to identify their structure. In B. subtilis, our preliminary observations suggest that one of the 3 CozE homologs regulates the activity of several PBPs and would be implicated in spore formation. Regarding TseB, we demonstrated that TseB is required for B. subtilis to achieve a proper cell shape. We also showed that TseB specifically interacts with the monofunctional transpeptidase PBP2a. In addition, we observed that TseB is also required for spore growth, a feature shared by PBP2a. We have recently tackled the function of the TseB homolog in S. pneumoniae. Our preliminary observations show that it localizes at the division septum where it specifically interacts with some PBPs. In addition, our analysis reveals that TseB is also required for the morphogenesis of the pneumococcal cell.
Since the beginning of the project, we have also developed a new method to label the new PG in S. pneumoniae and observed its synthesis by single-molecule localization microscopy. The unprecedented resolution and information provided has open new concepts for ovococci morphogenesis. This work lays foundations for the detailed characterization of peptidoglycan synthase regulators (TseB, MacP and CozE proteins). The same strategy is in progress for labeling B. subtilis peptidoglycan. Last, we have performed an in-silico analysis of cell division in around 1,000 genomes of firmicutes. This work expands the repertoire of proteins potentially involved in cell division and morphogenesis, suggesting new functional links, and paving the way for project follow-up.

Our project focusing on different regulators of the cell wall assembly in two unrelated bacteria should lead to a better understanding of the fundamental mechanism developed to spatially and temporally regulate this process during the cell cycle. Indeed, different actors and suites of reaction are at play to reach a proper cell-shape and to satisfy the respective developmental behaviors and mode of life of these different bacteria. The knowledge gained with these bacterial species will illustrate how nature has evolved a variety of mechanisms to achieve successful cell division and morphogenesis. This knowledge is also likely to open new concepts concerning the inner-workings of the cell wall assembly. By contributing to a deeper understanding of PG assembly in two model but unrelated bacteria, the present project is also expected to have a strong impact on the identification of novel targets, either individual proteins or specific protein-protein interactions, for therapeutic intervention against pathogenic bacteria. Importantly, it is clearly established that mutations in genes encoding PBPs are directly responsible for ß-lactam resistance of many strains. Developing strategies targeting regulators of PBPs could therefore represent a promising strategy to block the assembly of PG and re-sensitize these resistant strains.

1. Nanoscale Dynamics of Peptidoglycan Assembly during the Cell Cycle of Streptococcus pneumoniae. Trouve J, Zapun A, Arthaud C, Durmort C, Di Guilmi AM, Soederstroem B, Pelletier A, Grangeasse C, Bourgeois D, Wong Y-S, Morlot C. Current Biology (2021) S0960-9822(21)00576-5.
2. Impact of Serine/threonine kinases on the regulation of Sporulation in Bacillus subtilis. Pompéo F, Foulquier E and Galinier A, Frontiers in Microbiology (2021) Doi : 10.3389/fmicb.2021.697930
3. Recent progress in our understanding of peptidoglycan assembly in Firmicutes. Ducret and Grangeasse, Current Opinion in Microbiology (2021), 60, 44-50.
4. A comprehensive evolutionary scenario of cell division and associated processes in the firmicutes. Garcia PS, Duchemin W, Flandrois JP, Gribaldo S, Grangeasse C and Brochier-Armanet C. Molecular Biology and Evolution (2021) 38, 2396-2412.
5. A CozE homolog contributes to cell size homeostatis of Streptococcus pneumoniae. Stamsas GA, Restelli M, Ducret A, Freton C, Garcia PF, Havartstein LS, Straume D, Grangeasse C and Kjos M. mBio (2020) 11, e02461-20.

In bacteria, the cell wall is the key determinant of the shape and the physical integrity of the cell. The main component of this cell wall is the peptidoglycan (PG), a polymer that is continuously assemble during cell growth. Any defect in PG assembly is harmful for the viability of the bacterial cell. By consequence, it's not surprising that numerous antibiotics currently used to treat bacterial infections specifically target the enzymes involved in PG assembly, the Penicillin-Binding Proteins (PBPs). Among these antibiotics, one can cite the ß-lactams and the glycopeptides that are widely used nowadays. However, an increasing number of bacterial species are now resistant to these molecules. This issue represents one of the most serious problems in global health and the finding of new inhibitors of PG assembly is one of the most important challenges in medical research. The PBPs are a hallmark of PG assembly and they are required for the polymerization and cross-linking of the PG polymer. Little is known about the control of their activity during the bacterial cell growth but recent works suggest that the activity of PBPs could be regulated either negatively or positively by dedicated regulators. The main goal of this project is to identify new PBP regulators and to characterize their cellular function in two model bacteria, the human bacterial pathogen, Streptococcus pneumoniae, and the soil bacterium, Bacillus subtilis. These two gram-positive bacteria were chosen as models of study because they differ in their cell shape, developmental behavior and way of life. Indeed, B. subtilis is a rod-shape sporulating bacterium whereas S. pneumoniae is an ovoid-shape, non-sporulating pathogenic bacterium. It is thus extremely interesting to determine if the mode of action of these regulators found in the two bacteria is conserved or, by contrast, if the regulators operate differently in these two bugs. In addition, many genetic tools are available in these two species to perform our study. Another goal is to test if these regulators affect bacterial resistance to ß-lactams. Indeed, resistance to this type of antibiotics is often associated to mutations in PBPs of S. pneumoniae that is in the WHO list of priority pathogens for research and development of new antibiotics. Our preliminary observations suggest that several proteins (TseB, CozEa, CozEb, CozEc and MacP) likely serve as regulators of PBPs in these two bacteria. To reach our goals and to provide a comprehensive description of the role of these potential PBP regulators, we will focus on 3 specific aims. We plan to elaborate the interaction network and biochemical characterization of TseB, CozEa, CozEb, CozEc and MacP. We will also determine their physiological role, their spatio-temporal localization and their influence on the localization of PBPs. Last, we will analyze their impact on the assembly and the composition of PG and we will evaluate their impact on ß-lactam sensitivity in resistant strains. The project is established by four research groups with a clear leadership status and expert in the domain of cell morphogenesis and cell wall assembly in S. pneumoniae and B. subtilis. Our strategy will strongly rely on specific competencies of each partner and will benefit of the strong synergy already existing between our teams. To reach our goals, we will use an integrative combination of technics, going from bacterial genetics, computational approaches, biochemistry to time-lapse and electron microscopy and cutting-edge imaging methods. From a fundamental point of view, this project will contribute to a better understanding of the inner-workings of bacterial cell morphogenesis. In applied terms, it will open new avenues towards the rational design of new antibacterial drugs and new strategies to combat bacterial infections, notably by antibiotic resistant-strains.

Project coordinator

Monsieur Christophe GRANGEASSE (Microbiologie Moléculaire et Biochimie Structurale)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


LCB Laboratoire de chimie bactérienne
MICALIS MICrobiologie de l'ALImentation au service de la Santé
MMSB Microbiologie Moléculaire et Biochimie Structurale

Help of the ANR 485,446 euros
Beginning and duration of the scientific project: December 2019 - 36 Months

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